Biju George

miR133a regulates cardiomyocyte hypertrophy in diabetes

Diabetic cardiomyopathy, characterized by cardiac hypertrophy and contractile dysfunction, eventually leads to heart failure. We have previously shown that alterations of a number of key molecules are involved in producing cardiomyocyte hypertrophy in diabetes. The aim of the present study was to determine whether microRNAs (miRNA) play a role in mediating altered gene expression and structural/functional deficits in the heart in diabetes.

Transcriptional coactivator p300 regulates glucose-induced gene expression in endothelial cells.

Sustained hyperglycemia in diabetes causes alteration of a large number of transcription factors and mRNA transcripts, leading to tissue damage. We investigated whether p300, a transcriptional coactivator with histone acetyl transferase activity, regulates glucose-induced activation of transcription factors and subsequent upregulation of vasoactive factors and extracellular matrix (ECM) proteins in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated in varied glucose concentrations and were studied after p300 small interfering RNA (siRNA) transfection, p300 overexpression, or incubation with the p300 inhibitor curcumin. Histone H2AX phosphorylation and lysine acetylation were examined for oxidative DNA damage and p300 activation. Screening for transcription factors was performed with the Luminex system. Alterations of selected transcription factors were validated. mRNA expression of p300, endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), and fibronectin (FN) and its splice variant EDB(+)FN and FN protein production were analyzed. HUVECs in 25 mmol/l glucose showed increased p300 production accompanied by increased binding of p300 to ET-1 and FN promoters, augmented histone acetylation, H2AX phosphorylation, activation of multiple transcription factors, and increased mRNA expression of vasoactive factors and ECM proteins. p300 overexpression showed a glucose-like effect on the mRNA expression of ET-1, VEGF, and FN. Furthermore, siRNA-mediated p300 blockade or chemical inhibitor of p300 prevented such glucose-induced changes. Similar mRNA upregulation was also seen in the organ culture of vascular tissues, which was prevented by p300 siRNA transfection. Data from these studies suggest that glucose-induced p300 upregulation is an important upstream epigenetic mechanism regulating gene expression of vasoactive factors and ECM proteins in endothelial cells and is a potential therapeutic target for diabetic complications.

Regulation of cardiomyocyte hypertrophy in diabetes at the transcriptional level

Diabetic cardiomyopathy, structurally characterized by cardiomyocyte hypertrophy and increased extracellular matrix (ECM) protein deposition, eventually leads to heart failure. We investigated the role of transcriptional coactivator p300 and its interaction with myocyte enhancer factor 2 (MEF2) in diabetes-induced cardiomyocyte hypertrophy. Neonatal rat cardiomyocytes were exposed to variable levels of glucose. Cardiomyocytes were analyzed with respect to their size. mRNA expression of p300, MEF2A, MEF2C, atrial natriuretic polypeptide (ANP), brain natriuretic polypeptide (BNP), angiotensinogen (ANG), cAMP-responsive element binding protein-binding protein (CBP), and protein analysis of MEF2 were done with or without p300 blockade. We investigated the hearts of STZ-induced diabetic rats and compared them with age- and sex-matched controls after 1 and 4 mo of followup with or without treatment with p300 blocker curcumin. The results were that cardiomyocytes, exposed to 25 mM glucose for 48 h, showed cellular hypertrophy and augmented mRNA expression of ANP, BNP, and ANG, molecular markers of cardiac hypertrophy. Glucose caused a duration-dependent increase of mRNA and protein expression in MEF2A and MEF2C and transcriptional coactivator p300. Curcumin, a p300 blocker, and p300 siRNA prevented these abnormalities. Similarly, ANP, BNP, and ANG mRNA expression was significantly higher in the hearts of diabetic rats compared with the controls, in association with increased p300, MEF2A, and MEF2C expression. Treatment with p300 blocker curcumin prevented diabetes-induced upregulation of these transcripts. We concluded that data from these studies demonstrate a novel glucose-induced epigenetic mechanism regulating gene expression and cardiomyocyte hypertrophy in diabetes.

PARP mediates structural alterations in diabetic cardiomyopathy.

Diabetic cardiomyopathy is characterized by structural alterations such as cardiomyocyte hypertrophy, necrosis and focal fibrosis. Hyperglycemia-induced oxidative damage may play an important role in this pathogenetic process. Recent studies have shown that poly (ADP-ribose) polymerase (PARP) is activated in response to oxidative stress and cellular damage as well, plays a role in gene expression. This study investigated mechanisms of diabetes-induced, PARP-mediated development of structural alterations in the heart. Two models of diabetic complications were used to determine the role of PARP in oxidative stress, cardiac hypertrophy and fibrosis in the heart. PARP-1 knockout (PARP(-/-)) mice and their respective controls were fed a 30% galactose diet while male Sprague-Dawley rats were injected with streptozotocin and subsequently treated with PARP inhibitor 3-aminobenzamide (ABA). The in vivo experiments were verified in in vitro models which utilized both neonatal cardiomyocytes and endothelial cells. Our results indicate that hyperhexosemia caused upregulation of extracellular matrix proteins in association with increased transcriptional co-activator p300 levels, cardiomyocyte hypertrophy and increased oxidative stress. These pathogenetic changes were not observed in the PARP(-/-) mice and diabetic rats treated with ABA. Furthermore, these changes appear to be influenced by histone deacetylases. Similar results were obtained in isolated cardiomyocytes and endothelial cells. This study has elucidated for the first time a PARP-dependent, p300-associated pathway mediating the development of structural alterations in the diabetic heart.

Leptin and endothelin‐1 mediated increased extracellular matrix protein production and cardiomyocyte hypertrophy in diabetic heart disease

We investigated the role of leptin and its interaction with endothelin 1 (ET‐1) in fibronectin (FN) synthesis and cardiomyocyte hypertrophy, two characteristic features of diabetic cardiomyopathy.

Extracellular Matrix Proteins in Epiretinal Membranes and in Diabetic Retinopathy

Purpose: Non-vascular epiretinal membranes (ERM) and neovascular membrane in proliferative diabetic retinopathy (PDR) are recognized causes of visual impairment. Both ERMs and neovascular membranes in PDR consist of cellular components and extracellular matrix (ECM) proteins such as fibronectin (FN) and collagen. Transforming growth factor-β (TGF-β) and endothelin-1 (ET-1) regulate ECM protein production. In this study, we investigated ECM proteins and their regulators in ERMs and vitreous from PDR subjects and non-diabetic subjects undergoing vitrectomy. Methods: ERMs from non-diabetic subjects undergoing membrane peeling were collected. Vitreous samples from non-diabetic and PDR subjects undergoing vitrectomy were also collected and separated into solid pellets consisting of fibrovascular tissue and vitreous fluid. Real-time PCR was done for estimating mRNA levels of extracellular matrix proteins like collagen, FN, its splice variant extra-domain B containing FN (EDBFN), and their regulators, TGF-β and ET-1. ELISA was done to detect the EDBFN level in blood and vitreous from non-diabetic and PDR subjects undergoing vitrectomy. Results: ECM proteins, including FN, its splice variant EDBFN, and collagen were significantly upregulated in the ERMs and PDR compared to vitreous from both other two group. The levels were, however, higher in the ERM. ECM protein regulators like TGF-β and ET-1 were also elevated. FN and EDBFN show significant correlation with TGF-β in vitreous but not in ERMs. Plasma and vitreous EDBFN were elevated in the PDR subjects compared to non-diabetic subjects. Conclusions: Data from these studies show that ECM proteins such as EDBFN and collagen are upregulated in ERM and PDR, and are regulated by TGF-β. Elevated serum EDBFN in the PDR may potentially be further explored as a possible molecular marker for the early detection of diabetic end organ damages.

Genotoxic stress and activation of novel DNA repair enzymes in human endothelial cells and in the retinas and kidneys of streptozotocin diabetic rats

Mammalian excision repair cross‐complementing 1 (ERCC1) and ERCC4 (a.k.a xeroderma pigmentosum complementation group F) are nucleotide excision repair enzymes, which excise the 5′ end of damaged DNA. ERCC1 and ERCC4 have an interactive relationship with poly (adenosine diphosphate ribose) polymerase (PARP). We studied the role of ERCC1 and ERCC4 in glucose‐induced extracellular matrix protein production in human endothelial cells and in the retinas and kidneys of streptozotocin diabetic rats.

Identification of Serum Biomarkers in End Stage Liver Disease

Background: Progressive fibrosis and cirrhosis, clinically presenting as end-stage liver disease are common outcomes in alcoholic hepatitis as well as non-alcoholic fatty liver disease(NAFLD). In these processes, a series of changes occurs in liver tissues leading to cell death, remodeling, fibrosis and regeneration. The aim of this study is to identify potential novel biomarkers for non-invasive diagnosis of cirrhosis due to alcoholic etiology or NAFLD. Methods: Serum from patients with biopsy proven end-stage liver disease of various etiologies, namely NAFLD(n=9), al- cohol(n=5), and other end-stage liver diseases(n=6), who underwent liver transplant during the first six months of 2007 were utilized for retrospective analysis. Serum samples were also collected from a group of healthy volunteers (n=7). The samples were analysed using Luminex technology or ELISA for 27 biomarkers that are known to be involved in patho- logic processes such as cell death, regeneration and fibrosis. Results: Of the 27 serum markers examined, 16 were elevated in the serum in all groups with end-stage liver diseases compared with the control group. They include adipokines, apoptosis and inflammatory mediators and growth factors. In- terestingly, the serum of NAFLD patients showed significantly elevated HGF levels and trend towards increase in sFAS, TGF 1, TNFR-1, TNFR-2 and leptin. The level of serum markers showed excellent correlation with each other indicating a complex interdependent pathogenetic mechanism. Conclusions: The data from this study indicate that a large number of serum markers are altered in end-stage liver dis- eases. A panel of such markers may potentially be useful in assessing advanced fibrosis and cirrhosis in patients with chronic end stage liver diseases.

ERCC1 and XPF mediated upregulation of Fibronectin (FN) in Diabetes.

ERCC1 and XPF mediated upregulation of Fibronectin (FN) in Diabetes. BIJU GEORGE*, SHALI CHEN, BIAO FENG, JANE CHIU AND SUBRATA CHAKRABARTI. Dept. of Pathology, Univ. of Western Ontario, London, ON. Excision Repair Cross Complimenting1 (ERCC-1) and Xeroderma Pigmentosum group F (XPF) are important DNA repair enzymes. They play important roles in aging, glucose metabolism and oxidative stress and demonstrate interactive relationships with Poly (ADP-Ribose) polymerase (PARP), another DNA repair protein, upregulated in diabetes. We studied the role of ERCC1 and XPF in glucose induced FN production. Human Umbilical Vein endothelial cells were grown in low (5mM) and high glucose (25mM) containing media. They were also exposed to siRNA against ERCC1 and XPF, histone deacetylase blocker TSA, PARP blocker ABA and p300 blocker Curcumin. Quantitative RT-PCR were performed for ERCC1, XPF, FN, PARP and p300 mRNA. High glucose led to upregulation of ERCC1, XPF, FN, PAPR, p300 mRNA levels compared to the cells in low glucose. ERCC1 and XPF silencing reduced such upregulation. ABA and curcumin also reduced both basal and glucose induced ERCC1 and XPF expression. TSA however, only prevented glucose induced ERCC1 and XPF upregulation. Western blots of ERCC1 and XPF from the nuclear proteins showed similar changes. Data from this study indicated that glucose induced upregulation of ERCC1 and XPF may lead to increased FN production via PARP, P300 pathway. These novel mechanisms may play important roles in diabetic complications. These studies were supported in part by CDA and CIHR 116

Glucose-induced Alteration of Endothelial microRNA

ERCC1 and XPF mediated upregulation of Fibronectin (FN) in Diabetes. BIJU GEORGE*, SHALI CHEN, BIAO FENG, JANE CHIU AND SUBRATA CHAKRABARTI. Dept. of Pathology, Univ. of Western Ontario, London, ON. Excision Repair Cross Complimenting1 (ERCC-1) and Xeroderma Pigmentosum group F (XPF) are important DNA repair enzymes. They play important roles in aging, glucose metabolism and oxidative stress and demonstrate interactive relationships with Poly (ADP-Ribose) polymerase (PARP), another DNA repair protein, upregulated in diabetes. We studied the role of ERCC1 and XPF in glucose induced FN production. Human Umbilical Vein endothelial cells were grown in low (5mM) and high glucose (25mM) containing media. They were also exposed to siRNA against ERCC1 and XPF, histone deacetylase blocker TSA, PARP blocker ABA and p300 blocker Curcumin. Quantitative RT-PCR were performed for ERCC1, XPF, FN, PARP and p300 mRNA. High glucose led to upregulation of ERCC1, XPF, FN, PAPR, p300 mRNA levels compared to the cells in low glucose. ERCC1 and XPF silencing reduced such upregulation. ABA and curcumin also reduced both basal and glucose induced ERCC1 and XPF expression. TSA however, only prevented glucose induced ERCC1 and XPF upregulation. Western blots of ERCC1 and XPF from the nuclear proteins showed similar changes. Data from this study indicated that glucose induced upregulation of ERCC1 and XPF may lead to increased FN production via PARP, P300 pathway. These novel mechanisms may play important roles in diabetic complications. These studies were supported in part by CDA and CIHR 116