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-␣ inflammatory stimulus, which induced dramatic and sustained decreases in promoter H3K9me3 and Suv39h1 occupancy. Recruitment of corepressor HP1␣ 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.
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.
Nuclear factor -B (NF-B)-regulated inflammatory genes, such as TNF-␣ (tumor necrosis factor-␣), 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-B. Gene silencing of SET7/9 with small interfering RNAs in monocytes significantly inhibited TNF-␣-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-␣-induced recruitment of NF-B 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-␣-stimulated monocytes, the induction of 25% NF-B 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.NF-B is a pleiotropic transcription factor that plays a pivotal role in regulating multiple biological functions, such as inflammation, immunity, cell proliferation, and apoptosis (1, 2). NF-B plays an important role in the regulation of proinflammatory genes, such as TNF-␣ (tumor necrosis factor ␣) and MCP-1 (monocyte-chemoattractant protein-1), that are associated with several inflammatory diseases, including atherosclerosis, insulin resistance, metabolic syndrome, and diabetes and its complications (3-7). These genes also lead to monocyte activation associated with these inflammatory diseases.NF-B consists of homo-or heterodimers of different subunits, such as p50, p52, p65/RelA, RelB, and c-Rel, with p65/ RelA and p50 being the most common and well studied (1,8). In most unstimulated cells, NF-B resides in the cytoplasm in an inactive latent form complexed with its inhibitor subunit, IB␣. Multiple extracellular stimuli, including inflammatory cytokines, such as TNF-␣, and ligands of the receptor for advanced glycation end products (RAGE), 2 can induce NF-B activation by promoting IB␣ phosphorylation and its proteasomal degradation (6, 9). The released p65-p50 dimer then translocates to the nucleus, where it binds to the promoters of NF-B-dependent inflammatory genes, such as TNF-␣, MCP-1, and IL-6 (interleukin-6), to induce their expression (2). p65 protein is a key transcriptionally active component of NF-B whose transactivation potential is enhanced by several coactivators, including CREB-binding protein/p300, p/CAF, and SRC1 (10), which have histone acetyltransferase activity, and CARM1, which has arginine methyltransferase activity (11,12). Recently, we showed that histone H3 lysine acetylation is enriched...
TGF-1-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-1-induced gene expression in rat mesangial cells under normal and high-glucose (HG) conditions. TGF-1 increased the expression of the ECM-associated genes connective tissue growth factor, collagen-␣1 [⌱], 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-1 also increased expression of the H3K4 methyltransferase SET7/9 and recruitment to these promoters. SET7/9 gene silencing with siRNAs significantly attenuated TGF-1-induced ECM gene expression. Furthermore, a TGF-1 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-1-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.
Diabetic nephropathy (DN) is characterized by mesangial cell (MC) expansion and accumulation of extracellular matrixproteins. TGF- 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- can mediate MC survival and oxidative stress. TGF- 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- treatment triggered the nuclear exclusion of FoxO3a, significantly inhibited FoxO3a transcriptional activity, and markedly protected MC from apoptosis. A PI3K inhibitor blocked these TGF- 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- 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- can induce unopposed MC survival and oxidant stress in early DN, thereby accelerating renal disease.
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.
The mechanisms by which macrophages mediate the enhanced inflammation associated with diabetes complications are not completely understood. We used RNA sequencing to profile the transcriptome of bone marrow macrophages isolated from diabetic db/db mice and identified 1,648 differentially expressed genes compared with control db/+ mice. Data analyses revealed that diabetes promoted a proinflammatory, profibrotic, and dysfunctional alternatively activated macrophage phenotype possibly via transcription factors involved in macrophage function. Notably, diabetes altered levels of several long noncoding RNAs (lncRNAs). Because the role of lncRNAs in diabetes complications is unknown, we further characterized the function of lncRNA E330013P06, which was upregulated in macrophages from db/db and diet-induced insulin-resistant type 2 diabetic (T2D) mice, but not from type 1 diabetic mice. It was also upregulated in monocytes from T2D patients. E330013P06 was also increased along with inflammatory genes in mouse macrophages treated with high glucose and palmitic acid. E330013P06 overexpression in macrophages induced inflammatory genes, enhanced responses to inflammatory signals, and increased foam cell formation. In contrast, small interfering RNA–mediated E330013P06 gene silencing inhibited inflammatory genes induced by the diabetic stimuli. These results define the diabetic macrophage transcriptome and novel functional roles for lncRNAs in macrophages that could lead to lncRNA-based therapies for inflammatory diabetes complications.
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