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Role of the lysine-specific demethylase 1 in the proinflammatory phenotype of vascular smooth muscle cells of diabetic mice.

ABSTRACT: 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-alpha-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.

SUBMITTER: Reddy MA

PROVIDER: S-EPMC2693400 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Role of the lysine-specific demethylase 1 in the proinflammatory phenotype of vascular smooth muscle cells of diabetic mice.

Reddy Marpadga A MA Villeneuve Louisa M LM Wang Mei M Lanting Linda L Natarajan Rama R

Circulation research 20080807 6

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 th ...[more]

PMID: 18688044

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Project description:BackgroundAmong older adults, arterial aging is the major factor contributing to increased risk for cardiovascular disease-related morbidity and mortality. The chronic vascular inflammation that accompanies aging causes diffuse intimal-medial thickening of the arterial wall, thus increasing the vulnerability of aged vessels to vascular insults. Milk fat globule-epidermal growth factor 8 (MFG-E8) is a biomarker for aging arteries. This integrin-binding glycoprotein, induced by angiotensin II, facilitates vascular smooth muscle cell (VSMC) proliferation and invasion in aging vasculatures. This study investigated whether MFG-E8 directly mediates the initial inflammatory responses in aged arteries or VSMCs.MethodsA model of neointimal hyperplasia was induced in the common carotid artery (CCA) of aged mice to exacerbate age-associated vascular remodeling. Recombinant MFG-E8 (rMFG-E8) was administered to the injured artery using Pluronic gel to accentuate the effect on age-related vascular pathophysiology. The MFG-E8 level, leukocyte infiltration, and proinflammatory cell adhesion molecule (CAM) expression in the arterial wall were evaluated through immunohistochemistry. By using immunofluorescence and immunoblotting, the activation of the critical proinflammatory transcription factor nuclear factor (NF)-κB in the injured CCAs was analyzed. Immunofluorescence, immunoblotting, and quantitative real-time polymerase chain reaction were conducted using VSMCs isolated from the aortas of young and aged mice to assess NF-κB nuclear translocation, NF-κB-dependent gene expression, and cell proliferation. The extent of intimal-medial thickening in the injured vessels was analyzed morphometrically. Finally, Transwell migration assay was used to examine VSMC migration.ResultsEndogenous MFG-E8 expression in aged CCAs was significantly induced by ligation injury. Aged CCAs treated with rMFG-E8 exhibited increased leukocyte extravasation, CAM expression, and considerably increased NF-κB activation induced by rMFG-E8 in the ligated vessels. Exposure of early passage VSMCs from aged aortas to rMFG-E8 substantially increased NF-κB activation, proinflammatory gene expression, and cell proliferation. However, rMFG-E8 attenuated VSMC migration.ConclusionsMFG-E8 promoted the proinflammatory phenotypic shift of aged VSMCs and arteries, rendering the vasculature prone to vascular diseases. MFG-E8 may constitute a novel therapeutic target for retarding the aging processes in such vessels.

Project description:ObjectiveWe examined the role of syndecan-1 in modulating the phenotype of vascular smooth muscle cells in the context of endogenous inflammatory factors and altered microenvironments that occur in disease or injury-induced vascular remodeling.Methods and resultsVascular smooth muscle cells (vSMCs) display a continuum of phenotypes that can be altered during vascular remodeling. While the syndecans have emerged as powerful and complex regulators of cell function, their role in controlling vSMC phenotype is unknown. Here, we isolated vSMCs from wild type (WT) and syndecan-1 knockout (S1KO) mice. Gene expression and western blotting studies indicated decreased levels of α-smooth muscle actin (α-SMA), calponin, and other vSMC-specific differentiation markers in S1KO relative to WT cells. The spread area of the S1KO cells was found to be greater than WT cells, with a corresponding increase in focal adhesion formation, Src phosphorylation, and alterations in actin cytoskeletal arrangement. In addition, S1KO led to increased S6RP phosphorylation and decreased AKT and PKC-α phosphorylation. To examine whether these changes were present in vivo, isolated aortae from aged WT and S1KO mice were stained for calponin. Consistent with our in-vitro findings, the WT mice aortae stained higher for calponin relative to S1KO. When exposed to the inflammatory cytokine TNF-α, WT vSMCs had an 80% reduction in syndecan-1 expression. Further, with TNF-α, S1KO vSMCs produced increased pro-inflammatory cytokines relative to WT. Finally, inhibition of interactions between syndecan-1 and integrins αvβ3 and αvβ5 using the inhibitory peptide synstatin appeared to have similar effects on vSMCs as knocking out syndecan-1, with decreased expression of vSMC differentiation markers and increased expression of inflammatory cytokines, receptors, and osteopontin.ConclusionsTaken together, our results support that syndecan-1 promotes vSMC differentiation and quiescence. Thus, the presence of syndecan-1 would have a protective effect against vSMC dedifferentiation and this activity is linked to interactions with integrins αvβ3 and αvβ5.

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Project description:Wnt signaling regulates key aspects of diabetic vascular disease.We generated SM22-Cre;LRP6(fl/fl);LDLR(-/-) mice to determine contributions of Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6) in the vascular smooth muscle lineage of male low-density lipoprotein receptor-null mice, a background susceptible to diet (high-fat diet)-induced diabetic arteriosclerosis.As compared with LRP6(fl/fl);LDLR(-/-) controls, SM22-Cre;LRP6(fl/fl);LDLR(-/-) (LRP6-VKO) siblings exhibited increased aortic calcification on high-fat diet without changes in fasting glucose, lipids, or body composition. Pulse wave velocity (index of arterial stiffness) was also increased. Vascular calcification paralleled enhanced aortic osteochondrogenic programs and circulating osteopontin (OPN), a matricellular regulator of arteriosclerosis. Survey of ligands and Frizzled (Fzd) receptor profiles in LRP6-VKO revealed upregulation of canonical and noncanonical Wnts alongside Fzd10. Fzd10 stimulated noncanonical signaling and OPN promoter activity via an upstream stimulatory factor (USF)-activated cognate inhibited by LRP6. RNA interference revealed that USF1 but not USF2 supports OPN expression in LRP6-VKO vascular smooth muscle lineage, and immunoprecipitation confirmed increased USF1 association with OPN chromatin. ML141, an antagonist of cdc42/Rac1 noncanonical signaling, inhibited USF1 activation, osteochondrogenic programs, alkaline phosphatase, and vascular smooth muscle lineage calcification. Mass spectrometry identified LRP6 binding to protein arginine methyltransferase (PRMT)-1, and nuclear asymmetrical dimethylarginine modification was increased with LRP6-VKO. RNA interference demonstrated that PRMT1 inhibits OPN and TNAP, whereas PRMT4 supports expression. USF1 complexes containing the histone H3 asymmetrically dimethylated on Arg-17 signature of PRMT4 are increased with LRP6-VKO. Jmjd6, a demethylase downregulated with LRP6 deficiency, inhibits OPN and TNAP expression, USF1: histone H3 asymmetrically dimethylated on Arg-17 complex formation, and transactivation.LRP6 restrains vascular smooth muscle lineage noncanonical signals that promote osteochondrogenic differentiation, mediated in part via USF1- and arginine methylation-dependent relays.

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Role of the lysine-specific demethylase 1 in the proinflammatory phenotype of vascular smooth muscle cells of diabetic mice.

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Role of the lysine-specific demethylase 1 in the proinflammatory phenotype of vascular smooth muscle cells of diabetic mice.

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