Paras K. Mishra

MicroRNAs as a therapeutic target for cardiovascular diseases

•  Introduction •  Biogenesis of miRNAs and their regulatory mechanisms •  MiRNAs in cardiovascular diseases •  MiRNAs in hypertrophy •  MiRNAs in cardiac fibrosis •  MiRNAs in arrhythmia •  MiRNAs in myocardial infarction •  MiRNAs in heart failure •  MiRNAs in angiogenesis •  MiRNAs in cardiomyopathy •  MiRNAs in antherogenesis •  Role of Dicer in cardiomyopathy •  MiRNAs ‐ a new strategy for treatment of cardiovascular diseases ‐  MiRNA – an innovative therapeutic approach ‐  MiRNAs in stem cell therapy ‐  A new candidate in microRNomics •  Therapeutic challenges and their remedies ‐  Mode of delivery of miRNAs ‐  Role of microevironment •  Approaches of miRNAs in cardiovascular therapy

Homocysteine to hydrogen sulfide or hypertension.

Hyperhomocysteinemia, an increased level of plasma homocysteine, is an independent risk factor for the development of premature arterial fibrosis with peripheral and cerebro-vascular, neurogenic and hypertensive heart disease, coronary occlusion and myocardial infarction, as well as venous thromboembolism. It is reported that hyperhomocysteinemia causes vascular dysfunction by two major routes: (1) increasing blood pressure and, (2) impairing the vasorelaxation activity of endothelial-derived nitric oxide. The homocysteine activates metalloproteinases and induces collagen synthesis and causes imbalances of elastin/collagen ratio which compromise vascular elastance. The metabolites from hyperhomocysteinemic endothelium could modify components of the underlying muscle cells, leading to vascular dysfunction and hypertension. Homocysteine metabolizes in the body to produce H2S, which is a strong antioxidant and vasorelaxation factor. At an elevated level, homocysteine inactivates proteins by homocysteinylation including its endogenous metabolizing enzyme, cystathionine γ-lyase. Thus, reduced production of H2S during hyperhomocysteinemia exemplifies hypertension and vascular diseases. In light of the present information, this review focuses on the mechanism of hyperhomocysteinemia-associated hypertension and highlights the novel modulatory role of H2S to ameliorate hypertension.

H2S ameliorates oxidative and proteolytic stresses and protects the heart against adverse remodeling in chronic heart failure

Reactive oxygen and nitrogen species (ROS and RNS, respectively) generate nitrotyrosine and activate latent resident myocardial matrix metalloproteinases (MMPs). Although in chronic heart failure (CHF) there is robust increase in ROS, RNS, and MMP activation, recent data suggest that hydrogen sulfide (H(2)S, a strong antioxidant gas) is cardioprotective. However, the role of H(2)S in mitigating oxidative and proteolytic stresses in cardiac remodeling/apoptosis in CHF was unclear. To test the hypothesis that H(2)S ameliorated cardiac apoptosis and fibrosis by decreasing oxidative and proteolytic stresses, arteriovenous fistula (AVF) was created in wild-type (C57BL/6J) mice. The hearts were analyzed at 0, 2, and 6 wk after AVF. To reverse the remodeling, AVF mice were treated with NaHS (an H(2)S donor, 30 micromol/l in drinking water) at 8 and 10 wk. The levels of MMPs were measured by gelatin-gel zymography. The levels of nitrotyrosine, tissue inhibitors of metalloproteinase (TIMPs), beta(1)-integrin, and a disintegrin and metalloproteinase-12 (ADAM-12) were analyzed by Western blots. The levels of pericapillary and interstitial fibrosis were identified by Masson trichrome stains. The levels of apoptosis were measured by identifying the TdT-mediated dUTP nick end labeling (TUNEL)-positive cells and caspase-3 levels. The results suggested robust nitrotyrosine and MMP activation at 2 and 6 wk of AVF. The treatment with H(2)S donor mitigated nitrotyrosine generation and MMP activation (i.e., oxidative and proteolytic stresses). The levels of TIMP-1 and TIMP-3 were increased and TIMP-4 decreased in AVF hearts. The treatment with H(2)S donor reversed this change in TIMPs levels. The levels of ADAM-12, apoptosis, and fibrosis were robust and integrin were decreased in AVF hearts. The treatment with H(2)S donor attenuated the fibrosis, apoptosis, and decrease in integrin.

Hydrogen Sulfide Mitigates Cardiac Remodeling During Myocardial Infarction via Improvement of Angiogenesis

Exogenous hydrogen sulfide (H2S) leads to down-regulation of inflammatory responses and provides myocardial protection during acute ischemia/reperfusion injury; however its role during chronic heart failure (CHF) due to myocardial infarction (MI) is yet to be unveiled. We previously reported that H2S inhibits antiangiogenic factors such, as endostatin and angiostatin, but a little is known about its effect on parstatin (a fragment of proteinase-activated receptor-1, PAR-1). We hypothesize that H2S inhibits parstatin formation and promotes VEGF activation, thus promoting angiogenesis and significantly limiting the extent of MI injury. To verify this hypothesis MI was created in 12 week-old male mice by ligation of left anterior descending artery (LAD). Sham surgery was performed except LAD ligation. After the surgery mice were treated with sodium hydrogen sulfide (30 μmol/l NaHS, a donor for H2S, in drinking water) for 4 weeks. The LV tissue was analyzed for VEGF, flk-1 and flt-1, endostatin, angiostatin and parstatin. The expression of VEGF, flk-1 and flt-1 were significantly increased in treated mice while the level of endostatin, angiostatin and parstatin were decreased compared to in untreated mice. The echocardiography in mice treated with H2S showed the improvement of heart function compared to in untreated mice. The X-ray and Doppler blood flow measurements showed enhancement of cardiac-angiogenesis in mice treated with H2S. This observed cytoprotection was associated with an inhibition of anti-angiogenic proteins and stimulation of angiogenic factors. We established that administration of H2S at the time of MI ameliorated infarct size and preserved LV function during development of MI in mice. These results suggest that H2S is cytoprotective and angioprotective during evolution of MI.

Increased endogenous H2S generation by CBS, CSE, and 3MST gene therapy improves ex vivo renovascular relaxation in hyperhomocysteinemia

Hydrogen sulfide (H(2)S) has recently been identified as a regulator of various physiological events, including vasodilation, angiogenesis, antiapoptotic, and cellular signaling. Endogenously, H(2)S is produced as a metabolite of homocysteine (Hcy) by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). Although Hcy is recognized as vascular risk factor at an elevated level [hyperhomocysteinemia (HHcy)] and contributes to vascular injury leading to renovascular dysfunction, the exact mechanism is unclear. The goal of the current study was to investigate whether conversion of Hcy to H(2)S improves renovascular function. Ex vivo renal artery culture with CBS, CSE, and 3MST triple gene therapy generated more H(2)S in the presence of Hcy, and these arteries were more responsive to endothelial-dependent vasodilation compared with nontransfected arteries treated with high Hcy. Cross section of triple gene-delivered renal arteries immunostaining suggested increased expression of CD31 and VEGF and diminished expression of the antiangiogenic factor endostatin. In vitro endothelial cell culture demonstrated increased mitophagy during high levels of Hcy and was mitigated by triple gene delivery. Also, dephosphorylated Akt and phosphorylated FoxO3 in HHcy were reversed by H(2)S or triple gene delivery. Upregulated matrix metalloproteinases-13 and downregulated tissue inhibitor of metalloproteinase-1 in HHcy were normalized by overexpression of triple genes. Together, these results suggest that H(2)S plays a key role in renovasculopathy during HHcy and is mediated through Akt/FoxO3 pathways. We conclude that conversion of Hcy to H(2)S by CBS, CSE, or 3MST triple gene therapy improves renovascular function in HHcy.

MMP-2/TIMP-2/TIMP-4 versus MMP-9/TIMP-3 in transition from compensatory hypertrophy and angiogenesis to decompensatory heart failure*

Although matrix metalloproteinase (MMPs) and tissue inhibitor of metalloproteinase (TIMPs) play a vital role in tumour angiogenesis and TIMP-3 caused apoptosis, their role in cardiac angiogenesis is unknown. Interestingly, a disruption of co-ordinated cardiac hypertrophy and angiogenesis contributed to the transition to heart failure, however, the proteolytic and anti-angiogenic mechanisms of transition from compensatory hypertrophy to decompensatory heart failure were unclear. We hypothesized that after an aortic stenosis MMP-2 released angiogenic factors during compensatory hypertrophy and MMP-9/TIMP-3 released anti-angiogenic factors causing decompensatory heart failure. To verify this hypothesis, wild type (WT) mice were studied 3 and 8 weeks after aortic stenosis, created by banding the ascending aorta in WT and MMP-9-/- (MMP-9KO) mice. Cardiac function (echo, PV loops) was decreased at 8 weeks after stenosis. The levels of MMP-2 (western blot) increased at 3 weeks and returned to control level at 8 weeks, MMP-9 increased only at 8 weeks. TIMP-2 and -4 decreased at 3 and even more at 8 weeks. The angiogenic VEGF increased at 3 weeks and decreased at 8 weeks, the anti-angiogenic endostatin and angiostatin increased only at 8 weeks. CD-31 positive endothelial cells were more intensely labelled at 3 weeks than in sham operated or in 8 weeks banded mice. Vascularization, as estimated by x-ray angiography, was increased at 3 weeks and decreased at 8 weeks post-banding. Although the vast majority of studies were performed on control WT mice only, interestingly, MMP9-KO mice seemed to have increased vascular density 8 weeks after banding. These results suggested that there was increase in MMP-2, decrease in TIMP-2 and -4, increase in angiogenic factors and vascularization in compensatory hearts. However, in decompensatory hearts there was increase in MMP-9, TIMP-3, endostatin, angiostatin and vascular rarefaction.

Predictors and prevention of diabetic cardiomyopathy

Despite our cognizance that diabetes can enhance the chances of heart failure, causes multiorgan failure,and contributes to morbidity and mortality, it is rapidly increasing menace worldwide. Less attention has been paid to alert prediabetics through determining the comprehensive predictors of diabetic cardiomyopathy (DCM) and ameliorating DCM using novel approaches. DCM is recognized as asymptomatic progressing structural and functional remodeling in the heart of diabetics, in the absence of coronary atherosclerosis and hypertension. The three major stages of DCM are: (1) early stage, where cellular and metabolic changes occur without obvious systolic dysfunction; (2) middle stage, which is characterized by increased apoptosis, a slight increase in left ventricular size, and diastolic dysfunction and where ejection fraction (EF) is <50%; and (3) late stage, which is characterized by alteration in microvasculature compliance, an increase in left ventricular size, and a decrease in cardiac performance leading to heart failure. Recent investigations have revealed that DCM is multifactorial in nature and cellular, molecular, and metabolic perturbations predisposed and contributed to DCM. Differential expression of microRNA (miRNA), signaling molecules involved in glucose metabolism, hyperlipidemia, advanced glycogen end products, cardiac extracellular matrix remodeling, and alteration in survival and differentiation of resident cardiac stem cells are manifested in DCM. A sedentary lifestyle and high fat diet causes obesity and this leads to type 2 diabetes and DCM. However, exercise training improves insulin sensitivity, contractility of cardiomyocytes, and cardiac performance in type 2 diabetes. These findings provide new clues to diagnose and mitigate DCM. This review embodies developments in the field of DCM with the aim of elucidating the future perspectives of predictors and prevention of DCM.

Differential Expression of Dicer, miRNAs, and Inflammatory Markers in Diabetic Ins2+/- Akita Hearts

Diabetic cardiomyopathy is a leading cause of morbidity and mortality, and Insulin2 mutant (Ins2+/−) Akita is a genetic mice model of diabetes relevant to humans. Dicer, miRNAs, and inflammatory cytokines are associated with heart failure. However, the differential expression of miRNAs, dicer, and inflammatory molecules are not clear in diabetic cardiomyopathy of Akita. We measured the levels of miRNAs, dicer, pro-inflammatory tumor necrosis factor alpha (TNFα), and anti-inflammatory interleukin 10 (IL-10) in C57BL/6J (WT) and Akita hearts. The results revealed increased heart to body weight ratio and robust expression of brain natriuretic peptide (BNP: a hypertrophy marker) suggesting cardiac hypertrophy in Akita. The multiplex RT-PCR, qPCR, and immunoblotting showed up regulation of dicer, whereas miRNA array elicited spread down regulation of miRNAs in Akita including dramatic down regulation of let-7a, miR-130, miR-142-3p, miR-148, miR-338, miR-345-3p, miR-384-3p, miR-433, miR-450, miR-451, miR-455, miR-494, miR-499, miR-500, miR-542-3p, miR-744, and miR-872. Conversely, miR-295 is induced in Akita. Cardiac TNFα is upregulated at mRNA (RT-PCR and qPCR), protein (immunoblotting), and cellular (immunohistochemistry and confocal microscopy) levels, and is robust in hypertrophic cardiomyocytes suggesting direct association of TNFα with hypertrophy. Contrary to TNFα, cardiac IL-10 is downregulated in Akita. In conclusion, induction of dicer and TNFα, and attenuation of IL-10 and majority of miRNA are associated with cardiomyopathy in Akita and could be used for putative therapeutic target for heart failure in diabetics.

MicroRNAs Are Involved in Homocysteine-Induced Cardiac Remodeling

Elevated level of homocysteine (Hcy) called hyperhomocysteinemia (HHcy) is one of the major risk factors for chronic heart failure. Although the role of Hcy in cardiac remodeling is documented, the regulatory mechanism involved therein is still nebulous. MicroRNAs (miRNAs) and dicer have been implicated in regulation of cardiovascular diseases. Dicer is the only known enzyme involved in miRNA maturation. We investigated the involvement of dicer and miRNA in Hcy-induced cardiac remodeling. HL-1 cardiomyocytes were cultured in different doses of Hcy. Total RNA was isolated and RT-PCR and real-time PCR was performed for dicer, MMP-2,-9, TIMP-1,-3, and NOX-4. MiRNA microarray was used for analyzing the differential expression of miRNAs. Individual miRNA assay was also done. Western blotting was used to assess the MMP-9 expression in HHcy cardiomyocytes. The RT-PCR results suggest that dicer expression is enhanced in HHcy cardiomyocytes suggesting its involvement in cardiac remodeling caused due to high dose of Hcy. On the other hand, high dose of Hcy increased NOX-4 expression, a marker for oxidative stress. Additionally, HHcy cardiomyocytes showed elevated levels of MMP-2,-9 and TIMP-1,-3, and reduced expression of TIMP-4, suggesting cardiac remodeling due to oxidative stress. The miRNA microarray assay revealed differential expression of 11 miRNAs and among them miR-188 show dramatic downregulation. These findings suggest that dicer and miRNAs especially miR-188 are involved in Hcy-induced cardiac remodeling.

Stem cells as a therapeutic target for diabetes.

The rapidly increasing number of diabetes patients across the world poses a great challenge to the current therapeutic approach. The traditional method of exogenous supply of insulin has ephemeral effect and often causes lethal hypoglycemia that demands to develop a novel strategy. Recent investigations on regeneration of insulin producing cells (IPCs) revealed that in addition to primary source i.e., pancreatic beta cells, IPCs can be derived from several alternative sources including embryonic, adult, mesenchymal and hematopoietic stem cells via the process of proliferation, dedifferentiation, neogenesis, nuclear reprogramming and transdifferentiation. There is considerable success in insulin independency of diabetes patient after transplantation of whole pancreas and/or the islet cells. However, the major challenge for regenerative therapy is to obtain a large source of islet/beta cells donor. Recent advances in the directed differentiation of stem cells generated a promising hope for a better and permanent insulin independency for diabetes. In this review we discussed stem cells as a potential future therapeutic target for the treatment of diabetes and associated diseases.