Gkouskou K, Vasilogiannakopoulou T, Andreakos E, Davanos N, Gazouli M, Sanoudou D, Eliopoulos AG. COVID-19 enters the expanding network of apolipoprotein E4-related pathologies. Redox BiolRedox BiolRedox Biol. 2021;41:101938.Abstract
COVID-19 incidence and case fatality rates (CFR) differ among ethnicities, stimulating efforts to pinpoint genetic factors that could explain these phenomena. In this regard, the multiallelic apolipoprotein E (APOE) gene has recently been interrogated in the UK biobank cohort, demonstrating associations of the APOE epsilon4/epsilon4 genotype with COVID-19 severity and mortality. The frequency of the epsilon4 allele and thus the distribution of APOE epsilon4/epsilon4 genotype may differ among populations. We have assessed APOE genotypes in 1638 Greek individuals, based on haplotypes derived from SNP rs7412 and rs429358 and found reduced frequency of epsilon4/epsilon4 compared to the British cohort. Herein we discuss this finding in relation to CFR and hypothesize on the potential mechanisms linking APOE epsilon4/epsilon4 to severe COVID-19. We postulate that the metabolic deregulation ensued by APOE4, manifested by elevated cholesterol and oxidized lipoprotein levels, may be central to heightened pneumocyte susceptibility to infection and to exaggerated lung inflammation associated with the epsilon4/epsilon4 genotype. We also discuss putative dietary and pharmacological approaches for the prevention and management of COVID-19 in APOE epsilon4/epsilon4 individuals.
Valanti EK, Dalakoura-Karagkouni K, Siasos G, Kardassis D, Eliopoulos AG, Sanoudou D. Advances in biological therapies for dyslipidemias and atherosclerosis. MetabolismMetabolismMetabolism. 2021;116:154461.Abstract
Atherosclerosis is a multifactorial disease influenced by genetics, lifestyle and environmental factors. Despite therapeutic advances that reduce the risk of cardiovascular events, atherosclerosis-related diseases remain the leading cause of mortality worldwide. Precise targeting of genes involved in lipoprotein metabolism is an emerging approach for atherosclerosis prevention and treatment. This article focuses on the latest developments, clinical potential and current challenges of monoclonal antibodies, vaccines and genome/transcriptome modification strategies, including antisense oligonucleotides, genome/base editing and gene therapy. Multiple lipid lowering biological therapies have already been approved by the FDA with impressive results to date, while many more promising targets are being pursued in clinical trials or pre-clinical animal models.
Haghighi K, Gardner G, Vafiadaki E, Kumar M, Green LC, Ma J, Crocker JS, Koch S, Arvanitis DA, Bidwell P, et al. Impaired Right Ventricular Calcium Cycling Is an Early Risk Factor in R14del-Phospholamban Arrhythmias. J Pers MedJ Pers MedJ Pers Med. 2021;11.Abstract
The inherited mutation (R14del) in the calcium regulatory protein phospholamban (PLN) is linked to malignant ventricular arrhythmia with poor prognosis starting at adolescence. However, the underlying early mechanisms that may serve as prognostic factors remain elusive. This study generated humanized mice in which the endogenous gene was replaced with either human wild type or R14del-PLN and addressed the early molecular and cellular pathogenic mechanisms. R14del-PLN mice exhibited stress-induced impairment of atrioventricular conduction, and prolongation of both ventricular activation and repolarization times in association with ventricular tachyarrhythmia, originating from the right ventricle (RV). Most of these distinct electrocardiographic features were remarkably similar to those in R14del-PLN patients. Studies in isolated cardiomyocytes revealed RV-specific calcium defects, including prolonged action potential duration, depressed calcium kinetics and contractile parameters, and elevated diastolic Ca-levels. Ca-sparks were also higher although SR Ca-load was reduced. Accordingly, stress conditions induced after contractions, and inclusion of the CaMKII inhibitor KN93 reversed this proarrhythmic parameter. Compensatory responses included altered expression of key genes associated with Ca-cycling. These data suggest that R14del-PLN cardiomyopathy originates with RV-specific impairment of Ca-cycling and point to the urgent need to improve risk stratification in asymptomatic carriers to prevent fatal arrhythmias and delay cardiomyopathy onset.
Sousonis V, Sfakianaki T, Ntalianis A, Nanas I, Kontogiannis C, Aravantinos D, Kapelios C, Katsaros L, Nana M, Sampaziotis D, et al. Intracoronary Administration of Allogeneic Cardiosphere-Derived Cells Immediately Prior to Reperfusion in Pigs With Acute Myocardial Infarction Reduces Infarct Size and Attenuates Adverse Cardiac Remodeling. J Cardiovasc Pharmacol TherJ Cardiovasc Pharmacol TherJ Cardiovasc Pharmacol Ther. 2021;26:88-99.Abstract
BACKGROUND: Allogeneic cardiosphere-derived cells (CDCs) exert cardioprotective effects when administered intracoronarily after reperfusion in animal models of acute myocardial infarction (AMI). The "no-reflow" phenomenon develops rapidly post-reperfusion and may undermine the efficacy of cell therapy, due to poor cell delivery in areas of microvascular obstruction (MVO). We hypothesized that CDC-induced cardioprotection would be enhanced by cell administration prior to reperfusion, when microvasculature is still relatively intact, to facilitate widespread cell delivery within the ischemic area. METHODS AND RESULTS: We studied 81 farm pigs; 55 completed the specified protocols. A dose-optimization study in infarcted pigs demonstrated that the doses of 5 million and 10 million CDCs are the maximum safe doses that can be administered intracoronarily at 5 minutes prior to and at 5 minutes post-reperfusion, respectively, without aggravating MVO. Quantification of acute cell retention by polymerase chain reaction demonstrated that cell delivery prior to reperfusion resulted in higher cardiac cell retention compared to delivery post-reperfusion. We then performed a randomized, placebo-controlled study to assess the long-term efficacy of intracoronary infusion of 5 million allogeneic CDCs, delivered at 5 minutes prior to reperfusion, in a porcine model of AMI. The CDC therapy resulted in decreased scar size, improved regional systolic function, and attenuation of adverse cardiac remodeling (manifested as preserved global systolic function, preserved end-systolic volume, and decreased interstitial fibrosis) compared to placebo at 30 days post-MI. CONCLUSIONS: Dose-optimized intracoronary infusion of allogeneic CDCs prior to reperfusion in a porcine model of AMI is feasible, safe and confers long-term benefits.
Gkolfinopoulou C, Soukou F, Dafnis I, Kellici TF, Sanoudou D, Mavromoustakos T, Stratikos E, Chroni A. Structure-function analysis of naturally occurring apolipoprotein A-I L144R, A164S and L178P mutants provides insight on their role on HDL levels and cardiovascular risk. Cell Mol Life SciCell Mol Life SciCell Mol Life Sci. 2021;78:1523-1544.Abstract
Naturally occurring point mutations in apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein (HDL), may affect plasma HDL-cholesterol levels and cardiovascular risk. Here, we evaluated the effect of human apoA-I mutations L144R (associated with low HDL-cholesterol), L178P (associated with low HDL-cholesterol and increased cardiovascular risk) and A164S (associated with increased cardiovascular risk and mortality without low HDL-cholesterol) on the structural integrity and functions of lipid-free and lipoprotein-associated apoA-I in an effort to explain the phenotypes of subjects carrying these mutations. All three mutants, in lipid-free form, presented structural and thermodynamic aberrations, with apoA-I[L178P] presenting the greatest thermodynamic destabilization. Additionally, apoA-I[L178P] displayed reduced ABCA1-mediated cholesterol efflux capacity. When in reconstituted HDL (rHDL), apoA-I[L144R] and apoA-I[L178P] were more thermodynamically destabilized compared to wild-type apoA-I, both displayed reduced SR-BI-mediated cholesterol efflux capacity and apoA-I[L144R] showed severe LCAT activation defect. ApoA-I[A164S] was thermodynamically unaffected when in rHDL, but exhibited a series of functional defects. Specifically, it had reduced ABCG1-mediated cholesterol and 7-ketocholesterol efflux capacity, failed to reduce ROS formation in endothelial cells and had reduced capacity to induce endothelial cell migration. Mechanistically, the latter was due to decreased capacity of rHDL-apoA-I[A164S] to activate Akt kinase possibly by interacting with endothelial LOX-1 receptor. The impaired capacity of rHDL-apoA-I[A164S] to preserve endothelial function may be related to the increased cardiovascular risk for this mutation. Overall, our structure-function analysis of L144R, A164S and L178P apoA-I mutants provides insights on how HDL-cholesterol levels and/or atheroprotective properties of apoA-I/HDL are impaired in carriers of these mutations.
Gkouskou K, Vlastos I, Karkalousos P, Chaniotis D, Sanoudou D, Eliopoulos AG. The "Virtual Digital Twins" Concept in Precision Nutrition. Adv NutrAdv NutrAdv Nutr. 2020;11:1405-1413.Abstract
Nutritional and lifestyle changes remain at the core of healthy aging and disease prevention. Accumulating evidence underscores the impact of genetic, metabolic, and host gut microbial factors on individual responses to nutrients, paving the way for the stratification of nutritional guidelines. However, technological advances that incorporate biological, nutritional, lifestyle, and health data at an unprecedented scale and depth conceptualize a future where preventative dietary interventions will exceed stratification and will be highly individualized. We herein discuss how genetic information combined with longitudinal metabolomic, immune, behavioral, and gut microbial parameters, and bioclinical variables could define a digital replica of oneself, a "virtual digital twin," which could serve to guide nutrition in a personalized manner. Such a model may revolutionize the management of obesity and its comorbidities, and provide a pillar for healthy aging.
Efentakis P, Varela A, Chavdoula E, Sigala F, Sanoudou D, Tenta R, Gioti K, Kostomitsopoulos N, Papapetropoulos A, Tasouli A, et al. Levosimendan prevents doxorubicin-induced cardiotoxicity in time- and dose-dependent manner: implications for inotropy. Cardiovasc ResCardiovasc ResCardiovasc Res. 2020;116:576-591.Abstract
AIMS: Levosimendan (LEVO) a clinically-used inodilator, exerts multifaceted cardioprotective effects. Case-studies indicate protection against doxorubicin (DXR)-induced cardiotoxicity, but this effect remains obscure. We investigated the effect and mechanism of different regimens of levosimendan on sub-chronic and chronic doxorubicin cardiotoxicity. METHODS AND RESULTS: Based on preliminary in vivo experiments, rats serving as a sub-chronic model of doxorubicin-cardiotoxicity and were divided into: Control (N/S-0.9%), DXR (18 mg/kg-cumulative), DXR+LEVO (LEVO, 24 mug/kg-cumulative), and DXR+LEVO (acute) (LEVO, 24 mug/kg-bolus) for 14 days. Protein kinase-B (Akt), endothelial nitric oxide synthase (eNOS), and protein kinase-A and G (PKA/PKG) pathways emerged as contributors to the cardioprotection, converging onto phospholamban (PLN). To verify the contribution of PLN, phospholamban knockout (PLN-/-) mice were assigned to PLN-/-/Control (N/S-0.9%), PLN-/-/DXR (18 mg/kg), and PLN-/-/DXR+LEVO (ac) for 14 days. Furthermore, female breast cancer-bearing (BC) mice were divided into: Control (normal saline 0.9%, N/S 0.9%), DXR (18 mg/kg), LEVO, and DXR+LEVO (LEVO, 24 mug/kg-bolus) for 28 days. Echocardiography was performed in all protocols. To elucidate levosimendan's cardioprotective mechanism, primary cardiomyocytes were treated with doxorubicin or/and levosimendan and with N omega-nitro-L-arginine methyl ester (L-NAME), DT-2, and H-89 (eNOS, PKG, and PKA inhibitors, respectively); cardiomyocyte-toxicity was assessed. Single bolus administration of levosimendan abrogated DXR-induced cardiotoxicity and activated Akt/eNOS and cAMP-PKA/cGMP-PKG/PLN pathways but failed to exert cardioprotection in PLN-/- mice. Levosimendan's cardioprotection was also evident in the BC model. Finally, in vitro PKA inhibition abrogated levosimendan-mediated cardioprotection, indicating that its cardioprotection is cAMP-PKA dependent, while levosimendan preponderated over milrinone and dobutamine, by ameliorating calcium overload. CONCLUSION: Single dose levosimendan prevented doxorubicin cardiotoxicity through a cAMP-PKA-PLN pathway, highlighting the role of inotropy in doxorubicin cardiotoxicity.
Sorce S, Nuvolone M, Russo G, Chincisan A, Heinzer D, Avar M, Pfammatter M, Schwarz P, Delic M, Muller M, et al. Genome-wide transcriptomics identifies an early preclinical signature of prion infection. PLoS PathogPLoS PathogPLoS Pathog. 2020;16:e1008653.Abstract
The clinical course of prion diseases is accurately predictable despite long latency periods, suggesting that prion pathogenesis is driven by precisely timed molecular events. We constructed a searchable genome-wide atlas of mRNA abundance and splicing alterations during the course of disease in prion-inoculated mice. Prion infection induced PrP-dependent transient changes in mRNA abundance and processing already at eight weeks post inoculation, well ahead of any neuropathological and clinical signs. In contrast, microglia-enriched genes displayed an increase simultaneous with the appearance of clinical signs, whereas neuronal-enriched transcripts remained unchanged until the very terminal stage of disease. This suggests that glial pathophysiology, rather than neuronal demise, could be the final driver of disease. The administration of young plasma attenuated the occurrence of early mRNA abundance alterations and delayed signs in the terminal phase of the disease. The early onset of prion-induced molecular changes might thus point to novel biomarkers and potential interventional targets.
Gkouskou KK, Grammatikopoulou MG, Vlastos I, Sanoudou D, Eliopoulos AG. Genotype-guided dietary supplementation in precision nutrition. Nutr RevNutr RevNutr Rev. 2020.Abstract
Achieving adequate micronutrient status, while avoiding deficiencies, represents a challenge for people globally. Consequently, many individuals resort to oral nutrient supplementation (ONS) in order to correct suboptimal dietary intakes. Advances in the fields of nutrigenetics and nutritional genomics have identified differences in response to micronutrient supplementation according to genetic makeup, adding dietary supplement use to the clinician's toolkit in the precision nutrition era. This review focuses on published evidence linking genetic variants to the responses associated with some of the most popular dietary supplements. With an increasing number of health professionals becoming involved in the prescription of ONS, identifying and matching individuals to the appropriate dietary supplement according to their genotype is important for achieving optimal health benefits and micronutrient equilibrium, while reducing the adverse events and financial costs often associated with excessive ONS.
Vafiadaki E, Arvanitis DA, Eliopoulos AG, Kranias EG, Sanoudou D. The Cardioprotective PKA-Mediated Hsp20 Phosphorylation Modulates Protein Associations Regulating Cytoskeletal Dynamics. Int J Mol SciInt J Mol SciInt J Mol Sci. 2020;21.Abstract
The cytoskeleton has a primary role in cardiomyocyte function, including the response to mechanical stimuli and injury. The small heat shock protein 20 (Hsp20) conveys protective effects in cardiac muscle that are linked to serine-16 (Ser16) Hsp20 phosphorylation by stress-induced PKA, but the link between Hsp20 and the cytoskeleton remains poorly understood. Herein, we demonstrate a physical and functional interaction of Hsp20 with the cytoskeletal protein 14-3-3. We show that, upon phosphorylation at Ser16, Hsp20 translocates from the cytosol to the cytoskeleton where it binds to 14-3-3. This leads to dissociation of 14-3-3 from the F-actin depolymerization regulator cofilin-2 (CFL2) and enhanced F-actin depolymerization. Importantly, we demonstrate that the P20L Hsp20 mutation associated with dilated cardiomyopathy exhibits reduced physical interaction with 14-3-3 due to diminished Ser16 phosphorylation, with subsequent failure to translocate to the cytoskeleton and inability to disassemble the 14-3-3/CFL2 complex. The topological sequestration of Hsp20 P20L ultimately results in impaired regulation of F-actin dynamics, an effect implicated in loss of cytoskeletal integrity and amelioration of the cardioprotective functions of Hsp20. These findings underscore the significance of Hsp20 phosphorylation in the regulation of actin cytoskeleton dynamics, with important implications in cardiac muscle physiology and pathophysiology.
Papaioannou TG, Kalantzis C, Katsianos E, Sanoudou D, Vavuranakis M, Tousoulis D. Personalized Assessment of the Coronary Atherosclerotic Arteries by Intravascular Ultrasound Imaging: Hunting the Vulnerable Plaque. J Pers MedJ Pers MedJ Pers Med. 2019;9.Abstract
The term "vulnerable plaque" is commonly used to refer to an atherosclerotic plaque that is prone to rupture and the formation of thrombosis, which can lead to several cardiovascular and cerebrovascular events. Coronary artery atherosclerosis has a wide variety of different phenotypes among patients who may have a substantially variable risk for plaque rupture and cardiovascular events. Mounting evidence has proposed three distinctive histopathological mechanisms: plaque rupture, plaque erosion and calcified nodules. Studies have demonstrated the characteristics of plaques with high vulnerability such as the presence of a thin fibrous cap, a necrotic lipid-rich core, abundant infiltrating macrophages and neovascularization. However, traditional coronary angiographic imaging fails to determine plaque vulnerability features, and its ability to individualize treatment strategies is limited. In recent decades, catheter-based intravascular ultrasound imaging (IVUS) modalities have been developed to identify vulnerable plaques and ultimately vulnerable patients. The aim is to individualize prediction, prevention and treatment of acute coronary events based on the identification of specific features of high-risk atherosclerotic plaques, and to identify the most appropriate interventional procedures for their treatment. In this context, the aim of this review is to discuss how personalized assessment of coronary atherosclerotic arteries can be achieved by intravascular ultrasound imaging focusing on vulnerable plaque detection.
Nana-Leventaki E, Nana M, Poulianitis N, Sampaziotis D, Perrea D, Sanoudou D, Rontogianni D, Malliaras K. Cardiosphere-Derived Cells Attenuate Inflammation, Preserve Systolic Function, and Prevent Adverse Remodeling in Rat Hearts With Experimental Autoimmune Myocarditis. J Cardiovasc Pharmacol TherJ Cardiovasc Pharmacol TherJ Cardiovasc Pharmacol Ther. 2019;24:70-77.Abstract
BACKGROUND: Cardiosphere-derived cells (CDCs) have yielded promising efficacy signals in early-phase clinical trials of ischemic and nonischemic cardiomyopathy. The potential efficacy of CDCs in acute myocarditis, an inflammatory cardiomyopathy without effective therapy, remains unexplored. Given that CDCs produce regenerative, cardioprotective, anti-inflammatory, and anti-fibrotic effects (all of which could be beneficial in acute myocarditis), we investigated the efficacy of intracoronary delivery of CDCs in a rat model of experimental autoimmune myocarditis. METHODS: Lewis rats underwent induction of experimental autoimmune myocarditis by subcutaneous footpad injection of purified porcine cardiac myosin supplemented with Mycobacterium tuberculosis on days 1 and 7. On day 10, rats were randomly assigned to receive global intracoronary delivery of 500 000 CDCs or vehicle. Global intracoronary delivery was performed by injection of cells or vehicle into the left ventricular (LV) cavity during transient occlusion of the aortic root. Rats were euthanized 18 days after infusion. Cardiac volumes and systolic function were assessed by serial echocardiography, performed on days 1, 10, and 28. Myocardial inflammation, T-cell infiltration, and cardiac fibrosis were evaluated by histology. RESULTS: Experimental autoimmune myocarditis was successfully induced in 14/14 rats that completed follow-up. Left ventricular ejection fraction (LVEF) and volumes were comparable on days 1 and 10 between groups. CDC infusion resulted in increased LVEF (81.5% +/- 3% vs 65.4% +/- 8%, P < .001) and decreased LV end-systolic volume (43 +/- 15 vs 100 +/- 24 muL, P < .001) compared to placebo administration at 18 days post-infusion. Cardiosphere-derived cell infusion decreased myocardial inflammation (7.4% +/- 7% vs 20.7% +/- 4% of myocardium, P = .007), cardiac fibrosis (16.6% +/- 13% vs 38.1% +/- 3% of myocardium, P = .008), and myocardial T-cell infiltration (30.4 +/- 29 vs 125.8 +/- 49 cells per field, P = .005) at 18 days post-infusion compared to placebo administration. CONCLUSION: Intracoronary delivery of CDCs attenuates myocardial inflammation, T-cell infiltration, and fibrosis while preventing myocarditis-induced systolic dysfunction and adverse remodeling in rats with experimental autoimmune myocarditis.
Hof IE, van der Heijden JF, Kranias EG, Sanoudou D, de Boer RA, van Tintelen JP, van der Zwaag PA, Doevendans PA. Prevalence and cardiac phenotype of patients with a phospholamban mutation. Neth Heart JNeth Heart JNeth Heart J. 2019;27:64-69.Abstract
Pathogenic mutations in the phospholamban (PLN) gene may give rise to inherited cardiomyopathies due to its role in calcium homeostasis. Several PLN mutations have been identified, with the R14del mutation being the most prevalent cardiomyopathy-related mutation in the Netherlands. It is present in patients diagnosed with arrhythmogenic cardiomyopathy as well as dilated cardiomyopathy. Awareness of the phenotype of this PLN mutation is of great importance, since many carriers remain to be identified. Patients with the R14del mutation are characterised by older age at onset, low-voltage electrocardiograms and a high frequency of ventricular arrhythmias. Additionally, these patients have a poor prognosis often with left ventricular dysfunction and early-onset heart failure. Therefore, when there is a suspicion of a PLN mutation, cardiac and genetic screening is strongly recommended.
Valanti EK, Chroni A, Sanoudou D. The future of apolipoprotein E mimetic peptides in the prevention of cardiovascular disease. Curr Opin LipidolCurr Opin LipidolCurr Opin Lipidol. 2019;30:326-341.Abstract
PURPOSE OF REVIEW: This review aims to discuss the recent developments in the area of apolipoprotein E (apoE) mimetics and their therapeutic potential for treating cardiovascular disease, the leading cause of mortality worldwide. RECENT FINDINGS: Ongoing research efforts target the development of novel therapies that would not only reduce circulating levels of atherogenic lipoproteins, but could also increase high density lipoprotein cholesterol (HDL-C) levels and/or improve HDL function. Among them, synthetic peptides that mimic the structure of natural human apoE, a component of triglyceride-rich lipoproteins and HDL, have been designed and proven to be functionally similar to apoE. In specific, apoE mimetic peptides mediate hepatic clearance of circulating atherogenic lipoproteins, dramatically reduce plasma cholesterol, and lead to attenuation of atherosclerosis development in vivo. These peptides also exhibit pleiotropic antiatherogenic properties, such as macrophage cholesterol efflux capacity, as well as anti-inflammatory and antioxidative functions. SUMMARY: ApoE mimetics are undergoing preclinical and clinical evaluation with promising results to date that render them attractive candidates in cardiovascular disease prevention and treatment.
Chalatsa I, Arvanitis DA, Koulakiotis NS, Giagini A, Skaltsounis AL, Papadopoulou-Daifoti Z, Tsarbopoulos A, Sanoudou D. The Crocus sativus Compounds trans-Crocin 4 and trans-Crocetin Modulate the Amyloidogenic Pathway and Tau Misprocessing in Alzheimer Disease Neuronal Cell Culture Models. Front NeurosciFront NeurosciFront Neurosci. 2019;13:249.Abstract
Crocus sativus L. natural compounds have been extensively used in traditional medicine for thousands of years. Recent research evidence is now emerging in support of its therapeutic potential for different pathologies including neurodegenerative diseases. Herein, the C. sativus L. natural compounds trans-crocin 4 and trans-crocetin were selected for in depth molecular characterization of their potentially protective effects against Alzheimer's Disease (AD), utilizing two AD neuronal cell culture models (SH-SY5Y overexpressing APP and PC12 expressing hyperphosphorylated tau). Biologically relevant concentrations, ranging from 0.1 muM to 1 mM, applied for 24 h or 72 h, were well tolerated by differentiated wild type SH-SY5Y and PC12 cells. When tested on neuronally differentiated SH-SY5Y-APP both trans-crocin 4 and trans-crocetin had significant effects against amyloidogenic pathways. Trans-crocin 4 significantly decreased of beta-secretase, a key enzyme of the amyloidogenic pathway, and APP-C99, while it decreased gamma-secretases that generate toxic beta-amyloid peptides. Similarly, trans-crocetin treatment led to a reduction in beta- and gamma-secretases, as well as to accumulation of cellular AbetaPP. When tested on the neuronally differentiated PC12-htau cells, both compounds proved effective in suppressing the active forms of GSK3beta and ERK1/2 kinases, as well as significantly reducing total tau and tau phosphorylation. Collectively, our data demonstrate a potent effect of trans-crocin 4 and trans-crocetin in suppressing key molecular pathways of AD pathogenesis, rendering them a promising tool in the prevention and potentially the treatment of AD.
Papaioannou TG, Kalantzis C, Katsianos E, Sanoudou D, Vavuranakis M, Tousoulis D. Personalized assessment of the coronary atherosclerotic arteries by intravascular ultrasound imaging: Hunting the vulnerable plaque. Journal of Personalized Medicine [Internet]. 2019;9(1). Website
Abou-Saleh H, Zouein FA, El-Yazbi A, Sanoudou D, Raynaud C, Rao C, Pintus G, Dehaini H, Eid AH. The march of pluripotent stem cells in cardiovascular regenerative medicine. Stem Cell Res TherStem Cell Res TherStem Cell Res Ther. 2018;9:201.Abstract
Cardiovascular disease (CVD) continues to be the leading cause of global morbidity and mortality. Heart failure remains a major contributor to this mortality. Despite major therapeutic advances over the past decades, a better understanding of molecular and cellular mechanisms of CVD as well as improved therapeutic strategies for the management or treatment of heart failure are increasingly needed. Loss of myocardium is a major driver of heart failure. An attractive approach that appears to provide promising results in reducing cardiac degeneration is stem cell therapy (SCT). In this review, we describe different types of stem cells, including embryonic and adult stem cells, and we provide a detailed discussion of the properties of induced pluripotent stem cells (iPSCs). We also present and critically discuss the key methods used for converting somatic cells to pluripotent cells and iPSCs to cardiomyocytes (CMs), along with their advantages and limitations. Integrating and non-integrating reprogramming methods as well as characterization of iPSCs and iPSC-derived CMs are discussed. Furthermore, we critically present various methods of differentiating iPSCs to CMs. The value of iPSC-CMs in regenerative medicine as well as myocardial disease modeling and cardiac regeneration are emphasized.
Bidwell PA, Liu GS, Nagarajan N, Lam CK, Haghighi K, Gardner G, Cai WF, Zhao W, Mugge L, Vafiadaki E, et al. HAX-1 regulates SERCA2a oxidation and degradation. J Mol Cell CardiolJ Mol Cell CardiolJ Mol Cell Cardiol. 2018;114:220-233.Abstract
Ischemia/reperfusion injury is associated with contractile dysfunction and increased cardiomyocyte death. Overexpression of the hematopoietic lineage substrate-1-associated protein X-1 (HAX-1) has been shown to protect from cellular injury but the function of endogenous HAX-1 remains obscure due to early lethality of the knockout mouse. Herein we generated a cardiac-specific and inducible HAX-1 deficient model, which uncovered an unexpected role of HAX-1 in regulation of sarco/endoplasmic reticulum Ca-ATPase (SERCA2a) in ischemia/reperfusion injury. Although ablation of HAX-1 in the adult heart elicited no morphological alterations under non-stress conditions, it diminished contractile recovery and increased infarct size upon ischemia/reperfusion injury. These detrimental effects were associated with increased loss of SERCA2a. Enhanced SERCA2a degradation was not due to alterations in calpain and calpastatin levels or calpain activity. Conversely, HAX-1 overexpression improved contractile recovery and maintained SERCA2a levels. The regulatory effects of HAX-1 on SERCA2a degradation were observed at multiple levels, including intact hearts, isolated cardiomyocytes and sarcoplasmic reticulum microsomes. Mechanistically, HAX-1 ablation elicited increased production of reactive oxygen species at the sarco/endoplasic reticulum compartment, resulting in SERCA2a oxidation and a predisposition to its proteolysis. This effect may be mediated by NAPDH oxidase 4 (NOX4), a novel binding partner of HAX-1. Accordingly, NOX inhibition with apocynin abrogated the effects of HAX-1 ablation in hearts subjected to ischemia/reperfusion injury. Taken together, our findings reveal a role of HAX-1 in the regulation of oxidative stress and SERCA2a degradation, implicating its importance in calcium homeostasis and cell survival pathways.
Arvanitis DA, Vafiadaki E, Johnson DM, Kranias EG, Sanoudou D. The Histidine-Rich Calcium Binding Protein in Regulation of Cardiac Rhythmicity. Front PhysiolFront PhysiolFront Physiol. 2018;9:1379.Abstract
Sudden unexpected cardiac death (SCD) accounts for up to half of all-cause mortality of heart failure patients. Standardized cardiology tools such as electrocardiography, cardiac imaging, electrophysiological and serum biomarkers cannot accurately predict which patients are at risk of life-threatening arrhythmic episodes. Recently, a common variant of the histidine-rich calcium binding protein (HRC), the Ser96Ala, was identified as a potent biomarker of malignant arrhythmia triggering in these patients. HRC has been shown to be involved in the regulation of cardiac sarcoplasmic reticulum (SR) Ca(2+) cycling, by binding and storing Ca(2+) in the SR, as well as interacting with the SR Ca(2+) uptake and release complexes. The underlying mechanisms, elucidated by studies at the molecular, biochemical, cellular and intact animal levels, indicate that transversion of Ser96 to Ala results in abolishment of an HRC phosphorylation site by Fam20C kinase and dysregulation of SR Ca(2+) cycling. This is mediated through aberrant SR Ca(2+) release by the ryanodine receptor (RyR2) quaternary complex, due to the impaired HRC/triadin interaction, and depressed SR Ca(2+) uptake by the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA2) pump, due to the impaired HRC/SERCA2 interaction. Pharmacological intervention with KN-93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), in the HRC Ser96Ala mouse model, reduced the occurrence of malignant cardiac arrhythmias. Herein, we summarize the current evidence on the pivotal role of HRC in the regulation of cardiac rhythmicity and the importance of HRC Ser96Ala as a genetic modifier for arrhythmias in the setting of heart failure.
Valanti EK, Dalakoura-Karagkouni K, Sanoudou D. Current and Emerging Reconstituted HDL-apoA-I and HDL-apoE Approaches to Treat Atherosclerosis. J Pers MedJ Pers MedJ Pers Med. 2018;8.Abstract
Atherosclerosis affects millions of people worldwide. However, the wide variety of limitations in the current therapeutic options leaves much to be desired in future lipid-lowering therapies. For example, although statins, which are the first-line treatment for coronary heart disease (CHD), reduce the risk of cardiovascular events in a large percentage of patients, they lead to optimal levels of low density lipoprotein-cholesterol (LDL-C) in only about one-third of patients. A new promising research direction against atherosclerosis aims to improve lipoprotein metabolism. Novel therapeutic approaches are being developed to increase the levels of functional high density lipoprotein (HDL) particles. This review aims to highlight the atheroprotective potential of the in vitro synthesized reconstituted HDL particles containing apolipoprotein E (apoE) as their sole apolipoprotein component (rHDL-apoE). For this purpose, we provide: (1) a summary of the atheroprotective properties of native plasma HDL and its apolipoprotein components, apolipoprotein A-I (apoA-I) and apoE; (2) an overview of the anti-atherogenic functions of rHDL-apoA-I and apoA-I-containing HDL, i.e., natural HDL isolated from transgenic Apoa1(-/-) x Apoe(-/-) mice overexpressing human apoA-I (HDL-apoA-I); and (3) the latest developments and therapeutic potential of HDL-apoE and rHDL-apoE. Novel rHDL formulations containing apoE could possibly present enhanced biological functions, leading to improved therapeutic efficacy against atherosclerosis.
Theofilatos D, Fotakis P, Valanti E, Sanoudou D, Zannis V, Kardassis D. HDL-apoA-I induces the expression of angiopoietin like 4 (ANGPTL4) in endothelial cells via a PI3K/AKT/FOXO1 signaling pathway. MetabolismMetabolismMetabolism. 2018;87:36-47.Abstract
BACKGROUND: High Density Lipoprotein (HDL) and its main protein component, apolipoprotein A-I (apoA-I), have numerous atheroprotective functions on various tissues including the endothelium. Therapies based on reconstituted HDL containing apoA-I (rHDL-apoA-I) have been used successfully in patients with acute coronary syndrome, peripheral vascular disease or diabetes but very little is known about the genomic effects of rHDL-apoA-I and how they could contribute to atheroprotection. OBJECTIVE: The present study aimed to understand the endothelial signaling pathways and the genes that may contribute to rHDL-apoA-I-mediated atheroprotection. METHODS: Human aortic endothelial cells (HAECs) were treated with rHDL-apoA-I and their total RNA was analyzed with whole genome microarrays. Validation of microarray data was performed using multiplex RT-qPCR. The expression of ANGPTL4 in EA.hy926 endothelial cells was determined by RT-qPCR and Western blotting. The contribution of signaling kinases and transcription factors in ANGPTL4 gene regulation by HDL-apoA-I was assessed by RT-qPCR, Western blotting and immunofluorescence using chemical inhibitors or siRNA-mediated gene silencing. RESULTS: It was found that 410 transcripts were significantly changed in the presence of rHDL-apoA-I and that angiopoietin like 4 (ANGPTL4) was one of the most upregulated and biologically relevant molecules. In validation experiments rHDL-apoA-I, as well as natural HDL from human healthy donors or from transgenic mice overexpressing human apoA-I (TgHDL-apoA-I), increased ANGPTL4 mRNA and protein levels. ANGPTL4 gene induction by HDL was direct and was blocked in the presence of inhibitors for the AKT or the p38 MAP kinases. TgHDL-apoA-I caused phosphorylation of the transcription factor forkhead box O1 (FOXO1) and its translocation from the nucleus to the cytoplasm. Importantly, a FOXO1 inhibitor or a FOXO1-specific siRNA enhanced ANGPTL4 expression, whereas administration of TgHDL-apoA-I in the presence of the FOXO1 inhibitor or the FOXO1-specific siRNA did not induce further ANGPTL4 expression. These data suggest that FOXO1 functions as an inhibitor of ANGPTL4, while HDL-apoA-I blocks FOXO1 activity and induces ANGPTL4 through the activation of AKT. CONCLUSION: Our data provide novel insights into the global molecular effects of HDL-apoA-I on endothelial cells and identify ANGPTL4 as a putative mediator of the atheroprotective functions of HDL-apoA-I on the artery wall, with notable therapeutic potential.
Chalatsa I, Arvanitis DA, Mikropoulou EV, Giagini A, Papadopoulou-Daifoti Z, Aligiannis N, Halabalaki M, Tsarbopoulos A, Skaltsounis LA, Sanoudou D. Beneficial Effects of Sideritis scardica and Cichorium spinosum against Amyloidogenic Pathway and Tau Misprocessing in Alzheimer's Disease Neuronal Cell Culture Models. J Alzheimers DisJ Alzheimers DisJ Alzheimers Dis. 2018;64:787-800.Abstract
BACKGROUND: Natural products are a significantly underutilized source of potential treatments against human disease. Alzheimer's disease (AD) is a prime example of conditions that could be amenable to such treatments as suggested by recent findings. OBJECTIVE: Aiming to identify novel potentially therapeutic approaches against AD, we assessed the effects of Cichorium spinosum and Sideritis scardica extracts, both distinct components of the Mediterranean diet. METHODS/RESULTS: After the detailed characterization of the extracts' composition using LC-HRMS methods, they were evaluated on two AD neuronal cell culture models, namely the AbetaPP overexpressing SH-SY5Y-AbetaPP and the hyperphosphorylated tau expressing PC12-htau. Initially their effect on cell viability of SH-SY5Y and PC12 cells was examined, and subsequently their downstream effects on AbetaPP and tau processing pathways were investigated in the SH-SY5Y-AbetaPP and PC12-htau cells. We found that the S. scardica and C. spinosum extracts have similar effects on tau, as they both significantly decrease total tau, the activation of the GSK3beta, ERK1 and/or ERK2 kinases of tau, as well as tau hyperphosphorylation. Furthermore, both extracts appear to promote AbetaPP processing through the alpha, non-amyloidogenic pathway, albeit through partly different mechanisms. CONCLUSIONS: These findings suggest that C. spinosum and S. scardica could have a notable potential in the prevention and/or treatment of AD, and merit further investigations at the in vivo level.
Kalozoumi G, Kel-Margoulis O, Vafiadaki E, Greenberg D, Bernard H, Soreq H, Depaulis A, Sanoudou D. Glial responses during epileptogenesis in Mus musculus point to potential therapeutic targets. PLoS OnePLoS OnePLoS One. 2018;13:e0201742.Abstract
The Mesio-Temporal Lobe Epilepsy syndrome is the most common form of intractable epilepsy. It is characterized by recurrence of focal seizures and is often associated with hippocampal sclerosis and drug resistance. We aimed to characterize the molecular changes occurring during the initial stages of epileptogenesis in search of new therapeutic targets for Mesio-Temporal Lobe Epilepsy. We used a mouse model obtained by intra-hippocampal microinjection of kainate and performed hippocampal whole genome expression analysis at 6h, 12h and 24h post-injection, followed by multilevel bioinformatics analysis. We report significant changes in immune and inflammatory responses, neuronal network reorganization processes and glial functions, predominantly initiated during status epilepticus at 12h and persistent after the end of status epilepticus at 24h post-kainate. Upstream regulator analysis highlighted Cyba, Cybb and Vim as central regulators of multiple overexpressed genes implicated in glial responses at 24h. In silico microRNA analysis indicated that miR-9, miR-19b, miR-129, and miR-223 may regulate the expression of glial-associated genes at 24h. Our data support the hypothesis that glial-mediated inflammatory response holds a key role during epileptogenesis, and that microglial cells may participate in the initial process of epileptogenesis through increased ROS production via the NOX complex.
Liu GS, Zhu H, Cai WF, Wang X, Jiang M, Essandoh K, Vafiadaki E, Haghighi K, Lam CK, Gardner G, et al. Regulation of BECN1-mediated autophagy by HSPB6: Insights from a human HSPB6(S10F) mutant. AutophagyAutophagyAutophagy. 2018;14:80-97.Abstract
HSPB6/Hsp20 (heat shock protein family B [small] member 6) has emerged as a novel cardioprotector against stress-induced injury. We identified a human mutant of HSPB6 (HSPB6(S10F)) exclusively present in dilated cardiomyopathy (DCM) patients. Cardiac expression of this mutant in mouse hearts resulted in remodeling and dysfunction, which progressed to heart failure and early death. These detrimental effects were associated with reduced interaction of mutant HSPB6(S10F) with BECN1/Beclin 1, leading to BECN1 ubiquitination and its proteosomal degradation. As a result, autophagy flux was substantially inhibited and apoptosis was increased in HSPB6(S10F)-mutant hearts. In contrast, overexpression of wild-type HSPB6 (HSPB6 WT) not only increased BECN1 levels, but also competitively suppressed binding of BECN1 to BCL2, resulting in stimulated autophagy. Indeed, preinhibition of autophagy attenuated the cardioprotective effects of HSPB6 WT. Taken together, these findings reveal a new regulatory mechanism of HSPB6 in cell survival through its interaction with BECN1. Furthermore, Ser10 appears to be crucial for the protective effects of HSPB6 and transversion of this amino acid to Phe contributes to cardiomyopathy.
Tzimas C, Johnson DM, Santiago DJ, Vafiadaki E, Arvanitis DA, Davos CH, Varela A, Athanasiadis NC, Dimitriou C, Katsimpoulas M, et al. Impaired calcium homeostasis is associated with sudden cardiac death and arrhythmias in a genetic equivalent mouse model of the human HRC-Ser96Ala variant. Cardiovasc ResCardiovasc ResCardiovasc Res. 2017;113:1403-1417.Abstract
Aims: The histidine-rich calcium-binding protein (HRC) Ser96Ala variant has previously been identified as a potential biomarker for ventricular arrhythmias and sudden cardiac death in patients with idiopathic dilated cardiomyopathy. Herein, the role of this variant in cardiac pathophysiology is delineated through a novel mouse model, carrying the human mutation in the homologous mouse position. Methods and results: The mouse HRC serine 81, homologous to human HRC serine 96, was mutated to alanine, using knock-in gene targeting. The HRC-Ser81Ala mice presented increased mortality in the absence of structural or histological abnormalities, indicating that early death may be arrhythmia-related. Indeed, under stress-but not baseline-conditions, the HRC-Ser81Ala mice developed ventricular arrhythmias, whilst at the cardiomyocyte level they exhibited increased occurrence of triggered activity. Cardiac contraction was decreased in vivo, ex vivo, and in vitro. Additionally, Ca2+ transients and SR Ca2+ load were both reduced suggesting that cytosolic Ca2+ overload is not the underlying proarrhythmic mechanism. Interestingly, total SR Ca2+ leak was increased in HRC-Ser81Ala cardiomyocytes, without an increase in Ca2+ spark and wave frequency. However, Ca2+ wave propagation was significantly slower and the duration of the associated Na/Ca exchange current was increased. Moreover, action potential duration was also increased. Notably, Ca2+/Calmodulin kinase II (CaMKII) phosphorylation of the ryanodine receptor was increased, whilst KN-93, an inhibitor of CaMKII, reduced the occurrence of arrhythmias. Conclusions: The homologous mutation Ser81Ala in HRC in mice, corresponding to Ser96Ala in humans, is associated with sudden death and depressed cardiac function. Ventricular arrhythmias are related to abnormal Ca2+ cycling across the SR. The data further support a role for CaMKII with the perspective to treat arrhythmias through CaMKII inhibition.
Sorce S, Stocker R, Seredenina T, Holmdahl R, Aguzzi A, Chio A, Depaulis A, Heitz F, Olofsson P, Olsson T, et al. NADPH oxidases as drug targets and biomarkers in neurodegenerative diseases: What is the evidence?. Free Radic Biol MedFree Radic Biol MedFree Radic Biol Med. 2017;112:387-396.Abstract
Neurodegenerative disease are frequently characterized by microglia activation and/or leukocyte infiltration in the parenchyma of the central nervous system and at the molecular level by increased oxidative modifications of proteins, lipids and nucleic acids. NADPH oxidases (NOX) emerged as a novel promising class of pharmacological targets for the treatment of neurodegeneration due to their role in oxidant generation and presumably in regulating microglia activation. The unique function of NOX is the generation of superoxide anion (O2(*-)) and hydrogen peroxide (H2O2). However in the context of neuroinflammation, they present paradoxical features since O2(*-)/H2O2 generated by NOX and/or secondary reactive oxygen species (ROS) derived from O2(*-)/H2O2 can either lead to neuronal oxidative damage or resolution of inflammation. The role of NOX enzymes has been investigated in many models of neurodegenerative diseases by using either genetic or pharmacological approaches. In the present review we provide a critical assessment of recent findings related to the role of NOX in the CNS as well as how the field has advanced over the last 5 years. In particular, we focus on the data derived from the work of a consortium (Neurinox) funded by the European Commission's Programme 7 (FP7). We discuss the evidence gathered from animal models and human samples linking NOX expression/activity with neuroinflammation in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Creutzfeldt-Jakob disease as well as autoimmune demyelinating diseases like multiple sclerosis (MS) and chronic inflammatory demyelinating polyneuropathy (CIDP). We address the possibility to use measurement of the activity of the NOX2 isoform in blood samples as biomarker of disease severity and treatment efficacy in neurodegenerative disease. Finally we clarify key controversial aspects in the field of NOX, such as NOX cellular expression in the brain, measurement of NOX activity, impact of genetic deletion of NOX in animal models of neurodegeneration and specificity of NOX inhibitors.
Arvanitis DA, Vafiadaki E, Papalouka V, Sanoudou D. Muscle Lim Protein and myosin binding protein C form a complex regulating muscle differentiation. Biochim Biophys Acta Mol Cell ResBiochim Biophys Acta Mol Cell ResBiochim Biophys Acta Mol Cell Res. 2017;1864:2308-2321.Abstract
Muscle Lim Protein (MLP) is a protein with multiple functional roles in striated muscle physiology and pathophysiology. Herein, we demonstrate that MLP directly binds to slow, fast, and cardiac myosin-binding protein C (MyBP-C) during myogenesis, as shown by yeast two-hybrid and a range of protein-protein interaction assays. The minimal interacting domains involve MLP inter-LIM and MyBP-C [C4]. The interaction is sensitive to cytosolic Ca(2+) concentrations changes and to MyBP-C phosphorylation by PKA or CaMKII. Confocal microscopy of differentiating myoblasts showed MLP and MyBP-C colocalization during myoblast differentiation. Suppression of the complex formation with recombinant MyBP-C [C4] peptide overexpression, inhibited myoblast differentiation by 65%. Suppression of both MLP and MyBP-C expression in myoblasts by siRNA revealed negative synergistic effects on differentiation. The MLP/MyBP-C complex modulates the actin activated myosin II ATPase activity in vitro, which could interfere with sarcomerogenesis and myofilaments assembly during differentiation. Our data demonstrate a critical role of the MLP/MyBP-C complex during early myoblast differentiation. Its absence in muscles with mutations or aberrant expression of MLP or MyBP-C could be directly implicated in the development of cardiac and skeletal myopathies.
Pollak AJ, Haghighi K, Kunduri S, Arvanitis DA, Bidwell PA, Liu GS, Singh VP, Gonzalez DJ, Sanoudou D, Wiley SE, et al. Phosphorylation of serine96 of histidine-rich calcium-binding protein by the Fam20C kinase functions to prevent cardiac arrhythmia. Proc Natl Acad Sci U S AProc Natl Acad Sci U S AProc Natl Acad Sci U S A. 2017;114:9098-9103.Abstract
Precise Ca cycling through the sarcoplasmic reticulum (SR), a Ca storage organelle, is critical for proper cardiac muscle function. This cycling initially involves SR release of Ca via the ryanodine receptor, which is regulated by its interacting proteins junctin and triadin. The sarco/endoplasmic reticulum Ca ATPase (SERCA) pump then refills SR Ca stores. Histidine-rich Ca-binding protein (HRC) resides in the lumen of the SR, where it contributes to the regulation of Ca cycling by protecting stressed or failing hearts. The common Ser96Ala human genetic variant of HRC strongly correlates with life-threatening ventricular arrhythmias in patients with idiopathic dilated cardiomyopathy. However, the underlying molecular pathways of this disease remain undefined. Here, we demonstrate that family with sequence similarity 20C (Fam20C), a recently characterized protein kinase in the secretory pathway, phosphorylates HRC on Ser96. HRC Ser96 phosphorylation was confirmed in cells and human hearts. Furthermore, a Ser96Asp HRC variant, which mimics constitutive phosphorylation of Ser96, diminished delayed aftercontractions in HRC null cardiac myocytes. This HRC phosphomimetic variant was also able to rescue the aftercontractions elicited by the Ser96Ala variant, demonstrating that phosphorylation of Ser96 is critical for the cardioprotective function of HRC. Phosphorylation of HRC on Ser96 regulated the interactions of HRC with both triadin and SERCA2a, suggesting a unique mechanism for regulation of SR Ca homeostasis. This demonstration of the role of Fam20C-dependent phosphorylation in heart disease will open new avenues for potential therapeutic approaches against arrhythmias.
Bourdenx M, Koulakiotis NS, Sanoudou D, Bezard E, Dehay B, Tsarbopoulos A. Protein aggregation and neurodegeneration in prototypical neurodegenerative diseases: Examples of amyloidopathies, tauopathies and synucleinopathies. Prog NeurobiolProg NeurobiolProg Neurobiol. 2017;155:171-193.Abstract
Alzheimer's and Parkinson's diseases are the most prevalent neurodegenerative diseases that generate important health-related direct and indirect socio-economic costs. They are characterized by severe neuronal losses in several disease-specific brain regions associated with deposits of aggregated proteins. In Alzheimer's disease, beta-amyloid peptide-containing plaques and intraneuronal neurofibrillary tangles composed of hyperphosphorylated microtubule-associated protein tau are the two main neuropathological lesions, while Parkinson's disease is defined by the presence of Lewy Bodies that are intraneuronal proteinaceous cytoplasmic inclusions. alpha-Synuclein has been identified as a major protein component of Lewy Bodies and heavily implicated in the pathogenesis of Parkinson's disease. In the past few years, evidence has emerged to explain how these aggregate-prone proteins can undergo spontaneous self-aggregation, propagate from cell to cell, and mediate neurotoxicity. Current research now indicates that oligomeric forms are probably the toxic species. This article discusses recent progress in the understanding of the pathogenesis of these diseases, with a focus on the underlying mechanisms of protein aggregation, and emphasizes the pathophysiological molecular mechanisms leading to cellular toxicity. Finally, we present the putative direct link between beta-amyloid peptide and tau in causing toxicity in Alzheimer's disease as well as alpha-synuclein in Parkinson's disease, along with some of the most promising therapeutic strategies currently in development for those incurable neurodegenerative disorders.
Nuvolone M, Hermann M, Sorce S, Russo G, Tiberi C, Schwarz P, Minikel E, Sanoudou D, Pelczar P, Aguzzi A. Strictly co-isogenic C57BL/6J-Prnp-/- mice: A rigorous resource for prion science. J Exp MedJ Exp MedJ Exp Med. 2016;213:313-27.Abstract
Although its involvement in prion replication and neurotoxicity during transmissible spongiform encephalopathies is undisputed, the physiological role of the cellular prion protein (PrP(C)) remains enigmatic. A plethora of functions have been ascribed to PrP(C) based on phenotypes of Prnp(-/-) mice. However, all currently available Prnp(-/-) lines were generated in embryonic stem cells from the 129 strain of the laboratory mouse and mostly crossed to non-129 strains. Therefore, Prnp-linked loci polymorphic between 129 and the backcrossing strain resulted in systematic genetic confounders and led to erroneous conclusions. We used TALEN-mediated genome editing in fertilized mouse oocytes to create the Zurich-3 (ZH3) Prnp-ablated allele on a pure C57BL/6J genetic background. Genomic, transcriptional, and phenotypic characterization of Prnp(ZH3/ZH3) mice failed to identify phenotypes previously described in non-co-isogenic Prnp(-/-) mice. However, aged Prnp(ZH3/ZH3) mice developed a chronic demyelinating peripheral neuropathy, confirming the crucial involvement of PrP(C) in peripheral myelin maintenance. This new line represents a rigorous genetic resource for studying the role of PrP(C) in physiology and disease.
Tsompanidis A, Vafiadaki E, Bluher S, Kalozoumi G, Sanoudou D, Mantzoros CS. Ciliary neurotrophic factor upregulates follistatin and Pak1, causes overexpression of muscle differentiation related genes and downregulation of established atrophy mediators in skeletal muscle. MetabolismMetabolismMetabolism. 2016;65:915-25.Abstract
INTRODUCTION: The Ciliary Neurotrophic Factor (CNTF) is a pluripotent cytokine with anorexigenic actions in the hypothalamus that improves insulin sensitivity, increases energy expenditure and induces weight loss. Since CNTF also has an established myotrophic role, we sought to examine whether skeletal muscle contributes to the CNTF-induced metabolic improvement and identify the molecular mechanisms mediating these effects. METHODS: We used a mouse model of diet-induced obesity, to which high or low CNTF doses were administered for 7days. Whole transcriptome expression levels were analyzed in dissected soleus muscles using microarrays and data were then confirmed using qRT-PCR. RESULTS: We demonstrate that CNTF administration significantly downregulates leptin, while it upregulates follistatin and Pak1; a molecule associated with insulin sensitization in skeletal muscle. A significant overexpression of muscle differentiation related genes and downregulation of established atrophy mediators was observed. CONCLUSIONS: The overall gene expression changes suggest an indirect, beneficial effect of CNTF on metabolism, energy expenditure and insulin sensitivity, exerted by the pronounced stimulation of muscle growth, with similarities to the described effect of follistatin and the activation of the Akt pathway in skeletal muscle.
Bibli SI, Andreadou I, Chatzianastasiou A, Tzimas C, Sanoudou D, Kranias E, Brouckaert P, Coletta C, Szabo C, Kremastinos DT, et al. Cardioprotection by H2S engages a cGMP-dependent protein kinase G/phospholamban pathway. Cardiovasc ResCardiovasc ResCardiovasc Res. 2015;106:432-42.Abstract
AIMS: H2S is known to confer cardioprotection; however, the pathways mediating its effects in vivo remain incompletely understood. The purpose of the present study is to evaluate the contribution of cGMP-regulated pathways in the infarct-limiting effect of H2S in vivo. METHODS AND RESULTS: Anaesthetized rabbits were subjected to myocardial ischaemia (I)/reperfusion (R), and infarct size was determined in control or H2S-exposed groups. The H2S donor sodium hydrosulfide (NaHS, an agent that generates H2S) increased cardiac cGMP and reduced the infarct size. The cGMP-dependent protein kinase (PKG)-I inhibitor DT2 abrogated the protective effect of NaHS, whereas the control peptide TAT or l-nitroarginine methyl ester (l-NAME) did not alter the effect of NaHS. Moreover, the KATP channel inhibitor, glibenclamide, partially reversed the effects of NaHS, whereas inhibition of mitochondrial KATP did not modify the NaHS response. NaHS enhanced phosphorylation of phospholamban (PLN), in a PKG-dependent manner. To further investigate the role of PLN in H2S-mediated cardioprotection, wild-type and PLN KO mice underwent I/R. NaHS did not exert cardioprotection in PLN KO mice. Unlike what was observed in rabbits, genetic or pharmacological inhibition of eNOS abolished the infarct-limiting effect of NaHS in mice. CONCLUSIONS: Our findings demonstrate (i) that administration of NaHS induces cardioprotection via a cGMP/PKG/PLN pathway and (ii) contribution of nitric oxide to the H2S response is species-specific.
Vafiadaki E, Arvanitis DA, Sanoudou D. Muscle LIM Protein: Master regulator of cardiac and skeletal muscle functions. GeneGeneGene. 2015;566:1-7.Abstract
Muscle LIM Protein (MLP) has emerged as a key regulator of striated muscle physiology and pathophysiology. Mutations in cysteine and glycine-rich protein 3 (CSRP3), the gene encoding MLP, are causative of human cardiomyopathies, whereas altered expression patterns are observed in human failing heart and skeletal myopathies. In vitro and in vivo evidences reveal a complex and diverse functional role of MLP in striated muscle, which is determined by its multiple interacting partners and subcellular distribution. Experimental evidence suggests that MLP is implicated in both myogenic differentiation and myocyte cytoarchitecture, although the full spectrum of its intracellular roles still unfolds.
Tzimas C, Terrovitis J, Lehnart SE, Kranias EG, Sanoudou D. Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibition ameliorates arrhythmias elicited by junctin ablation under stress conditions. Heart RhythmHeart RhythmHeart Rhythm. 2015;12:1599-610.Abstract
BACKGROUND: Aberrant calcium signaling is considered one of the key mechanisms contributing to arrhythmias, especially in the context of heart failure. In human heart failure, there is significant down-regulation of the sarcoplasmic reticulum (SR) protein junctin, and junctin deficiency in mice is associated with stress-induced arrhythmias. OBJECTIVE: The purpose of this study was to determine whether the increased SR Ca(2+) leak and arrhythmias associated with junctin ablation may be associated with increased calcium/calmodulin-dependent protein kinase II (CaMKII) activity and phosphorylation of the cardiac ryanodine receptor (RyR2) and whether pharmacologic inhibition of CaMKII activity may prevent these arrhythmias. METHODS: Using a combination of biochemical, cellular, and in vivo approaches, we tested the ability of KN-93 to reverse aberrant CaMKII phosphorylation of RyR2. Specifically, we performed protein phosphorylation analysis, in vitro cardiomyocyte contractility and Ca(2+) kinetics, and in vivo ECG analysis in junctin-deficient mice. RESULTS: In the absence of junctin, RyR2 channels displayed CaMKII-dependent hyperphosphorylation. Notably, CaMKII inhibition by KN-93 reduced the in vivo incidence of stress-induced ventricular tachycardia by 65% in junctin null mice. At the cardiomyocyte level, KN-93 reduced the percentage of junctin null cells exhibiting spontaneous Ca(2+) aftertransients and aftercontractions under stress conditions by 35% and 37%, respectively. At the molecular level, KN-93 blunted the CaMKII-mediated hyperphosphorylation of RyR2 and phospholamban under stress conditions. CONCLUSION: Our data suggest that CaMKII inhibition is effective in preventing arrhythmogenesis in the setting of junctin ablation through modulation of both SR Ca(2+) release and uptake. Thus, it merits further investigation as promising molecular therapy.
Kaldara E, Sanoudou D, Adamopoulos S, Nanas JN. Outpatient management of chronic heart failure. Expert Opin PharmacotherExpert Opin PharmacotherExpert Opin Pharmacother. 2015;16:17-41.Abstract
INTRODUCTION: Heart failure (HF) treatment attracts a share of intensive research because of its poor HF prognosis. In the past decades, the prognosis of HF has improved considerably, mainly as a consequence of the progress that has been made in the pharmacological management of HF. AREAS COVERED: This article reviews the outpatient pharmacological management of chronic HF due to left ventricular systolic dysfunction and offers recommendations on the use of various drugs. In addition, the present article attempts to provide practical therapeutic algorithms based on current clinical strategies. EXPERT OPINION: Continued research directed toward identifying factors associated with high pharmacotherapy guideline adherence and understanding of variants that influence response to drugs will hopefully halt or reverse the major pathophysiological mechanisms involved in this syndrome.
Herrmann US, Sonati T, Falsig J, Reimann RR, Dametto P, O'Connor T, Li B, Lau A, Hornemann S, Sorce S, et al. Prion infections and anti-PrP antibodies trigger converging neurotoxic pathways. PLoS PathogPLoS PathogPLoS Pathog. 2015;11:e1004662.Abstract
Prions induce lethal neurodegeneration and consist of PrPSc, an aggregated conformer of the cellular prion protein PrPC. Antibody-derived ligands to the globular domain of PrPC (collectively termed GDL) are also neurotoxic. Here we show that GDL and prion infections activate the same pathways. Firstly, both GDL and prion infection of cerebellar organotypic cultured slices (COCS) induced the production of reactive oxygen species (ROS). Accordingly, ROS scavenging, which counteracts GDL toxicity in vitro and in vivo, prolonged the lifespan of prion-infected mice and protected prion-infected COCS from neurodegeneration. Instead, neither glutamate receptor antagonists nor inhibitors of endoplasmic reticulum calcium channels abolished neurotoxicity in either model. Secondly, antibodies against the flexible tail (FT) of PrPC reduced neurotoxicity in both GDL-exposed and prion-infected COCS, suggesting that the FT executes toxicity in both paradigms. Thirdly, the PERK pathway of the unfolded protein response was activated in both models. Finally, 80% of transcriptionally downregulated genes overlapped between prion-infected and GDL-treated COCS. We conclude that GDL mimic the interaction of PrPSc with PrPC, thereby triggering the downstream events characteristic of prion infection.
Haghighi K, Pritchard TJ, Liu GS, Singh VP, Bidwell P, Lam CK, Vafiadaki E, Das P, Ma J, Kunduri S, et al. Human G109E-inhibitor-1 impairs cardiac function and promotes arrhythmias. J Mol Cell CardiolJ Mol Cell CardiolJ Mol Cell Cardiol. 2015;89:349-59.Abstract
A hallmark of human and experimental heart failure is deficient sarcoplasmic reticulum (SR) Ca-uptake reflecting impaired contractile function. This is at least partially attributed to dephosphorylation of phospholamban by increased protein phosphatase 1 (PP1) activity. Indeed inhibition of PP1 by transgenic overexpression or gene-transfer of constitutively active inhibitor-1 improved Ca-cycling, preserved function and decreased fibrosis in small and large animal models of heart failure, suggesting that inhibitor-1 may represent a potential therapeutic target. We recently identified a novel human polymorphism (G109E) in the inhibitor-1 gene with a frequency of 7% in either normal or heart failure patients. Transgenic mice, harboring cardiac-specific expression of G109E inhibitor-1, exhibited decreases in contractility, Ca-kinetics and SR Ca-load. These depressive effects were relieved by isoproterenol stimulation. Furthermore, stress conditions (2Hz +/- Iso) induced increases in Ca-sparks, Ca-waves (60% of G109E versus 20% in wild types) and after-contractions (76% of G109E versus 23% of wild types) in mutant cardiomyocytes. Similar findings were obtained by acute expression of the G109E variant in adult cardiomyocytes in the absence or presence of endogenous inhibitor-1. The underlying mechanisms included reduced binding of mutant inhibitor-1 to PP1, increased PP1 activity, and dephosphorylation of phospholamban at Ser16 and Thr17. However, phosphorylation of the ryanodine receptor at Ser2808 was not altered while phosphorylation at Ser2814 was increased, consistent with increased activation of Ca/calmodulin-dependent protein kinase II (CaMKII), promoting aberrant SR Ca-release. Parallel in vivo studies revealed that mutant mice developed ventricular ectopy and complex ventricular arrhythmias (including bigeminy, trigeminy and ventricular tachycardia), when challenged with isoproterenol. Inhibition of CaMKII activity by KN-93 prevented the increased propensity to arrhythmias. These findings suggest that the human G109E inhibitor-1 variant impairs SR Ca-cycling and promotes arrhythmogenesis under stress conditions, which may present an additional insult in the compromised function of heart failure carriers.
Karakikes I, Stillitano F, Nonnenmacher M, Tzimas C, Sanoudou D, Termglinchan V, Kong CW, Rushing S, Hansen J, Ceholski D, et al. Correction of human phospholamban R14del mutation associated with cardiomyopathy using targeted nucleases and combination therapy. Nat CommunNat CommunNat Commun. 2015;6:6955.Abstract
A number of genetic mutations is associated with cardiomyopathies. A mutation in the coding region of the phospholamban (PLN) gene (R14del) is identified in families with hereditary heart failure. Heterozygous patients exhibit left ventricular dilation and ventricular arrhythmias. Here we generate induced pluripotent stem cells (iPSCs) from a patient harbouring the PLN R14del mutation and differentiate them into cardiomyocytes (iPSC-CMs). We find that the PLN R14del mutation induces Ca(2+) handling abnormalities, electrical instability, abnormal cytoplasmic distribution of PLN protein and increases expression of molecular markers of cardiac hypertrophy in iPSC-CMs. Gene correction using transcription activator-like effector nucleases (TALENs) ameliorates the R14del-associated disease phenotypes in iPSC-CMs. In addition, we show that knocking down the endogenous PLN and simultaneously expressing a codon-optimized PLN gene reverses the disease phenotype in vitro. Our findings offer novel strategies for targeting the pathogenic mutations associated with cardiomyopathies.
Sanoudou D, Kolokathis F, Arvanitis D, Al-Shafai K, Krishnamoorthy N, Buchan RJ, Walsh R, Tsiapras D, Barton PJ, Cook SA, et al. Genetic modifiers to the PLN L39X mutation in a patient with DCM and sustained ventricular tachycardia?. Glob Cardiol Sci PractGlob Cardiol Sci PractGlob Cardiol Sci Pract. 2015;2015:29.
Pitychoutis PM, Sanoudou D, Papandreou M, Nasias D, Kouskou M, Tomlinson CR, Tsonis PA, Papadopoulou-Daifoti Z. Forced swim test induces divergent global transcriptomic alterations in the hippocampus of high versus low novelty-seeker rats. Hum GenomicsHum GenomicsHum Genomics. 2014;8:4.Abstract
BACKGROUND: Many neuropsychiatric disorders, including stress-related mood disorders, are complex multi-parametric syndromes. Susceptibility to stress and depression is individually different. The best animal model of individual differences that can be used to study the neurobiology of affect regards spontaneous reactions to novelty. Experimentally, when naive rats are exposed to the stress of a novel environment, they display a highly variable exploratory activity and are classified as high or low responders (HR or LR, respectively). Importantly, HR and LR rats do not seem to exhibit a substantial differentiation in relation to their 'depressive-like' status in the forced swim test (FST), a widely used animal model of 'behavioral despair'. In the present study, we investigated whether FST exposure would be accompanied by phenotype-dependent differences in hippocampal gene expression in HR and LR rats. RESULTS: HR and LR rats present a distinct behavioral pattern in the pre-test session but develop comparable depressive-like status in the second FST session. At 24 h following the second FST session, HR and LR rats (stressed and unstressed controls) were sacrificed and hippocampal samples were independently analyzed on whole rat genome Illumina arrays. Functional analysis into pathways and networks was performed using Ingenuity Pathway Analysis (IPA) software. Notably, hippocampal gene expression signatures between HR and LR rats were markedly divergent, despite their comparable depressive-like status in the FST. These molecular differences are reflected in both the extent of transcriptional remodeling (number of significantly changed genes) and the types of molecular pathways affected following FST exposure. A markedly higher number of genes (i.e., 2.28-fold) were statistically significantly changed following FST in LR rats, as compared to their HR counterparts. Notably, genes associated with neurogenesis and synaptic plasticity were induced in the hippocampus of LR rats in response to FST, whereas in HR rats, FST induced pathways directly or indirectly associated with induction of apoptotic mechanisms. CONCLUSIONS: The markedly divergent gene expression signatures exposed herein support the notion that the hippocampus of HR and LR rats undergoes distinct transcriptional remodeling in response to the same stress regimen, thus yielding a different FST-related 'endophenotype', despite the seemingly similar depressive-like phenotype.
Kalozoumi G, Yacoub M, Sanoudou D. MicroRNAs in heart failure: Small molecules with major impact. Glob Cardiol Sci PractGlob Cardiol Sci PractGlob Cardiol Sci Pract. 2014;2014:79-102.Abstract
MicroRNAs (miRNAs) have emerged as potent modulators of mammalian gene expression, thereby broadening the spectrum of molecular mechanisms orchestrating human physiological and pathological cellular functions. Growing evidence suggests that these small non-coding RNA molecules are pivotal regulators of cardiovascular development and disease. Importantly, multiple miRNAs have been specifically implicated in the onset and progression of heart failure, thus providing a new platform for battling this multi-faceted disease. This review introduces the basic concepts of miRNA biology, describes representative examples of miRNAs associated with multiple aspects of HF pathogenesis, and explores the prognostic, diagnostic and therapeutic potential of miRNAs in the cardiology clinic.
Vafiadaki E, Arvanitis DA, Papalouka V, Terzis G, Roumeliotis TI, Spengos K, Garbis SD, Manta P, Kranias EG, Sanoudou D. Muscle lim protein isoform negatively regulates striated muscle actin dynamics and differentiation. FEBS JFEBS JFEBS J. 2014;281:3261-79.Abstract
Muscle lim protein (MLP) has emerged as a critical regulator of striated muscle physiology and pathophysiology. Mutations in cysteine and glycine-rich protein 3 (CSRP3), the gene encoding MLP, have been directly associated with human cardiomyopathies, whereas aberrant expression patterns are reported in human cardiac and skeletal muscle diseases. Increasing evidence suggests that MLP has an important role in both myogenic differentiation and myocyte cytoarchitecture, although the full spectrum of its intracellular roles has not been delineated. We report the discovery of an alternative splice variant of MLP, designated as MLP-b, showing distinct expression in neuromuscular disease and direct roles in actin dynamics and muscle differentiation. This novel isoform originates by alternative splicing of exons 3 and 4. At the protein level, it contains the N-terminus first half LIM domain of MLP and a unique sequence of 22 amino acids. Physiologically, it is expressed during early differentiation, whereas its overexpression reduces C2C12 differentiation and myotube formation. This may be mediated through its inhibition of MLP/cofilin-2-mediated F-actin dynamics. In differentiated striated muscles, MLP-b localizes to the sarcomeres and binds directly to Z-disc components, including alpha-actinin, T-cap and MLP. The findings of the present study unveil a novel player in muscle physiology and pathophysiology that is implicated in myogenesis as a negative regulator of myotube formation, as well as in differentiated striated muscles as a contributor to sarcomeric integrity.
Valanti E, Tsompanidis A, Sanoudou D. Pharmacogenomics in the development and characterization of atheroprotective drugs. Methods Mol BiolMethods Mol BiolMethods Mol Biol. 2014;1175:259-300.Abstract
Atherosclerosis is the main cause of cardiovascular disease (CVD) and can lead to stroke, myocardial infarction, and death. The clinically available atheroprotective drugs aim mainly at reducing the levels of circulating low-density lipoprotein (LDL), increasing high-density lipoprotein (HDL), and attenuating inflammation. However, the cardiovascular risk remains high, along with morbidity, mortality, and incidence of adverse drug events. Pharmacogenomics is increasingly contributing towards the characterization of existing atheroprotective drugs, the evaluation of novel ones, and the identification of promising, unexplored therapeutic targets, at the global molecular pathway level. This chapter presents highlights of pharmacogenomics investigations and discoveries that have contributed towards the elucidation of pharmacological atheroprotection, while opening the way to new therapeutic approaches.
Totary-Jain H, Sanoudou D, Ben-Dov IZ, Dautriche CN, Guarnieri P, Marx SO, Tuschl T, Marks AR. Reprogramming of the microRNA transcriptome mediates resistance to rapamycin. J Biol ChemJ Biol ChemJ Biol Chem. 2013;288:6034-44.Abstract
The mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation that is often deregulated in cancer. Inhibitors of mTOR, including rapamycin and its analogues, are being evaluated as antitumor agents. For their promise to be fulfilled, it is of paramount importance to identify the mechanisms of resistance and develop novel therapies to overcome it. Given the emerging role of microRNAs (miRNAs) in tumorigenesis, we hypothesized that miRNAs could play important roles in the response of tumors to mTOR inhibitors. Long-term rapamycin treatment showed extensive reprogramming of miRNA expression, characterized by up-regulation of miR-17-92 and related clusters and down-regulation of tumor suppressor miRNAs. Inhibition of members of the miR-17-92 clusters or delivery of tumor suppressor miRNAs restored sensitivity to rapamycin. This study identifies miRNAs as new downstream components of the mTOR-signaling pathway, which may determine the response of tumors to mTOR inhibitors. It also identifies potential markers to assess the efficacy of treatment and provides novel therapeutic targets to treat rapamycin-resistant tumors.
Pitychoutis PM, Kokras N, Sanoudou D, Dalla C, Papadopoulou-Daifoti Z. Pharmacogenetic considerations for late life depression therapy. Expert Opin Drug Metab ToxicolExpert Opin Drug Metab ToxicolExpert Opin Drug Metab Toxicol. 2013;9:989-99.Abstract
INTRODUCTION: Geriatric depression is a heterogeneous disorder with a complex genetic background. Current first-line treatment of depression is associated with a lower therapeutic outcome in aged depressed patients, when compared to younger subjects. Research which has explored this inadequate response has highlighted several factors which have come into play with the pharmacogenetics of antidepressants in the elderly being a particular area of interest. AREAS COVERED: The authors perform a critical review of the English language articles from PubMed using search terms such as late-life/geriatric depression, antidepressants, pharmacogenetics, pharmacogenomics, pharmacokinetic, genetic, genotype, remission, therapy, treatment and polymorphism. EXPERT OPINION: The emerging clinical and pharmacogenetic data are slowly unveiling the importance of the genome - age interaction in antidepressant response. This data introduces a critical new parameter in personalized medicine. A profound analysis of the age factor in the pharmacogenetics of antidepressant response is imperative, in order to elucidate the clinical significance of these findings and thereby improve patient treatment in the elderly.
Vafiadaki E, Arvanitis DA, Sanoudou D, Kranias EG. Identification of a protein phosphatase-1/phospholamban complex that is regulated by cAMP-dependent phosphorylation. PLoS OnePLoS OnePLoS One. 2013;8:e80867.Abstract
In human and experimental heart failure, the activity of the type 1 phosphatase is significantly increased, associated with dephosphorylation of phospholamban, inhibition of the sarco(endo)plasmic reticulum Ca(2+) transport ATPase (SERCA2a) and depressed function. In the current study, we investigated the molecular mechanisms controlling protein phosphatase-1 activity. Using recombinant proteins and complementary in vitro binding studies, we identified a multi-protein complex centered on protein phosphatase-1 that includes its muscle specific glycogen-targeting subunit GM and substrate phospholamban. GM interacts directly with phospholamban and this association is mediated by the cytosolic regions of the proteins. Our findings suggest the involvement of GM in mediating formation of the phosphatase-1/GM/phospholamban complex through the direct and independent interactions of GM with both protein phosphatase-1 and phospholamban. Importantly, the protein phosphatase-1/GM/phospholamban complex dissociates upon protein kinase A phosphorylation, indicating its significance in the beta-adrenergic signalling axis. Moreover, protein phosphatase-1 activity is regulated by two binding partners, inhibitor-1 and the small heat shock protein 20, Hsp20. Indeed, human genetic variants of inhibitor-1 (G147D) or Hsp20 (P20L) result in reduced binding and inhibition of protein phosphatase-1, suggesting aberrant enzymatic regulation in human carriers. These findings provide insights into the mechanisms underlying fine-tuned regulation of protein phosphatase-1 and its impact on the SERCA2/phospholamban interactome in cardiac function.
Elmaghawry M, Migliore F, Mohammed N, Sanoudou D, Alhashemi M. Science and practice of arrhythmogenic cardiomyopathy: A paradigm shift. Glob Cardiol Sci PractGlob Cardiol Sci PractGlob Cardiol Sci Pract. 2013;2013:63-79.
Kalozoumi G, Tzimas C, Sanoudou D. The expanding role of epigenetics. Glob Cardiol Sci PractGlob Cardiol Sci PractGlob Cardiol Sci Pract. 2012;2012:7.
Kassem HS, Girolami F, Sanoudou D. Molecular genetics made simple. Glob Cardiol Sci PractGlob Cardiol Sci PractGlob Cardiol Sci Pract. 2012;2012:6.Abstract
Genetics have undoubtedly become an integral part of biomedical science and clinical practice, with important implications in deciphering disease pathogenesis and progression, identifying diagnostic and prognostic markers, as well as designing better targeted treatments. The exponential growth of our understanding of different genetic concepts is paralleled by a growing list of genetic terminology that can easily intimidate the unfamiliar reader. Rendering genetics incomprehensible to the clinician however, defeats the very essence of genetic research: its utilization for combating disease and improving quality of life. Herein we attempt to correct this notion by presenting the basic genetic concepts along with their usefulness in the cardiology clinic. Bringing genetics closer to the clinician will enable its harmonious incorporation into clinical care, thus not only restoring our perception of its simple and elegant nature, but importantly ensuring the maximal benefit for our patients.
Sakellariou A, Sanoudou D, Spyrou G. Combining multiple hypothesis testing and affinity propagation clustering leads to accurate, robust and sample size independent classification on gene expression data. BMC BioinformaticsBMC BioinformaticsBMC Bioinformatics. 2012;13:270.Abstract
BACKGROUND: A feature selection method in microarray gene expression data should be independent of platform, disease and dataset size. Our hypothesis is that among the statistically significant ranked genes in a gene list, there should be clusters of genes that share similar biological functions related to the investigated disease. Thus, instead of keeping N top ranked genes, it would be more appropriate to define and keep a number of gene cluster exemplars. RESULTS: We propose a hybrid FS method (mAP-KL), which combines multiple hypothesis testing and affinity propagation (AP)-clustering algorithm along with the Krzanowski & Lai cluster quality index, to select a small yet informative subset of genes. We applied mAP-KL on real microarray data, as well as on simulated data, and compared its performance against 13 other feature selection approaches. Across a variety of diseases and number of samples, mAP-KL presents competitive classification results, particularly in neuromuscular diseases, where its overall AUC score was 0.91. Furthermore, mAP-KL generates concise yet biologically relevant and informative N-gene expression signatures, which can serve as a valuable tool for diagnostic and prognostic purposes, as well as a source of potential disease biomarkers in a broad range of diseases. CONCLUSIONS: mAP-KL is a data-driven and classifier-independent hybrid feature selection method, which applies to any disease classification problem based on microarray data, regardless of the available samples. Combining multiple hypothesis testing and AP leads to subsets of genes, which classify unknown samples from both, small and large patient cohorts with high accuracy.
Sanoudou D, Mountzios G, Arvanitis DA, Pectasides D. Array-based pharmacogenomics of molecular-targeted therapies in oncology. Pharmacogenomics JPharmacogenomics JPharmacogenomics J. 2012;12:185-96.Abstract
The advent of microarrays over the past decade has transformed the way genome-wide studies are designed and conducted, leading to an unprecedented speed of acquisition and amount of new knowledge. Microarray data have led to the identification of molecular subclasses of solid tumors characterized by distinct oncogenic pathways, as well as the development of multigene prognostic or predictive models equivalent or superior to those of established clinical parameters. In the field of molecular-targeted therapy for cancer, in particular, the application of array-based methodologies has enabled the identification of molecular targets with 'key' roles in neoplastic transformation or tumor progression and the subsequent development of targeted agents, which are most likely to be active in the specific molecular setting. Herein, we present a summary of the main applications of whole-genome expression microarrays in the field of molecular-targeted therapies for solid tumors and we discuss their potential in the clinical setting. An emphasis is given on deciphering the molecular mechanisms of drug action, identifying novel therapeutic targets and suitable agents to target them with, and discovering molecular markers/signatures that predict response to therapy or optimal drug dose for each patient.
Psarras S, Mavroidis M, Sanoudou D, Davos CH, Xanthou G, Varela AE, Panoutsakopoulou V, Capetanaki Y. Regulation of adverse remodelling by osteopontin in a genetic heart failure model. Eur Heart JEur Heart JEur Heart J. 2012;33:1954-63.Abstract
AIMS: Desmin, the muscle-specific intermediate filament protein, is a major target in dilated cardiomyopathy and heart failure in humans and mice. The hallmarks of desmin-deficient (des(-/-)) mice pathology include pronounced myocardial degeneration, extended fibrosis, and osteopontin (OPN) overexpression. We sought to identify the molecular and cellular events regulating adverse cardiac remodelling in des(-/-) mice and their potential link to OPN. METHODS AND RESULTS: In situ hybridization, histology, and immunostaining demonstrated that inflammatory cells and not cardiomyocytes were the source of OPN. RNA profile comparison revealed that activation of inflammatory pathways, sustained by innate immunity mechanisms, predominated among all changes occurring in degenerating des(-/-) myocardium. The expression of the most highly up-regulated genes (OPN: 226x, galectin-3: 26x, osteoactivin/Gpnmb/DC-HIL: 160x and metalloprotease-12: 98x) was associated with heart infiltrating macrophages. To evaluate the role of OPN, we generated des(-/-)OPN(-/-) mice and compared their cardiac function and remodelling indices with those of des(-/-). Osteopontin promoted cardiac dysfunction in this model since des(-/-)OPN(-/-) mice showed 53% improvement of left ventricular function, paralleled to an up to 44% reduction in fibrosis. The diminished fibrotic response in the absence of OPN could be partly mediated by a dramatic reduction in myocardial galectin-3 levels, associated with an impaired galectin-3 secretion by OPN-deficient infiltrating macrophages. CONCLUSION: Cardiomyocyte death due to desmin deficiency leads to inflammation and subsequent overexpression of a series of remodelling modulators. Among them, OPN seems to be a major regulator of des(-/-) adverse myocardial remodelling and it functions at least by potentiating galectin-3 up-regulation and secretion.
Totary-Jain H, Sanoudou D, Dautriche CN, Schneller H, Zambrana L, Marks AR. Rapamycin resistance is linked to defective regulation of Skp2. Cancer ResCancer ResCancer Res. 2012;72:1836-43.Abstract
The mammalian target of rapamycin (mTOR) plays a role in controlling malignant cellular growth. mTOR inhibitors, including rapamycin (sirolimus), are currently being evaluated in cancer trials. However, a significant number of tumors are rapamycin resistant. In this study, we report that the ability of rapamycin to downregulate Skp2, a subunit of the ubiquitin protein ligase complex, identifies tumors that are sensitive to rapamycin. RNA interference (RNAi)-mediated silencing of Skp2 in human tumor cells increased their sensitivity to rapamycin in vitro and inhibited the growth of tumor xenografts in vivo. Our findings suggest that Skp2 levels are a key determinant of antitumor responses to mTOR inhibitors, highlighting a potentially important pharmacogenomic marker to predict sensitivity to rapamycin as well as Skp2 silencing strategies for therapeutic purposes.
Sakellariou A, Sanoudou D, Spyrou G. Investigating the minimum required number of genes for the classification of neuromuscular disease microarray data. IEEE Trans Inf Technol BiomedIEEE Trans Inf Technol BiomedIEEE Trans Inf Technol Biomed. 2011;15:349-55.Abstract
The discovery of potential microarray markers, which will expedite molecular diagnosis/prognosis and provide reliable results to clinical decision-making and treatment selection for patients, is of paramount importance. Feature selection techniques, which aim at minimizing the dimensionality of the microarray data by keeping the most statistically significant genes, are a powerful approach toward this goal. In this paper, we investigate the minimum required subsets of genes, which best classify neuromuscular disease data. For this purpose, we implemented a methodology pipeline that facilitated the use of multiple feature selection methods and subsequent performance of data classification. Five feature selection methods on datasets from ten different neuromuscular diseases were utilized. Our findings reveal subsets of very small number of genes, which can successfully classify normal/disease samples. Interestingly, we observe that similar classification results may be obtained from different subsets of genes. The proposed methodology can expedite the identification of small gene subsets with high-classification accuracy that could ultimately be used in the genetics clinics for diagnostic, prognostic, and pharmacogenomic purposes.
Qian J, Vafiadaki E, Florea SM, Singh VP, Song W, Lam CK, Wang Y, Yuan Q, Pritchard TJ, Cai W, et al. Small heat shock protein 20 interacts with protein phosphatase-1 and enhances sarcoplasmic reticulum calcium cycling. Circ ResCirc ResCirc Res. 2011;108:1429-38.Abstract
BACKGROUND: Heat shock proteins (Hsp) are known to enhance cell survival under various stress conditions. In the heart, the small Hsp20 has emerged as a key mediator of protection against apoptosis, remodeling, and ischemia/reperfusion injury. Moreover, Hsp20 has been implicated in modulation of cardiac contractility ex vivo. The objective of this study was to determine the in vivo role of Hsp20 in the heart and the mechanisms underlying its regulatory effects in calcium (Ca) cycling. METHODS AND RESULTS: Hsp20 overexpression in intact animals resulted in significant enhancement of cardiac function, coupled with augmented Ca cycling and sarcoplasmic reticulum Ca load in isolated cardiomyocytes. This was associated with specific increases in phosphorylation of phospholamban (PLN) at both Ser16 and Thr17, relieving its inhibition of the apparent Ca affinity of SERCA2a. Accordingly, the inotropic effects of Hsp20 were abrogated in cardiomyocytes expressing nonphosphorylatable PLN (S16A/T17A). Interestingly, the activity of type 1 protein phosphatase (PP1), a known regulator of PLN signaling, was significantly reduced by Hsp20 overexpression, suggesting that the Hsp20 stimulatory effects are partially mediated through the PP1-PLN axis. This hypothesis was supported by cell fractionation, coimmunoprecipitation, and coimmunolocalization studies, which revealed an association between Hsp20, PP1, and PLN. Furthermore, recombinant protein studies confirmed a physical interaction between AA 73 to 160 in Hsp20 and AA 163 to 330 in PP1. CONCLUSIONS: Hsp20 is a novel regulator of sarcoplasmic reticulum Ca cycling by targeting the PP1-PLN axis. These findings, coupled with the well-recognized cardioprotective role of Hsp20, suggest a dual benefit of targeting Hsp20 in heart disease.
Arvanitis DA, Vafiadaki E, Sanoudou D, Kranias EG. Histidine-rich calcium binding protein: the new regulator of sarcoplasmic reticulum calcium cycling. J Mol Cell CardiolJ Mol Cell CardiolJ Mol Cell Cardiol. 2011;50:43-9.Abstract
The histidine-rich calcium binding protein (HRC) is a novel regulator of sarcoplasmic reticulum (SR) Ca(2+)-uptake, storage and release. Residing in the SR lumen, HRC binds Ca(2+) with high capacity but low affinity. In vitro phosphorylation of HRC affects ryanodine affinity of the ryanodine receptor (RyR), suggesting a functional role of HRC on SR Ca(2+)-release. Indeed, acute HRC overexpression in isolated rodent cardiomyocytes decreases Ca(2+)-induced Ca(2+)-release, increases SR Ca(2+)-load, and impairs contractility. The HRC effects on RyR may be regulated by the Ca(2+)-sensitivity of its interaction with triadin. However, HRC also affects the SR Ca(2+)-ATPase, as shown by HRC overexpression in transgenic mouse hearts, which resulted in reduced SR Ca(2+)-uptake rates, cardiac remodeling and hypertrophy. In fact, in vitro generated evidence suggests that HRC directly interacts with SR Ca(2+)-ATPase2, supporting a dual role of HRC in Ca(2+)-homeostasis: regulation of both SR Ca(2+)-uptake and Ca(2+)-release. Furthermore, HRC plays an important role in myocyte differentiation and in antiapoptotic cardioprotection against ischemia/reperfusion induced cardiac injury. Interestingly, HRC has been linked with familiar cardiac conduction disease and an HRC polymorphism was shown to associate with malignant ventricular arrhythmias in the background of idiopathic dilated cardiomyopathy. This review summarizes studies, which have established the critical role of HRC in Ca(2+)-homeostasis, suggesting its importance in cardiac physiology and pathophysiology.
Wang HS, Arvanitis DA, Dong M, Niklewski PJ, Zhao W, Lam CK, Kranias EG, Sanoudou D. SERCA2a superinhibition by human phospholamban triggers electrical and structural remodeling in mouse hearts. Physiol GenomicsPhysiol GenomicsPhysiol Genomics. 2011;43:357-64.Abstract
Phospholamban (PLN), the reversible inhibitor of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a), is a key regulator of myocyte Ca(2+) cycling with a significant role in heart failure. We previously showed that the single amino acid difference between human and mouse PLN results in increased inhibition of Ca(2+) cycling and cardiac remodeling and attenuated stress responses in transgenic mice expressing the human PLN (hPLN) in the null background. Here we dissect the molecular and electrophysiological processes triggered by the superinhibitory hPLN in the mouse. Using a multidisciplinary approach, we performed global gene expression analysis, electrophysiology, and mathematical simulations on hPLN mice. We identified significant changes in a series of Na(+) and K(+) homeostasis genes/proteins (including Kcnd2, Scn9a, Slc8a1) and ionic conductance (including L-type Ca(2+) current, Na(+)/Ca(2+) exchanger, transient outward K(+) current). Simulation analysis suggests that this electrical remodeling has a critical role in rescuing cardiac function by improving sarcoplasmic reticulum Ca(2+) load and overall Ca(2+) dynamics. Furthermore, multiple structural and transcription factor gene expression changes indicate an ongoing structural remodeling process, favoring hypertrophy and myogenesis while suppressing apoptosis and progression to heart failure. Our findings expand current understanding of the hPLN function and provide additional insights into the downstream implications of SERCA2a superinhibition in the mammalian heart.
Luo J, Schumacher M, Scherer A, Sanoudou D, Megherbi D, Davison T, Shi T, Tong W, Shi L, Hong H, et al. A comparison of batch effect removal methods for enhancement of prediction performance using MAQC-II microarray gene expression data. Pharmacogenomics JPharmacogenomics JPharmacogenomics J. 2010;10:278-91.Abstract
Batch effects are the systematic non-biological differences between batches (groups) of samples in microarray experiments due to various causes such as differences in sample preparation and hybridization protocols. Previous work focused mainly on the development of methods for effective batch effects removal. However, their impact on cross-batch prediction performance, which is one of the most important goals in microarray-based applications, has not been addressed. This paper uses a broad selection of data sets from the Microarray Quality Control Phase II (MAQC-II) effort, generated on three microarray platforms with different causes of batch effects to assess the efficacy of their removal. Two data sets from cross-tissue and cross-platform experiments are also included. Of the 120 cases studied using Support vector machines (SVM) and K nearest neighbors (KNN) as classifiers and Matthews correlation coefficient (MCC) as performance metric, we find that Ratio-G, Ratio-A, EJLR, mean-centering and standardization methods perform better or equivalent to no batch effect removal in 89, 85, 83, 79 and 75% of the cases, respectively, suggesting that the application of these methods is generally advisable and ratio-based methods are preferred.
Mountzios G, Dimopoulos MA, Soria JC, Sanoudou D, Papadimitriou CA. Histopathologic and genetic alterations as predictors of response to treatment and survival in lung cancer: a review of published data. Crit Rev Oncol HematolCrit Rev Oncol HematolCrit Rev Oncol Hematol. 2010;75:94-109.Abstract
Lung carcinogenesis is considered to be the result of composite environmental, genetic and epigenetic changes. Despite the fact that many of the genetic alterations, including loss of heterozygocity in the 3p chromosome locus and point mutations in the tumor-suppressor genes TP53 and retinoblastoma (RB1), occur in nearly all histopathologic types of lung cancer, the frequency and the "timing" of their occurrence seems to differ between small-cell lung cancer (SCLC) cells, that are characterized by neuroendocrine differentiation, and non-small-cell lung cancer (NSCLC) cells. Although loss of cell-cycle control is the crucial molecular event in both types, the mechanism by which it provokes oncogenesis differs significantly between SCLC and NSCLC. Importantly, some of these molecular events, including DNA-damage response and epidermal growth factor receptor (EGFR) mutations are valuable in predicting response to conventional chemotherapy or molecular-targeted agents as well as in the prognosis of patients that harbor these alterations. In the current review we report on the best characterized histopathologic and genetic changes in NSCLC and SCLC in relation to each histological subtype and we discuss their predictive and prognostic implications.
Mountzios G, Sanoudou D, Syrigos KN. Clinical pharmacogenetics in oncology: the paradigm of molecular targeted therapies. Curr Pharm DesCurr Pharm DesCurr Pharm Des. 2010;16:2184-93.Abstract
Even though treatment of several types of solid tumors has improved in the past few years with the introduction of molecular targeted agents in the therapeutic armamentarium of the medical oncologist, response rates to these agents are generally modest. Increasing evidence is now revealing that genetic factors are affecting patients' response to these therapeutic agents as well as the frequency and intensity of toxic reactions. Importantly, pharmacogenetic analysis is now required for the administration of several molecular targeted agents in clinical practice. For the vast majority of these agents, however, data remain purely experimental. Herein, we provide an overview of the genetic changes (mutations and polymorphisms) that have been associated with response to treatment with anticancer molecular targeted agents. Special emphasis is given on molecules (monoclonal antibodies and tyrosine kinase inhibitors) that target critical mediators in the epidermal growth factor receptor (EGFR), the human epidermal growth factor receptor 2 (HER2/ERBB2/NEU) and the vascular endothelial growth factor receptor (VEGFR) pathways. The true clinical utility of these applications remains to be proven in future prospective, randomized clinical trials in large patient cohorts of all different ethnic backgrounds.
Sanoudou D, Duka A, Drosatos K, Hayes KC, Zannis VI. Role of Esrrg in the fibrate-mediated regulation of lipid metabolism genes in human ApoA-I transgenic mice. Pharmacogenomics JPharmacogenomics JPharmacogenomics J. 2010;10:165-79.Abstract
We have used a new ApoA-I transgenic mouse model to identify by global gene expression profiling, candidate genes that affect lipid and lipoprotein metabolism in response to fenofibrate treatment. Multilevel bioinformatical analysis and stringent selection criteria (2-fold change, 0% false discovery rate) identified 267 significantly changed genes involved in several molecular pathways. The fenofibrate-treated group did not have significantly altered levels of hepatic human APOA-I mRNA and plasma ApoA-I compared with the control group. However, the treatment increased cholesterol levels to 1.95-fold mainly due to the increase in high-density lipoprotein (HDL) cholesterol. The observed changes in HDL are associated with the upregulation of genes involved in phospholipid biosynthesis and lipid hydrolysis, as well as phospholipid transfer protein. Significant upregulation was observed in genes involved in fatty acid transport and beta-oxidation, but not in those of fatty acid and cholesterol biosynthesis, Krebs cycle and gluconeogenesis. Fenofibrate changed significantly the expression of seven transcription factors. The estrogen receptor-related gamma gene was upregulated 2.36-fold and had a significant positive correlation with genes of lipid and lipoprotein metabolism and mitochondrial functions, indicating an important role of this orphan receptor in mediating the fenofibrate-induced activation of a specific subset of its target genes.
Vujasinovic T, Zampera AS, Jackers P, Sanoudou D, Depaulis A. In silico dynamic molecular interaction networks for the discovery of new therapeutic targets. Curr Pharm DesCurr Pharm DesCurr Pharm Des. 2010;16:2241-51.Abstract
Systems biology has emerged as a major trend in biological research during the past decade. As living organisms are described in more and more detail, it aims at filling the gap between understanding basic molecular processes and complex biological systems in which new properties often emerge from the combination of these elementary processes. This approach culminates in the development of computer-based mathematical models of physiological and pathophysiological processes. We review the state of the art in dynamic modelling, with emphasis on two complementary approaches: the modelling of small systems that is mostly developed by academic teams and aims at understanding generic biological properties, and the modelling of large systems that is mostly implemented by industrial companies and aims at the generation of new therapeutic strategies. We also provide an example of such large-scale modelling applied to the identification of drug targets for neurodegeneration.
Vafiadaki E, Arvanitis DA, Kranias EG, Sanoudou D. Pharmacogenetically tailored treatments for heart disease. Curr Pharm DesCurr Pharm DesCurr Pharm Des. 2010;16:2194-213.Abstract
Heart disease represents the primary cause of death worldwide, with mortality rates being predicted to remain constant within the next couple of decades. Cardiac disease treatment currently includes the administration of drugs, predominantly aiming at improving heart performance, through controlling heart rhythm, blood pressure, as well as reducing cholesterol and blood clotting. Despite, however, the medical advances that have lead towards a better understanding of heart disease pathophysiology and the development of new therapeutic approaches, the degree of success of the available drug therapies varies among patients. The existence of polymorphisms in a number of genes has been shown to result in differences in pharmacokinetics, pharmacodynamics and drug metabolism and have therefore been associated with response to drug treatment. The occurrence of adverse drug reactions that may lead to drug-induced toxicity represents another factor influencing outcome of therapeutic treatment. While the influence of genetic polymorphisms in patient's response to heart disease drugs is being unveiled, the rapidly evolving field of pharmacogenetics is promising to aid clinicians in choosing the best suited drug/dose for each patient and the pharmaceutical companies in the design of better targeted, more effective new chemical compounds. In the near future individualized, targeted therapy will become part of clinical care routine maximizing patient therapeutic benefits and minimizing risks of adverse effects.
Sanoudou D. "Pharmacogenomics: achievements, challenges and prospects, for patients, pharmaceutical industries and healthcare systems". Curr Pharm DesCurr Pharm DesCurr Pharm Des. 2010;16:2182-3.
Al-Qusairi L, Weiss N, Toussaint A, Berbey C, Messaddeq N, Kretz C, Sanoudou D, Beggs AH, Allard B, Mandel JL, et al. T-tubule disorganization and defective excitation-contraction coupling in muscle fibers lacking myotubularin lipid phosphatase. Proc Natl Acad Sci U S AProc Natl Acad Sci U S AProc Natl Acad Sci U S A. 2009;106:18763-8.Abstract
Skeletal muscle contraction is triggered by the excitation-contraction (E-C) coupling machinery residing at the triad, a membrane structure formed by the juxtaposition of T-tubules and sarcoplasmic reticulum (SR) cisternae. The formation and maintenance of this structure is key for muscle function but is not well characterized. We have investigated the mechanisms leading to X-linked myotubular myopathy (XLMTM), a severe congenital disorder due to loss of function mutations in the MTM1 gene, encoding myotubularin, a phosphoinositide phosphatase thought to have a role in plasma membrane homeostasis and endocytosis. Using a mouse model of the disease, we report that Mtm1-deficient muscle fibers have a decreased number of triads and abnormal longitudinally oriented T-tubules. In addition, SR Ca(2+) release elicited by voltage-clamp depolarizations is strongly depressed in myotubularin-deficient muscle fibers, with myoplasmic Ca(2+) removal and SR Ca(2+) content essentially unaffected. At the molecular level, Mtm1-deficient myofibers exhibit a 3-fold reduction in type 1 ryanodine receptor (RyR1) protein level. These data reveal a critical role of myotubularin in the proper organization and function of the E-C coupling machinery and strongly suggest that defective RyR1-mediated SR Ca(2+) release is responsible for the failure of muscle function in myotubular myopathy.
Papalouka V, Arvanitis DA, Vafiadaki E, Mavroidis M, Papadodima SA, Spiliopoulou CA, Kremastinos DT, Kranias EG, Sanoudou D. Muscle LIM protein interacts with cofilin 2 and regulates F-actin dynamics in cardiac and skeletal muscle. Mol Cell BiolMol Cell BiolMol Cell Biol. 2009;29:6046-58.Abstract
The muscle LIM protein (MLP) and cofilin 2 (CFL2) are important regulators of striated myocyte function. Mutations in the corresponding genes have been directly associated with severe human cardiac and skeletal myopathies, and aberrant expression patterns have often been observed in affected muscles. Herein, we have investigated whether MLP and CFL2 are involved in common molecular mechanisms, which would promote our understanding of disease pathogenesis. We have shown for the first time, using a range of biochemical and immunohistochemical methods, that MLP binds directly to CFL2 in human cardiac and skeletal muscles. The interaction involves the inter-LIM domain, amino acids 94 to 105, of MLP and the amino-terminal domain, amino acids 1 to 105, of CFL2, which includes part of the actin depolymerization domain. The MLP/CFL2 complex is stronger in moderately acidic (pH 6.8) environments and upon CFL2 phosphorylation, while it is independent of Ca(2+) levels. This interaction has direct implications in actin cytoskeleton dynamics in regulating CFL2-dependent F-actin depolymerization, with maximal depolymerization enhancement at an MLP/CFL2 molecular ratio of 2:1. Deregulation of this interaction by intracellular pH variations, CFL2 phosphorylation, MLP or CFL2 gene mutations, or expression changes, as observed in a range of cardiac and skeletal myopathies, could impair F-actin depolymerization, leading to sarcomere dysfunction and disease.
Vafiadaki E, Arvanitis DA, Pagakis SN, Papalouka V, Sanoudou D, Kontrogianni-Konstantopoulos A, Kranias EG. The anti-apoptotic protein HAX-1 interacts with SERCA2 and regulates its protein levels to promote cell survival. Mol Biol CellMol Biol CellMol Biol Cell. 2009;20:306-18.Abstract
Cardiac contractility is regulated through the activity of various key Ca(2+)-handling proteins. The sarco(endo)plasmic reticulum (SR) Ca(2+) transport ATPase (SERCA2a) and its inhibitor phospholamban (PLN) control the uptake of Ca(2+) by SR membranes during relaxation. Recently, the antiapoptotic HS-1-associated protein X-1 (HAX-1) was identified as a binding partner of PLN, and this interaction was postulated to regulate cell apoptosis. In the current study, we determined that HAX-1 can also bind to SERCA2. Deletion mapping analysis demonstrated that amino acid residues 575-594 of SERCA2's nucleotide binding domain are required for its interaction with the C-terminal domain of HAX-1, containing amino acids 203-245. In transiently cotransfected human embryonic kidney 293 cells, recombinant SERCA2 was specifically targeted to the ER, whereas HAX-1 selectively concentrated at mitochondria. On triple transfections with PLN, however, HAX-1 massively translocated to the ER membranes, where it codistributed with PLN and SERCA2. Overexpression of SERCA2 abrogated the protective effects of HAX-1 on cell survival, after hypoxia/reoxygenation or thapsigargin treatment. Importantly, HAX-1 overexpression was associated with down-regulation of SERCA2 expression levels, resulting in significant reduction of apparent ER Ca(2+) levels. These findings suggest that HAX-1 may promote cell survival through modulation of SERCA2 protein levels and thus ER Ca(2+) stores.
Vafiadaki E, Papalouka V, Arvanitis DA, Kranias EG, Sanoudou D. The role of SERCA2a/PLN complex, Ca(2+) homeostasis, and anti-apoptotic proteins in determining cell fate. Pflugers ArchPflugers ArchPflugers Arch. 2009;457:687-700.Abstract
Intracellular calcium is a major coordinator of numerous aspects of cellular physiology, including muscle contractility and cell survival. In cardiac muscle, aberrant Ca(2+) cycling has been implicated in a range of pathological conditions including cardiomyopathies and heart failure. The sarco(endo)plasmic reticulum Ca(2+) transport adenosine triphosphatase (SERCA2a) and its regulator phospholamban (PLN) have a central role in modulating Ca(2+) homeostasis and, therefore, cardiac function. Herein, we discuss the mechanisms through which SERCA2a and PLN control cardiomyocyte function in health and disease. Emphasis is placed on our newly identified PLN-binding partner HS-1-associated protein X-1 (HAX-1), which has an anti-apoptotic function and presents with numerous similarities to Bcl-2. Recent evidence indicates that proteins of the Bcl-2 family can influence ER Ca(2+) content, a critical determinant of cellular sensitivity to apoptosis. The discovery of the PLN/HAX-1 interaction therefore unveils an important new link between Ca(2+) homeostasis and cell survival, with significant therapeutic potential.
Arvanitis DA, Sanoudou D, Kolokathis F, Vafiadaki E, Papalouka V, Kontrogianni-Konstantopoulos A, Theodorakis GN, Paraskevaidis IA, Adamopoulos S, Dorn, G. W. 2nd, et al. The Ser96Ala variant in histidine-rich calcium-binding protein is associated with life-threatening ventricular arrhythmias in idiopathic dilated cardiomyopathy. Eur Heart JEur Heart JEur Heart J. 2008;29:2514-25.Abstract
AIMS: To investigate whether genetic variants of the histidine-rich calcium (HRC)-binding protein are associated with idiopathic dilated cardiomyopathy (DCM) and its progression. METHODS AND RESULTS: We screened 123 idiopathic DCM patients and 96 healthy individuals by single-strand conformation polymorphism analysis and direct sequencing for genetic variants in HRC. Six polymorphisms were detected: Leu35Leu (A/G), Ser43Asn (G/A), Ser96Ala (T/G), Glu202_Glu203insGlu (-/GAG), Asp261del (GAT/-), and an in-frame insertion of 51 amino acids at His321. The analysis of their frequencies did not reveal any significant correlation with DCM development. However, the Ser96Ala polymorphism exhibited a statistically significant correlation with the occurrence of life-threatening ventricular arrhythmias. During a follow-up of 4.02 +/- 2.4 years, the risk for ventricular arrhythmias was higher (HR, 9.620; 95% CI, 2.183-42.394; P = 0.003) in the Ala/Ala patients, compared with Ser/Ser homozygous patients. On multivariable Cox regression analysis, the Ser96Ala polymorphism was the only significant genetic arrythmogenesis predictor in DCM patients (HR, 4.191; 95% CI, 0.838-20.967; P = 0.018). CONCLUSION: The Ser96Ala genetic variant of HRC is associated with life-threatening ventricular arrhythmias in idiopathic DCM and may serve as an independent predictor of susceptibility to arrhythmogenesis in the setting of DCM.
Garbis SD, Tyritzis SI, Roumeliotis T, Zerefos P, Giannopoulou EG, Vlahou A, Kossida S, Diaz J, Vourekas S, Tamvakopoulos C, et al. Search for potential markers for prostate cancer diagnosis, prognosis and treatment in clinical tissue specimens using amine-specific isobaric tagging (iTRAQ) with two-dimensional liquid chromatography and tandem mass spectrometry. J Proteome ResJ Proteome ResJ Proteome Res. 2008;7:3146-58.Abstract
This study aimed to identify candidate new diagnosis and prognosis markers and medicinal targets of prostate cancer (PCa), using state of the art proteomics. A total of 20 prostate tissue specimens from 10 patients with benign prostatic hyperplasia (BPH) and 10 with PCa (Tumour Node Metastasis [TNM] stage T1-T3) were analyzed by isobaric stable isotope labeling (iTRAQ) and two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) approaches using a hybrid quadrupole time-of-flight system (QqTOF). The study resulted in the reproducible identification of 825 nonredundant gene products (p < or = 0.05) of which 30 exhibited up-regulation (> or =2-fold) and another 35 exhibited down-regulation (< or =0.5-fold) between the BPH and PCa specimens constituting a major contribution toward their global proteomic assessment. Selected findings were confirmed by immunohistochemical analysis of prostate tissue specimens. The proteins determined support existing knowledge and uncover novel and promising PCa biomarkers. The PCa proteome found can serve as a useful aid for the identification of improved diagnostic and prognostic markers and ultimately novel chemopreventive and therapeutic targets.
Arvanitis DA, Vafiadaki E, Fan GC, Mitton BA, Gregory KN, Del Monte F, Kontrogianni-Konstantopoulos A, Sanoudou D, Kranias EG. Histidine-rich Ca-binding protein interacts with sarcoplasmic reticulum Ca-ATPase. Am J Physiol Heart Circ PhysiolAm J Physiol Heart Circ PhysiolAm J Physiol Heart Circ Physiol. 2007;293:H1581-9.Abstract
Depressed cardiac Ca cycling by the sarcoplasmic reticulum (SR) has been associated with attenuated contractility, which can progress to heart failure. The histidine-rich Ca-binding protein (HRC) is an SR component that binds to triadin and may affect Ca release through the ryanodine receptor. HRC overexpression in transgenic mouse hearts was associated with decreased rates of SR Ca uptake and delayed relaxation, which progressed to hypertrophy with aging. The present study shows that HRC may mediate part of its regulatory effects by binding directly to sarco(endo)plasmic reticulum Ca-ATPase type 2 (SERCA2) in cardiac muscle, which is confirmed by coimmunostaining observed under confocal microscopy. This interaction involves the histidine- and glutamic acid-rich domain of HRC (320-460 aa) and the part of the NH(2)-terminal cation transporter domain of SERCA2 (74-90 aa) that projects into the SR lumen. The SERCA2-binding domain is upstream from the triadin-binding region in human HRC (609-699 aa). Specific binding between HRC and SERCA was verified by coimmunoprecipitation and pull-down assays using human and mouse cardiac homogenates and by blot overlays using glutathione S-transferase and maltose-binding protein recombinant proteins. Importantly, increases in Ca concentration were associated with a significant reduction of HRC binding to SERCA2, whereas they had opposite effects on the HRC-triadin interaction in cardiac homogenates. Collectively, our data suggest that HRC may play a key role in the regulation of SR Ca cycling through its direct interactions with SERCA2 and triadin, mediating a fine cross talk between SR Ca uptake and release in the heart.
Drosatos K, Sanoudou D, Kypreos KE, Kardassis D, Zannis VI. A dominant negative form of the transcription factor c-Jun affects genes that have opposing effects on lipid homeostasis in mice. J Biol ChemJ Biol ChemJ Biol Chem. 2007;282:19556-64.Abstract
c-Jun is a transcription factor activated by phosphorylation by the stress-activated protein kinase/c-Jun N-terminal kinase pathway in response to extracellular signals and cytokines. We show that adenovirus-mediated gene transfer of the dominant negative form of c-Jun (dn-c-Jun) in C57BL/6 mice increased greatly apoE hepatic mRNA and plasma levels, increased plasma cholesterol, triglyceride, and very low density lipoprotein levels, and resulted in the accumulation of discoidal high density lipoprotein particles. A similar but more severe phenotype was generated by overexpression of the mouse apoE in C57BL/6 mice, suggesting that dyslipidemia induced by dn-c-Jun was the result of apoE overexpression. Unexpectedly, infection of apoE(-/-) mice with adenovirus expressing dn-c-Jun reduced plasma cholesterol by 70%, suggesting that dn-c-Jun affected other genes that control plasma cholesterol levels. To identify these genes, we performed whole genome expression analysis (34,000 genes) of isolated livers from two groups of five apoE(-/-) mice, infected with adenoviruses expressing either the dn-c-Jun or the green fluorescence protein. Bioinformatic analysis and Northern blotting validation revealed that dn-c-Jun increased 40-fold the apoE mRNA and reduced by 70% the Scd-1 (stearoyl-CoA-desaturase 1) mRNA. The involvement of Scd-1 in lowering plasma cholesterol was confirmed by restoration of high cholesterol levels of apoE(-/-) mice following coinfection with adenoviruses expressing dn-c-Jun and Scd-1. In conclusion, dn-c-Jun appears to trigger two opposing events in mice that affect plasma cholesterol and triglyceride levels as follows: one results in apoE overexpression and triggers dyslipidemia and the other results in inhibition of Scd-1 and offsets dyslipidemia.
Vafiadaki E, Sanoudou D, Arvanitis DA, Catino DH, Kranias EG, Kontrogianni-Konstantopoulos A. Phospholamban interacts with HAX-1, a mitochondrial protein with anti-apoptotic function. J Mol BiolJ Mol BiolJ Mol Biol. 2007;367:65-79.Abstract
Phospholamban (PLN) is a key regulator of Ca(2+) homeostasis and contractility in the heart. Its regulatory effects are mediated through its interaction with the sarcoplasmic reticulum Ca(2+)-ATPase, (SERCA2a), resulting in alterations of its Ca(2+)-affinity. To identify additional proteins that may interact with PLN, we used the yeast-two-hybrid system to screen an adult human cardiac cDNA library. HS-1 associated protein X-1 (HAX-1) was identified as a PLN-binding partner. The minimal binding regions were mapped to amino acid residues 203-245 for HAX-1 and residues 16-22 for PLN. The interaction between the two proteins was confirmed using GST-HAX-1, bound to the glutathione-matrix, which specifically adsorbed native PLN from human or mouse cardiac homogenates, while in reciprocal binding studies, recombinant His-HAX-1 bound GST-PLN. Kinetic studies using surface plasmon resonance yielded a K(D) of approximately 1 muM as the binding affinity for the PLN/HAX-1 complex. Phosphorylation of PLN by cAMP-dependent protein kinase reduced binding to HAX-1, while increasing concentrations of Ca(2+) diminished the PLN/HAX-1 interaction in a dose-dependent manner. HAX-1 concentrated to mitochondria, but upon transient co-transfection of HEK 293 cells with PLN, HAX-1 redistributed and co-localized with PLN at the endoplasmic reticulum. Analysis of the anti-apoptotic function of HAX-1 revealed that the presence of PLN enhanced the HAX-1 protective effects from hypoxia/reoxygenation-induced cell death. These findings suggest a possible link between the Ca(2+) handling by the sarcoplasmic reticulum and cell survival mediated by the PLN/HAX-1 interaction.
Sanoudou D, Corbett MA, Han M, Ghoddusi M, Nguyen MA, Vlahovich N, Hardeman EC, Beggs AH. Skeletal muscle repair in a mouse model of nemaline myopathy. Hum Mol GenetHum Mol GenetHum Mol Genet. 2006;15:2603-12.Abstract
Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is a variably severe neuromuscular disorder for which no effective treatment is available. Although a number of genes have been identified in which mutations can cause NM, the pathogenetic mechanisms leading to the phenotypes are poorly understood. To address this question, we examined gene expression patterns in an NM mouse model carrying the human Met9Arg mutation of alpha-tropomyosin slow (Tpm3). We assessed five different skeletal muscles from affected mice, which are representative of muscles with differing fiber-type compositions, different physiological specializations and variable degrees of pathology. Although these same muscles in non-affected mice showed marked variation in patterns of gene expression, with diaphragm being the most dissimilar, the presence of the mutant protein in nemaline muscles resulted in a more similar pattern of gene expression among the muscles. This result suggests a common process or mechanism operating in nemaline muscles independent of the variable degrees of pathology. Transcriptional and protein expression data indicate the presence of a repair process and possibly delayed maturation in nemaline muscles. Markers indicative of satellite cell number, activated satellite cells and immature fibers including M-Cadherin, MyoD, desmin, Pax7 and Myf6 were elevated by western-blot analysis or immunohistochemistry. Evidence suggesting elevated focal repair was observed in nemaline muscle in electron micrographs. This analysis reveals that NM is characterized by a novel repair feature operating in multiple different muscles.
Gazda HT, Kho AT, Sanoudou D, Zaucha JM, Kohane IS, Sieff CA, Beggs AH. Defective ribosomal protein gene expression alters transcription, translation, apoptosis, and oncogenic pathways in Diamond-Blackfan anemia. Stem CellsStem CellsStem Cells. 2006;24:2034-44.Abstract
Diamond-Blackfan anemia (DBA) is a broad developmental disease characterized by anemia, bone marrow (BM) erythroblastopenia, and an increased incidence of malignancy. Mutations in ribosomal protein gene S19 (RPS19) are found in approximately 25% of DBA patients; however, the role of RPS19 in the pathogenesis of DBA remains unknown. Using global gene expression analysis, we compared highly purified multipotential, erythroid, and myeloid BM progenitors from RPS19 mutated and control individuals. We found several ribosomal protein genes downregulated in all DBA progenitors. Apoptosis genes, such as TNFRSF10B and FAS, transcriptional control genes, including the erythropoietic transcription factor MYB (encoding c-myb), and translational genes were greatly dysregulated, mostly in diseased erythroid cells. Cancer-related genes, including RAS family oncogenes and tumor suppressor genes, were significantly dysregulated in all diseased progenitors. In addition, our results provide evidence that RPS19 mutations lead to codownregulation of multiple ribosomal protein genes, as well as downregulation of genes involved in translation in DBA cells. In conclusion, the altered expression of cancer-related genes suggests a molecular basis for malignancy in DBA. Downregulation of c-myb expression, which causes complete failure of fetal liver erythropoiesis in knockout mice, suggests a link between RPS19 mutations and reduced erythropoiesis in DBA.
Haslett JN, Kang PB, Han M, Kho AT, Sanoudou D, Volinski JM, Beggs AH, Kohane IS, Kunkel LM. The influence of muscle type and dystrophin deficiency on murine expression profiles. Mamm GenomeMamm GenomeMamm Genome. 2005;16:739-48.Abstract
The phenotypic differences among Duchenne muscular dystrophy patients, mdx mice, and mdx(5cv) mice suggest that despite the common etiology of dystrophin deficiency, secondary mechanisms have a substantial influence on phenotypic severity. The differential response of various skeletal muscles to dystrophin deficiency supports this hypothesis. To explore these differences, gene expression profiles were generated from duplicate RNA targets extracted from six different skeletal muscles (diaphragm, soleus, gastrocnemius, quadriceps, tibialis anterior, and extensor digitorum longus) from wild-type, mdx, and mdx(5cv) mice, resulting in 36 data sets for 18 muscle samples. The data sets were compared in three different ways: (1) among wild-type samples only, (2) among all 36 data sets, and (3) between strains for each muscle type. The molecular profiles of soleus and diaphragm separate significantly from the other four muscle types and from each other. Fiber-type proportions can explain some of these differences. These variations in wild-type gene expression profiles may also reflect biomechanical differences known to exist among skeletal muscles. Further exploration of the genes that most distinguish these muscles may help explain the origins of the biomechanical differences and the reasons why some muscles are more resistant than others to dystrophin deficiency.
Kang PB, Kho AT, Sanoudou D, Haslett JN, Dow CP, Han M, Blasko JM, Lidov HG, Beggs AH, Kunkel LM. Variations in gene expression among different types of human skeletal muscle. Muscle NerveMuscle NerveMuscle Nerve. 2005;32:483-91.Abstract
There is a consistent variation in the response of different skeletal muscle groups to mutations in genes known to cause muscular dystrophy, yet these muscles appear histologically similar. To better understand these phenotypic differences, we analyzed gene expression patterns in control muscle specimens obtained from four sites at autopsy: deltoid, quadriceps, gastrocnemius, and tibialis anterior (TA). A total of 35 muscle samples from nine individuals (four pediatric and five geriatric) were studied. Factors potentially influencing gene expression in the different samples included individuality, age, muscle type, gender, cause of death, postmortem interval, and ethnicity. The first three factors, in decreasing order, were found to have a significant impact on the stratification of muscle specimens. A novel analytic method, using a second round of normalization, was used to elicit differences between muscle types. This approach may be extended to a broader survey, potentially elucidating a molecular classification of the skeletal muscles.
Greenberg SA, Pinkus JL, Pinkus GS, Burleson T, Sanoudou D, Tawil R, Barohn RJ, Saperstein DS, Briemberg HR, Ericsson M, et al. Interferon-alpha/beta-mediated innate immune mechanisms in dermatomyositis. Ann NeurolAnn NeurolAnn Neurol. 2005;57:664-78.Abstract
Dermatomyositis has been modeled as an autoimmune disease largely mediated by the adaptive immune system, including a local humorally mediated response with B and T helper cell muscle infiltration, antibody and complement-mediated injury of capillaries, and perifascicular atrophy of muscle fibers caused by ischemia. To further understand the pathophysiology of dermatomyositis, we used microarrays, computational methods, immunohistochemistry and electron microscopy to study muscle specimens from 67 patients, 54 with inflammatory myopathies, 14 with dermatomyositis. In dermatomyositis, genes induced by interferon-alpha/beta were highly overexpressed, and immunohistochemistry for the interferon-alpha/beta inducible protein MxA showed dense staining of perifascicular, and, sometimes all myofibers in 8/14 patients and on capillaries in 13/14 patients. Of 36 patients with other inflammatory myopathies, 1 patient had faint MxA staining of myofibers and 3 of capillaries. Plasmacytoid dendritic cells, potent CD4+ cellular sources of interferon-alpha, are present in substantial numbers in dermatomyositis and may account for most of the cells previously identified as T helper cells. In addition to an adaptive immune response, an innate immune response characterized by plasmacytoid dendritic cell infiltration and interferon-alpha/beta inducible gene and protein expression may be an important part of the pathogenesis of dermatomyositis, as it appears to be in systemic lupus erythematosus.
Liadaki K, Kho AT, Sanoudou D, Schienda J, Flint A, Beggs AH, Kohane IS, Kunkel LM. Side population cells isolated from different tissues share transcriptome signatures and express tissue-specific markers. Exp Cell ResExp Cell ResExp Cell Res. 2005;303:360-74.Abstract
Side Population (SP) cells, isolated from murine adult bone marrow (BM) based on the exclusion of the DNA dye Hoechst 33342, exhibit potent hematopoietic stem cell (HSC) activity when compared to Main Population (MP) cells. Furthermore, SP cells derived from murine skeletal muscle exhibit both hematopoietic and myogenic potential in vivo. The multipotential capacity of SP cells isolated from variable tissues is supported by an increasing number of studies. To investigate whether the SP phenotype is associated with a unique transcriptional profile, we characterized gene expression of SP cells isolated from two biologically distinct tissues, bone marrow and muscle. Comparison of SP cells with differentiated MP cells within a tissue revealed that SP cells are in an active transcriptional and translational status and underexpress genes reflecting tissue-specific functions. Direct comparison of gene expression of SP cells isolated from different tissues identified genes common to SP cells as well as genes specific to SP cells within a particular tissue and further define a muscle and bone marrow environment. This study reports gene expression of muscle SP cells, common features and differences between SP cells isolated from muscle and bone marrow, and further identifies common signaling pathways that might regulate SP cell functions.
Barnes CM, Huang S, Kaipainen A, Sanoudou D, Chen EJ, Eichler GS, Guo Y, Yu Y, Ingber DE, Mulliken JB, et al. Evidence by molecular profiling for a placental origin of infantile hemangioma. Proc Natl Acad Sci U S AProc Natl Acad Sci U S AProc Natl Acad Sci U S A. 2005;102:19097-102.Abstract
The origin of the pathogenic endothelial cells in common infantile hemangioma is unknown. We show here that the transcriptomes of human placenta and infantile hemangioma are sufficiently similar to suggest a placental origin for this tumor, expanding on recent immunophenotypical studies that have suggested this possibility [North, P. E., et al. (2001) Arch. Dermatol. 137, 559-570]. The transcriptomes of placenta, hemangioma, and eight normal and diseased tissues were compared by hierarchical and nonhierarchical clustering analysis of >7,800 genes. We found that the level of transcriptome similarity between placenta and hemangioma exceeded that of any other tissue compared and paralleled that observed between a given tissue and its derived tumor, such as normal and cancerous lung. The degree of similarity was even greater when a subset of endothelial cell-specific genes was analyzed. Genes preferentially expressed in both placenta and hemangiomas were identified, including 17-beta hydroxysteroid dehydrogenase type 2 and tissue factor pathway inhibitor 2. These data demonstrate the value of global molecular profiling of tissues as a tool for hypothesis-driven research. Furthermore, it suggests that the unique self-limited growth of infantile hemangioma may, in fact, mirror the lifetime of placental endothelium.
Sanoudou D, Vafiadaki E, Arvanitis DA, Kranias E, Kontrogianni-Konstantopoulos A. Array lessons from the heart: focus on the genome and transcriptome of cardiomyopathies. Physiol GenomicsPhysiol GenomicsPhysiol Genomics. 2005;21:131-43.Abstract
Our understanding of the cardiovascular system has evolved through the years by extensive studies emphasizing the identification of the molecular and physiological mechanisms involved in its normal function and disease pathogenesis. Major discoveries have been made along the way. However, the majority of this work has focused on specific genes or pathways rather than integrative approaches. In cardiomyopathies alone, over 30 different loci have shown mutations with varying inheritance patterns, yet mostly coding for structural proteins. The emergence of microarrays in the early 1990s paved the way to a new era of cardiovascular research. Microarrays dramatically accelerated the rhythm of discoveries by giving us the ability to simultaneously study thousands of genes in a single experiment. In the field of cardiovascular research, microarrays are having a significant contribution, with the majority of work focusing on end-stage cardiomyopathies that lead to heart failure. Novel molecular mechanisms have been identified, known pathways are seen under new light, disease subgroups begin to emerge, and the effects of various drugs are molecularly dissected. This cross-study data comparison concludes that consistent energy metabolism gene expression changes occur across dilated, hypertrophic, and ischemic cardiomyopathies, while Ca2+ homeostasis changes are prominent in the first two cardiomyopathies, and structural gene expression changes accompany mostly the dilated form. Gene expression changes are further correlated to disease genetics. The future of microarrays in the cardiomyopathy field is discussed with an emphasis on optimum experimental design and on applications in diagnosis, prognosis, and drug discovery.
Sanoudou D, Kang PB, Haslett JN, Han M, Kunkel LM, Beggs AH. Transcriptional profile of postmortem skeletal muscle. Physiol GenomicsPhysiol GenomicsPhysiol Genomics. 2004;16:222-8.Abstract
Autopsy specimens are often used in molecular biological studies of disease pathophysiology. However, few analyses have focused specifically on postmortem changes in skeletal muscles, and almost all of those investigate protein or metabolic changes. Although some structural and enzymatic changes have been described, the sequence of transcriptional events associated with these remains unclear. We analyzed a series of new and preexisting human skeletal muscle data sets on approximately 12,500 genes and expressed sequence tags (ESTs) generated by the Affymetrix U95Av2 GeneChips from seven autopsy and seven surgical specimens. Remarkably, postmortem specimens (up to 46 h) revealed a significant and prominent upregulation of transcripts involved with protein biosynthesis. Additional upregulated transcripts are associated with cellular responses to oxidative stress, hypoxia, and ischemia; however, only a subset of genes in these pathways was affected. Overexpression was also seen for apoptosis-related, cell cycle regulation/arrest-related, and signal transduction-related genes. No major gene expression differences were seen between autopsy specimens with <20-h and 34- to 46-h postmortem intervals or between pediatric and adult cases. These data demonstrate that, likely in response to hypoxia and oxidative stress, skeletal muscle undergoes a highly active transcriptional, and possibly, translational phase during the initial 46-h postmortem interval. Knowledge of these changes is important for proper interpretation of gene expression studies utilizing autopsy specimens.
Tomczak KK, Marinescu VD, Ramoni MF, Sanoudou D, Montanaro F, Han M, Kunkel LM, Kohane IS, Beggs AH. Expression profiling and identification of novel genes involved in myogenic differentiation. FASEB JFASEB JFASEB J. 2004;18:403-5.Abstract
Skeletal muscle differentiation is a complex, highly coordinated process that relies on precise temporal gene expression patterns. To better understand this cascade of transcriptional events, we used expression profiling to analyze gene expression in a 12-day time course of differentiating C2C12 myoblasts. Cluster analysis specific for time-ordered microarray experiments classified 2895 genes and ESTs with variable expression levels between proliferating and differentiating cells into 22 clusters with distinct expression patterns during myogenesis. Expression patterns for several known and novel genes were independently confirmed by real-time quantitative RT-PCR and/or Western blotting and immunofluorescence. MyoD and MEF family members exhibited unique expression kinetics that were highly coordinated with cell-cycle withdrawal regulators. Among genes with peak expression levels during cell cycle withdrawal were Vcam1, Itgb3, Itga5, Vcl, as well as Ptger4, a gene not previously associated with the process of myogenesis. One interesting uncharacterized transcript that is highly induced during myogenesis encodes several immunoglobulin repeats with sequence similarity to titin, a large sarcomeric protein. These data sets identify many additional uncharacterized transcripts that may play important functions in muscle cell proliferation and differentiation and provide a baseline for comparison with C2C12 cells expressing various mutant genes involved in myopathic disorders.
Sanoudou D, Frieden LA, Haslett JN, Kho AT, Greenberg SA, Kohane IS, Kunkel LM, Beggs AH. Molecular classification of nemaline myopathies: "nontyping" specimens exhibit unique patterns of gene expression. Neurobiol DisNeurobiol DisNeurobiol Dis. 2004;15:590-600.Abstract
Nemaline myopathy (NM) is a slowly progressive or nonprogressive neuromuscular disorder caused by mutations in genes encoding skeletal muscle sarcomeric thin filament proteins. It is characterized by great heterogeneity at the clinical, histopathological, and genetic level. Although multiple molecular pathways are commonly affected in all NM patients, little is known about the molecular characteristics of muscles from patients in different NM subgroups. We have analyzed a group of global gene expression data sets for transcriptional patterns characteristic of particular nemaline myopathy classes. Differential expression between disease subgroups was primarily seen in mitochondrial-, structural-, and transcription-related genes. Multiple lines of evidence support the hypothesis that muscles from cases with "nontyping" NM, although clinically classified as typical NM, share a unique pathophysiological state and are characterized by distinct patterns of gene expression. Determination of the specific molecular differences in NM subgroups may eventually lead to improved prognostic determinations and treatment of these patients.
Mahler-Araujo MB, Sanoudou D, Tingby O, Liu L, Coleman N, Ichimura K, Collins VP. Structural genomic abnormalities of chromosomes 9 and 18 in myxopapillary ependymomas. J Neuropathol Exp NeurolJ Neuropathol Exp NeurolJ Neuropathol Exp Neurol. 2003;62:927-35.Abstract
Myxopapillary ependymomas (MPEs) are low-grade neuroepithelial tumors typically occurring in the conus-cauda equina-filum terminale region. Limited molecular and cytogenetic analysis of MPEs has not demonstrated consistent abnormalities. In an attempt to clarify the chromosomal status of these tumors and identify commonly aberrant regions in the genome we have combined 3 molecular/cyto/genetic methods to study 17 MPEs. Comparative genomic hybridization of 7/17 tumors identified concurrent gain on chromosomes 9 and 18 as the most frequent finding. The majority of the 17 tumors were also studied using microsatellite analysis with marker spanning the whole chromosomes 9 and 18 and interphase-FISH with centromeric probes for both chromosomes. Our combined results were consistent with concurrent gain in both chromosomes 9 and 18 in 11/17 cases, gain of either chromosome 9 or 18 and imbalance in the other chromosome in 3/17 tumors and allelic imbalances of chromosomes 9 or 18 in 3/17 and 1/17 tumors, respectively. Other abnormalities observed included gain of chromosomes 3, 4, 7, 8, 11, 13, 17q, 20, and X and loss of chromosomes 10 and 22. Our findings represent some steps towards understanding the molecular mechanisms involved in the development of MPE.
Nimgaonkar A, Sanoudou D, Butte AJ, Haslett JN, Kunkel LM, Beggs AH, Kohane IS. Reproducibility of gene expression across generations of Affymetrix microarrays. BMC BioinformaticsBMC BioinformaticsBMC Bioinformatics. 2003;4:27.Abstract
BACKGROUND: The development of large-scale gene expression profiling technologies is rapidly changing the norms of biological investigation. But the rapid pace of change itself presents challenges. Commercial microarrays are regularly modified to incorporate new genes and improved target sequences. Although the ability to compare datasets across generations is crucial for any long-term research project, to date no means to allow such comparisons have been developed. In this study the reproducibility of gene expression levels across two generations of Affymetrix GeneChips (HuGeneFL and HG-U95A) was measured. RESULTS: Correlation coefficients were computed for gene expression values across chip generations based on different measures of similarity. Comparing the absolute calls assigned to the individual probe sets across the generations found them to be largely unchanged. CONCLUSION: We show that experimental replicates are highly reproducible, but that reproducibility across generations depends on the degree of similarity of the probe sets and the expression level of the corresponding transcript.
Ryan MM, Ilkovski B, Strickland CD, Schnell C, Sanoudou D, Midgett C, Houston R, Muirhead D, Dennett X, Shield LK, et al. Clinical course correlates poorly with muscle pathology in nemaline myopathy. NeurologyNeurologyNeurology. 2003;60:665-73.Abstract
OBJECTIVE: To report pathologic findings in 124 Australian and North American cases of primary nemaline myopathy. METHODS: Results of 164 muscle biopsies from 124 Australian and North American patients with primary nemaline myopathy were reviewed, including biopsies from 19 patients with nemaline myopathy due to alpha-actin (ACTA1) mutations and three with mutations in alpha-tropomyosin(SLOW) (TPM3). For each biopsy rod number per fiber, percentage of fibers with rods, fiber-type distribution of rods, and presence or absence of intranuclear rods were documented. RESULTS: Rods were present in all skeletal muscles and diagnosis was possible at all ages. Most biopsies contained nemaline bodies in more than 50% of fibers, although rods were seen only on electron microscopy in 10 patients. Rod numbers and localization correlated poorly with clinical severity. Frequent findings included internal nuclei and increased fiber size variation, type 1 fiber predominance and atrophy, and altered expression of fiber type specific proteins. Marked sarcomeric disruption, increased glycogen deposition, and intranuclear rods were associated with more severe clinical phenotypes. Serial biopsies showed progressive fiber size variation and increasing numbers of rods with time. Pathologic findings varied widely in families with multiple affected members. CONCLUSIONS: Very numerous nemaline bodies, glycogen accumulation, and marked sarcomeric disruption were common in nemaline myopathy associated with mutations in skeletal alpha-actin. Nemaline myopathy due to mutations in alpha-tropomyosin(SLOW) was characterized by preferential rod formation in, and atrophy of, type 1 fibers. Light microscopic features of nemaline myopathy correlate poorly with disease course. Electron microscopy may correlate better with disease severity and genotype.
Haslett JN, Sanoudou D, Kho AT, Han M, Bennett RR, Kohane IS, Beggs AH, Kunkel LM. Gene expression profiling of Duchenne muscular dystrophy skeletal muscle. NeurogeneticsNeurogeneticsNeurogenetics. 2003;4:163-71.Abstract
The primary cause of Duchenne muscular dystrophy (DMD) is a mutation in the dystrophin gene, leading to absence of the corresponding protein, disruption of the dystrophin-associated protein complex, and substantial changes in skeletal muscle pathology. Although the primary defect is known and the histological pathology well documented, the underlying molecular pathways remain in question. To clarify these pathways, we used expression microarrays to compare individual gene expression profiles for skeletal muscle biopsies from DMD patients and unaffected controls. We have previously published expression data for the 12,500 known genes and full-length expressed sequence tags (ESTs) on the Affymetrix HG-U95Av2 chips. Here we present comparative expression analysis of the 50,000 EST clusters represented on the remainder of the Affymetrix HG-U95 set. Individual expression profiles were generated for biopsies from 10 DMD patients and 10 unaffected control patients. Two methods of statistical analysis were used to interpret the resulting data (t-test analysis to determine the statistical significance of differential expression and geometric fold change analysis to determine the extent of differential expression). These analyses identified 183 probe sets (59 of which represent known genes) that differ significantly in expression level between unaffected and disease muscle. This study adds to our knowledge of the molecular pathways that are altered in the dystrophic state. In particular, it suggests that signaling pathways might be substantially involved in the disease process. It also highlights a large number of unknown genes whose expression is altered and whose identity therefore becomes important in understanding the pathogenesis of muscular dystrophy.
Sanoudou D, Haslett JN, Kho AT, Guo S, Gazda HT, Greenberg SA, Lidov HG, Kohane IS, Kunkel LM, Beggs AH. Expression profiling reveals altered satellite cell numbers and glycolytic enzyme transcription in nemaline myopathy muscle. Proc Natl Acad Sci U S AProc Natl Acad Sci U S AProc Natl Acad Sci U S A. 2003;100:4666-71.Abstract
The nemaline myopathies (NMs) are a clinically and genetically heterogeneous group of disorders characterized by nemaline rods and skeletal muscle weakness. Mutations in five sarcomeric thin filament genes have been identified. However, the molecular consequences of these mutations are unknown. Using Affymetrix oligonucleotide microarrays, we have analyzed the expression patterns of >21,000 genes and expressed sequence tags in skeletal muscles of 12 NM patients and 21 controls. Multiple complementary approaches were used for data analysis, including geometric fold analysis, two-tailed unequal variance t test, hierarchical clustering, relevance network, and nearest-neighbor analysis. We report the identification of high satellite cell populations in NM and the significant down-regulation of transcripts for key enzymes of glucose and glycogen metabolism as well as a possible regulator of fatty acid metabolism, UCP3. Interestingly, transcript level changes of multiple genes suggest possible changes in Ca(2+) homeostasis. The increased expression of multiple structural proteins was consistent with increased fibrosis. This comprehensive study of downstream molecular consequences of NM gene mutations provides insights in the cellular events leading to the NM phenotype.
Greenberg SA, Sanoudou D, Haslett JN, Kohane IS, Kunkel LM, Beggs AH, Amato AA. Molecular profiles of inflammatory myopathies. NeurologyNeurologyNeurology. 2002;59:1170-82.Abstract
OBJECTIVE: To describe the use of large-scale gene expression profiles to distinguish broad categories of myopathy and subtypes of inflammatory myopathies (IM) and to provide insight into the pathogenesis of inclusion body myositis (IBM), polymyositis, and dermatomyositis. METHODS: Using Affymetrix GeneChip microarrays, the authors measured the simultaneous expression of approximately 10,000 genes in muscle specimens from 45 patients in four major disease categories (dystrophy, congenital myopathy, inflammatory myopathy, and normal). The authors separately analyzed gene expression in 14 patients limited to the three major subtypes of IM. Bioinformatics techniques were used to classify specimens with similar expression profiles based on global patterns of gene expression and to identify genes with significant differential gene expression compared with normal. RESULTS: Ten of 11 patients with IM, all normals and nemaline myopathies, and 10 of 12 patients with Duchenne muscular dystrophy were correctly classified by this approach. The various subtypes of inflammatory myopathies have distinct gene expression signatures. Specific sets of immune-related genes allow for molecular classification of patients with IBM, polymyositis, and dermatomyositis. Analysis of differential gene expression identifies as relevant to disease pathogenesis previously reported cytokines, major histocompatibility complex class I and II molecules, granzymes, and adhesion molecules, as well as newly identified members of these categories. Increased expression of actin cytoskeleton genes is also identified. CONCLUSIONS: The molecular profiles of muscle tissue in patients with inflammatory myopathies are distinct and represent molecular signatures from which diagnostic insight may follow. Large numbers of differentially expressed genes are rapidly identified.
Haslett JN, Sanoudou D, Kho AT, Bennett RR, Greenberg SA, Kohane IS, Beggs AH, Kunkel LM. Gene expression comparison of biopsies from Duchenne muscular dystrophy (DMD) and normal skeletal muscle. Proc Natl Acad Sci U S AProc Natl Acad Sci U S AProc Natl Acad Sci U S A. 2002;99:15000-5.Abstract
The primary cause of Duchenne muscular dystrophy (DMD) is a mutation in the dystrophin gene leading to the absence of the corresponding RNA transcript and protein. Absence of dystrophin leads to disruption of the dystrophin-associated protein complex and substantial changes in skeletal muscle pathology. Although the histological pathology of dystrophic tissue has been well documented, the underlying molecular pathways remain poorly understood. To examine the pathogenic pathways and identify new or modifying factors involved in muscular dystrophy, expression microarrays were used to compare individual gene expression profiles of skeletal muscle biopsies from 12 DMD patients and 12 unaffected control patients. Two separate statistical analysis methods were used to interpret the resulting data: t test analysis to determine the statistical significance of differential expression and geometric fold change analysis to determine the extent of differential expression. These analyses identified 105 genes that differ significantly in expression level between unaffected and DMD muscle. Many of the differentially expressed genes reflect changes in histological pathology. For instance, immune response signals and extracellular matrix genes are overexpressed in DMD muscle, an indication of the infiltration of inflammatory cells and connective tissue. Significantly more genes are overexpressed than are underexpressed in dystrophic muscle, with dystrophin underexpressed, whereas other genes encoding muscle structure and regeneration processes are overexpressed, reflecting the regenerative nature of the disease.
Sanoudou D, Beggs AH. Clinical and genetic heterogeneity in nemaline myopathy--a disease of skeletal muscle thin filaments. Trends Mol MedTrends Mol MedTrends Mol Med. 2001;7:362-8.Abstract
The term nemaline myopathy (NM) encompasses a heterogeneous group of disorders of primary skeletal muscle weakness characterized by the presence of nemaline rods in muscles of affected individuals. Disease severity is variable and unpredictable, with prognosis ranging from neonatal death to almost normal motor function. Recent advances in the identification of NM disease genes demonstrate that NM is a disease of the skeletal muscle sarcomere and, in particular, of the thin filaments. These findings are starting to alter the approach that neurologists and geneticists take to diagnosing and counseling patients with NM, and could lead to insights into specific directed therapies in the future.
Sanoudou D, Tingby O, Ferguson-Smith MA, Collins VP, Coleman N. Analysis of pilocytic astrocytoma by comparative genomic hybridization. Br J CancerBr J CancerBr J Cancer. 2000;82:1218-22.Abstract
Very little is known about genetic abnormalities involved in the development of pilocytic astrocytoma, the most frequently occurring brain tumour of childhood. We have analysed 48 pilocytic astrocytoma specimens using comparative genomic hybridization. Only five of 41 tumours from children showed abnormalities detectable by comparative genomic hybridization, and in each case this represented gain of a single chromosome. Interestingly, two of seven tumours from adults showed abnormalities, which were multiple and relatively complex. Six of the seven tumours showing abnormalities were from female patients (two adults and four children). The most frequently detectable abnormality was gain of 9q34.1-qter, which was present in three cases (two adult and one paediatric).
Li YC, Lee C, Sanoudou D, Hseu TH, Li SY, Lin CC. Interstitial colocalization of two cervid satellite DNAs involved in the genesis of the Indian muntjac karyotype. Chromosome ResChromosome ResChromosome Res. 2000;8:363-73.Abstract
A number of repetitive DNA clones were generated from PCR amplifications of Indian muntjac genomic DNA using primer sequences derived from a white tailed deer satellite II DNA sequence. One clone (Mmv-0.7) was characterized and shown to be a cervid satellite II DNA clone. Multiple colored FISH studies with cervid satellite I (C5) and this satellite II clone (Mmv-0.7) to Chinese muntjac metaphase chromosomes localized both satellite DNAs at the pericentromeric regions of all chromosomes except for chromosome 3 and the Y chromosome, whereas chromosome 3 exhibited pericentromeric satellite II DNA only. Where distinguishable, the pericentromeric satellite II signals appeared terminally oriented with respect to satellite I. Six pairs of Chinese muntjac autosomes had interstitial satellite I sites with four of these autosomal pairs (chromosomes 1, 2 and two other smaller autosomal pairs) also exhibiting interstitial satellite II signals. An interstitial site on the X chromosome was found to have satellite II signals. For the Indian muntjac chromosomes, FISH studies revealed a pericentromeric hybridization for satellites I and II as well as 27 distinct interstitial hybridization sites, each having at least one of the satellite DNAs. These data were used to more precisely define the chromosome fusion-associated breakpoints that presumably led to the formation of the present-day Indian muntjac karyotype. It further hints at the possibility that the Indian muntjac karyotype may have evolved directly from a 2n = 70 ancestral karyotype rather than from an intermediate 2n = 46 Chinese muntjac-like karyotype.
Goike HM, Asplund AC, Pettersson EH, Liu L, Sanoudou D, Collins VP. Acquired rearrangement of an amplified epidermal growth factor receptor (EGFR) gene in a human glioblastoma xenograft. J Neuropathol Exp NeurolJ Neuropathol Exp NeurolJ Neuropathol Exp Neurol. 1999;58:697-701.Abstract
Amplification of the epidermal growth factor receptor (EGFR) occurs in about 40% of human glioblastomas. In half of these cases, rearrangements of the amplified gene result in aberrant transcripts and proteins. The most frequent rearrangement affects the external domain of the receptor and results in nonbinding of ligand and constitutive activity. Less frequent rearrangements involve changes resulting in the loss of cytoplasmic amino acid sequences necessary for downregulation of the receptor following ligand binding. Here we report the development and selection for a rearranged amplified EGF receptor, which lacks cytoplasmic amino acid sequences in a human glioblastoma xenograft. An identical aberration has previously been reported in glioblastoma tissue. The patient tumor material, as well as the first passages of the xenograft showed amplification of the EGFR gene, but no evidence of gene rearrangement or an aberrant transcript. Interphase FISH data show the amplified gene on double minutes. Between passages 3 and 16, the growth rate of the xenograft almost doubled, the rearranged amplicon became dominant, as did the aberrant transcript, indicating selection under these conditions.
Ding X, Sanoudou D, Yang F, Chen H. [Determination of trisomy 9p by molecular cytogenetic technology]. Zhonghua Yi Xue Yi Chuan Xue Za ZhiZhonghua Yi Xue Yi Chuan Xue Za ZhiZhonghua Yi Xue Yi Chuan Xue Za Zhi. 1999;16:383-5.Abstract
OBJECTIVE: To use molecular cytogenetic techniques for the determination of complex chromosomal aberrations that could not be distinguished by conventional cytogenetic method. METHODS: Chromosome painting, comparative genomic hybridization (CGH) and color banding chromosome analysis (RxFISH) were used to identify a case with chromosome 9 aberration by G-banding. RESULTS: The patient has a karyotype of 46,XX,9p+ by G-banding. Both chromosomes 9 were uniformly painted, including the extra segment on one of the 9p alleles. CGH revealed a duplication of the entire 9p short arm. After analysis with RxFISH the patient's karyotype could be accurately described as 46,XX,dup9p (p11-->p24::p24-->qter). CONCLUSION: The present report shows that some late technological developments in the field of cytogenetics can facilitate the study of the diseases linked to complex chromosomal aberrations and may find significant application in basic and clinical medical studies.
Griffin DK, Sanoudou D, Adamski E, McGiffert C, O'Brien P, Wienberg J, Ferguson-Smith MA. Chromosome specific comparative genome hybridisation for determining the origin of intrachromosomal duplications. J Med GenetJ Med GenetJ Med Genet. 1998;35:37-41.Abstract
Chromosome specific comparative genome hybridisation (CGH) is a novel approach for the detection of cytogenetic abnormalities. It combines flow sorting of chromosomes, degenerate oligonucleotide primed (DOP)-PCR and a modified comparative genome hybridisation (CGH) technique to define the site and extent of intrachromosomal duplications. Chromosome specific paint probes for aberrant chromosomes and their normal homologues from four subjects with unbalanced duplications within chromosomes 2p11-15, 3q25-26, 5q34-qter, and 12q23-24.2 were made. They were then cohybridised on normal metaphase spreads and the ratio of their relative intensities of hybridisation analysed. The results were compared to those of similar experiments where regular CGH was performed on the same four patients. We provide evidence that this method can detect duplications and deficiencies which might be missed by conventional CGH, as the ratio of hybridisation of abnormal/normal DNA is 2:1 rather than 3:2. It is the method of choice where mosaicism is present or where only one of several homologous chromosomes is duplicated. Furthermore, it suggests that DOP-PCR amplifies all or most of the euchromatic regions of the genome equally.