|Year : 2016 | Volume
| Issue : 4 | Page : 22-34
|Date of Web Publication||8-Apr-2016|
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
. Poster Presentations. J Pract Cardiovasc Sci 2016;2, Suppl S1:22-34
Post-transcriptional Regulation of 3-Hydroxy-3-Methyl Glutaryl-Coenzyme A Reductase Gene by MiRNA-27a
Abrar A. Khan, Vinayak Gupta, Kalyani Ananthamohan, Vikas Arige, Anil Kiran Chokkalla, Nitish R. Mahapatra
Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, Tamil Nadu, India
Dyslipidemia is a strong predictor of cardiovascular diseases including Essential Hypertension (EH). 3-Hydroxy-3-Methyl Glutaryl-Coenzyme A reductase (Hmgcr), coding for the rate-limiting enzyme in the cholesterol biosynthesis, is an important candidate gene for EH. However, the regulation of Hmgcr, especially at the post-transcriptional level, is incompletely understood. We set out to explore the possible roles of miRNAs in the regulation of Hmgcr. In silico predictions coupled with systematic functional analysis revealed specific interaction of miR-27a with mouse Hmgcr 3'-UTR in mouse liver fibroblasts AML-12. Furthermore, miR-27a showed a significant negative correlation with Hmgcr expression in cultured AML-12 cells as well as rat liver, kidney and skeletal muscle tissues. Next, we quantified the expression of miR-27a and Hmgcr protein levels in liver tissues of genetically hypertensive blood pressure high (BPH) and genetically hypotensive blood pressure low (BPL) mice. Liver tissues from BPH showed elevated Hmgcr protein levels and diminished miR-27a levels as compared to BPL. Interestingly, cholesterol depletion in AML-12 cells resulted in down-regulation of endogenous miR-27a while augmenting the Hmgcr protein levels. In addition, miR-27a expression was elevated in AML-12 cells under hypoxic conditions. This study highlights the role of miR-27a in the post-transcriptional regulation of Hmgcr, thereby implicating its plausible role in cholesterol homeostasis and hypoxia-like pathological conditions.
Monocyte to Macrophage Differentiation in Arterial Diseases is Inhibited by Diosgenin: An In vitro Study
Ambika Binesh, Sivasitambaram Niranjali Devaraj 1 , Devaraj Halagowder
Unit of Biochemistry, Department of Zoology, University of Madras, 1 Department of Biochemistry, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India
Differentiation of human monocyte into macrophage is the earliest and vital episode for the progressive story of atherosclerosis. Impediment of this inceptive cellular phenomenon should form the basic line of defense in the prevention and treatment of inflammatory cascades that could be the cause of arterial ailments. Diosgenin, a member of steroidal sapogenin found in several plants including Solanum, Trigonella and Dioscorea species, has favorable effects on lipid metabolism, anti-inflammatory and antioxidant activity. In this study, we investigated the efficacy of diosgenin in inhibiting this differentiation, thus preventing the atherosclerotic progression. Lipoproteins (LDL + VLDL) were isolated from human plasma, oxidized in vitro and used to differentiate monocyte into macrophage. Addition of oxidized lipoproteins to monocyte cell line THP 1, upregulated the macrophage markers like CD 68 and CD 36, inflammatory molecules like PPARγ, COX-2, NFκB, suggesting the conversion of monocyte to macrophage. Expression of c-kit (tyrosine kinase receptor) also advocates the macrophage differentiation. Diosgenin downregulated this differentiation and thus could prevent the arterial diseases.
Effect of Oligomeric Proanthocyanidins on Isoproterenol Induced Left Ventricular Remodeling After Myocardial Infarction: An In vivo Study
R. Ashokkumar, S. Jamuna, M. S. Sakeena Sadullah, S. Niranjali Devaraj
Department of Biochemistry, University of Madras, Chennai, Tamil Nadu, India
Extracellular matrix (ECM) is an active microenvironment and a major contributor for the adverse left ventricular remodeling that leads to myocardial infarction (MI), via activation of matrix metalloproteinases (MMPs) that enhances collagenase activity leading to myocyte loss and formation of scar. Deposition of degraded ECM materials inaccessible from the region of the MI is clearly detrimental to ventricular function and contributory to adverse outcomes of post-MI. Therapeutic options targeting ECM degradation during post-MI may contribute beneficial effects, in part, via reductions in ECM deposition. Anthocyanidins intake has been associated with reduced cardiac risk. Oligomeric proanthocyanidins possess anti-oxidant, anti-inflammatory and anti-apoptotic effects. The aim of the present study is to investigate the cardio protective effect of Oligomeric proanthocyanidins (OPC) against left ventricular remodeling after myocardial infarction (MI). Remodeling of left ventricle achieved by ECM proteins and Matrix metallo proteinases were examined. In this study, biochemical analysis such as LDH, CK, SOD, CAT and MDA content were determined. OPC could significantly attenuate the elevation of LDH, CK and MDA activity and elevated the levels of SOD and CAT. We also analyzed the protein expression of Collagen Type I & III and MMP-2 & 9 in post MI by Immunofluorescence study. Western blot analysis showed that OPC effectively decreases the expression of MMP-2 & 9. All of the above results substantiate the cardioprotective effect of OPC against left ventricular remodeling post MI.
Role of Mitochondrial Transcription Factors in Essential Hypertension
Bhargavi Natarajan, Manish Jain 1 , Madhu Dikshit 1 , Manoj K. Barthwal 1 , Nitish R. Mahapatra
Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, Tamil Nadu, 1 Division of Pharmacology, Central Drug Research Institute, Lucknow, Uttar Pradesh, India
Mitochondria are the site of oxidative phosphorylation, generating energy in the form of ATP. Failure to produce the required amount of ATP (mitochondrial dysfunction) affects cardiac output and may lead to heart failure. The rate of ATP synthesis in liver and brain of SHR (Spontaneously Hypertensive Rat) is observed to be lesser than WKY (Wistar-Kyoto Normotensive rat) counterparts. Recent studies indicate that more than 50 mitochondrial genes are differentially expressed between SHR and WKY. Mitochondria, being ancestral proteobacteria, contain a 16 kb circular genome coding for 13 proteins of the electron transport chain, tRNAs and rRNAs. Mitochondrial gene expression is enabled through nuclear-encoded genes. Notably, TFAM, TFBM1 and TFBM2 are transcription factors forming the basal transcription machinery in the mitochondria. We hypothesize that mitochondrial dysfunction in SHR might be, in part, due to differential expression of these transcription factors genes. Therefore, we sought to determine an expression profile of these genes in various tissues of SHR and WKY and explain the differences. We found that the expression of TFAM and TFB1M are higher in SHR liver, kidney and brain while TFB2M is higher in corresponding WKY tissues. Sequencing of the ~1.5 kb promoter regions of TFAM and TFB2M of SHR and WKY revealed absence of any genetic variations. However, multiple polymorphisms were found in TFB1M promoter region of SHR (TFB1M_SHR) creating binding sites for transcription factors NRF2, AML1, IRF2 and c-Rel. Furthermore, 6 CA repeat deletion is found in the TFB1M_SHR promoter. TheTFB1M_SHR promoter activity was two-fold higher than that of WKY. This observation of inverse correlation between CA repeats and promoter activity is consistent with previous reports. In conclusion, this study provides molecular basis for differential expression of TFB1M in the rat models of genetic hypertension.
Deciphering the Cardiovascular Disease Biomarkers Using Systems Biological Methodologies
Fazal Khan 1,2,3 , Peter Natesan Pushparaj 1 , Kalamegam Gauthaman 1 , Adel Abuzenadah 1,2 , Taha A. Kumosani 3 , Mohammed Al Qahtani 1
1 Center of Excellence in Genomic Medicine Research, Faculty of Applied Medical Sciences, 2 Center of Innovation in Personalized Medicine, 3 Department of Biochemistry, Faculty of Science, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
Incidence of cardiovascular diseases (CVD) increases with age. Although it is well known that risk factors such as obesity, hypertension and dyslipidaemia predispose to CVD the underlying molecular mechanisms and pathways are still not fully understood. We in the present study attempt to identify the genes/biomarkers in CVD which will further help us to understand the disease process and lead to improved therapeutic outcomes. Ingenuity Pathway Analysis (IPA) knowledge base (Ingenuity Systems, Qiagen, USA) was used to identify differentially expressed genes in CVD, and they were further clarified with Fisher's Exact Test (P < 0.01) and Benjamini Hochberg Multiple Testing Correction (P < 0.01). These genes were then subjected to core analysis in IPA to deduce the differential modulation of various canonical pathways, biological and toxicological functions, upstream regulators and novel molecular networks. Nearly 682 genes were significantly (P < 0.01) regulated in CVD, mostly involved in the signalling pathways of G-protein coupled receptor, cardiac hypertrophy, acute phase response and glucocorticoid receptor (P < 0.01). The top upstream regulators in CVD were TNF-α, TGFβ1, PPARγ, LEP and APOE. Additionally, a cluster of 140 genes that significantly (P < 0.01) regulate cardiac fibrosis, hypertrophy, infarction, arteriopathy and arrhythmia were deduced. G-protein coupled receptor and cAMP-mediated signaling play critical roles in CVD. Interestingly, most of the identified genes are also involved in diabetes mellitus clearly indicating disease associations with underlying common molecular pathways.
Terminalia Arjuna Partially Ameliorates the Monocrotaline Induced Pulmonary Artery Hypertension
Himanshu Meghwani, P. Prabhakar, M. P. Hote 1 , S. Seth 2 , R. Ray 3 , S. K. Maulik
Departments of Pharmacology, 1 CTVS, 2 Cardiology, 3 Pathology, All India Institute of Medical Sciences, New Delhi, India
Pulmonary artery hypertension (PAH) is defined as increased mean pulmonary artery pressure (>25 mm of Hg) with normal capillary wedge pressure. Reactive oxygen species (ROS) mediated injury is one of the important mechanisms involved in initiation and progression of PAH in humans. Stem bark of Terminalia Arjuna (TA) is considered as cardio-protective due to its anti-oxidant, anti-inflammatory and cardiotonic properties. Thus, we hypothesized TA could attenuate the monocrotaline induced PAH in rats. 28 female Wistar rats were randomized into 4 groups (n = 6), Control: rats were injected 1 ml/kg normal saline subcutaneously (s.c.) on day1; PAH: rats were administered monocrotaline (MCT) 50 mg/kg s.c. on day1; TA250: rats were administered with MCT 50 mg/kg s.c and water extract of TA (250 mg/kg) was administered orally from day 28-42. Echocardiography was done on day1, 28 & 42. Rats were sacrificed after hemodynamic measurements, heart and lungs were removed. Right ventricular hypertrophy was measured; lungs were kept in formalin for histopathology and liquid nitrogen for oxidative stress parameters. Monocrotaline caused increase in right ventricular systolic pressure (RVSP) (p < 0.001), right ventricular hypertrophy index (p < 0.001), right ventricular outflow tract dimension ratio aortic dimension (RVoTD/AoD) (p < 0.001), lung weight to body weight (p < 0.05) and % medial wall thickness in vessels with external diameter <100 ΅m (p < 0.001) and decrease in pulmonary artery acceleration time ratio ejection time (PAAT/ET) (p < 0.001) after 42 days. Monocrotaline caused increase in lung TBARS level (p < 0.001) while decrease in SOD (p < 0.005), GSH (p < 0.001) and catalase levels (p < 0.001). Increase in SOD (p < 0.05) and catalase levels (p < 0.05) were observed in lungs after treatment with Terminalia extract.
Exploring the Role of Vitamin D on Insulin Resistance: A Mechanistic Study in Cardiomyoblast Cells
Hina Lateef Nizami, Sanjay K. Banerjee
Drug Discovery Research Center, Translational Health Science and Technology Institute, Faridabad, Haryana, India
Metabolic syndrome, which includes hyperglycemia, insulin resistance and hypertension, is a strong risk factor for cardiovascular diseases. Among the various associated risk factors and complications, vitamin D deficiency is gaining attention as being strongly associated with incidence of type 2 diabetes, hypertension and coronary artery disease. Vitamin D deficiency has been reported to be highly prevalent in India. However, questions prevail over the mechanistic basis of the role of vitamin D in these disorders and also regarding the possible benefit of vitamin D supplementation in such cases. We observed that SD rats that were fed with vitamin D-deficient diet had insulin resistance and significantly higher blood glucose levels than those fed with normal (vitamin D-sufficient) diet. We further explored this observation by performing an in-vitro study where we used a model of insulin resistance induced by palmitate in cultured rat cardiomyoblasts (H9C2 cell line). Insulin resistance was observed in terms of reduced glucose uptake in the cardiomyocytes using a fluorescent glucose analog (2-NBDG). However, when the cells were treated with 10nM calcitriol (1, 25-OH vitamin D3) for 24 hours prior to induction of insulin resistance, the glucose uptake in cells was significantly improved than untreated group. The results indicate that vitamin D has a positive role in regulating glucose homeostasis in heart, in the setting of insulin resistance. We aim to further explore the molecular basis of this observation, and whether these results have any translational potential remains to be seen.
Oligomeric Proanthocyanidins Mediated Antioxidant Intervention of the OxLDL-Induced Oxidative Stress in H9c2 Rat Cardiomyoblasts
S. Jamuna, R. Ashokkumar, M. S. Sakeena Sadullah, S. Niranjali Devaraj
Department of Biochemistry, University of Madras, Chennai, Tamil Nadu, India
Cardiac extracellular matrix (ECM) is comprised of matrix proteins - where cardiac myocytes, fibroblasts, leukocytes, and cardiac vascular cells reside. Accumulation of Oxidized LDL (OxLDL) in the cardiac cells, an etiology for atherosclerotic lesion development via ECM remodeling, facilitates biomechanical signals in response to pathophysiological conditions. This event leads to matrix degradation and plaque instability. Oligomeric proanthocyanidins (OPC) have been known for various therapeutic properties including cardioprotective, anti-oxidant and anti-inflammatory. This study has been designed to explore the oxidative stress caused by OxLDL on H9C2 (2-1) cardiomyoblast as a model, specifically on the effect of ECM and interfilamental remodeling. H9C2 (2-1) cells, exposed to OxLDL and the release of ROS, expression of angiogenic marker LOX-1, NFkB p65- inflammatory marker were analyzed. OxLDL induced H9C2 cells showed a significant increase in ROS release, transcriptional upregulation of LOX-1 and NFkB p65 gene. In addition, an increased expression of MMPs, Cytoskeletal interfilamental proteins (Desmin and Vimentin) and ECM proteins (Collagen Type I & III) was found to be elevated in OxLDL treated cells, whereas cells treated with OPC impede the oxidative stress and cytotoxicity. Thus, the present study demonstrates the protective effect of OPC in attenuating the deleterious effects of OxLDL which directly induces oxidative stress and matrix derangement.
Human Wharton's Jelly Stem Cells and Cardiac Differentiation
Kalamegam Gauthaman 1 , Peter N. Pushparaj 1 , Adel Abuzenadah 1,2 , Adeel Chaudhary 1 , Mohammed Al-Qahtani 1
1 Centre of Excellence in Genomic Medicine Research, Faculty of Applied Medical Sciences, King Abdulaziz University, 2 Center of Innovation in Personalized Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
0Many different stem cells such as the human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) have been successfully differentiated into cardiac tissues. However, the problem of teratoma associated with pluripotent stem cells (hESCs, iPSCs) makes them less desirable for clinical translation compared to MSCs. Of the various MSCs the human umbilical cord derived Wharton's jelly stem cells (hWJSCs) have been identified to have several advantages. As such we attempted differentiation of hWJSCs towards cardiac lineage to understand whether hWJSCs would be better than other cells types. Human umbilical cords were obtained following ethical approval and primary cultures of hWJSCs were established and characterized. hWJSCs were for four weeks either in the presence or absence of 5-azacytidine and TGF-β1. Changes in cell morphology as well as expression of cardiac related proteins (immunofluorescence) were analyzed. Primary cultures of hWJSCs demonstrated their characteristic short spindle shaped fibroblasts and expressed MSC related positive CD markers. In the presence of 10΅M azacytidine and TGF-β1 for 72 h, the hWJSCs showed decreases in proliferation, had flattened morphology that were positive for sarcomeric protein, cardiac actin, and connexin 43 following a differentiation period of four weeks. hWJSCs were successfully differentiated towards cardiac lineage in vitro. These cells are relatively young compared to other currently available MSCs, are less hypo-immunogenic, have prolonged stemness and are non-tumorigeneic thus making them an ideal choice for use in the management of cardiovascular diseases.
Development of Monoclonal Antibody Against Oxidized Apo A1 for Early Diagnosis of Cardiovascular Risks in Humans
K. Lokeshwaran, N. S. Jayaprakash, M. A. Vijayalakshmi, Krishnan Venkataraman
Advanced Centre for Bioseparation Technology, VIT University, Vellore, Tamil Nadu, India
High Density Lipoprotein (HDL) plays an important role in Cardio Vascular system. They are involved in the removal of cholesterol from periphery and transport them to liver to be converted into bile acids which is excreted. In addition HDL has anti-inflammatory, anti-apoptotic, anti-thrombotic funcions which are beneficial for the cardiovascular system. Thus HDL is considered as cardio protective and the cholesterol which binds to HDL is considered as good cholesterol. It has been documented that high levels of HDL-Cholesterol is inversely correlated with cardiovascular risks in humans. ApoA1 is a major protein component of high density lipoprotein (HDL) and it is involved in reverse cholesterol transport (RCT) by transporting cholesterol from peripheral tissues like arteries is taken back to liver for excretion thus reducing the risk of atherosclerosis. Under chronic inflammatory conditions ApoA1 undergoes oxidation and gets modified at several amino residues in the primary sequence which renders the HDL dysfunctional. The dysfunctional HDL further contributes for worsening of the cardiovascular diseases resulting in poor prognosis. Thus the quality of HDL is more important than the quantity of HDL. In order to measure the levels of oxidized ApoA1, currently mass spectrometric methods are being used for the measurement of oxidized ApoA1. However the mass spectrometry methods are time consuming, expensive and cannot be routinely used in clinical settings. In this work, we have developed a monoclonal antibody which is specific to oxidized ApoA1 in order to be used robustly and routinely in the clinical settings. Specifically we have used a synthetic chlorinated peptide of ApoA1 as the antigen and subsequently developed ELISA based methods for detecting the extent of chlorinated Tyr-192 of Apo A1 in myocardial infarction patients. A successful development of this antibody may pave way for the utilizing this Anti-Chloro-Tyr 192 containing ApoA1 antibody for both early diagnosis and prognosis.
Novel Mechanisms in Wound Healing in the Heart: A Critical Role for Discoidin Domain Receptor 2 in Cardiac Fibroblast Response to Injury
U. Mereena George, K. Shivakumar
Division of Cellular and Molecular Cardiology, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala, India
Cardiac fibroblasts, the only intracardiac source of type I and type III collagens, provide a good experimental model to dissect hitherto unknown molecular interactions between pro-fibrotic factors that could potentially culminate in fibrogenesis. Discoidin domain receptor 2 (DDR2), a mesenchymal cell-specific collagen receptor tyrosine kinase, is known for its manifold roles, including matrix turnover, cell proliferation and differentiation. The present study probed the regulatory links between three critical fibrosis-related genes, Angiotensin II, DDR2 and collagen type 1, in cardiac fibroblasts. Angiotensin II was found to enhance DDR2 protein and mRNA expression in rat cardiac fibroblasts. Angiotensin II also stimulated collagen protein and mRNA expression in these cells. Importantly, the stimulatory effects of Angiotensin II on DDR2 and collagen were inter-dependent as siRNA-mediated silencing of one abolished the other, showing that DDR2 and collagen are locked in a cycle of mutual regulation in Angiotensin II-treated cardiac fibroblasts. It was also found that Angiotensin II promotes ERK1/2 phosphorylation, the inhibition of which attenuated Angiotensin II-stimulation of collagen. Further, DDR2 knockdown by RNA interference prevented Angiotensin II-induced ERK1/2 phosphorylation, indicating that DDR2-dependent ERK1/2 activation enhances collagen expression in cardiac fibroblasts exposed to Angiotensin II. Functionally, wound healing assay revealed compromised wound closure in DDR2-silenced cells exposed to Ang II, indicating that DDR2 is essential for the wound healing ability of cardiac fibroblasts. The specific localization of DDR2 on fibroblasts and its regulatory role in collagen gene expression and wound healing identify it as a potential drug target in the control of cardiac fibrogenesis.
Effect of Different Calorie Diets on the Metabolic Status of p47phox Knockout Mice
Nageswararao Kanuri, Sanjay C. Rebello, Jitendra S. Kanshana,
Priya Pathak, Anand P. Gupta 1 , Jiaur R. Gayen 1 , Kumaravelu J, Madhu Dikshit
Pharmacology Division, CSIR-Central Drug Research Institute, 1 Pharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India
Objective: The present study was undertaken to systematically evaluate parameters associated with the development of insulin resistance (IR) in p47phox knockout (p47phox-/-) mice. Research Design and Methods: Wild type (WT) and p47phox-/- mice fed on commercially available rodent low fat low caloric diet (LF) [Altromin], low fat high caloric diet (LF-H) or high fat high caloric diet (HF) [Research Diet] for five weeks, were monitored to assess their energy homeostasis, glucose intolerance and lipid metabolism. Results: As compared to WT controls, p47phox-/- mice kept on LF diet showed alterations in insulin signaling and glucose tolerance, while animals on LF-H diet exhibited altered hepatic glucose metabolism with changes in insulin signaling and glucose tolerance. Exacerbated changes in the above mentioned parameters were evident in mice on HF diet. Conclusion: The results obtained indicated that p47phox-/- mice kept on high calorie diets were predisposed to IR complications, suggesting important regulatory role of ROS/p47phox in energy metabolism.
Inhibition of Toll Like Receptor 4 Attenuated Cardiac Hypertrophy in Rats
Parmeshwar Katare, Pankaj Bagul, Sanjay Kumar Banerjee
Drug Discovery Research Centre, Translational Health Science and Technology Institute, Faridabad, Haryana, India
Background: Toll like receptor 4 (TLR 4) identifies various endogenous and exogenous molecules and act as mediators of innate immune system by producing various cytokines. TLR 4 plays an important role in many diseases like diabetes, cardiac hypertrophy and heart failure. Therefore, the primary objective of the present study was to find the role of TLR4 in hypertrophic heart. Method: Cardiac hypertrophy was induced in male SD rats after administration of isoproterenol (5 mg/kg/day) through subcutaneous route for 14 days. TLR 4 antagonist was administered through osmotic pump for a period of 14 days (20 ug/day). On 15th day, animals were sacrificed and heart tissue was collected. Heart tissue homogenate was used for measuring mitochondrial health as well as oxidative stress parameters. Cryopreserved heart tissue samples were used for the estimation of gene and protein expression. Results: Cardiac hypertrophy was confirmed by measuring heart weight/body weight ratio as well as ANP, BNP and beta-MHC expression in heart. There was marked increase in the TLR 4 expression in heart tissue in cardiac hypertrophy model. TLR 4 inhibition significantly decreased heart weight/body weight ratio and ANP, BNP and beta-MHC expression in cardiac hypertrophy and restored the disturbed cellular antioxidant flux. Mitochondrial changes that observed in hypertrophy heart were also normalized after administration of TLR4 antagonist. Conclusion: From the present study, we have concluded that TLR 4 may be involved in the pathogenesis of hypertrophic cardiomyopathy. Therefore, TLR 4 inhibition by pharmacological agents could be an effective intervention strategy to reduce cardiac hypertrophy.
Exploring Effect of Thalidomide Exposure on Fetal Heart
Pavitra Kumar, Harish Kumar A 1 , Apurva Thilak 1 , Anuran Ghosh 1 ,
Devi Rethinam Sunderesan, Priyadarshan Kathirvel,
Kavitha Sankaranarayanan, Suvro Chatterjee
AU-KBC Research Centre, Anna University, 1 Department of Biotechnology,
Anna University, Chennai, Tamil Nadu, India
Thalidomide, the notorious teratogen was introduced as a sedative drug in the late 1950s. In 1961, it was withdrawn from the clinical use due to teratogenicity and neuropathy. Congenital heart defects were also found as one of the major birth defects under thalidomide teratogenicity. However, mechanistic insight of thalidomide implications in the heart development has not been studied so far. In present study, we have explored the effect of thalidomide on heart looping and its manifestation in the function of the heart. Morphological, histological, biochemical estimation of ROS, measurement of field potential and altered gene expression study has been done for heart isolated from chick embryo. Thalidomide effect, unlike other teratogens, is specific to time of exposer such as in chick it is HH8 stage which is an intermediate stage during heart looping. Thalidomide treated embryos showed multiple morphological defects in heart looping which further leads to weakening of heart wall and accumulation of blood in cardiac wall in the form of lump. We reported increased level of peroxynitrite, ROS, H 2 O 2 and superoxide indicating the oxidative stress in thalidomide caused lump in cardiac wall. The study concludes that thalidomide exposer during early stages of embryonic development causes morphological defects in heart, which further leads to malformation and malfunctions of heart.
Decoding the Interleukin 33 Receptor Signalling in Cardiac Inflammation: A Systems Based Approach
Peter Natesan Pushparaj 1 , Kalamegam Gauthaman 1 , Adel Abuzenadah 1,2 , Adeel Chaudhary 1 , Mohammed Al Qahtani 1
1 Center of Excellence in Genomic Medicine Research, Faculty of Applied Medical Sciences, King Abdulaziz University, 2 Center of Innovation in Personalized Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
Interleukin-33 receptor (IL-33R) or ST2 is expressed in various cell types including cardiac fibroblasts (CF) and cardiomyocytes (CM). Interleukin-33 (IL-33), an IL-1 family of cytokine, binds with IL-33R to activate an array of intracellular signalling molecules and regulates various cellular and molecular processes. In the present study, we decode the role of IL-33R/IL-33 system in cardiac inflammation using systems based approaches. We identified 163 genes to be differentially regulated by IL-33R/IL-33 axis using Ingenuity Pathway Analysis (IPA) knowledge base (Ingenuity Systems, Qiagen, USA). These genes were further clarified using Fisher's Exact Test (P < 0.05) and Benjamini Hochberg Multiple Testing Correction (P < 0.05) and subjected to core analysis using IPA to decipher the role of IL33R/IL-33 axis in various canonical pathways, biological and toxicological functions, upstream regulators and novel molecular networks. IL33R/IL-33 axis regulates High Mobility Group-BI (HMGB1) signalling, pattern recognition receptors, functions of fibroblasts, macrophages, and endothelial cells, cytokine-chemokine signaling networks and hepatic cholestasis. This axis is essential for the induction of genes implicated in cardiac necrosis, fibrosis, infarction, hypertrophy and inflammation. Ten unique genes namely, IFN-ɣ, IL-1ί, IL-2, IL-4, IRAK4, IL-17A, ITGAM, KLF2, TBX21 and TGFί1 are specifically involved in the regulation of cardiac inflammation. IL-33R/IL-33 axis differentially regulates cardiac inflammation, where induction of IL-2, IL-4, IL-17A, TGFί1, KLF2 and ITGAM decrease cardiac inflammation and offer cardiac protection, while IFN-ɣ, IL-1ί, and IRAK4 lead to cardiac inflammation. Thus IL33R/IL-33 axis might influence translational outcome and needs careful exploration using in vitro/in vivo bioassays.
Interleukin-1 Receptor Associated Kinase Mediates Angiotensin II Induced Vascular Smooth Muscle Cells Proliferation
Preeti Maurya, Santosh Reddy, M. K. Barthwal
Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India
Introduction: IRAK play an important role in innate immunity and inflammation pathways and is activated through Toll like receptors (TLRs). In hypertension, Angiotensin II (Ang II) induces vascular dysfunction. Present study evaluated the role of IRAK1/4 on Angiotensin II induced smooth muscle cell proliferation (SMC), migration and free radical generation. Methods and Results: SMCs were treated with increasing concentration of Ang II (10 nM, 100nM and 1000 nM) and proliferation was evaluated by XTT assay, flow cytometry cell cycle analysis, cell counting by Neubauer hemocytometer chamber and Western blot of PCNA in the presence or absence of IRAK1/4 (3 μM) inhibitor. Western blot for MAPK phosphorylation and flow cytometry for cell cycle analysis were done in presence of the p38MAPK inhibitor (SB203580, 10 μM), ERK inhibitor (U0126 (10 μM) and IRAK1/4 inhibitor. To check the cell migration in ANG II stimulated SMCs in IRAK1/4 inhibitor or vehicle treated cells, scratch wound assay was performed. Further, reactive oxygen species generation was also evaluated in the presence of different ROS inhibitors (NAC, DPI and VAS) and IRAK1/4 inhibitor by DCF-DA. Ang II induced SMCs proliferation was decreased in the presence of IRAK1/4 inhibitor as determined by XTT assay and cell cycle analysis (P > 0.001), cell counting method (p > 0.01) and PCNA expression (p > 0.05). After 15 minutes of Ang II treatment activation of P38MAPK (P > 0.001) and ERK MAPK (p > 0.001) was observed which was significantly attenuated in the presence of IRAK1/4 inhibitor. In cell migration experiments, Ang II stimulated VSMCs migration was significantly inhibited in the presence of IRAK1/4 inhibitor at 24h (P > 0.01) and 48h (P > 0.05). IRAK1/4 inhibitor pre-treatment decreased ROS generation (P > 0.01) in Ang II stimulated SMCs. Conclusion: Present findings suggest that IRAK plays an important role in smooth muscle cell proliferation, migration and ROS generation.
Effects of Herbal Culinary Supplements on the Proliferation of Cardiomyoblasts
A. Mangala Gowri, S. Priya 1 , V. Perasiriyan 1 , V. S. Vadivu
Department of Animal Biotechnology, Centre for Stem Cell Research and Regenerative Medicine, Madras Veterinary College, 1 College of Food and Dairy Technology, Tamil Nadu Veterinary and Animal Science University, Chennai, Tamil Nadu, India
Adult stem cells show reduced restorative potential for tissue regeneration as age advances and are more vulnerable to oxidative stress resulting in reduced ability to heal the tissue damages. In cardiovascular diseases there is a correlation between a reduction in peripheral blood endothelial cells and many risk factors for CVD. Growing literature evidences states that prominent roles of certain nutraceuticals, flavonoids have prominent roles in the proliferation and maintenance of a continuous replacement of stem cells required for healthy self renewal of mature cells in tissues. Certain Indian phytochemicals are well known to have positive effect on heart muscular function were used for the analysing their activity on cardiomyoblasts in vitro. A combination of coriander, hibiscus flower petals and pomegranate extract powder was prepared and used in different concentrations on cultures of different age chicken cardiomyocytes from embryonic to adults (8 weeks). The antioxidant activity of the cultured supernatant analyzed showed increased activity in extract supplemented culture. The cellular proliferation also showed a positive correlation to the concentration of herbal extract supplemented cultures (p < 0.05). The Population doubling time (PDT) was recorded during different culture days have shown significant correlation to the proliferation Index (PI). The result of the preliminary preclinical study further supports the concept that natural compounds have the ability to stimulate and stem cell proliferation in culture. The enhanced antioxidant potential in herbal supplementation recommends herbal incorporation in food preparation as a healthy prophylaxis to cardio vascular diseases.
Poly L-Lactide-Co-Caprolactone Coated Drug-Eluting Stents for Sustained Release of Hydrophobic Drug
Purandhi Roopmani, Swaminathan Sethuraman, Uma Maheswari Krishnan
Centre for Nanotechnology and Advanced Biomaterials, SASTRA University, Thanjavur, Tamil Nadu, India
Drug eluting stents are an effective non-surgical intervention for the treatment of cardiovascular diseases. It is a localized drug delivery system, which treats the diseased artery as well as supports the weak vessel wall. Sustained drug release at the stented site is achieved with the help of a polymer coating that acts as a carrier in drug eluting stents. The polymer coating on the stent plays an important role in maintaining the drug release under therapeutic level with less toxicity in vascular space. The present study explores poly(L-lactide-co-caprolactone) (70:30) copolymer for the stent applications and as a potential carrier for hydrophobic drugs. PLCL has been characterized for their elastic behavior to reduce the coating defects. The morphology of polymer and drug coated stent was analyzed by scanning electron microscopy. Uniform drug distribution in polymer is determined with the help of confocal microscopy by using model drug. In-vitro studies have also been performed to evaluate the biocompatibility and hemo-compatibility of PLCL films using human umbilical vein endothelial cells and platelets.
Angiopoietin-2 Mediates Thrombin-induced Monocyte Adhesion and Endothelial Permeability
K. Rathnakumar, Soniya Savant 1 , Hemant Giri, Beate Fisslthaler 2 ,
Ingrid Fleming 2 , Uma Ram 3 , Hellmut G. Augustin 1,4 , Madhulika Dixit
Department of Biotechnology, Indian Institute of Technology Madras, Chennai, Tamil Nadu, India, 1 Division of Vascular Oncology and Metastasis, German Cancer Research Center Heidelberg (DKFZ-ZMBH Alliance), Heidelberg, 2 Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt, 3 Seethapathy Clinic and Hospital, Chennai, 4 Vascular Biology and Tumor Angiogenesis, Medical Faculty Mannheim (CBTM), Heidelberg University, Heidelberg
Thrombin imparts an inflammatory phenotype to the endothelium by promoting increased monocyte adhesion and vascular permeability. However, the molecular players that govern these events are incompletely understood. The aim of this study was to determine whether Angiopoietin-2 (Ang-2), an endothelial specific molecule has a role, if any, in regulating inflammatory signals initiated by thrombin.In time course experiments, thrombin-stimulated Ang-2 up-regulation, peaked prior to the expression of adhesion molecule ICAM-1 in human umbilical vein derived endothelial cells (HUVECs). Knockdown of Ang-2 blocked both thrombin induced monocyte adhesion and ICAM-1 expression (flow cytometry). In addition, assessment of vascular leakage (Miles assay) demonstrated that Ang-2-/- mice display defective vascular leakage when treated with thrombin. Mechanistically, thrombin activated the tyrosine phosphatase SHP2 by inducing its phosphorylation at Tyr542 and the down regulation of endogenous SHP2 attenuated both thrombin-induced Ang-2 expression and release as well as monocyte adhesion. Down regulation of the adaptor protein Gab1, a co-activator of SHP2, as well as over-expression of Gab1 mutant incapable of interacting with SHP2 (YFGab1), both inhibited thrombin mediated effects. Thrombin induced Gab1-SHP2 axis was necessary for downstream activation of p38 MAPK, which in turn, was required for Ang-2 expression. The data establishes an essential role of Gab1/SHP2/p38MAPK signaling pathway and Ang-2 in regulating thrombin-induced monocyte adhesion and vascular leakage.
Are Food Colorants the Unknown Culprits of Glucose Metabolic Dysregulation?
Balakrishnan Rekha, Ganesan Velmurugan, Sivakumar Anusha, Subbiah Ramasamy
Department of Molecular Biology, School of Biological Sciences, Madurai Kamaraj University, Madurai, Tamila Nadu, India
Caramel colours are among the most widely used colour additives worldwide, regularly consumed through food stuffs. 4-methyl-imidazole (4-MEI), a potential carcinogen, is an undesirable by-product of caramelization. Notable concentration of 4-MEI is detected in most of the consumables including branded soft drinks, blended whiskey, sauces, etc. Hence, consumers including children are at high risk of 4-MEI exposure. No significant risk level (NSRL) which intimates the lifetime average daily exposure associated with a 1-in-100,000 cancer risk, and for 4-MEI is set at 29 μg/day. By considering its carcinogenecity, California Environmental Protection Agency under proposition 65 law entails warning alerts in beverages which has ≥29 ΅g/355 ml. In the present study we investigated the chronic effect of 4-MEI on glucose homeostasis. Balb/c mice were administered with 4-MEI (7.5 ΅g kg-1 body weight) orally via gavage, daily, for 42 days. 4-MEI administered animals exhibited significant hypoglycemia as confirmed by glucose and insulin tolerance test. Pyruvate tolerance test, G6pase,Fbpase, and Pepck declined expression indicated deregulation in gluconeogenesis. Histopathological analysis of pancreas showcased β-cell hyperplasia, leading to increased insulin production also confirmed with C-peptide levels and increased insulin mRNA levels. In addition, 4-MEI induced the levels of serum hepatic markers and significant reduction in protein level of G6Pase and PEPCK. Furthermore, glycogen content and glycogen synthase mRNA level was elevated this also infers reduced activity of G6Pase. Thus findings of this present study illustrate that chronic intake of 4-MEI induces increased proliferation of β-cells leads to dysregulation in gluconeogenesis and glycogenolysis leading to hypoglycemic condition.
Therapeutics Against Cardiac Remodeling: "InsP3Ring" Target
Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India
In heart, Inositol 1,4,5-triphosphate receptor-type 2 (InsP3R2) is a ligand-gated Ca2+ channel predominantly expressed and localized in the nuclear membrane. InsP3R2 mediated bi-directional Ca 2+ mobilization regulates diverse cellular processes including arrhythmias and nuclear signaling in the heart. Cardiac specific deletion of InsP3R2 is cardio protective and not susceptible to endothelin-1 induced arrhythmia. Activated Ca 2+-calmodulin kinase IIδ (CaMKIIδ) and calcineurin-Nuclear Factor Activated in T cells (NFATc), the two major signaling pathways are the leading causes of a diverse pathophysiology of the heart. The multifunctional CaMKIIδ and calcineurin are effectors of Ca2+ and calmodulin and activation of these two pathways are primarily depends on InsP3R2 mediated Ca 2+ mobilization in the heart and not by beat-to-beat Ca 2+ fluctuations during excitation-contraction coupling (ECC). The activated CaMKIIδ modulates diverse targets including InsP3R2 in a negative feedback and increases nuclear Ca 2+ to activate hypertrophic gene expression. We hypothesized that intervention of InsP3R2 Ca 2+ channel activity by competitive and blocking inhibitions would be a novel therapeutics against cardiac remodeling. In competitive inhibition,the InsP3R2 specific peptide (around CaMKIIδ target site) acts as sponge and traps the CaMKIIδ molecules to inhibit CaMKIIδ signaling pathway inducing cellular hypertrophy. In blocking inhibition the InsP3R2 specific polyclonal antibody binds to InsP3R2, inhibits its Ca 2+ channel and the activation of CaMKIIδ pathway leading to prevention of cellular hypertrophy. Our data demonstrate that, the InsP3R2 specific peptide and the antibody inhibit cellular hypertrophy in-vitro. We further evaluate using different heart targeting recombinant peptides and scFv (single chain) antibodies in adult cardiomyocytes and rabbit models.
Angiotensin II Potentiates the Left Ventricle Remodeling Process in the High Fat Diet Fed Mice
S. S. Reddy, Anant Jaiswal, Preeti Maurya, M. K. Barthwal
Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India
Introduction: Present study was undertaken to assess the effect of high fat diet and Angiotensin II in Ventricular remodelling in mice. Methods: Male C57Bl/6J mice were fed with 10% Kcal low fat diet (LFD) or 45% Kcal high fat diet (HFD) from Research Diet, USA to develop insulin resistance. At the end of 16 weeks of HFD feeding, they were infused with Ang II (1500 ng/kg/min, s.c) via osmotic pump (1007D, ALZET, USA) for 7 days and then, subjected to ultrasound imaging (Visualsonics1100, Canada) to monitor the changes in the left ventricle. Further, heart was harvested for gravimetric parameters, gene expression analysis by Real time PCR and histological studies by Picrosirius red staining for studying collagen deposition. Results: Animals fed HFD diet for 16 weeks demonstrated increased food intake, body weight gain, hyperglycemia, hyperlipidaemia, hyperinsulinemia and alteration in insulin sensitivity indices and endothelial dysfunction and; moderate left ventricular remodeling. However, HFD animals infused with Ang II showed aggravated loss of left ventricular function associated with increase in InterVentricular Septum Thickness (IVST), Left Ventricle Internal Diameter (LVID), Left Ventricle Posterior Wall Thickness (LVPWT), LV mass, heart weight to body weight ratio, interstitial collagen deposition and; decrease in ejection fraction and fractional shortening in comparison with the HFD fed animals. We also found an increase in iNOS mRNA levels after Ang II infusion for 21 days in LFD fed animals. Conclusion: HFD fed animals treated with Angiotensin II evidenced elevated biometric, gravimetric and haemodynamic parameters; and interstitial collagen depositon compared to HFD alone.
Role of Subsarcolemmal and Interfibrillar Mitochondria in the Regulation of H 2 S Cardioprotection Against Myocardial Ischemia Reperfusion Injury
A. Shakila Banu, Gino A. Kurian
School of Chemical and Biotechnology, SASTRA University, Thanjavur,
Tamil Nadu, India
Hydrogen sulfide (H 2 S), a novel third endogenous gaseotransmitter, proven to be cardioprotective against ischemia reperfusion (I/R) induced injury and apoptosis, primarily by preserving the mitochondria. Recently researchers observed the presence of two populations of mitochondria viz., interfibrillar (IFM) and subsarcolemmal (SSM) in the heart, which behaves distinctly during physiology and pathology. Thus the present study is designed to understand the specific action of H 2 S conditioning on these subpopulation in exerting its cardioprotection. Male Wistar rats were subjected to I/R injury with or without H 2 S treatment during pre-/post-ischemic period and analysed infarct size, cardiac injury markers release, mitochondria physiology, ETC enzymes activity and mitochondrial respiration. Apoptosis was determined using DNA fragmentation assay. H 2 S pre- and post- ischemic treatment shows cardioprotection confirmed by improved LVEDP and reduced infarct size (>50%). Among the subpopulation, IFM shows higher complex I and complex III activities. H 2 S pre-conditioning attenuates I/R induced decline in complex I, complex IV activities and regained complex II activity in IFM but not in SSM. Diminished complex I, III and IV activities in I/R rat heart were recovered with H 2 S POC in both SSM and IFM. In addition, H 2 S pre- and post-ischemic treatments, irrespective of the energy status, the mitochondrial membrane potential was near to the normal level. SSM from rat hearts exposed to H 2 S_IPC showed higher swelling than IFM in non-energized and succinate energized conditions, unlike H 2 S_POC, where both shows substantial reduced swelling. H 2 S_IPC recovered phosphorylation efficiency in SSM; but both SSM and IFM were recovered in H 2 S_POC. H 2 S pre-conditioning exerts its cardioprotective effect by preserving the IFM subpopulation, however H 2 S post- conditioning could recover both IFM and SSM.
Alterations in the Expression Pattern of Protein Kinase C Isoforms in Age Associated Cardiac Hypertrophy
Department of Zoology, University of Calcutta, Kolkata, West Bengal, India
Cardiovascular disease is the leading cause of death worldwide. Cardiac hypertrophy is an important compensatory mechanism of the heart in response to diverse pathophysiological stimuli. While adaptive hypertrophy is associated with improved cardiac function, prolonged pathological stimuli often results in transition from adaptive to maladaptive cardiac hypertrophy. Age is a major risk factor for cardiovascular diseases. Cardiomyocyte hypertrophy, apoptosis and compromised contractile function increase significantly with advancing age. Among various signaling pathways involved in promoting cardiac hypertrophy protein kinase-C (PKC) has been identified as an important component used by myocytes in response to a variety of stimuli. Cardiomyocytes express multiple PKC-isoforms (α, δ, ε etc.) with distinct biological functions. Several reports have also shown their association with different cardiac diseases. The goal of the present study is to examine the pattern of PKC isoform expression in physiological and pathological cardiac hypertrophy during the course of aging. Our results indicate distinct age related changes in PKC epsilon (ε) activation pattern during hypertrophy. Changes in expression of other PKC isoforms did not correlate with age in various experimental groups. Interestingly, induction pattern of PKC epsilon is different with advancing age during pathological hypertrophy and exercise withdrawal physiological hypertrophy group that showed signs of compromised cardiac function. Few reports suggested a link between cardiomyocyte autophagy and cardiac hypertrophy. Also, autophagic activity is gradually decreased in aging hearts. Hence, further investigation is needed to more completely understand the role of PKC epsilon in regulating autophagy during aging associated cardiac hypertrophy.
A Novel Non-active Site Inhibition of Carbonic Anhydrase by Acacetin: Implications for Cardioprotection
Subhalakshmi Kalyanavenkataraman, Pandurangan Nanjan, Asoke Banerji, Bipin G. Nair, Geetha B. Kumar
Amrita School of Biotechnology, Amrita University, Kollam, Kerala, India
Acacetin, a partial methylated flavone, exhibits a novel binding pattern with human Carbonic Anhydrase II (CA II), and inhibits its activity in vitro with an IC50 of 0.9μM. Molecular modeling studies indicate, that unlike the hydrophobic face exchanges of commonly known CA II inhibitors, acacetin is unique in that it targets the hydrophilic face of the protein through non-active site interactions, inhibiting proton shuttling and catalysis. The present study demonstrates that acacetin forms hydrogen bonds with Ala23, Lys24 and Arg246 present in the hydrophilic face of the enzyme. The binding of acacetin to Arg246, interferes with the electron transfer from Arg246 to Glu106, which then transfers it to the Zn 2+ -bound water that is crucial for catalysis. Furthermore, cell based studies demonstrated that acacetin also inhibited the intracellular CA activity in H9c2 rat embryonic myocardial cells. Additionally, qRT-PCR studies also indicated that acacetin significantly decreased the expression of CAII. The inhibition of CAi in H9c2 cells prevents the intracellular Na + load (due to lack of H + extrusion), resulting in a decrease in the Ca 2+ influx, which subsequently prevents the depolarization of the mitochondrial membrane potential. Thus, acacetin, reduces the vulnerability of the mitochondria to the intracellular Ca 2+ by inhibiting the prognostic molecular marker CAi, making it a potential molecule in therapeutics related to cardiomyocyte hypertrophy.
Role of 5-Azacytidine on Cardiac Stem Cells Differentiation: Promising Insight towards Cardiac Therapeutics
Tarun Pant, M. Reddy Sailaja, Shailendra Narayan, K. Prakash, C. Ramya, Anuradha Dhanasekaran
Centre for Biotechnology, Anna University, Chennai, Tamil Nadu, India
Cardiovascular diseases (CVDs), particularly myocardial infarction, have emerged as major cause of worldwide mortality and morbidity. Resident cardiac stem cells (CSCs), due to their allogenic and non-immunogenic nature, act as ideal candidates for treatment of CVDs .The present study aims at exploring the role of 5-Azacytidine on differentiation potential of cardiosphere derived cells(CDCs) to cardiomyocytes. Cardiac stem cells were isolated by explant method with intermediate Cardiosphere step and subsequent derivation of Cardiosphere derived cells. The enhanced cardiac lineage commitment of CDCs in comparison to explants outgrowth was shown by high expression of early cardiac lineage markers such as Isl-1, GATA-4, Nkx2.5 and CD90. Further, the pretreatment of CDCs with 10΅M 5-Azacytidine resulted in high differentiation to cardiomyocytes. Immunofluorescence analysis of 5-Azacytidine treated and untreated CDCs revealed a higher expression of cardiomyocyte markers- α-sarcomeric actin and Nkx2.5 in treated CDCs. The preliminary results confirm the existence of adult cardiac stem cells in the mammalian heart with better efficacy in treating myocardial disorders when compared to other stem cell types. Further investigation of 5-azacytidine action under hypoxic conditions will give a better understanding on therapeutic efficiency of the compound on CDCs differentiation potential.
Anti Atherogenic Effect of Gentiana Lutea and its Component Isovitexin
Potunuru Uma Rani, Rushendhiran Kesavan, Shivam Chandel,
Swapna Upadhyaya, Raghu Ganugula 1 , Hemant Giri, Giriraj Sahu,
Uday P. Kumar 1 , Bhanuprakash Reddy 1 , Gordana Joksic 2 , Amal K. Bera, Madhulika Dixit
Department of Biotechnology, Indian Institute of Technology Madras, Chennai, Tamil Nadu, 1 National Institute of Nutrition, Hyderabad, Telangana, India, 2 Department of Physical Chemistry, Vinca Institute of Nuclear Sciences, Belgrade, Serbia
Phytotherapy under Complementary and Alternative Medicine play a significant role in cardiovascular health and diseases. One such phytoproduct is Gentiana lutea (GL) aqueous root extract and its component isovitexin (ISO) and suggestive of exhibiting anti atherosclerotic properties. In the present study we have investigated the effect of GL and ISO on endothelial inflammation, smooth muscle cell migration and the onset and progression of atherosclerosis in streptozotocin (STZ) induced diabetic rats. Our results show that both GL extract and ISO, block leukocyte adhesion and generation of reactive oxygen species in human umbilical vein endothelial cells (HUVECs) and rat aortic smooth muscle cells (RASMCs) following TNF-alpha and platelet derived growth factor-BB (PDGF-BB) challenges respectively. Both the extract and isovitexin blocked TNF-α induced expression of ICAM-1 and VCAM-1in HUVECs. PDGF-BB induced migration of RASMCs and phospholipase C-γ activation were also abrogated by GL extract and isovitexin. Both extract and ISO inhibited PDGF-BB mediated intracellular calcium rise in RASMCs as determined by Fura-2 based ratio metric measurements. Supplementation of regular diet with 2% GL root powder for STZ rats, reduced total cholesterol in blood. Oil red O staining demonstrated decreased lipid accumulation in aortic wall. Medial thickness and deposition of collagen in the aortic segment of diabetic rats were also reduced upon supplementation. Immunohistochemistry demonstrated reduced expression of vascular cell adhesion molecule-1 (VCAM-1), inducible nitric oxide synthase (iNOS), and vascular endothelial cadherin (VE-cadherin) in aortic segments of diabetic rats following GL treatment. Thus, our results support anti-atherosclerotic effect of GL root extract and isovitexin.
Role of Matrix Metalloproteinases Isolated from Pseudomonas Fluorescensmigula in Cardiovascular Diseases
B. Usharani, P. David Jawahar
University of Madras, Chennai, Tamil Nadu, India
Matrix metalloproteinases (MMPs) are extracellular enzymes that are important in many physiologic and pathologic processes. Their activity is regulated mainly by tissue inhibitors of metalloproteinases (TIMPs). MMPs expression is related with the classical cardiovascular risk factors as well as with inflammation. They play a central role in atherosclerosis, plaque formation, platelet aggregation, acute coronary syndrome, restenosis, aortic aneurysms and peripheral vascular disease. The aim of the review is to present literature data on the role of MMPs matrix ECM metalloprotease isolated from Pseudomonas fluorescens and their inhibitors in cardiovascular disease. The specific regulation of this MMP by miR-146a was further confirmed at the secretion and enzymatic activity levels, as well as after anti-miR-mediated miR-146a inhibition. The Key future areas of research for the cardiovascular matrix metalloproteinase field revealed that the enzyme cleaves the fibrin clots and the molecular weight of the protein determined by SDS- Profile as 35KDa. The MMPs are down regulated by miR 146 gene expression analysis. These findings suggest that miR-146a and MMPs isolated from Pseudomonas fluorescensmigula might be a new and promising therapeutic tool for treating cardiac disorders associated with enhanced inflammation in the heart. The prominent spots were identified on 2-D Electrophoresis subjected to Nano LC/MS analysis, identified as Metalloprotease and the designed structure was validated by Ramachandran plot analysis with consensus sequence and the amplified PCR product 185 bp genes of 35kDa of metalloprotease respectively. The phylogenetic tree was constructed, Homology search, alignment of multiple amino acid sequences confirmed a member of metalloprotease. Our findings suggested that the reported MMP of Pseudomonas fluorescensmigula plays important role to cleave the fibrin clots in the heart, artries, lungs and valves and can be used as effective enzyme for the Heart medication approved by WHO concern.
Sirtuin 6 Transcriptionally Regulates mTOR Signaling in Diabetic Heart
Venkatraman Ravi, Faiz Ahamed, Sneha Mishra, Ullas Kolthur-Seetharam 1 , Nagalingam Ravi Sundaresan
Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru, Karnataka, 1 Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai, Maharashtra, India
Dysregulated mechanistic target of rapamycin (mTOR) signaling is associated with cardiovascular diseases including diabetic cardiomyopathy. In diabetic heart, mTOR signaling is hyperactive and has been implicated in several pathologies such as inflammation, cardiomyocytes hypertrophy, sarcomere abnormalities and autophagy defects. However, the reason for the hyperactivation of mTOR signaling is not well understood at the molecular level. In this study, we describe, for the first time that Sirtuin 6 (SIRT6), one of the seven mammalian homologs of NAD+ dependent deacetylases, directly regulates mTOR signaling at the transcriptional level. SIRT6 is a nuclear localized chromatin-associated deacetylase that has histone H3K9, H3K56 deacetylase activity and it is also known to repress several transcription factors such as HIF-1α, NF-κB, c-Myc and FoxO. In type 2 diabetic mice heart, we found that SIRT6 levels were significantly low, which is correlated with increased activity of mTOR. Further analysis of SIRT6-deficient heart lysates revealed increased mRNA and protein levels of mTOR and its downstream targets, p70S6K, 4eBP1, and RPS6. Chromatin Immunoprecipitation (ChIP) analysis indicated that SIRT6 binds to the promoter of mTOR pathway genes. Further, promoter analysis of the mTOR pathway genes and subsequent ChIP experiments revealed that SIRT6 regulates mTOR-signaling genes via the transcription factor Sp1. We also found that increased mTOR signaling resulted in defective autophagy in SIRT6-deficients hearts. Taken together the findings suggest a critical role for SIRT6 in the regulation of mTOR signaling and can be targeted for treating diabetic cardiomyopathy.
Mechanism of Regulation of Mouse Renalase Gene Transcription by Epinephrine
Venkatasubramaniam Sundaramurthy, Ananthamohan Kalyani, Parshuram J. Sonawane, Nitish R. Mahapatra
Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, Tamil Nadu, India
Renalase is a recently discovered flavoprotein that is mainly expressed in kidney and heart. It is emerging as an important regulator of cardiovascular, metabolic and renal diseases. Despite its important role in the regulation of blood pressure and association with several disorders, the mechanism of its transcriptional regulation remains largely unknown. Previous studies have shown that renalase deficiency in human and animal models are associated with elevated circulating catecholamines. Therefore, we started by investigating the expression levels of renalase in the mouse models of hypertension. Analysis of renalase protein and mRNA levels showed a marked increase in the expression of renalase in the BPL kidney samples. In order to determine the epinephrine-mediated expression of renalase, we transfected mouse renalase promoter-reporter plasmids into N2a cells, which showed an increase in renalase expression on treatment with increasing concentrations of epinephrine. This prompted us to identify possible transcription factors which regulated renalase gene expression through epinephrine. Hence, we undertook a systematic analysis of the mouse renalase proximal promoter region (~1.0 kb upstream of the transcription initiation site) to identify cis-elements that may regulate mouse renalase gene expression. Progressive mouse renalase 5'-promoter-deletion/luciferase reporter constructs were generated, and were transiently transfected into different cell types to identify the cis-elements. We identified two promoter domains (-677 bp to -569 bp and -569 to -230 bp) which are crucial for directing the basal renalase gene expression in several cell types. Computational analysis of these crucial promoter domains indicated potential binding sites for several transcription factors including STAT3, Sp1, YY1 and CREB. In conclusion, this study provides new insights to the mechanism of regulation of mouse renalase gene expression by epinephrine and has implications for pathological conditions that involve excess catecholamines in the circulation.
Multiple Transcription Factors and MicroRNAs Interplay to Govern the Expression of Monoamine Oxidase B
Vikas Arige, Anshu Agarwal, Vinayak Gupta, Abrar Ali Khan, Bhargavi Natarajan, Kalyani Ananthamohan, Nitish R. Mahapatra
Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, Tamil Nadu, India
Monoamine oxidase B (MAO-B) is a flavoenzyme, located on the outer membrane of mitochondria. It is involved in the catabolism of dopamine, benzylamine and phenylethylamine. Altered levels of MAO-B are known to be associated with many neurological diseases and very recently, with type 2-diabetes. Hence, understanding the mechanism of regulation of MAO-B under basal and pathological conditions is crucial. We are currently studying the role of transcription factors and miRNAs in regulating the expression of MAO-B. In order to identify the MAO-B core promoter region, deletion/reporter constructs of the promoter were generated and transfected into various cell lines. Stringent in silico analysis of MAO-B core promoter (-144/+25) revealed putative binding sites for transcription factors Sp1, Egr1 and CREB. Co-transfection of MAO-B core promoter reporter construct with Sp1, Egr1 and CREB expression plasmids independently resulted in increased MAO-B promoter activity. In corroboration, knockdown of endogenous Sp1, Egr1 and CREB repressed the MAO-B promoter activity. In vitro interaction of Sp1 and Egr1 with the MAO-B promoter was confirmed by gel shift assays, whereas the in vivo interaction of Sp1, Egr1 and CREB with the MAO-B promoter was confirmed by chromatin immunoprecipitation (ChIP).The 3'-UTR of MAO-B harbored putative binding sites for microRNAs-miR-1224 and miR-300. Co-transfection of 3'-UTR reporter construct with miR-1224 or miR-300 expression plasmids resulted in decreased MAO-B 3'-UTR reporter activity. Interestingly, miR-1224 also binds to the 3'-UTR of Sp1 and regulates its expression, whereas miR-300 is predicted to bind to the 3'UTR of Egr1. These findings demonstrate the regulation of MAO-B expression by several transcription factors and miRNAs.
CardioGenBase: A Literature Based Multi-Omics Database for Major Cardiovascular Diseases
Alexandar V, Pradeep G. Nayar 1 , R. Murugesan, Beaulah Mary, Darshana P, Shiek S. S. J. Ahmed 2
Faculty of Allied Health Sciences, 1 Department of Cardiology, Chettinad Super Specialty Hospital, 2 Department of Computational Biology, Chettinad Academy of Research and Education, Kelambakkam, Tamil Nadu, India
Cardiovascular diseases (CVDs) are the leading cause of deaths worldwide. Among the CVDs, cerebrovascular disease, coronary artery disease (CAD), hypertensive heart disease, inflammatory heart disease, ischemic heart disease and rheumatic heart disease are considered major cardiovascular conditions that are responsible for increased mortality. Though, several research and vast amount of multi-omics data are accumulated in the field of cardiovascular research, yet the understanding of key mechanistic aspects of these cardiovascular conditions remain uncovered. Hence, a comprehensive online resource tool is requisite to comprehend previous research findings and to draw novel methodology for understanding disease pathophysiology. In this study, we have developed a literature-based database, CardioGenBase, collecting gene-disease association from PubMed and MEDLINE. The database covers major cardiovascular diseases which contains ~1500 cardiovascular disease genes from ~24000 research articles. In addition to literature evidence, ontology, pathways, single nucleotide polymorphism, protein-protein interaction network, normal gene expression, protein expressions in various body fluids and tissues are provided. Further, tools like gene-disease association finder and gene expression finder are made available for the users with figures, tables, maps and venn diagram to fit their needs. To our knowledge, CardioGenBase is the only database to provide gene-disease association for above mentioned major cardiovascular conditions in a single portal. CardioGenBase is a vital online resource to support genome-wide analysis, genetic, epigenetic and pharmacological studies.
Correlation of Oil with Cardiac Health in South Tamil Nadu
Department of Biochemistry, V.V. Vanniaperumal College for Women, Virudhunagar, Tamil Nadu, India
The primary oil consumption pattern of various families of South Tamil Nadu was studied. A total number of 163 families were selected for this study and a survey was conducted about their cooking oil and about the general health of their family members. A special enquiry about various types of heart ailments was also made out of 163 families, thirty two families used gingelley oil as their primary cooking oil, thirty six families used groundnut oil, thirty two families used refined oil, forty three families used palm oil, and twenty families used more than two oils. Statistical analysis revealed that the prevalence of the percentage of cardiac ailment in gingelley oil using families is 12.5%, that of groundnut oil using families is 8.5%, refined oil using families was 28.12%, palm oil using families 30.23%, more than two oils was 35%. The economical status of the families were middle and lower middle class. The hospitals involved for this study were Government Hospital, Sathur and Government Hospital, Virudhunagar, Tamil Nadu. The study reveals that Groundnut oil and Gingelley oil are good for our heart than the Refined oil and Palm oil.
Incessant Left Ventricular Tachycardia of Unusual Etiology
Praloy Chakraborty, H S Isser, Sudheer Arava 1 , Kausik Mandal 2
Department of Cardiology, Vardhaman Mahavir Medical College and Safdarjang Hospital, 1 Department of Pathology, All India Institute of Medical Science, New Delhi, 2 Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
A 40 years male patient presented with incessant left ventricular tachycardia for 10 days. The arrhythmia was controlled with combination of antiarrhythmic therapy (Amiodarone and phenytoin). His echocardiography was normal. Contrast enhanced cardiac MRI showed abnormal sub-endocardial and mural late gadolinium enhancement (LGE) with in apical inferior and apical septal wall of left ventricle. CECT chest, cardiac PET-CT and Coronary angiography was normal. Mantoux test showed 20 mm x 20 mm induration after 72 hours and The QuantiFERON-TB Gold test (QFT-G) was within normal range. Endomyocardial biopsy showed myocytes loss and fatty replacement. A diagnosis of left dominant arrhythmogenic cardiomyopathy (LDAC) was made. Genetic analysis using Next Generation sequencing (NGS) technique and targeting genes associated with arrhythmogenic cardiomyopathy showed that individual harboured a variation (p.Thr277Ser) caused by a substitution (c.829A>T) in exon 10 of the TMEM43 gene. His mother did not have this variation by mutation analysis study and father was not available for gene testing. Two out five in-silico prediction tools suggested that The Thr277 residue is well conserved and the change is predicted to be damaging. Protein modelling of the mutant protein to check the deleterious effect of the sequence variation was not possible in view of lack of availability of structure of the wild type protein in the database. Any homologous protein structure with up to 80% homology with TMEM 43 protein was also not available. Thus, it is uncertain if the c.829A [T (p.T277S) variant is related to his ARVC presentation. To conclude we report a case of incessant left ventricular tachycardia due to unusual form of arrhythmogenic cardiomyopathy (LDAC) associated with variation in TMEM43 gene.
MiRNAs in Congenital Heart Diseases and its Expression in Septal Defects Patients of South Indian Origin
Sambantham Shanmugam, Sambhavi Bhagavatheeswaran,
Vinu Ramachandran, Malathi Raghunathan, Sowmya Ramanan 1 ,
Cherian K. M 1 , Anandan Balakrishnan
Department of Genetics, Dr. ALMPGIBMS, University of Madras, Taramani Campus, 1 Frontier Life Line Hospital, Chennai, Tamil Nadu, India
Congenital heart disease (CHD) is a multi-factorial disease that arises from developmental errors in cardiac morphogenesis. CHD represents a major cause for perinatal death and occurs in ~1% (1 in 1000 live births) of newborns. Septal defect like atrial septal defect-ASD and ventricular septal defect-VSD, the most common types of CHD, results from a combination of environmental and genetic factors. The miRNAs as governors of gene expression during cardiovascular development and disease, modulate the effects of critical cardiac regulatory proteins to control the balance between differentiation and proliferation. Also, these highly conserved miRNAs are dynamically expressed during distinct phases of early cardiogenesis. miR-1 was shown to be very important in embryonic heart development and deletion of miR-1-2 caused VSD and other cardiac anomalies. This study was to identify and characterize the expression of miRNAs (miR-1, -133, -208a and -155a) that might be involved in the development of septal defects in CHD subjects. miRNAs were isolated from 20 tissue samples of CHD patients and 3 control tissue samples from Frontier Life Line Hospital. The expression levels of candidate miRNAs were quantified using qRT-PCR. Compared with control samples, patients with septal defects showed increased expression of miR-1, -155a and -133a except for miR-208a. The upregulation of miR-1,-133 and -155a is associated with septal defects in CHD patients. However, detailed studies are required to understand the role of these miRNAs and mechanism of action on its downstream targets in order to exploit its efficacy in developing a miRNA-based therapy module for CHD.
Lipoprotein Associated Phospholipase A2: A Surrogate Marker of Coronary Artery Disease in Diabetic Patients
Saswati Das 1,2
Departments of 1 Biochemistry and 2 Cardiology, Maulana Azad Medical College and G.B. Pant Hospital, New Delhi, India
Lipoprotein-associated phospholipase A2 [LpPLA2] is known as an emerging marker of coronary artery disease (CAD). However its role and levels have not been documented clearly in diabetic patients with CAD in Indians. The aim of this study was to explore if LpplA2 can be established as a surrogate marker of CAD in diabetic patients and compare it with other established markers like hs-CRP. Sixty individuals with angiographically proven CAD and 30 healthy individuals matched for age & sex were studied. CAD patients were divided into two groups based on presence (n = 30) [Group I] and absence (n = 30) [group II] of type 2 diabetes mellitus (DM). The serum levels of LpPLA2, hs-CRP were measured by ELISA. Angiographic clinical vessel scoring was also done for all the patients. Both groups of CAD with and without DM had significantly higher levels of LpPLA2 (Group I-408.48 +/- 38.96 ng/ml, Group II 272.88 +/- 34.21ng/ml respectively) and hsCRP (Group I-10.61 +/- 1.34 mg/l, Group II-5.75 +/- 2.59 mg/l respectively) when compared with healthy control subjects (LpPLA2 =200.82 +/- 20.97ng/ml & hsCRP=1.89 +/- 1.34mg/1) [p < 0.001]. LpplA2 levels between the two CAD groups were highly significant(p < 0.001), levels being maximum for CAD with type 2 diabetes (Group I) which could be due an increase in its substrate sLDL and oxidised LDL in DM. Angiographic clinical vessel score of CAD severity was also higher in CAD with DM. LpPLA2 levels correlated strongly (r = 0.763,p < 0.001) with the angiographic clinical vessel score in diabetes patients with CAD while hsCRP has moderate correlation with the vessel score (r = 0.475, p < 0.01). LpPLA2 and hsCRP elevation is increased with patients of type 2 diabetes mellitus with CAD as compared to only CAD patients and LpPLA2 levels increase with the severity of CAD in diabetes patients. Measurement of LpPLA2 may be considered as a surrogate marker for better prediction of cardiovascular risk in diabetes patients.
Is microRNA 196a2: A Susceptible Factor for Cardiovascular Diseases (CVD) in South Indians?
P. Sundaravadivel, D. D. Venkatesh Babu, Aparna Murali, S. Ramasamy
Department of Molecular Biology, School of Biological Sciences, Madurai Kamaraj University, Madurai, Tamil Nadu, India
Cardiovascular diseases (CVD's) are major cause of morbidity and mortality throughout the world. It is complex and multifactorial, where dyslipidemia, hypertension and diabetes were proved to be major risk factors. Scientific community has been working towards identification of novel biomarkers that could possibly help in early detection of these fatal conditions. MicroRNA's are small non-coding RNA molecules involved in post-transcriptional modifications and emerging as potential biomarker for CVD's. It is very important for a microRNA to be produced at optimal levels and to maintain its structure, to be able to regulate post transcription and its own binding to the target miRNA gene. Since, they are produced from precursor microRNA gene (pre-miRNA), polymorphism at these genes might affect the levels of miRNA produced. Single nucleotide polymorphisms (SNP) occurring at pre-miRNA genes, might have a significant role in determining the susceptibility/resistance of a disease in population specific manner. A polymorphism (T/C) at microRNA 196a2 (rs11614913) has been reported to be associated with increased risk of CVD in type 2 diabetes patients in Western population. Indian population is highly heterogeneous at genetic level as they kept themselves isolated from rest of the world for several thousands of years. Hence, we sought to investigate the role of microRNA 196a2 (rs11614913) in South Indian population in an ethnically and geographically matched case-control study (Cases = 206; Controls = 87). We believe that outcome of this study will helps in further understanding of CVD pathogenesis.
A Novel Mutation Reported in TnnI3 Gene in Hypertropic Cardiomyopathic Patients in Indian Population
Syed Abrar Ahmad, Chandrakant Chavan 1 , Rajesh Badani 2 ,
Department of Zoology, Savitribai Phule Pune University, 1 Department of Cardiology, Bharti Hospital Katraj, 2 Department of Cardiology, Aditya Birla Hospital, Pune, Maharashtra, India
In India, cardiovascular diseases (CVD) are largest cause of mortality in different regions of country. Hypertropic cardiomyopathy (HCM) is dominant disease type in nature, with a prevalence of 1: 500 in general population. HCM is mostly identified as LVH without other complications related to same output. Myosin binding protein C (MYBPC) is key player in gene associated mutations in HCM patients. About 15% of all reported mutations are located on this gene. Troponin I (TNNI3) and Myosin light chain 2 (MYL2) are other HCM related genes. The Institutional Human Ethics Committee approved the present study. HCM patients were identified at cardiology unit Bharti hospital, Pune. Written informed consent was obtained from all the patients and their relatives. 10ml blood sample was withdrawn from the HCM patients from Bharti hospital, Pune, followed by DNA extraction, PCR amplication. All samples were screened by SSCP technique. Samples showing abnormal bands were processed for DNA sequencing. Screening of all exons in MYBPC3, TNNI3, TPM1 and MYl2 genes in 10 HCM Probands revealed a novel SNP in exon 04 of TNNI3 gene. This polymorphism produced C>T transition (ATC/ATT). Proband is of 19 years and shows asymmetrical septal hypertrophy, while his father is also phenotypically positive for HCM. The novel SNP causes splicing regulation mainly by diminishing Exonic splicing enhancer (ESE) and protein domain abolition in pfam00992 protein at CDD 201540. This novel SNP is synonymous and shows a high disease risk
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