Cardiovascular Research

Smyd1/Bop is an evolutionary conserved histone methyltransferase previously shown by conventional knockout to be critical for embryonic heart development. To further explore the mechanism(s) in a cell autonomous context, we conditionally ablated Smyd1 in the first and second heart fields of mice using a knock-in (KI) Nkx2.5-cre driver. Robust deletion of floxed-Smyd1 in cardiomyocytes and the outflow tract (OFT) resulted in embryonic lethality at E9.5, truncation of the OFT and right ventricle, and additional defects consistent with impaired expansion and proliferation of the second heart field (SHF). Using a transgenic (Tg) Nkx2.5-cre driver previously shown to not delete in the SHF and OFT, early embryonic lethality was bypassed and both ventricular chambers were formed; however, reduced cardiomyocyte proliferation and other heart defects resulted in later embryonic death at E11.5-12.5. Proliferative impairment prior to both early and mid-gestational lethality was accompanied by dysregulation of transcripts critical for endoplasmic reticulum (ER) stress. Mid-gestational death was also associated with impairment of oxidative stress defense-a phenotype highly similar to the previously characterized knockout of the Smyd1-interacting transcription factor, skNAC. We describe a potential feedback mechanism in which the stress response factor Tribbles3/TRB3, when directly methylated by Smyd1, acts as a co-repressor of Smyd1-mediated transcription. Our findings suggest that Smyd1 is required for maintaining cardiomyocyte proliferation at minimally two different embryonic heart developmental stages, and its loss leads to linked stress responses that signal ensuing lethality.

Genetic or nutritional deficiencies in homocysteine (Hcy) metabolism lead to hyperhomocysteinemia (HHcy) and cause endothelial dysfunction, a hallmark of atherosclerosis. In addition to Hcy, related metabolites accumulate in HHcy but their role in endothelial dysfunction is unknown. Here, we examine how Hcy-thiolactone, N-Hcy-protein, and Hcy affect gene expression and molecular pathways in human umbilical vein endothelial cells. We used microarray technology, real-time quantitative polymerase chain reaction, and bioinformatic analysis with PANTHER, DAVID, and Ingenuity Pathway Analysis (IPA) resources. We identified 47, 113, and 30 mRNAs regulated by N-Hcy-protein, Hcy-thiolactone, and Hcy, respectively, and found that each metabolite induced a unique pattern of gene expression. Top molecular pathways affected by Hcy-thiolactone were chromatin organization, one-carbon metabolism, and lipid-related processes [−log(P value) = 20–31]. Top pathways affected by N-Hcy-protein and Hcy were blood coagulation, sulfur amino acid metabolism, and lipid metabolism [−log(P value)] = 4–11; also affected by Hcy-thiolactone, [−log(P value) = 8–14]. Top disease related to Hcy-thiolactone, N-Hcy-protein, and Hcy was ‘atherosclerosis, coronary heart disease’ [−log(P value) = 9–16]. Top-scored biological networks affected by Hcy-thiolactone (score = 34–40) were cardiovascular disease and function; those affected by N-Hcy-protein (score = 24–35) were ‘small molecule biochemistry, neurological disease,’ and ‘cardiovascular system development and function’; and those affected by Hcy (score = 25–37) were ‘amino acid metabolism, lipid metabolism,’ ‘cellular movement, and cardiovascular and nervous system development and function.’ These results indicate that each Hcy metabolite uniquely modulates gene expression in pathways important for vascular homeostasis and identify new genes and pathways that are linked to HHcy-induced endothelial dysfunction and vascular disease.

Background After surgical resection of hepatocellular carcinoma (HCC), recurrence is common, especially in patients presenting with vascular invasion or multifocal disease after curative surgery. Consequently, we examined the expression pattern and prognostic value of miR-19b in samples from these patients. Methods We performed a miRNA microarray to detect differential expression of microRNAs (miRNAs) in 5 paired samples of HCC and non-tumoral adjacent liver tissue and a quantitative real-time polymerase chain reaction (PCR) analysis to validate the results in 81 paired samples of HCC and adjacent non-tumoral liver tissues. We examined the associations of miR-19b expression with clinicopathological parameters and survival. MiR-19b was knocked down in Hep3B and an mRNA microarray was performed to detect the affected genes. Results In both the miRNA microarray and real-time PCR, miR-19b was significantly overexpressed in the HCC tumor compared with adjacent non-tumor liver tissues (P < 0.001). The expression of miR-19b was significantly higher in patients who were disease-free 2 years after surgery (P < 0.001). High miR-19b expression levels were associated with higher α-fetoprotein levels (P = 0.017). In the log-rank test, high miR-19b was associated with better disease-free survival (median survival 37.107 vs. 11.357; P = 0.022). In Cox multivariate analysis, high miR-19b predicted better disease-free survival and overall survival (hazards ratio [HR] = 0.453, 95 % confidence interval [CI] = 0.245–0.845, P = 0.013; HR = 0.318, CI = 0.120–0.846, P = 0.022, respectively). N-myc downstream regulated 1 (NDRG1) was downregulated, while epithelial cell adhesion molecule (EPCAM), hypoxia-inducible factor 1-alpha (HIF1A), high-mobility group protein B2 (HMGB2), and mitogen activated protein kinase 14 (MAPK14) were upregulated when miR-19b was knocked down in Hep3B. Conclusions The overexpression of miR-19b was significantly correlated with better disease-free and overall survival in patients with HCC presenting with vascular invasion or multifocal disease after curative surgery. MiR-19b may influence the expression of NDRG1, EPCAM, HMGB2, HIF1A, and MAPK14.

Dietary methionine restriction (MR) in rodents increased lifespan despite higher heart-to-body weight ratio (w/w) and hyperhomocysteinemia, which are symptoms associated with increased risk for cardiovascular disease. We investigated this paradoxical effect of MR on cardiac function using young, old, and apolipoprotein E-deficient (ApoE-KO) mice. Indeed, MR animals exhibited higher heart-to-body weight ratio (w/w) and hyperhomocysteinemia with a molecular pattern consistent with cardiac stress while maintaining the integrity of cardiac structure. Baseline cardiac function, which was measured by non-invasive electrocardiography (ECG), showed that young MR mice had prolonged QRS intervals compared with control-fed (CF) mice, whereas old and ApoE-KO mice showed similar results for both groups. Following £]-adrenergic challenge, responses of MR mice were either similar or attenuated compared with CF mice. Cardiac contractility, which was measured by isolated heart retrograde perfusion, was similar in both groups of old mice. Finally, the MR diet induced secretion of cardioprotective hormones, adiponectin and fibroblast growth factor 21 (FGF21), in MR mice with concomitant alterations in cardiac metabolic molecular signatures. Our findings demonstrate that MR diet does not alter cardiac function in mice despite the presence of hyperhomocysteinemia because of the adaptive responses of increased adiponectin and FGF21 levels.

Purpose: To compare the clinical features and gene expression patterns of the physiologic development of retinal vessels and oxygen-induced retinopathy (OIR) in a mouse model, with the aim of identifying differential regulators of physiologic and pathological angiogenesis in the retina. Results: The sequential orders and patterns of vasculature development in normal mice and the OIR models were significantly different. In brief, in the early days (P1 to P7) for normal mice, retinal vessels grew from the optic disc into the non-vascularized retina in a radial fashion. In the hyperoxic stage of the OIR model, the main central retina became devoid of a vascular network, and when the mice returned to the normoxic room, the vessels grew from peripheral perfused areas toward the center of the retina, but the development of intermediate and deep layers of vasculature was significantly delayed. Gene profiling at three critical time points (P8, P12, and P13) showed that 162 probes were upregulated to ≥1.5-fold or downregulated to ≤0.67-fold at one or more time points in the OIR model compared to the controls. In the 45 upregulated genes for the P8-O/P8-N group, enriched genes were mainly related to cytoskeleton formation, whereas the 62 upregulated genes for P13-O/P13-N participated in various pathological processes. In the physiologic conditions on P9, however, 135 genes were upregulated compared with P30; the gap junction and Fc gamma R-mediated phagocytosis were the two main enriched pathways for these genes. Fifty-three probes, including vascular endothelium growth factor A, annexin A2, and endothelin 2, changed at P13-O but not at P9-N, and these changed genes might reflect the modulation of pathological neovascularization. Conclusions: Angiogenesis in physiologic and pathological conditions is characterized by the differential presentation of vasculature and gene expression patterns. Investigation of those genes unique to the OIR model may help develop new strategies and therapies for intervening in retinal neovascularization.

The serine/threonine protein kinase paralogs ROCK1 & 2 have been implicated as essential modulators of angiogenesis; however their paralog-specific roles in endothelial function are unknown. shRNA knockdown of ROCK1 or 2 in endothelial cells resulted in a significant disruption of in vitro capillary network formation, cell polarization, and cell migration compared to cells harboring non-targeting control shRNA plasmids. Knockdowns led to alterations in cytoskeletal dynamics due to ROCK1 & 2-mediated reductions in actin isoform expression, and ROCK2-specific reduction in myosin phosphatase and cofilin phosphorylation. Knockdowns enhanced cell survival and led to ROCK1 & 2-mediated reduction in caspase 6 and 9 cleavage, and a ROCK2-specific reduction in caspase 3 cleavage. Microarray analysis of ROCK knockdown lines revealed overlapping and unique control of global transcription by the paralogs, and a reduction in the transcriptional regulation of just under 50% of VEGF responsive genes. Finally, paralog knockdown in xenograft angiosarcoma tumors resulted in a significant reduction in tumor formation. Our data reveals that ROCK1 & 2 exhibit overlapping and unique roles in normal and dysfunctional endothelial cells, that alterations in cytoskeletal dynamics are capable of overriding mitogen activated transcription, and that therapeutic targeting of ROCK signaling may have profound impacts for targeting angiogenesis.

Proliferation and migration of vascular smooth muscle cells (VSMCs) can cause atherosclerosis and neointimal formation. MicroRNAs have been shown to regulate cell proliferation and phenotype transformation. We discovered abundant expression of microRNA-195 in VSMCs and conducted a series of studies to identify its function in the cardiovascular system. MicroRNA-195 expression was initially found to be altered when VSMCs were treated with oxidized low-density lipoprotein (oxLDL) in a non-replicated microRNA array experiment. Using cellular studies, we found that microRNA-195 reduced VSMC proliferation, migration, and synthesis of IL-1£], IL-6, and IL-8. Using bioinformatics prediction and experimental studies, we showed that microRNA-195 could repress the expression of Cdc42, CCND1, and FGF1 genes. Using a rat model, we found that the microRNA-195 gene, introduced by adenovirus, substantially reduced neointimal formation in a balloon-injured carotid artery. In situ hybridization confirmed the presence of microRNA-195 in the treated arteries but not in control arteries. Immunohistochemistry experiments showed abundant Cdc42 in the neointima of treated arteries. We showed that microRNA-195 plays a role in the cardiovascular system by inhibiting VSMC proliferation, migration, and proinflammatory biomarkers. MicroRNA-195 may have the potential to reduce neointimal formation in patients receiving stenting or angioplasty.

Myxomatous mitral valve prolapse (MVP) is the most common cardiac valvular abnormality in industrialized countries and a leading cause of mitral valve surgery for isolated mitral regurgitation. The key role of valvular interstitial cells (VICs) during mitral valve development and homeostasis has been recently suggested, however little is known about the molecular pathways leading to MVP. We aim to characterize bone morphogenetic protein 4 (BMP4) as a cellular regulator of mitral VIC activation towards a pathologic synthetic phenotype and to analyze the cellular phenotypic changes and extracellular matrix (ECM) reorganization associated with the development of myxomatous MVP. Microarray analysis showed significant up regulation of BMP4-mediated signaling molecules in myxomatous MVP when compared to controls. Histological analysis and cellular characterization suggest that during myxomatous MVP development, healthy quiescent mitral VICs undergo a phenotypic activation via up regulation of BMP4-mediated pathway. In vitro hBMP4 treatment of isolated human mitral VICs mimics the cellular activation and ECM remodeling as seen in MVP tissues. The present study characterizes the cell biology of mitral VICs in physiological and pathological conditions and provides insights into the molecular and cellular mechanisms mediated by BMP4 during MVP. The ability to test and control the plasticity of VICs using different molecules may help in developing new diagnostic and therapeutic strategies for myxomatous MVP.

Although bone marrow endothelial progenitor cell (EPC)-based therapies improve the symptoms in patients with ischemic heart disease, their limited plasticity and decreased function in patients with existing heart disease limit the full benefit of EPC therapy for cardiac regenerative medicine. We hypothesized that reprogramming mouse or human EPCs, or both, using small molecules targeting key epigenetic repressive marks would lead to a global increase in active gene transcription, induce their cardiomyogenic potential, and enhance their inherent angiogenic potential. Mouse Lin-Sca1(+)CD31(+) EPCs and human CD34(+) cells were treated with inhibitors of DNA methyltransferases (5-Azacytidine), histone deacetylases (valproic acid), and G9a histone dimethyltransferase. A 48-hour treatment led to global increase in active transcriptome, including the reactivation of pluripotency-associated and cardiomyocyte-specific mRNA expression, whereas endothelial cell-specific genes were significantly upregulated. When cultured under appropriate differentiation conditions, reprogrammed EPCs showed efficient differentiation into cardiomyocytes. Treatment with epigenetic-modifying agents show marked increase in histone acetylation on cardiomyocyte and pluripotent cell-specific gene promoters. Intramyocardial transplantation of reprogrammed mouse and human EPCs in an acute myocardial infarction mouse model showed significant improvement in ventricular functions, which was histologically supported by their de novo cardiomyocyte differentiation and increased capillary density and reduced fibrosis. Importantly, cell transplantation was safe and did not form teratomas. Taken together, our results suggest that epigenetically reprogrammed EPCs display a safe, more plastic phenotype and improve postinfarct cardiac repair by both neocardiomyogenesis and neovascularization.