Xiaoke Yin

Proteomics Characterization of Extracellular Space Components in the Human Aorta

The vascular extracellular matrix (ECM) is essential for the structural integrity of the vessel wall and also serves as a substrate for the binding and retention of secreted products of vascular cells as well as molecules coming from the circulation. Although proteomics has been previously applied to vascular tissues, few studies have specifically targeted the vascular ECM and its associated proteins. Thus, its detailed composition remains to be characterized. In this study, we describe a methodology for the extraction of extracellular proteins from human aortas and their identification by proteomics. The approach is based on (a) effective decellularization to enrich for scarce extracellular proteins, (b) successful solubilization and deglycosylation of ECM proteins, and (c) relative estimation of protein abundance using spectral counting. Our three-step extraction approach resulted in the identification of 103 extracellular proteins of which one-third have never been reported in the proteomics literature of vascular tissues. In particular, three glycoproteins (podocan, sclerostin, and agrin) were identified for the first time in human aortas at the protein level. We also identified extracellular adipocyte enhancer-binding protein 1, the cartilage glycoprotein asporin, and a previously hypothetical protein, retinal pigment epithelium (RPE) spondin. Moreover, our methodology allowed us to screen for proteolysis in the aortic samples based on the identification of proteolytic enzymes and their corresponding degradation products. For instance, we were able to detect matrix metalloproteinase-9 by mass spectrometry and relate its presence to degradation of fibronectin in a clinical specimen. We expect this proteomics methodology to further our understanding of the composition of the vascular extracellular environment, shed light on ECM remodeling and degradation, and provide insights into important pathological processes, such as plaque rupture, aneurysm formation, and restenosis.

Short Communication: Asymmetric Dimethylarginine Impairs Angiogenic Progenitor Cell Function in Patients With Coronary Artery Disease Through a MicroRNA-21–Dependent Mechanism

Rationale: The endogenous nitric oxide synthase inhibitor asymmetrical dimethylarginine (ADMA) is increased in patients with coronary artery disease and may regulate function of circulating angiogenic progenitor cells (APCs) by small regulatory RNAs. Objectives: To study the role of microRNAs in ADMA-mediated impairment of APCs. Methods and Results: By using microarray analyses, we established microRNA expression profiles of human APCs. We used ADMA to induce APC dysfunction and found 16 deregulated microRNAs. We focused on miR-21, which was 3-fold upregulated by ADMA treatment. Overexpression of miR-21 in human APCs impaired migratory capacity. To identify regulated miR-21 targets, we used proteome analysis, using difference in-gel electrophoresis followed by mass spectrometric analysis of regulated proteins. We found that transfection of miR-21 precursors significantly repressed superoxide dismutase 2 in APCs, which resulted in increased intracellular reactive oxygen species concentration and impaired nitric oxide bioavailability. MiR-21 further repressed sprouty-2, leading to Erk Map kinase–dependent reactive oxygen species formation and APC migratory defects. Small interference RNA–mediated superoxide dismutase 2 or sprouty-2 reduction also increased reactive oxygen species formation and impaired APC migratory capacity. ADMA-mediated reactive oxygen species formation and APC dysfunction was rescued by miR-21 blockade. APCs from patients with coronary artery disease and high ADMA plasma levels displayed >4-fold elevated miR-21 levels, low superoxide dismutase 2 expression, and impaired migratory capacity, which could be normalized by miR-21 antagonism. Conclusions: We identified a novel miR-21–dependent mechanism of ADMA-mediated APC dysfunction. MiR-21 antagonism therefore emerges as an interesting strategy to improve dysfunctional APCs in patients with coronary artery disease.

Extracellular Matrix Composition and Remodeling in Human Abdominal Aortic Aneurysms: A Proteomics Approach

Abdominal aortic aneurysms (AAA) are characterized by pathological remodeling of the aortic extracellular matrix (ECM). However, besides the well-characterized elastolysis and collagenolysis little is known about changes in other ECM proteins. Previous proteomics studies on AAA focused on cellular changes without emphasis on the ECM. In the present study, ECM proteins and their degradation products were selectively extracted from aneurysmal and control aortas using a solubility-based subfractionation methodology and analyzed by gel-liquid chromatography-tandem MS and label-free quantitation. The proteomics analysis revealed novel changes in the ECM of AAA, including increased expression as well as degradation of collagen XII, thrombospondin 2, aortic carboxypeptidase-like protein, periostin, fibronectin and tenascin. Proteomics also confirmed the accumulation of macrophage metalloelastase (MMP-12). Incubation of control aortic tissue with recombinant MMP-12 resulted in the extensive fragmentation of these glycoproteins, most of which are novel substrates of MMP-12. In conclusion, our proteomics methodology allowed the first detailed analysis of the ECM in AAA and identified markers of pathological ECM remodeling related to MMP-12 activity.

Vascular Smooth Muscle Cell Calcification Is Mediated by Regulated Exosome Secretion

RATIONALE Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. OBJECTIVE The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. METHODS AND RESULTS Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. CONCLUSIONS This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention.

Combined metabolomic and proteomic analysis of human atrial fibrillation.

OBJECTIVES We sought to decipher metabolic processes servicing the increased energy demand during persistent atrial fibrillation (AF) and to ascertain whether metabolic derangements might instigate this arrhythmia. BACKGROUND Whereas electrical, structural, and contractile remodeling processes are well-recognized contributors to the self-perpetuating nature of AF, the impact of cardiac metabolism upon the persistence/initiation of this resilient arrhythmia has not been explored in detail. METHODS Human atrial appendage tissues from matched cohorts in sinus rhythm (SR), from those who developed AF post-operatively, and from patients in persistent AF undergoing cardiac surgery were analyzed using a combined metabolomic and proteomic approach. RESULTS High-resolution proton nuclear magnetic resonance (NMR) spectroscopy of cardiac tissue from patients in persistent AF revealed a rise in beta-hydroxybutyrate, the major substrate in ketone body metabolism, along with an increase in ketogenic amino acids and glycine. These metabolomic findings were substantiated by proteomic experiments demonstrating differential expression of 3-oxoacid transferase, the key enzyme for ketolytic energy production. Notably, compared with the SR cohort, the group susceptible to post-operative AF showed a discordant regulation of energy metabolites. Combined principal component and linear discriminant analyses of metabolic profiles from proton NMR spectroscopy correctly classified more than 80% of patients at risk of AF at the time of coronary artery bypass grafting. CONCLUSIONS The present study characterized the metabolic adaptation to persistent AF, unraveling a potential role for ketone bodies, and demonstrated that discordant metabolic alterations are evident in individuals susceptible to post-operative AF.

Proteomics Analysis of Cardiac Extracellular Matrix Remodeling in a Porcine Model of Ischemia/Reperfusion Injury

Background— After myocardial ischemia, extracellular matrix (ECM) deposition occurs at the site of the focal injury and at the border region. Methods and Results— We have applied a novel proteomic method for the analysis of ECM in cardiovascular tissues to a porcine model of ischemia/reperfusion injury. ECM proteins were sequentially extracted and identified by liquid chromatography tandem mass spectrometry. For the first time, ECM proteins such as cartilage intermediate layer protein 1, matrilin-4, extracellular adipocyte enhancer binding protein 1, collagen &agr;-1(XIV), and several members of the small leucine-rich proteoglycan family, including asporin and prolargin, were shown to contribute to cardiac remodeling. A comparison in 2 distinct cardiac regions (the focal injury in the left ventricle and the border region close to the occluded coronary artery) revealed a discordant regulation of protein and mRNA levels; although gene expression for selected ECM proteins was similar in both regions, the corresponding protein levels were much higher in the focal lesion. Further analysis based on >100 ECM proteins delineated a signature of early- and late-stage cardiac remodeling with transforming growth factor-&bgr;1 signaling at the center of the interaction network. Finally, novel cardiac ECM proteins identified by proteomics were validated in human left ventricular tissue acquired from ischemic cardiomyopathy patients at cardiac transplantation. Conclusion— Our findings reveal a biosignature of early- and late-stage ECM remodeling after myocardial ischemia/reperfusion injury, which may have clinical utility as a prognostic marker and modifiable target for drug discovery.

Macrophage MicroRNA-155 Promotes Cardiac Hypertrophy and Failure

Background— Cardiac hypertrophy and subsequent heart failure triggered by chronic hypertension represent major challenges for cardiovascular research. Beyond neurohormonal and myocyte signaling pathways, growing evidence suggests inflammatory signaling pathways as therapeutically targetable contributors to this process. We recently reported that microRNA-155 is a key mediator of cardiac inflammation and injury in infectious myocarditis. Here, we investigated the impact of microRNA-155 manipulation in hypertensive heart disease. Methods and Results— Genetic loss or pharmacological inhibition of the leukocyte-expressed microRNA-155 in mice markedly reduced cardiac inflammation, hypertrophy, and dysfunction on pressure overload. These alterations were macrophage dependent because in vivo cardiomyocyte-specific microRNA-155 manipulation did not affect cardiac hypertrophy or dysfunction, whereas bone marrow transplantation from wild-type mice into microRNA-155 knockout animals rescued the hypertrophic response of the cardiomyocytes and vice versa. In vitro, media from microRNA-155 knockout macrophages blocked the hypertrophic growth of stimulated cardiomyocytes, confirming that macrophages influence myocyte growth in a microRNA-155-dependent paracrine manner. These effects were at least partly mediated by the direct microRNA-155 target suppressor of cytokine signaling 1 (Socs1) because Socs1 knockdown in microRNA-155 knockout macrophages largely restored their hypertrophy-stimulating potency. Conclusions— Our findings reveal that microRNA-155 expression in macrophages promotes cardiac inflammation, hypertrophy, and failure in response to pressure overload. These data support the causative significance of inflammatory signaling in hypertrophic heart disease and demonstrate the feasibility of therapeutic microRNA targeting of inflammation in heart failure.

Proteomics identifies thymidine phosphorylase as a key regulator of the angiogenic potential of colony-forming units and endothelial progenitor cell cultures.

Endothelial progenitor cell (EPC) cultures and colony-forming units (CFUs) have been extensively studied for their therapeutic and diagnostic potential. Recent data suggest a role for EPCs in the release of proangiogenic factors. To identify factors secreted by EPCs, conditioned medium from EPC cultures and CFUs was analyzed using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer combined with offline peptide separation by nanoflow liquid chromatography. Results were verified by RT-PCR and multiplex cytokine assays and complemented by a cellular proteomic analysis of cultured EPCs and CFUs using difference in-gel electrophoresis. This extensive proteomic analysis revealed the presence of the proangiogenic factor thymidine phosphorylase (TP). Functional experiments demonstrated that inhibition of TP by 5-bromo-6-amino-uracil or gene silencing resulted in a significant increase in basal and oxidative stress-induced apoptosis, whereas supplementation with 2-deoxy-d-ribose-1-phosphate (dRP), the enzymatic product of TP, abrogated this effect. Moreover, dRP produced in EPC cultures stimulated endothelial cell migration in a paracrine manner, as demonstrated by gene-silencing experiments in transmigration and wound repair assays. RGD peptides and inhibitory antibodies to integrin &agr;v&bgr;3 attenuated the effect of conditioned medium from EPC cultures on endothelial migration. Finally, the effect of TP on angiogenesis was investigated by implantation of Matrigel plugs in mice. In these in vivo experiments, dRP strongly promoted neovascularization. Our data support the concept that EPCs exert their proangiogenic activity in a paracrine manner and demonstrate a key role of TP activity in their survival and proangiogenic potential.

The hypoxia-inducible microRNA cluster miR-199a∼214 targets myocardial PPARδ and impairs mitochondrial fatty acid oxidation.

Peroxisome proliferator-activated receptor δ (PPARδ) is a critical regulator of energy metabolism in the heart. Here, we propose a mechanism that integrates two deleterious characteristics of heart failure, hypoxia and a metabolic shift toward glycolysis, involving the microRNA cluster miR-199a∼214 and PPARδ. We demonstrate that under hemodynamic stress, cardiac hypoxia activates DNM3os, a noncoding transcript that harbors the microRNA cluster miR-199a∼214, which shares PPARδ as common target. To address the significance of miR-199a∼214 induction and concomitant PPARδ repression, we performed antagomir-based silencing of both microRNAs and subjected mice to biomechanical stress to induce heart failure. Remarkably, antagomir-treated animals displayed improved cardiac function and restored mitochondrial fatty acid oxidation. Taken together, our data suggest a mechanism whereby miR-199a∼214 actively represses cardiac PPARδ expression, facilitating a metabolic shift from predominant reliance on fatty acid utilization in the healthy myocardium toward increased reliance on glucose metabolism at the onset of heart failure.

Terminal Differentiation, Advanced Organotypic Maturation, and Modeling of Hypertrophic Growth in Engineered Heart Tissue

Rationale: Cardiac tissue engineering should provide “realistic” in vitro heart muscle models and surrogate tissue for myocardial repair. For either application, engineered myocardium should display features of native myocardium, including terminal differentiation, organotypic maturation, and hypertrophic growth. Objective: To test the hypothesis that 3D-engineered heart tissue (EHT) culture supports (1) terminal differentiation as well as (2) organotypic assembly and maturation of immature cardiomyocytes, and (3) constitutes a methodological platform to investigate mechanisms underlying hypertrophic growth. Methods and Results: We generated EHTs from neonatal rat cardiomyocytes and compared morphological and molecular properties of EHT and native myocardium from fetal, neonatal, and adult rats. We made the following key observations: cardiomyocytes in EHT (1) gained a high level of binucleation in the absence of notable cytokinesis, (2) regained a rod-shape and anisotropic sarcomere organization, (3) demonstrated a fetal-to-adult gene expression pattern, and (4) responded to distinct hypertrophic stimuli with concentric or eccentric hypertrophy and reexpression of fetal genes. The process of terminal differentiation and maturation (culture days 7–12) was preceded by a tissue consolidation phase (culture days 0–7) with substantial cardiomyocyte apoptosis and dynamic extracellular matrix restructuring. Conclusions: This study documents the propensity of immature cardiomyocytes to terminally differentiate and mature in EHT in a remarkably organotypic manner. It moreover provides the rationale for the utility of the EHT technology as a methodological bridge between 2D cell culture and animal models.