Vajir Malek

Histone H2AK119 and H2BK120 mono-ubiquitination modulate SET7/9 and SUV39H1 in type 1 diabetes-induced renal fibrosis.

Hyperglycaemia-induced expression of extracellular matrix (ECM) components plays a major role in the development of diabetic nephropathy (DN). The epigenetic mechanisms that modulate ECM gene expression in DN remain unclear. Therefore, we examined the role of histone H2A and H2B monoubiquitination on epigenetic chromatin marks, such as histone H3 lysine dimethylation (H3K4Me2, H3K9Me2 and H3K79Me2) in type 1 diabetic rat kidney. Hyperglycaemia increased collagen deposition and Col1a1 gene expression. In whole kidney of diabetic animals, both H2AK119 mono-ubiquitination (H2AK119Ub) and H2BK120 mono-ubiquitination (H2BK120Ub) were found to be increased, whereas, in glomeruli of diabetic animals, expression of both H2AK119Ub and H2BK120Ub was reduced. Changes in ubiquitin proteasome system components like increased Rnf2 (H2A-specific E3 ligase) and decreased H2A- and H2B-specific deubiquitinases (ubiquitin-specific proteases 7, 16, 21 and 22) were also observed. Globally increased levels of chromatin marks associated with active genes (H3K4Me2 and H3K79Me2) and decreased levels of repressive marks (H3K9Me2) were also observed. Hyperglycaemia also increased the protein expression of SET7/9 and decreased the expression of SUV39H1. We also showed the decreased occupancy of H2AK119Ub and H2BK120Ub on the promoters of Set7/9 and Suv39h1 in diabetic kidney. In addition, methylation marks regulated by H2AK119Ub (H3K27Me2 and H3K36Me2) and H2BK120Ub (H3K4Me2 and H3K79Me2) were also found to be altered on the promoters of Set7/9 and Suv39h1 Taken together, these results show the functional role of H2AK119Ub and H2BK120Ub in regulating histone H3K4Me2 and H3K9Me2 through modulating the expression of SET7/9 and SUV39H1 in the development of diabetic renal fibrosis.

Differential regulation of angiotensin converting enzyme 2 and nuclear factor-κB by angiotensin II receptor subtypes in type 2 diabetic kidney.

Angiotensin II (Ang II) acts through Angiotensin Converting Enzyme (ACE)/Ang II type 1 receptor (AT1R) axis to promote renal failure whereas the Ang II type 2 receptor (AT2R)/Angiotensin Converting Enzyme 2 (ACE2)/Ang1-7/Mas axis constitutes the protective arm of Renin Angiotensin System (RAS). Though Ang II has been known to activate the Nuclear Factor-κB (NF-κB) signalling pathway through different receptor subtype(s) in different tissues under various diseases, the subtype orchestrating this stimulation in type 2 diabetic kidney remains elusive. ACE2, a protective monocarboxypeptidase, responsible for conversion of Ang II to Ang1-7, opposes the deleterious effects of RAS pathway but how its expression is altered with blockade of AT1R and AT2R is not yet known. Hence, the present study was conceived to understand the regulation of NF-κB and ACE2 by using specific AT1 and AT2 receptor antagonists in non-genetic model of type 2 diabetic nephropathy. Our results show that the AT1R and AT2R antagonists lead to the repression and activation of NF-κB signalling pathway, respectively which suggests the role of AT1R in NF-κB activation. The blockade of AT2R led to an increase in ACE2 expression, which may be a compensatory response to the drastically increased inflammatory mediators and oxidative stress in the diabetic kidney. To the best of our knowledge, this is the first study showing the differential regulation of NF-κB and ACE2 by Ang II receptor subtypes and thus this study improves our understanding regarding regulation of inflammatory cascade and ACE2 by AT1R and AT2R in type 2 diabetic kidney, which may help in designing novel strategies to combat the disease in future.

Insulin sensitizing and cardioprotective effects of Esculetin and Telmisartan combination by attenuating Ang II mediated vascular reactivity and cardiac fibrosis.

The combination of the angiotensin receptor blockers (ARBs) with other synthetic and natural molecules has been reported to have better safety profile and therapeutic efficacy in prevention of diabetes and its associated complications than their monotherapy. Driven by the aforementioned facts, this study was conceived to evaluate the potential additive effect of combination of Telmisartan and Esculetin in prevention of insulin resistance and associated cardiac fibrosis. Recently, we have reported that Esculetin prevented cardiovascular dysfunction associated with insulin resistance (IR) and type 2 diabetes. Insulin resistance was developed by high fat diet (HFD) feeding to Wistar rats. Telmisartan and Esculetin were administered at 10 mg/kg/day and 50 mg/kg/day doses (P.O, 2 weeks), respectively either alone or in combination. Plasma biochemical analyses, vascular reactivity and immunohistochemical experiments were performed to assess the beneficial effect of Telmisartan, Esculetin and their combination on insulin resistance and associated cardiac fibrosis. The study results showed that, co-administered Telmisartan and Esculetin ameliorated the pathological features like metabolic perturbation, morphometric alterations, vascular hyper responsiveness, extracellular matrix accumulation and the expression of fibronectin and TGF-β more effectively than monotherapy in HFD fed rats. Hence, the study urges us to conclude that the solution to IR and associated cardiovascular dysfunction may lie in the Telmisartan and Esculetin combination therapy.

Neprilysin inhibitors: A new hope to halt the diabetic cardiovascular and renal complications?

Diabetes is an enormous and ever-growing calamity and a global public health threat of the 21st century. Besides insulin and oral hypoglycaemic drugs, blockage of the renin-angiotensin system (RAS) denotes a key pharmacotherapy for the management of cardiovascular (CVD) and chronic kidney diseases (CKD), which are the leading causes of disability and death among diabetic patients. Neprilysin (NEP) inhibition, auxiliary to RAS blockage increases the bioavailability of natriuretic peptides and benefits the cardio-renal system. Omapatrilat, a dual angiotensin-converting enzyme (ACE) and NEP inhibitor has been reported to show superior anti-hypertensive, anti-atherosclerotic, insulin-sensitizing, cardiovascular and renoprotective effects to ACE inhibitors in experimental animal models for diabetes. In clinical trials on hypertensive subjects Omapatrilat increased the risk of angioedema due to which its further development as anti-hypertensive drug was hampered. This event prompted the development of angiotensin receptor neprilysin inhibitors (ARNi). The first representative of ARNi, LCZ696 (Sacubitril/ Valsartan) halted cardiovascular and renal functional decline and hence protected against CKD and CVD. Recently, LCZ696 was approved by U.S. Food and Drug Administration for the treatment of heart failure. This concise review intends to summarise the currently available reports on NEPi as a therapeutic intervention to treat CVD and CKD associated with diabetes.

Diminazene aceturate prevents nephropathy by increasing glomerular ACE2 and AT2 receptor expression in a rat model of type1 diabetes

One of the protective actions of angiotensin converting enzyme‐2 (ACE2) is the inactivation of angiotensin II. Expression and activity of ACE2 was reduced in glomeruli of diabetic patients and in animal models of diabetes. Recently the potential role of recombinant ACE2 administration in preventing diabetic nephropathy (DN) has been shown. Here we have tested the effects of the ACE2 activator, diminazene aceturate (DIZE), in a model of DN.Background and Purpose Angiotensin converting enzyme 2 (ACE2) shows its protective actions through degrading Ang II. ACE2 activity and its expression was found to be reduced in glomeruli of diabetic patients as well as in animal models of diabetes. Several recent studies have also showed the potential role of recombinant ACE2 administration in preventing diabetic nephropathy (DN). Hence, we hypothesized that ACE2 activator, diminazene aceturate (DIZE) may be beneficial in preventing DN. Experimental Approach Male Wistar rats were rendered diabetic using a single dose of Streptozotocin (55mg kg-1, i.p.). After 4 weeks, diabetic animals were grouped in to respective groups and the drug, DIZE, low dose (5mg kg-1day-1), high dose (15mg kg-1day-1) and high dose in presence of AT2 blocker (PD123319, 10mg kg-1day-1) was administered. At the end, kidneys from all the groups were collected and processed separately for glomerular isolation, protein isolation, mRNA extraction and for immunohistochemical studies. Key Results DIZE administration restored the ACE2 expression in glomeruli and increased the AT2 expression in whole kidney and isolated glomeruli of diabetic animals. DIZE administration, reduced the Ang II levels and increased Ang 1-7 levels in diabetic kidney. However, AT2 blockade, reversed all these actions of DIZE. Conclusion and Implications DIZE treatment reduced the diabetes induced renal damage through inhibiting fibrosis and apotosis. Moreover, DIZE failed to show its protective actions in presence of AT2 blocker. Taken together, these results suggest the protective role of DIZE in preventing DN through ACE2/Ang 1-7/AT2 axis.

Diminazene aceturate prevents type 1 diabetic nephropathy through increasing glomerular ACE2 and AT2 receptor expression

One of the protective actions of angiotensin converting enzyme‐2 (ACE2) is the inactivation of angiotensin II. Expression and activity of ACE2 was reduced in glomeruli of diabetic patients and in animal models of diabetes. Recently the potential role of recombinant ACE2 administration in preventing diabetic nephropathy (DN) has been shown. Here we have tested the effects of the ACE2 activator, diminazene aceturate (DIZE), in a model of DN.Background and Purpose Angiotensin converting enzyme 2 (ACE2) shows its protective actions through degrading Ang II. ACE2 activity and its expression was found to be reduced in glomeruli of diabetic patients as well as in animal models of diabetes. Several recent studies have also showed the potential role of recombinant ACE2 administration in preventing diabetic nephropathy (DN). Hence, we hypothesized that ACE2 activator, diminazene aceturate (DIZE) may be beneficial in preventing DN. Experimental Approach Male Wistar rats were rendered diabetic using a single dose of Streptozotocin (55mg kg-1, i.p.). After 4 weeks, diabetic animals were grouped in to respective groups and the drug, DIZE, low dose (5mg kg-1day-1), high dose (15mg kg-1day-1) and high dose in presence of AT2 blocker (PD123319, 10mg kg-1day-1) was administered. At the end, kidneys from all the groups were collected and processed separately for glomerular isolation, protein isolation, mRNA extraction and for immunohistochemical studies. Key Results DIZE administration restored the ACE2 expression in glomeruli and increased the AT2 expression in whole kidney and isolated glomeruli of diabetic animals. DIZE administration, reduced the Ang II levels and increased Ang 1-7 levels in diabetic kidney. However, AT2 blockade, reversed all these actions of DIZE. Conclusion and Implications DIZE treatment reduced the diabetes induced renal damage through inhibiting fibrosis and apotosis. Moreover, DIZE failed to show its protective actions in presence of AT2 blocker. Taken together, these results suggest the protective role of DIZE in preventing DN through ACE2/Ang 1-7/AT2 axis.

Telmisartan and thiorphan combination treatment attenuates fibrosis and apoptosis in preventing diabetic cardiomyopathy

Aims LCZ696, a first-generation dual angiotensin receptor-neprilysin inhibitor (ARNi), is effective in treating heart failure patients. However, the role of ARNis in treating diabetic cardiomyopathy is poorly understood. This study evaluates the efficacy of a novel combination of telmisartan [angiotensin receptor blocker (ARB)] and thiorphan [neprilysin inhibitor (NEPi)] in ameliorating diabetic cardiomyopathy while, at the same time, exploring the relevant underlying molecular mechanism(s). Methods and results Diabetes was induced by administration of streptozotocin (55 mg/kg, i.p.) in male Wistar rats. After 4 weeks, diabetic rats were subjected to either thiorphan (0.1 mg/kg/day, p.o.) or telmisartan (10 mg/kg/day, p.o.) monotherapy, or their combination, for a period of 4 weeks. Metabolic and morphometric alterations, failing ventricular functions, and diminished baroreflex indicated development of diabetic cardiac complications. Apart from morphometric alterations, all pathological consequences were prevented by telmisartan and thiorphan combination therapy. Diabetic rats exhibited significant modulation of the natriuretic peptide system, a key haemodynamic regulator; this was normalized by combination therapy. Histopathological studies showed augmented myocardial fibrosis, demonstrated by increased % PSR-positive area, with combination therapy giving the best improvement in these indices. More importantly, the combination of thiorphan and telmisartan was superior in attenuating inflammatory (NF-κB/MCP-1), profibrotic (TGF-β/Smad7) and apoptotic (PARP/Caspase-3) cascades compared to respective monotherapies when treating rats with diabetic cardiomyopathy. In addition, diabetic heart chromatin was in a state of active transcription, indicated by increased histone acetylation (H2AK5Ac, H2BK5Ac, H3K9Ac, and H4K8Ac) and histone acetyltransferase (PCAF and Ac-CBP) levels. Interestingly, combination treatment was sufficiently potent to normalize these alterations. Conclusion The protective effect of novel ARB and NEPi combination against diabetic cardiomyopathy can be attributed to inhibition of inflammatory, profibrotic, and apoptotic cascades. Moreover, reversal of histone acetylation assists its protective effect.

Telmisartan and esculetin combination ameliorates type 2 diabetic cardiomyopathy by reversal of H3, H2A, and H2B histone modifications

OBJECTIVES: Although cardioprotective effects of telmisartan are well explored, its effects on epigenetic alterations associated with type 2 diabetic (T2D) cardiomyopathy remain unmapped. Thus, the present study was designed to evaluate the potential of esculetin and telmisartan combination to reverse histone posttranslational modifications (PTMs) in curbing T2D cardiomyopathy. MATERIALS AND METHODS: T2D was induced by high-fat diet feeding along with low dose of streptozotocin (35 mg/kg, I.P) in male Wistar rats. T2D rats were treated with either telmisartan (10 mg/kg/day, P.O) or esculetin (50 mg/kg/day doses, P.O) or their combination for 2 weeks. Biochemical estimations, vascular reactivity, immunohistochemistry, and western blotting experiments were performed to evaluate the effects of the treatment in T2D cardiomyopathy. RESULTS: Esculetin and telmisartan combination alleviated the pathological features of T2D cardiomyopathy including metabolic perturbations, morphometric alterations, altered vascular reactivity, increased Keap1 and fibronectin expression more effectively than their respective monotherapy. This is the first report showing that telmisartan attenuates increased level of histone PTMs such as H3K9me2, H3K9Ac, H2AK119Ub, and H2BK120Ub in heart of T2D rats. The combination regimen showed a more significant reduction in augmented histone PTMs associated with T2D cardiomyopathy than their independent treatments. CONCLUSIONS: The present study demonstrates that esculetin and telmisartan combination can be an advanced pharmacological approach to ameliorate T2D cardiomyopathy which could be partially attributed to its ability to reverse the epigenetic alterations.

Esculetin ameliorates vascular perturbation by intervening in the occupancy of H2BK120Ub at At1, At2, Tgfβ1 and Mcp1 promoter gene in thoracic aorta of IR and T2D rats

Micro and macro vascular complications under diabetic condition are the responses to pathological stimuli exerted by up regulated renin angiotensin system (RAS) via deteriorating vascular physiology. Up-regulated RAS could influence in the adaptive mechanisms of target tissues to alter the abundance of angiotensin II type 1 receptor (AT1) and angiotensin II type 2 receptor (AT2). Such differential regulation of AT1 and AT2 have been reported to be associated with post-translational histone modifications (PTHMs). Additionally, recent evidences provide, esculetin (6,7-dihydroxycoumarin) reverses post-translational histone modifications (PTHMs) in diabetic cardiomyopathy and nephropathy. On account of these evidences, we further pursued this study to investigate the effects of esculetin on PTHMs in progressive vascular complications under insulin resistance (IR) and type 2 diabetic (T2D) conditions. Esculetin treatment in both IR and T2D conditions substantially improved vascular reactivity, increased eNos and decreased Vcam1 mRNA levels, and reduced collagen deposition in rat thoracic aorta. Further, the fold changes in At1 and At2 receptor mRNA in IR and T2D were reversed by esculetin treatment. Modifications in histone H2B lysine 120 monoubiquitination (H2BK120Ub) were also reversed in esculetin treatment group. Further, modification in the occupancy of H2BK120Ub at At1a, At2, Tgfβ and Mcp1 promoter genes was evidenced by treatment with esculetin. Taken together, our investigation concluded with the involvement of esculetin in the amelioration of vascular perturbation by modifying H2BK120Ub along with occupancy at At1, At2, Tgfβ1 and Mcp1 promoter gene.