Sudhiranjan Gupta

Circulating miRNAs as Potential Marker for Pulmonary Hypertension

MircoRNAs (miRNAs) are small non-coding RNAs that govern the gene expression and, play significant role in the pathogenesis of heart failure. The detection of miRNAs in circulation of pulmonary hypertensive (PH) human subjects remains elusive. In the current study, we determined the pattern of miRNAs of mild-to-severe human PH subjects and, compared them with the control subjects by miRNA array. Blood was obtained using fluoroscopic and waveform guided catheterization from the distal (pulmonary artery) port of the catheter. A total 40 human subjects were included in the study and, the degree of PH was determined by mean pulmonary arterial pressure. Among several miRNAs in the array, we validated 14 miRNAs and, the data were consistent with the array profile. We identified several novel downregulated miRNAs (miR-451, miR-1246) and upregulated miRNAs (miR-23b, miR-130a and miR-191) in the circulation of PH subjects. Our study showed novel set of miRNAs which are dysregulated in PH and, are directly proportional to the degree of PH. These miRNAs may be considered as potential biomarker for early detection of PH.

NF‐κB mediated miR‐26a regulation in cardiac fibrosis

Micro‐RNAs (miRNAs) are a class of small non‐coding RNAs, recently emerged as a post‐transcriptional regulator having a key role in various cardiac pathologies. Among them, cardiac fibrosis that occurs as a result from an imbalance of extracellular matrix proteins turnover and is a highly debilitating process that eventually lead to organ dysfunction. An emerging theme on is that miRNAs participate in feedback loop with transcription factors that regulate their transcription. NF‐κB, a key transcription factor regulator controls a series of gene program in various cardiac diseases through positive and negative feedback mechanism. But, NF‐κB mediated miRNA regulation in cardiac fibrosis remains obscure. Bioinformatics analysis revealed that miR‐26a has targets collagen I and CTGF and possesses putative NF‐κB binding element in its promoter region. Here, we show that inhibition of NF‐κB in cardiac fibroblast restores miR‐26a expression, attenuating collagen I, and CTGF gene expression in the presence of Ang II, conferring a feedback regulatory mechanism in cardiac fibrosis. The target genes for miR‐26a were confirmed using 3′‐UTR luciferase reporter assays for collagen I and CTGF genes. Using NF‐κB reporter assays, we determine that miR‐26a overexpression inhibits NF‐κB activity. Finally, we show that miR‐26a expression is restored along with the attenuation of collagen I and CTGF genes in cardiac specific IkBa triple mutant transgenic mice (preventing NF‐κB activation) subjected to 4 weeks transverse aortic banding (TAC), compared to wild type (WT) mice. The data indicate a potential role of miR‐26a in cardiac fibrosis and, offer novel therapeutic intervention. J. Cell. Physiol. 228: 1433–1442, 2013.

Thymosin Beta 4 Prevents Oxidative Stress by Targeting Antioxidant and Anti-Apoptotic Genes in Cardiac Fibroblasts

Rationale Thymosin beta-4 (Tβ4) is a ubiquitous protein with diverse functions relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory responses. The effecter molecules targeted by Tβ4 for cardiac protection remains unknown. The purpose of this study is to determine the molecules targeted by Tβ4 that mediate cardio-protection under oxidative stress. Methods Rat neonatal fibroblasts cells were exposed to hydrogen peroxide (H2O2) in presence and absence of Tβ4 and expression of antioxidant, apoptotic and pro-fibrotic genes was evaluated by quantitative real-time PCR and western blotting. Reactive oxygen species (ROS) levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant and antiapoptotic genes were silenced by siRNA transfections in cardiac fibroblasts and the effect of Tβ4 on H2O2-induced profibrotic events was evaluated. Results Pre-treatment with Tβ4 resulted in reduction of the intracellular ROS levels induced by H2O2 in the cardiac fibroblasts. This was associated with an increased expression of antioxidant enzymes Cu/Zn superoxide dismutase (SOD) and catalase and reduction of Bax/Bcl2 ratio. Tβ4 treatment reduced the expression of pro-fibrotic genes [connective tissue growth factor (CTGF), collagen type-1 (Col-I) and collagen type-3 (Col-III)] in the cardiac fibroblasts. Silencing of Cu/Zn-SOD and catalase gene triggered apoptotic cell death in the cardiac fibroblasts, which was prevented by treatment with Tβ4. Conclusion This is the first report that exhibits the targeted molecules modulated by Tβ4 under oxidative stress utilizing the cardiac fibroblasts. Tβ4 treatment prevented the profibrotic gene expression in the in vitro settings. Our findings indicate that Tβ4 selectively targets and upregulates catalase, Cu/Zn-SOD and Bcl2, thereby, preventing H2O2-induced profibrotic changes in the myocardium. Further studies are warranted to elucidate the signaling pathways involved in the cardio-protection afforded by Tβ4.

Circulating miRNA as novel markers for diastolic dysfunction

MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression. Though their significance is unclear, pioneer profiling studies have attributed specific serum miRNA signatures to different disease conditions. The diagnostic potential of miRNA detection in human plasma for cardiovascular disorders is beginning to be recognized as important. In this study, we examined miRNA profiling in isolated diastolic dysfunction (DD) with preserved systolic function to identify promising candidate miRNAs. The presence of these miRNAs was tested in stable patients with isolated DD, patients with stable compensated dilated cardiomyopathy (DCM—systolic plus diastolic dysfunction) and those with decompensated congestive heart failure secondary to dilated cardiomyopathy (DCM–CHF—systolic plus diastolic dysfunction). We identified new circulating miRNAs (miR-454, miR-500, miR-1246, miR-142-3p) which showed distinct patterns of expression in patients with diastolic dysfunction. The presence or absence of systolic dysfunction does not seem to affect this trend. MiR-454 and miR-500 are downregulated in diastolic dysfunction. MiR-1246 is upregulated in diastolic dysfunction. MiR-142-3p is downregulated in DCM and DCM–CHF groups but not in the DD group. The expression of miR-124-5p is highly upregulated in DCM but not in DD and DCM–CHF groups. We therefore propose that these circulating miRNAs may serve as novel biomarkers for diastolic dysfunction because in all of these patients the only common factor was diastolic dysfunction.

NF-κB mediated miR-21 regulation in cardiomyocytes apoptosis under oxidative stress

Abstract Oxidative stress, defined as an excess production of reactive oxygen species (ROS), is shown to play an important role in the pathophysiology of cardiac remodeling including cell death and contractile dysfunction. Therefore, the balance between ROS production and removal of excess ROS is essential in maintaining the redox state and homeostasis balance in the cell. The increased ROS further activates nuclear factor-κB (NF-κB), a redox-sensitive transcription factor and promotes cell death. Recently, microRNAs (miRNAs) have been identified as critical regulators of various pathophysiological processes of cardiac remodeling; however, NF-κB-mediated miRNAs role in cardiomyocytes under oxidative stress remains undetermined. The miR-21 has been implicated in diverse cardiac remodeling; but, NF-κB-mediated miR-21 modulation in oxidative stress is currently unknown. Neonatal cardiomyocytes were transfected with IκBα mutant, miR-21 mimetic, and inhibitors separately, and were challenged with H2O2. The target gene, programmed cell death 4 (PDCD4), ROS activity, and NF-κB translocation were analyzed. Our results indicated that NF-κB positively regulated miR-21 expression under oxidative stress, and PDCD4 was a direct target for miR-21. NF-κB further regulated the expression of PDCD4 in H2O2-induced oxidative stress. Moreover, H2O2-induced ROS activity and cardiomyocytes apoptosis were partly protected by overexpression of miR-21 and displayed an important role in ROS-mediated cardiomyocytes injury. We evaluated a critical role of NF-κB-mediated miR-21 modulation in H2O2-induced oxidative stress in cardiomyocytes by targeting PDCD4. Our data may provide a new insight of miR-21s role in cardiac diseases primarily mediated by ROS.

Thymosin Beta 4 Protects Cardiomyocytes from Oxidative Stress by Targeting Anti-Oxidative Enzymes and Anti-Apoptotic Genes

Background Thymosin beta-4 (Tβ4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. The mechanism by which Tβ4 modulates cardiac protection under oxidative stress is not known. The purpose of this study is to dissect the cardioprotective mechanism of Tβ4 on H2O2 induced cardiac damage. Methods Rat neonatal cardiomyocytes with or without Tβ4 pretreatment were exposed to H2O2 and expression of antioxidant, apoptotic, and anti-inflammatory genes was evaluated by quantitative real-time PCR and western blotting. ROS levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant, anti-inflammatory and antiapoptotic genes were silenced by siRNA transfections in neonatal cardiomyocytes and effect of Tβ4 on H2O2-induced cardiac damage was evaluated. Results Pre-treatment of Tβ4 resulted in reduction of the intracellular ROS levels induced by H2O2 in cardiomyocytes. Tβ4 pretreatment also resulted in an increase in the expression of antiapoptotic proteins and reduction of Bax/BCl2 ratio in the cardiomyocytes. Pretreatment with Tβ4 resulted in stimulating the expression of antioxidant enzymes copper/zinc SOD and catalase in cardiomyocytes at both transcription and translation levels. Tβ4 treatment resulted in the increased expression of anti-apoptotic and anti-inflammatory genes. Silencing of Cu/Zn SOD and catalase gene resulted in apoptotic cell death in the cardiomyocytes which was prevented by treatment with Tβ4. Conclusion This is the first report that demonstrates the effect of Tβ4 on cardiomyocytes and its capability to selectively upregulate anti-oxidative enzymes, anti-inflammatory genes, and antiapoptotic enzymes in the neonatal cardiomyocytes thus preventing cell death thereby protecting the myocardium. Tβ4 treatment resulted in decreased oxidative stress and inflammation in the myocardium under oxidative stress.

Modulation of miRNAs in Pulmonary Hypertension

MicroRNAs (miRNAs) have emerged as a new class of posttranscriptional regulators of many cardiac and vascular diseases. They are a class of small, noncoding RNAs that contributes crucial roles typically through binding of the 3′-untranslated region of mRNA. A single miRNA may influence several signaling pathways associated with cardiac remodeling by targeting multiple genes. Pulmonary hypertension (PH) is a rare disorder characterized by progressive obliteration of pulmonary (micro) vasculature that results in elevated vascular resistance, leading to right ventricular hypertrophy (RVH) and RV failure. The pathology of PH involves vascular cell remodeling including pulmonary arterial endothelial cell (PAEC) dysfunction and pulmonary arterial smooth muscle cell (PASMC) proliferation. There is no cure for this disease. Thus, novel intervention pathways that govern PH induced RVH may result in new treatment modalities. Current therapies are limited to reverse the vascular remodeling. Recent studies have demonstrated the roles of various miRNAs in the pathogenesis of PH and pulmonary disorders. This review provides an overview of recent discoveries on the role of miRNAs in the pathogenesis of PH and discusses the potential for miRNAs as therapeutic targets and biomarkers of PH at clinical setting.

NF-κB-mediated miR-30b regulation in cardiomyocytes cell death by targeting Bcl-2

Angiotensin II(Ang II)-stimulated cardiomyocytes hypertrophy and apoptosis are associated with nuclear factor-κB (NF-κB) activation. NF-κB, a redox-sensitive transcription factor, contributes a critical role in cell death, but, Ang II-stimulated NF-κB-mediated cardiomyocytes apoptosis remains less understood. Recently, microRNAs (miRNAs) have been shown to be critical regulators in various cardiac remodeling processes; however, NF-κB-mediated miRNA’s role in cardiomyocytes apoptosis remains undetermined. The miR-30b has been implicated in diverse cardiac remodeling; but, NF-κB-mediated miR-30b modulation in Ang II-induced cardiomyocytes death is currently unknown. In the present study, neonatal cardiomyocytes were pretreated with SN50, a selective cell permeable peptide inhibitor of NF-κB, or transfected with miR-30b mimetic and inhibitors separately, and then challenged with Ang II. The target gene, Bcl-2, and NF-κB transcriptional activity were analyzed. Our results demonstrated that NF-κB positively regulated miR-30b expression in Ang II-induced cardiomyocytes apoptosis, and Bcl-2 was a direct target for miR-30b. NF-κB further regulated the expression of Bcl-2 in the above setting. Furthermore, Ang II-induced cardiomyocytes apoptosis rescued by inhibiting either NF-κB or miR-30b provided an important role in cardiomyocytes cell death. We evaluated a critical role of NF-κB-mediated miR-30b modulation in Ang II-stimulated cardiomyocytes targeting Bcl-2. Our data may provide a new insight of miR-30b’s role in myocardial infarction or ischemia.

Thymosin β4 and cardiac protection: implication in inflammation and fibrosis

Thymosin beta 4 (Tβ4) is a ubiquitous protein with diverse biological functions. The effecter molecules targeted by Tβ4 in cardiac protection remain unknown. We summarize previously published work showing that treatment with Tβ4 in the myocardial infarction setting improves cardiac function by activating Akt phosphorylation, promoting the ILK–Pinch–Parvin complex, and suppressing NF‐κB and collagen synthesis. In the presence of Wortmannin, Tβ4 showed minimal cardiac protection. In vitro findings revealed that pretreatment with Tβ4 resulted in reduction of intracellular ROS in the cardiac fibroblasts and was associated with increased expression of antioxidant enzymes, reduction of Bax/Bcl2 ratio, and attenuation of profibrotic genes. Silencing of Cu/Zn‐SOD, catalase, and Bcl2 genes abrogated the protective effect of Tβ4. Our findings suggest that Tβ4 improves cardiac function by enhancing Akt and ILK activation and suppressing NF‐κB activity and collagen synthesis. Furthermore, Tβ4 selectively upregulates catalase, Cu/Zn‐SOD, and Bcl2, thereby protecting cardiac fibroblasts from H2O2‐induced oxidative damage. Further studies are warranted to elucidate the signaling pathway(s) involved in the cardiac protection afforded by Tβ4.

NF-κB-Mediated Integrin-Linked Kinase Regulation in Angiotensin II-Induced Pro-fibrotic Process in Cardiac Fibroblasts

AIMS Cardiac fibrosis is a final outcome of many clinical conditions that lead to cardiac failure and is characterized by a progressive substitution of cellular elements by extracellular-matrix proteins, such as collagen type I, collagen type II, connective tissue growth factor (CTGF), etc. The aim of this study was to identify the mechanisms responsible for angiotensin II (Ang II)-stimulated cardiac fibrosis using rat neonatal cardiac fibroblasts. MAIN METHODS Neonatal fibroblasts were transfected with IκBα mutant, constitutively active (ca) integrin-linked kinase (ILK), dominant negative of ILK and small interfering RNA (siRNA) of ILK in the presence and absence of Ang-II stimulation. The pro-fibrotic gene expression and protein levels were determined by quantitative real time PCR and western blotting using their specific probes and antibodies. NF-κB translocation was determined by immunocytochemistry and confocal microscopy images were analyzed. KEY FINDINGS Our results indicate that overexpression of ILK promotes a pro-fibrotic process by upregulating collagen type I and CTGF genes via activation of nuclear factor-κB (NF-κB) in cardiac fibroblasts. Inactivation of either NF-κB by the super-repressor IκBα or ILK by siRNA significantly attenuates the pro-fibrotic process. Moreover, ILK overexpression triggers NF-κB-p65 translocation to the nucleus, and ILK inhibition prevents the translocation in cardiac fibroblasts stimulated with Ang II. SIGNIFICANCE Our data suggest that the Ang II-stimulated pro-fibrotic process is regulated by a complex mechanism involving crosstalk between ILK and NF-κB activation. This dual mechanism may play a critical role in the progression of cardiac fibrosis.