High glucose induces Smad activation via the transcriptional coregulator p300 and contributes to cardiac fibrosis and hypertrophy.

BACKGROUND: Despite advances in the treatment of heart failure, mortality remains high, particularly in individuals with diabetes. Activated transforming growth factor beta (TGF-ß) contributes to the pathogenesis of the fibrotic interstitium observed in diabetic cardiomyopathy. We hypothesized that high glucose enhances the activity of the transcriptional co-activator p300, leading to the activation of TGF-ß via acetylation of Smad2; and that by inhibiting p300, TGF-ß activity will be reduced and heart failure prevented in a clinically relevant animal model of diabetic cardiomyopathy. METHODS: p300 activity was assessed in H9c2 cardiomyoblasts under normal glucose (5.6 mmol/L-NG) and high glucose (25 mmol/L-HG) conditions. 3H-proline incorporation in cardiac fibroblasts was also assessed as a marker of collagen synthesis. The role of p300 activity in modifying TGF-ß activity was investigated with a known p300 inhibitor, curcumin or p300 siRNA in vitro, and the functional effects of p300 inhibition were assessed using curcumin in a hemodynamically validated model of diabetic cardiomyopathy - the diabetic TG m(Ren-2)27 rat. RESULTS: In vitro, H9c2 cells exposed to HG demonstrated increased p300 activity, Smad2 acetylation and increased TGF-ß activity as assessed by Smad7 induction (all p < 0.05 c/w NG). Furthermore, HG induced 3H-proline incorporation as a marker of collagen synthesis (p < 0.05 c/w NG). p300 inhibition, using either siRNA or curcumin reduced p300 activity, Smad acetylation and TGF-ß activity (all p < 0.05 c/w vehicle or scrambled siRNA). Furthermore, curcumin therapy reduced 3H-proline incorporation in HG and TGF-ß stimulated fibroblasts (p < 0.05 c/w NG). To determine the functional significance of p300 inhibition, diabetic Ren-2 rats were randomized to receive curcumin or vehicle for 6 weeks. Curcumin treatment reduced cardiac hypertrophy, improved diastolic function and reduced extracellular matrix production, without affecting glycemic control, along with a reduction in TGF-ß activity as assessed by Smad7 activation (all p < 0.05 c/w vehicle treated diabetic animals). CONCLUSIONS: These findings suggest that high glucose increases the activity of the transcriptional co-regulator p300, which increases TGF-ß activity via Smad2 acetylation. Modulation of p300 may be a novel strategy to treat diabetes induced heart failure.

The Goto Kakizaki rat: Impact of age upon changes in cardiac and renal structure, function.

BACKGROUND: Patients with diabetes are at a high risk for developing cardiac dysfunction in the absence of coronary artery disease or hypertension, a condition known as diabetic cardiomyopathy. Contributing to heart failure is the presence of diabetic kidney disease. The Goto-Kakizaki (GK) rat is a non-obese, non-hypertensive model of type 2 diabetes that, like humans, shares a susceptibility locus on chromosome 10. Herein, we perform a detailed analysis of cardio-renal remodeling and response to renin angiotensin system blockade in GK rats to ascertain the validity of this model for further insights into disease pathogenesis. METHODS: Study 1: Male GK rats along with age matched Wistar control animals underwent longitudinal assessment of cardiac and renal function for 32 weeks (total age 48 weeks). Animals underwent regular echocardiography every 4 weeks and at sacrifice, early (~24 weeks) and late (~48 weeks) timepoints, along with pressure volume loop analysis. Histological and molecular characteristics were determined using standard techniques. Study 2: the effect of renin angiotensin system (RAS) blockade upon cardiac and renal function was assessed in GK rats. Finally, proteomic studies were conducted in vivo and in vitro to identify novel pathways involved in remodeling responses. RESULTS: GK rats developed hyperglycaemia by 12 weeks of age (p<0.01 c/w Wistar controls). Echocardiographic assessment of cardiac function demonstrated preserved systolic function by 48 weeks of age. Invasive studies demonstrated left ventricular hypertrophy, pulmonary congestion and impaired diastolic function. Renal function was preserved with evidence of hyperfiltration. Cardiac histological analysis demonstrated myocyte hypertrophy (p<0.05) with evidence of significant interstitial fibrosis (p<0.05). RT qPCR demonstrated activation of the fetal gene program, consistent with cellular hypertrophy. RAS blockade resulted in a reduction blood pressure(P<0.05) cardiac interstitial fibrosis (p<0.05) and activation of fetal gene program. No significant change on either systolic or diastolic function was observed, along with minimal impact upon renal structure or function. Proteomic studies demonstrated significant changes in proteins involved in oxidative phosp4horylation, mitochondrial dysfunction, beta-oxidation, and PI3K/Akt signalling (all p<0.05). Further, similar changes were observed in both LV samples from GK rats and H9C2 cells incubated in high glucose media. CONCLUSION: By 48 weeks of age, the diabetic GK rat demonstrates evidence of preserved systolic function and impaired relaxation, along with cardiac hypertrophy, in the presence of hyperfiltration and elevated protein excretion. These findings suggest the GK rat demonstrates some, but not all features of diabetes induced "cardiorenal" syndrome. This has implications for the use of this model to assess preclinical strategies to treat cardiorenal disease.

BReast CAncer susceptibility gene 2 deficiency exacerbates oxidized LDL-induced DNA damage and endothelial apoptosis.

Mutations in the tumor suppressor gene BRCA2 (BReast CAncer susceptibility gene 2) predispose carriers to breast, ovarian, and other cancers. In response to DNA damage, BRCA2 participates in homology-directed DNA damage repair to maintain genome stability. Genome-wide association studies have identified an association between BRCA2 single nucleotide polymorphisms and plasma-lipid levels and lipid deregulation in humans. To date, DNA damage, apoptosis, and lipid deregulation are recognized as central pathways for endothelial dysfunction and atherosclerosis; however, the role of BRCA2 in endothelial dysfunction remains to be elucidated. To determine the role of BRCA2 in endothelial dysfunction, BRCA2 was silenced in human umbilical vein endothelial cells (ECs) and assessed for markers of DNA damage, apoptosis, and endothelial function following oxidized low-density lipoprotein (oxLDL) treatment. OxLDL was found to induce significant reactive oxygen species (ROS) production in BRCA2-silenced ECs. This increase in ROS production was associated with exacerbated DNA damage evidenced by increased expression and activation of DNA double-stranded break (DSB) marker γH2AX and reduced RAD51-foci formation-an essential regulator of DSB repair. Increased DSBs were associated with enhanced expression and activation of pro-apoptotic p53 and significant apoptosis in oxLDL-treated BRCA2-silenced ECs. Loss of BRCA2 in ECs was further associated with oxLDL-induced impaired tube-forming potential and eNOS expression. Collectively, the data reveals, for the first time, a novel role of BRCA2 as a regulator of EC survival and function in the setting of oxLDL treatment in vitro. Additionally, the data provide important clues regarding the potential susceptibility of BRCA2 mutation carriers to endothelial dysfunction, atherosclerosis, and other cardiovascular diseases.

Endothelial-specific Loss of IFT88 Promotes Endothelial-to-Mesenchymal Transition and Exacerbates Bleomycin-induced Pulmonary Fibrosis.

Intraflagellar transport protein 88 (Ift88) is required for ciliogenesis and shear stress-induced dissolution of cilia in embryonic endothelial cells coincides with endothelial-to-mesenchymal transition (EndMT) in the developing heart. EndMT is also suggested to underlie heart and lung fibrosis, however, the mechanism linking endothelial Ift88, its effect on EndMT and organ fibrosis remains mainly unexplored. We silenced Ift88 in endothelial cells (ECs) in vitro and generated endothelial cell-specific Ift88-knockout mice (Ift88endo) in vivo to evaluate EndMT and its contribution towards organ fibrosis, respectively. Ift88-silencing in ECs led to mesenchymal cells-like changes in endothelial cells. The expression level of the endothelial markers (CD31, Tie-2 and VE-cadherin) were significantly reduced with a concomitant increase in the expression level of mesenchymal markers (αSMA, N-Cadherin and FSP-1) in Ift88-silenced ECs. Increased EndMT was associated with increased expression of profibrotic Collagen I expression and increased proliferation in Ift88-silenced ECs. Loss of Ift88 in ECs was further associated with increased expression of Sonic Hedgehog signaling effectors. In vivo, endothelial cells isolated from the heart and lung of Ift88endo mice demonstrated loss of Ift88 expression in the endothelium. The Ift88endo mice were born in expected Mendelian ratios without any adverse cardiac phenotypes at baseline. Cardiac and pulmonary endothelial cells isolated from the Ift88endo mice demonstrated signs of EndMT and bleomycin treatment exacerbated pulmonary fibrosis in Ift88endo mice. Pressure overload stress in the form of aortic banding did not reveal a significant difference in cardiac fibrosis between Ift88endo mice and control mice. Our findings demonstrate a novel association between endothelial cilia with EndMT and cell proliferation and also show that loss of endothelial cilia-associated increase in EndMT contributes specifically towards pulmonary fibrosis.

Heart Failure With Preserved Ejection Fraction in Diabetes: Mechanisms and Management.

Diabetes mellitus (DM) is a major cause of heart failure in the Western world, either secondary to coronary artery disease or from a distinct entity known as "diabetic cardiomyopathy." Furthermore, heart failure with preserved ejection fraction (HFpEF) is emerging as a significant clinical problem for patients with DM. Current clinical data suggest that between 30% and 40% of patients with HFpEF suffer from DM. The typical structural phenotype of the HFpEF heart consists of endothelial dysfunction, increased interstitial and perivascular fibrosis, cardiomyocyte stiffness, and hypertrophy along with advanced glycation end products deposition. There is a myriad of mechanisms that result in the phenotypical HFpEF heart including impaired cardiac metabolism and substrate utilization, altered insulin signalling leading to protein kinase C activation, advanced glycated end products deposition, prosclerotic cytokine activation (eg, transforming growth factor-ß activation), along with impaired nitric oxide production from the endothelium. Moreover, recent investigations have focused on the role of endothelial-myocyte interactions. Despite intense research, current therapeutic strategies have had little effect on improving morbidity and mortality in patients with DM and HFpEF. Possible explanations for this include a limited understanding of the role that direct cell-cell communication or indirect cell-cell paracrine signalling plays in the pathogenesis of DM and HFpEF. Additionally, integrins remain another important mediator of signals from the extracellular matrix to cells within the failing heart and might play a significant role in cell-cell cross-talk. In this review we discuss the characteristics and mechanisms of DM and HFpEF to stimulate potential future research for patients with this common, and morbid condition.

Valproic Acid Induces Endothelial-to-Mesenchymal Transition-Like Phenotypic Switching.

Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, is a widely used anticonvulsant drug that is currently undergoing clinical evaluation for anticancer therapy due to its anti-angiogenic potential. Endothelial cells (ECs) can transition into mesenchymal cells and this form of EC plasticity is called endothelial-to-mesenchymal transition (EndMT), which is widely implicated in several pathologies including cancer and organ fibrosis. However, the effect of VPA on EC plasticity and EndMT remains completely unknown. We report herein that VPA-treatment significantly inhibits tube formation, migration, nitric oxide production, proliferation and migration in ECs. A microscopic evaluation revealed, and qPCR, immunofluorescence and immunoblotting data confirmed EndMT-like phenotypic switching as well as an increased expression of pro-fibrotic genes in VPA-treated ECs. Furthermore, our data confirmed important and regulatory role played by TGFß-signaling in VPA-induced EndMT. Our qPCR array data performed for 84 endothelial genes further supported our findings and demonstrated 28 significantly and differentially regulated genes mainly implicated in angiogenesis, endothelial function, EndMT and fibrosis. We, for the first time report that VPA-treatment associated EndMT contributes to the VPA-associated loss of endothelial function. Our data also suggest that VPA based therapeutics may exacerbate endothelial dysfunction and EndMT-related phenotype in patients undergoing anticonvulsant or anticancer therapy, warranting further investigation.

Sirtuin 1 activation attenuates cardiac fibrosis in a rodent pressure overload model by modifying Smad2/3 transactivation.

Aims: Transforming growth factor ß1 (TGF-ß1) is a prosclerotic cytokine involved in cardiac remodelling leading to heart failure (HF). Acetylation/de-acetylation of specific lysine residues in Smad2/3 has been shown to regulate TGF-ß signalling by altering its transcriptional activity. Recently, the lysine de-acetylase sirtuin 1 (SIRT1) has been shown to have a cardioprotective effect; however, SIRT1 expression and activity are paradoxically reduced in HF. Herein, we investigate whether pharmacological activation of SIRT1 would induce cardioprotection in a pressure overload model and assess the impact of SIRT1 activation on TGF-ß signalling and the fibrotic response. Methods and results: Eight weeks old male C57BL/6 mice were randomized to undergo sham surgery or transverse aortic constriction (TAC) to induce pressure overload. Post-surgery, animals were further randomized to receive SRT1720 or vehicle treatment. Echocardiography, pressure-volume loops, and histological analysis revealed an impairment in cardiac function and deleterious left ventricular remodelling in TAC-operated animals that was improved with SRT1720 treatment. Genetic ablation and cell culture studies using a Smad-binding response element revealed SIRT1 to be a specific target of SRT1720 and identified Smad2/3 as a SIRT1 specific substrate. Conclusion: Overall, our data demonstrate that Smad2/3 is a specific SIRT1 target and suggests that pharmacological activation of SIRT1 may be a novel therapeutic strategy to prevent/reverse HF via modifying Smad activity.

Suppression of NLRP3 Inflammasome Activation Ameliorates Chronic Kidney Disease-Induced Cardiac Fibrosis and Diastolic Dysfunction.

Cardiac fibrosis is a common finding in patients with chronic kidney disease. Here, we investigate the cardio-renal effects of theracurmin, a novel formulation of the polyphenolic compound curcumin, in a rat model of chronic kidney disease. Briefly, Sprague-Dawley rats were randomized to undergo sham or subtotal nephrectomy (SNx) surgery. At 3 weeks post surgery, SNx animals were further randomized to received theracurmin via once daily oral gavage or vehicle for 5 consecutive weeks. At 8 weeks post surgery, cardiac function was assessed via echocardiography and pressure volume loop analysis, followed by LV and renal tissue collection for analysis. SNx animals developed key hallmarks of renal injury including hypertension, proteinuria, elevated blood urea nitrogen, and glomerulosclerosis. Renal injury in SNx animals was also associated with significant diastolic dysfunction, macrophage infiltration, and cardiac NLRP3 inflammasome activation. Treatment of SNx animals with theracurmin improved structural and functional manifestations of cardiac injury associated with renal failure and also attenuated cardiac NLRP3 inflammasome activation and mature IL-1ß release. Taken together, our findings suggest a significant role for the NLRP3 inflammasome in renal injury-induced cardiac dysfunction and presents inflammasome attenuation as a unique strategy to prevent adverse cardiac remodeling in the setting of chronic kidney disease.

Substoichiometric inhibition of transthyretin misfolding by immune-targeting sparsely populated misfolding intermediates: a potential diagnostic and therapeutic for TTR amyloidoses.

Wild-type and mutant transthyretin (TTR) can misfold and deposit in the heart, peripheral nerves, and other sites causing amyloid disease. Pharmacological chaperones, Tafamidis(®) and diflunisal, inhibit TTR misfolding by stabilizing native tetrameric TTR; however, their minimal effective concentration is in the micromolar range. By immune-targeting sparsely populated TTR misfolding intermediates (i.e. monomers), we achieved fibril inhibition at substoichiometric concentrations. We developed an antibody (misTTR) that targets TTR residues 89-97, an epitope buried in the tetramer but exposed in the monomer. Nanomolar misTTR inhibits fibrillogenesis of misfolded TTR under micromolar concentrations. Pan-specific TTR antibodies do not possess such fibril inhibiting properties. We show that selective targeting of misfolding intermediates is an alternative to native state stabilization and requires substoichiometric concentrations. MisTTR or its derivative may have both diagnostic and therapeutic potential.

Cardiovascular effects of incretins in diabetes.

Recent years have seen an enormous increase in the number of therapeutic agents available for lowering blood glucose levels in people with type 2 diabetes. Among these agents, the incretin mimetics glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists and dipeptidyl peptidase 4 (DPP4) inhibitors have received particular attention for the potential of these interventions to positively impact on cardiovascular outcomes. Although the results of large-scale cardiovascular outcome trials eagerly are anticipated, an increasing body of literature from preclinical and early phase clinical studies has indicated that both GLP-1R agonists and DPP4 inhibitors may exert glucose-independent cardiovascular effects. Despite its role in glucose homeostasis, the GLP-1R is surprisingly widely distributed throughout the body, including in the heart. GLP-1 may exert its effects through both receptor-dependent and receptor-independent mechanisms and through the actions of both the intact peptide and its metabolites. In addition, DPP4 inhibition not only augments the circulating levels of incretin hormones, but it also holds the capacity to augment the activity of other biologically important substrates, most notably the small protein stromal cell-derived factor 1 alpha. Whether these collective functions will act to reduce cardiovascular events in patients remains to be determined.