Marpadga A. Reddy

Epigenetic histone H3 lysine 9 methylation in metabolic memory and inflammatory phenotype of vascular smooth muscle cells in diabetes

Diabetic patients continue to develop inflammation and vascular complications even after achieving glycemic control. This poorly understood “metabolic memory” phenomenon poses major challenges in treating diabetes. Recent studies demonstrate a link between epigenetic changes such as chromatin histone lysine methylation and gene expression. We hypothesized that H3 lysine-9 tri-methylation (H3K9me3), a key repressive and relatively stable epigenetic chromatin mark, may be involved in metabolic memory. This was tested in vascular smooth muscle cells (VSMC) derived from type 2 diabetic db/db mice. These cells exhibit a persistent atherogenic and inflammatory phenotype even after culture in vitro. ChIP assays showed that H3K9me3 levels were significantly decreased at the promoters of key inflammatory genes in cultured db/db VSMC relative to control db/+ cells. Immunoblotting demonstrated that protein levels of the H3K9me3 methyltransferase Suv39h1 were also reduced in db/db VSMC. Furthermore, db/db VSMC were hypersensitive to TNF-α inflammatory stimulus, which induced dramatic and sustained decreases in promoter H3K9me3 and Suv39h1 occupancy. Recruitment of corepressor HP1α was also reduced under these conditions in db/db cells. Overexpression of SUV39H1 in db/db VSMC reversed this diabetic phenotype. Conversely, gene silencing of SUV39H1 with shRNAs in normal human VSMC (HVSMC) increased inflammatory genes. HVSMC cultured in high glucose also showed increased inflammatory gene expression and decreased H3K9me3 at their promoters. These results demonstrate protective roles for H3K9me3 and Suv39h1 against the preactivated state of diabetic VSMC. Dysregulation of epigenetic histone modifications may be a major underlying mechanism for metabolic memory and sustained proinflammatory phenotype of diabetic cells.

Role of the Histone H3 Lysine 4 Methyltransferase, SET7/9, in the Regulation of NF-κB-dependent Inflammatory Genes RELEVANCE TO DIABETES AND INFLAMMATION

Nuclear factor κ-B (NF-κB)-regulated inflammatory genes, such as TNF-α (tumor necrosis factor-α), play key roles in the pathogenesis of inflammatory diseases, including diabetes and the metabolic syndrome. However, the nuclear chromatin mechanisms are unclear. We report here that the chromatin histone H3-lysine 4 methyltransferase, SET7/9, is a novel coactivator of NF-κB. Gene silencing of SET7/9 with small interfering RNAs in monocytes significantly inhibited TNF-α-induced inflammatory genes and histone H3-lysine 4 methylation on these promoters, as well as monocyte adhesion to endothelial or smooth muscle cells. Chromatin immunoprecipitation revealed that SET7/9 small interfering RNA could reduce TNF-α-induced recruitment of NF-κB p65 to inflammatory gene promoters. Inflammatory gene induction by ligands of the receptor for advanced glycation end products was also attenuated in SET7/9 knockdown monocytes. In addition, we also observed increased inflammatory gene expression and SET7/9 recruitment in macrophages from diabetic mice. Microarray profiling revealed that, in TNF-α-stimulated monocytes, the induction of 25% NF-κB downstream genes, including the histone H3-lysine 27 demethylase JMJD3, was attenuated by SET7/9 depletion. These results demonstrate a novel role for SET7/9 in inflammation and diabetes.

Key Role of Src Kinase in S100B-induced Activation of the Receptor for Advanced Glycation End Products in Vascular Smooth Muscle Cells

The receptor for advanced glycation end products (RAGE) and its ligands have been implicated in the activation of oxidant stress and inflammatory pathways in vascular smooth muscle cells (VSMCs) leading to the initiation and augmentation of atherosclerosis. Here we report that non-receptor Src tyrosine kinase and the membrane protein caveolin-1 (Cav-1) play a key role in the activation of RAGE by S100B in VSMCs. S100B increased the activation of Src kinase and tyrosine phosphorylation of caveolin-1 in VSMCs. A RAGE-specific antibody blocked both these effects. An inhibitor of Src kinase, PP2, significantly blocked S100B-induced activation of Src kinase, mitogen-activated protein kinases, transcription factors NF-κB and STAT3, superoxide production, tyrosine phosphorylation of Cav-1, VSMC migration, and expression of the pro-inflammatory genes monocyte chemotactic protein-1 and interleukin-6. Cholesterol depletion also inhibited S100B-induced effects indicating the requirement for intact caveolae in RAGE-specific signaling. Nucleofection of either a Src dominant negative mutant, or a Cav-1 mutant lacking the scaffolding domain, or Cav-1 short hairpin RNA significantly reduced S100B-induced inflammatory gene expression in VSMCs. Furthermore, VSMCs derived from insulin-resistant and diabetic db/db mice displayed increased RAGE expression, Src activation, and migration compared with those from control db/+ mice. The RAGE antibody blocked enhanced migration in db/db cells. These studies demonstrate for the first time that, in VSMCs, Src kinase and Cav-1 play important roles in RAGE-mediated inflammatory gene expression and migration, key events associated with diabetic vascular complications.

Enhanced Levels of microRNA-125b in Vascular Smooth Muscle Cells of Diabetic db/db Mice Lead to Increased Inflammatory Gene Expression by Targeting the Histone Methyltransferase Suv39h1

OBJECTIVE Diabetes remains a major risk factor for vascular complications that seem to persist even after achieving glycemic control, possibly due to “metabolic memory.” Using cultured vascular smooth muscle cells (MVSMC) from type 2 diabetic db/db mice, we recently showed that decreased promoter occupancy of the chromatin histone H3 lysine-9 methyltransferase Suv39h1 and the associated repressive epigenetic mark histone H3 lysine-9 trimethylation (H3K9me3) play key roles in sustained inflammatory gene expression. Here we examined the role of microRNAs (miRs) in Suv39h1 regulation and function in MVSMC from diabetic mice. RESEARCH DESIGN AND METHODS We used luciferase assays with Suv39h1 3′untranslated region (UTR) reporter constructs and Western blotting of endogenous protein to verify that miR-125b targets Suv39h1. We examined the effects of Suv39h1 targeting on inflammatory gene expression by quantitative real time polymerase chain reaction (RT-qPCR), and H3K9me3 levels at their promoters by chromatin immunoprecipitation assays. RESULTS We observed significant upregulation of miR-125b with parallel downregulation of Suv39h1 protein (predicted miR-125b target) in MVSMC cultured from diabetic db/db mice relative to control db/+. miR-125b mimics inhibited both Suv39h1 3′UTR luciferase reporter activity and endogenous Suv39h1 protein levels. Conversely, miR-125b inhibitors showed opposite effects. Furthermore, miR-125b mimics increased expression of inflammatory genes, monocyte chemoattractant protein-1, and interleukin-6, and reduced H3K9me3 at their promoters in nondiabetic cells. Interestingly, miR-125b mimics increased monocyte binding to db/+ MVSMC toward that in db/db MVSMC, further imitating the proinflammatory diabetic phenotype. In addition, we found that the increase in miR-125b in db/db VSMC is caused by increased transcription of miR-125b-2. CONCLUSIONS These results demonstrate a novel upstream role for miR-125b in the epigenetic regulation of inflammatory genes in MVSMC of db/db mice through downregulation of Suv39h1.

Epigenetic mechanisms in diabetic vascular complications

There has been a rapid increase in the incidence of diabetes as well the associated vascular complications. Both genetic and environmental factors have been implicated in these pathologies. Increasing evidence suggests that epigenetic factors play a key role in the complex interplay between genes and the environment. Actions of major pathological mediators of diabetes and its complications such as hyperglycaemia, oxidant stress, and inflammatory factors can lead to dysregulated epigenetic mechanisms that affect chromatin structure and gene expression. Furthermore, persistence of this altered state of the epigenome may be the underlying mechanism contributing to a metabolic memory that results in chronic inflammation and vascular dysfunction in diabetes even after achieving glycaemic control. Further examination of epigenetic mechanisms by also taking advantage of recently developed next-generation sequencing technologies can provide novel insights into the pathology of diabetes and its complications and lead to the discovery of much needed new drug targets for these diseases. In this review, we highlight the role of epigenetics in diabetes and its vascular complications, and recent technological advances that have significantly accelerated the field.

Epigenetic Histone Methylation Modulates Fibrotic Gene Expression

TGF-β1-induced expression of extracellular matrix (ECM) genes plays a major role in the development of chronic renal diseases such as diabetic nephropathy. Although many key transcription factors are known, mechanisms involving the nuclear chromatin that modulate ECM gene expression remain unclear. Here, we examined the role of epigenetic chromatin marks such as histone H3 lysine methylation (H3Kme) in TGF-β1-induced gene expression in rat mesangial cells under normal and high-glucose (HG) conditions. TGF-β1 increased the expression of the ECM-associated genes connective tissue growth factor, collagen-α1[Ι], and plasminogen activator inhibitor-1. Increased levels of chromatin marks associated with active genes (H3K4me1, H3K4me2, and H3K4me3), and decreased levels of repressive marks (H3K9me2 and H3K9me3) at these gene promoters accompanied these changes in expression. TGF-β1 also increased expression of the H3K4 methyltransferase SET7/9 and recruitment to these promoters. SET7/9 gene silencing with siRNAs significantly attenuated TGF-β1-induced ECM gene expression. Furthermore, a TGF-β1 antibody not only blocked HG-induced ECM gene expression but also reversed HG-induced changes in promoter H3Kme levels and SET7/9 occupancy. Taken together, these results show the functional role of epigenetic chromatin histone H3Kme in TGF-β1-mediated ECM gene expression in mesangial cells under normal and HG conditions. Pharmacologic and other therapies that reverse these modifications could have potential renoprotective effects for diabetic nephropathy.

Epigenetic mechanisms in diabetic complications and metabolic memory

The incidence of diabetes and its associated micro- and macrovascular complications is greatly increasing worldwide. The most prevalent vascular complications of both type 1 and type 2 diabetes include nephropathy, retinopathy, neuropathy and cardiovascular diseases. Evidence suggests that both genetic and environmental factors are involved in these pathologies. Clinical trials have underscored the beneficial effects of intensive glycaemic control for preventing the progression of complications. Accumulating evidence suggests a key role for epigenetic mechanisms such as DNA methylation, histone post-translational modifications in chromatin, and non-coding RNAs in the complex interplay between genes and the environment. Factors associated with the pathology of diabetic complications, including hyperglycaemia, growth factors, oxidant stress and inflammatory factors can lead to dysregulation of these epigenetic mechanisms to alter the expression of pathological genes in target cells such as endothelial, vascular smooth muscle, retinal and cardiac cells, without changes in the underlying DNA sequence. Furthermore, long-term persistence of these alterations to the epigenome may be a key mechanism underlying the phenomenon of ‘metabolic memory’ and sustained vascular dysfunction despite attainment of glycaemic control. Current therapies for most diabetic complications have not been fully efficacious, and hence a study of epigenetic mechanisms that may be involved is clearly warranted as they can not only shed novel new insights into the pathology of diabetic complications, but also lead to the identification of much needed new drug targets. In this review, we highlight the emerging role of epigenetics and epigenomics in the vascular complications of diabetes and metabolic memory.

Role of the Akt/FoxO3a Pathway in TGF-β1–Mediated Mesangial Cell Dysfunction: A Novel Mechanism Related to Diabetic Kidney Disease

Diabetic nephropathy (DN) is characterized by mesangial cell (MC) expansion and accumulation of extracellular matrix proteins. TGF-beta is increased in MC under diabetic conditions and in DN and activates key signaling pathways, including the phosphoinositide-3-kinase/Akt (PI3K/Akt) pathway. FoxO transcription factors play roles in cell survival and oxidative stress and are negatively regulated by Akt-mediated phosphorylation. We tested whether phosphorylation-mediated inactivation of FoxO3a by TGF-beta can mediate MC survival and oxidative stress. TGF-beta treatment significantly increased levels of p-Akt (activation) and p-FoxO3a (inactivation) in cultured MC. This FoxO3a inactivation was accompanied by significant decreases in the expression of two key FoxO3a target genes, the proapoptotic Bim and antioxidant manganese superoxide dismutase in MC. TGF-beta treatment triggered the nuclear exclusion of FoxO3a, significantly inhibited FoxO3a transcriptional activity, and markedly protected MC from apoptosis. A PI3K inhibitor blocked these TGF-beta effects. It is interesting that p-Akt and p-FoxO3A levels also were increased in renal cortical tissues from rats and mice at 2 wk after the induction of diabetes by streptozotocin, thus demonstrating in vivo significance. In summary, TGF-beta and diabetes can increase FoxO3a phosphorylation and transcriptional inactivation via PI3K/Akt. These new results suggest that Akt/FoxO pathway regulation may be a novel mechanism by which TGF-beta can induce unopposed MC survival and oxidant stress in early DN, thereby accelerating renal disease.

Enhanced Proatherogenic Responses in Macrophages and Vascular Smooth Muscle Cells Derived From Diabetic db/db Mice

Diabetes is associated with enhanced inflammatory responses and cardiovascular complications such as atherosclerosis. However, it is unclear whether similar responses are present in cells derived from experimental animal models of diabetes. We examined our hypothesis that macrophages and short-term cultured vascular smooth muscle cells (VSMCs) derived from obese, insulin-resistant, and diabetic db/db mice would exhibit increased proatherogenic responses relative to those from control db/+ mice. We observed that macrophages from db/db mice exhibit significantly increased expression of key inflammatory cytokines and chemokines as well as arachidonic acid–metabolizing enzymes cyclooxygenase-2 and 12/15-lipoxygenase that generate inflammatory lipids. Furthermore, VSMCs derived from db/db mice also showed similar enhanced expression of inflammatory genes. Expression of inflammatory genes was also significantly increased in aortas derived from db/db mice. Both macrophages and VSMCs from db/db mice demonstrated significantly increased oxidant stress, activation of key signaling kinases, and transcription factors cAMP response element–binding protein and nuclear factor-κB, involved in the regulation of atherogenic and inflammatory genes. Interestingly, VSMCs from db/db mice displayed enhanced migration as well as adhesion to WEHI mouse monocytes relative to db/+. Thus, the diabetic milieu and a potential hyperglycemic memory can induce aberrant behavior of vascular cells. These new results demonstrate that monocyte/macrophages and VSMCs derived from db/db mice display a “preactivated” and proinflammatory phenotype associated with the pathogenesis of diabetic vascular dysfunction and atherosclerosis.

Role of the Lysine-Specific Demethylase 1 in the Proinflammatory Phenotype of Vascular Smooth Muscle Cells of Diabetic Mice

Insulin resistance and type 2 diabetes are major risk factors for vascular complications. Vascular smooth muscle cells (VSMCs) derived from db/db mice, an established mouse model of type 2 diabetes, displayed enhanced inflammatory gene expression and proatherogenic responses. We examined the hypothesis that aberrant epigenetic chromatin events may the underlying mechanism for this persistent dysfunctional behavior and “memory” of the diabetic cells. Chromatin immunoprecipitation assays showed that levels of histone H3 lysine 4 dimethylation (H3K4me2), a key chromatin mark associated with active gene expression, were significantly elevated at the promoters of the inflammatory genes monocyte chemoattractant protein-1 and interleukin-6 in db/db VSMCs relative to db/+ cells. Tumor necrosis factor-&agr;-induced inflammatory gene expression, H3K4me2 levels, and recruitment of RNA polymerase II at the gene promoters were also enhanced in db/db VSMCs, demonstrating the formation of open chromatin poised for transcriptional activation in diabetes. On the other hand, protein levels of lysine-specific demethylase1 (LSD1), which negatively regulates H3K4 methylation and its occupancy at these gene promoters, were significantly reduced in db/db VSMCs. High glucose (25 mmol/L) treatment of human VSMCs also increased inflammatory genes with parallel increases in promoter H3K4me2 levels and reduced LSD1 recruitment. LSD1 gene silencing with small interfering RNAs significantly increased inflammatory gene expression and enhanced VSMC-monocyte binding in nondiabetic VSMCs. In contrast, overexpression of LSD1 in diabetic db/db VSMCs inhibited their enhanced inflammatory gene expression. These results demonstrate novel functional roles for LSD1 and H3K4 methylation in VSMCs and inflammation. Dysregulation of their actions may be a major mechanism for vascular inflammation and metabolic memory associated with diabetic complications.