Uremia does not affect neointima formation in mice.
ABSTRACT: Atherosclerotic cardiovascular disease is a major complication of chronic kidney disease (CKD). CKD leads to uremia, which modulates the phenotype of aortic smooth muscle cells (SMCs). Phenotypic modulation of SMCs plays a key role in accelerating atherosclerosis. We investigated the hypothesis that uremia potentiates neointima formation in response to vascular injury in mice. Carotid wire injury was performed on C57BL/6 wt and apolipoprotein E knockout (Apoe -/-) mice two weeks after induction of uremia by 5/6 nephrectomy. Wire injury led to neointima formation and downregulation of genes encoding classical SMC markers (i.e., myocardin, ?-smooth muscle actin, SM22-alpha, and smooth muscle myosin heavy chain) in both wt and Apoe -/- mice. Contrary to our expectations, uremia did not potentiate neointima formation, nor did it affect intimal lesion composition as judged from magnetic resonance imaging and histological analyses. Also, there was no effect of uremia on SMC marker gene expression in the injured carotid arteries, suggesting that there may be different effects of uremia on SMCs in different vascular beds. In conclusion, uremia does not accelerate neointima formation in response to wire injury of the carotid artery in mice.
Project description:Interferon regulatory factor 8 (IRF8), a member of the IRF transcription factor family, was recently implicated in vascular diseases. In the present study, using the mouse left carotid artery wire injury model, we unexpectedly observed that the expression of IRF8 was greatly enhanced in smooth muscle cells (SMCs) by injury. Compared with the wild-type controls, IRF8 global knockout mice exhibited reduced neointimal lesions and maintained SMC marker gene expression. We further generated SMC-specific IRF8 transgenic mice using an SM22?-driven IRF8 plasmid construct. In contrast to the knockout mice, mice with SMC-overexpressing IRF8 exhibited a synthetic phenotype and enhanced neointima formation. Mechanistically, IRF8 inhibited SMC marker gene expression through regulating serum response factor (SRF) transactivation in a myocardin-dependent manner. Furthermore, a coimmunoprecipitation assay indicated a direct interaction of IRF8 with myocardin, in which a specific region of myocardin was essential for recruiting acetyltransferase p300. Altogether, IRF8 is crucial in modulating SMC phenotype switching and neointima formation in response to vascular injury via direct interaction with the SRF/myocardin complex.
Project description:OBJECTIVE:Smooth muscle cells (SMCs) contribute to neointima formation after vascular injury. Although ?-catenin expression is induced after injury, whether its function is essential in SMCs for neointimal growth is unknown. Moreover, although inhibitors of ?-catenin have been developed, their effects on SMC growth have not been tested. We assessed the requirement for SMC ?-catenin in short-term vascular homeostasis and in response to arterial injury and investigated the effects of ?-catenin inhibitors on vascular SMC growth. APPROACH AND RESULTS:We used an inducible, conditional genetic deletion of ?-catenin in SMCs of adult mice. Uninjured arteries from adult mice lacking SMC ?-catenin were indistinguishable from controls in terms of structure and SMC marker gene expression. After carotid artery ligation, however, vessels from mice lacking SMC ?-catenin developed smaller neointimas, with lower neointimal cell proliferation and increased apoptosis. SMCs lacking ?-catenin showed decreased mRNA expression of Mmp2, Mmp9, Sphk1, and S1pr1 (genes that promote neointima formation), higher levels of Jag1 and Gja1 (genes that inhibit neointima formation), decreased Mmp2 protein expression and secretion, and reduced cell invasion in vitro. Moreover, ?-catenin inhibitors PKF118-310 and ICG-001 limited growth of mouse and human vascular SMCs in a dose-dependent manner. CONCLUSIONS:SMC ?-catenin is dispensable for maintenance of the structure and state of differentiation of uninjured adult arteries, but is required for neointima formation after vascular injury. Pharmacological ?-catenin inhibitors hinder growth of human vascular SMCs. Thus, inhibiting ?-catenin has potential as a therapy to limit SMC accumulation and vascular obstruction.
Project description:The objective of this study is to investigate the role and underlying mechanism of Olfactomedin 2 (Olfm2) in smooth muscle cell (SMC) phenotypic modulation and vascular remodeling.Platelet-derived growth factor-BB induces Olfm2 expression in primary SMCs while modulating SMC phenotype as shown by the downregulation of SMC marker proteins. Knockdown of Olfm2 blocks platelet-derived growth factor-BB-induced SMC phenotypic modulation, proliferation, and migration. Conversely, Olfm2 overexpression inhibits SMC marker expression. Mechanistically, Olfm2 promotes the interaction of serum response factor with the runt-related transcription factor 2 that is induced by platelet-derived growth factor-BB, leading to a decreased interaction between serum response factor and myocardin, causing a repression of SMC marker gene transcription and consequently SMC phenotypic modulation. Animal studies show that Olfm2 is upregulated in balloon-injured rat carotid arteries. Knockdown of Olfm2 effectively inhibits balloon injury-induced neointima formation. Importantly, knockout of Olfm2 in mice profoundly suppresses wire injury-induced neointimal hyperplasia while restoring SMC contractile protein expression, suggesting that Olfm2 plays a critical role in SMC phenotypic modulation in vivo.Olfm2 is a novel factor mediating SMC phenotypic modulation. Thus, Olfm2 may be a potential target for treating injury-induced proliferative vascular diseases.
Project description:Abnormal proliferation and migration of vascular smooth muscle cells (SMCs) are the key events in the progression of neointima formation in response to vascular injury. The goal of this study is to investigate the functional role of a potent oncogene yes-associated protein (YAP) in SM phenotypic modulation in vitro and in vivo.In vitro cell culture and in vivo in both mouse and rat arterial injury models YAP expression is significantly induced and correlated with the vascular SMC synthetic phenotype. Overexpression of YAP promotes SMC migration and proliferation while attenuating SM contractile gene expression. Conversely, knocking down endogenous YAP in SMCs upregulates SM gene expression but attenuates SMC proliferation and migration. Consistent with this, knocking down YAP expression in a rat carotid balloon injury model and genetic deletion of YAP, specifically, in vascular SMCs in mouse after carotid artery ligation injury attenuates injury-induced SM phenotypic switch and neointima formation.YAP plays a novel integrative role in SM phenotypic modulation by inhibiting SM-specific gene expression while promoting SM proliferation and migration in vitro and in vivo. Blocking the induction of YAP would be a potential therapeutic approach for ameliorating vascular occlusive diseases.
Project description:Angioplasty and stent delivery are performed to treat atherosclerotic vascular disease but often cause deleterious neointimal lesion formation. Previously, growth factor receptor-bound protein 2 (Grb2), an intracellular linker protein, was shown to be essential for neointima formation and for p38 mitogen-activated protein kinase (MAPK) activation in vascular smooth muscle cells (SMCs). In this study, the role of vascular SMC p38alpha MAPK in neointimal development was examined.Compound transgenic mice were generated with doxycycline-inducible SMC-specific expression of dominant-negative p38alpha MAPK (DN-p38alpha). Doxycycline treatment resulted in the expression of DN-p38alpha mRNA and protein in transgenic arteries. Doxycycline-treated compound transgenic mice were resistant to neointima formation 21 days after carotid injury and showed reduced arterial p38 MAPK activation. To explore the mechanism by which p38alpha MAPK promotes neointima formation, an in vitro SMC culture system was used. Inhibition of p38alpha MAPK in cultured SMCs by treatment with SB202190 or small interfering RNA blocked platelet-derived growth factor-induced SMC proliferation, DNA replication, phosphorylation of the retinoblastoma protein, and induction of minichromosome maintenance protein 6.SMC p38alpha MAPK activation is required for neointima formation, perhaps because of its ability to promote retinoblastoma protein phosphorylation and minichromosome maintenance protein 6 expression.
Project description:Vascular remodeling as a result of smooth muscle cell (SMC) proliferation and neointima formation is a major medical challenge in cardiovascular intervention. However, antineointima drugs often indistinguishably block re-endothelialization, an essential step toward successful vascular repair, because of their nonspecific effect on endothelial cells (ECs). The objective of this study is to identify a therapeutic target that differentially regulates SMC and EC proliferation.Using both rat balloon injury and mouse wire injury models, we identified CTP synthase 1 (CTPS1) as one of the potential targets that may be used for developing therapeutics for treating neointima-related disorders. CTPS1 was induced in proliferative SMCs in vitro and neointima SMCs in vivo. Blockade of CTPS1 expression by small hairpin RNA or activity by cyclopentenyl cytosine suppressed SMC proliferation and neointima formation. Surprisingly, cyclopentenyl cytosine had much less effect on EC proliferation. Of importance, blockade of CTPS1 in vivo sustained the re-endothelialization as a result of induction of CTP synthesis salvage pathway enzymes nucleoside-diphosphate kinase A and B in ECs. Diphosphate kinase B seemed to preserve EC proliferation via use of extracellular cytidine to synthesize CTP. Indeed, blockade of both CTPS1 and diphosphate kinase B suppressed EC proliferation in vitro and the re-endothelialization in vivo.Our study uncovered a fundamental difference in CTP biosynthesis between SMCs and ECs during vascular remodeling, which provided a novel strategy by using cyclopentenyl cytosine or other CTPS1 inhibitors to selectively block SMC proliferation without disturbing or even promoting re-endothelialization for effective vascular repair after injury.
Project description:RATIONALE:Vascular smooth muscle cell (SMC) migration is an important pathological process in several vascular occlusive diseases, including atherosclerosis and restenosis, both of which are accelerated by diabetes mellitus. OBJECTIVE:To determine the mechanisms of abnormal vascular SMC migration in type 2 diabetes, the obese Zucker rat (ZO), a model of obesity and insulin resistance, was studied. METHODS AND RESULTS:In culture, ZO aortic SMCs showed a significant increase in Nox4 mRNA and protein levels compared with the control lean Zucker rat (ZL). The sarco-/endoplasmic reticulum Ca(2+) ATPase (SERCA) nitrotyrosine-294,295 and cysteine-674 (C674)-SO(3)H were increased in ZO SMCs, indicating oxidant stress. Unlike ZL SMC, nitric oxide (NO) failed to inhibit serum-induced SMC migration in ZO. Transfection of Nox4 small interference RNA or overexpression of SERCA2b wild type, but not C674S mutant SERCA, restored the response to NO. Knockdown of Nox4 also decreased SERCA oxidation in ZO SMCs. In addition, transforming growth factor-?1 via Smad2 was necessary and sufficient to upregulate Nox4, oxidize SERCA, and block the antimigratory action of NO in ZO SMCs. Corresponding to the results in cultured SMCs, immunohistochemistry confirmed that Nox4 and SERCA C674-SO(3)H were significantly increased in ZO aorta. After common carotid artery injury, knockdown of Nox4 by adenoviral Nox4 short hairpin RNA decreased Nox4 and SERCA C674-SO(3)H staining and significantly decreased injury-induced neointima. CONCLUSION:These studies indicate that the upregulation of Nox4 by transforming growth factor-?1 in ZO SMCs is responsible for the impaired response to NO by a mechanism involving the oxidation of SERCA C674. Knockdown of Nox4 inhibits oxidation of SERCA, as well as neointima formation, after ZO common carotid artery injury.
Project description:Smooth muscle cells (SMCs) in normal blood vessels exist in a highly differentiate state characterized by expression of SMC-specific contractile proteins ("contractile phenotype"). Following blood vessel injury in vivo or when cultured in vitro in the presence of multiple growth factors, SMC undergo a phenotype switch characterized by the loss of contractile markers and appearance of expression of non-muscle proteins ("proliferative phenotype"). While a number of factors have been reported to modulate this process, its regulation remains uncertain. Here we show that induction of SMC FGF signaling inhibits TGF? signaling and converts contractile SMCs to the proliferative phenotype. Conversely, inhibition of SMC FGF signaling induces TGF? signaling converting proliferating SMCs to the contractile phenotype, even in the presence of various growth factors in vitro or vascular injury in vivo. The importance of this signaling cross-talk is supported by in vivo data that show that an SMC deletion of a pan-FGF receptor adaptor Frs2? (fibroblast growth factor receptor substrate 2 alpha) in mice profoundly reduces neointima formation and vascular remodelling following carotid artery ligation. These results demonstrate that FGF-TGF? signaling antagonism is the primary regulator of the SMC phenotype switch. Manipulation of this cross-talk may be an effective strategy for treatment of SMC-proliferation related diseases.
Project description:In the previous report, Meox1 was found to promote SMCs phenotypic modulation and injury-induced vascular remodeling by regulating the FAK-ERK1/2-autophagy signaling cascade (Wu et al., 2017) . Here, we presented new original data on the involvement of Mesoderm/mesenchyme homeobox gene l (Meox1) in balloon-injury-induced neointima formation of rat. In rat carotid artery balloon injury model to induce vascular remodeling, Meox1 was induced in vascular smooth muscle cell (SMCs) of rat carotid arteries. Most proliferating cell nuclear antigen (PCNA)-positive cells also expressed Meox1. These data suggested that Meox1 may be involved in SMCs proliferation during injury-induced neointima formation. Furthermore, knocked down its expression in injured arteries by adenoviral delivery of Meox1 short hairpin RNA (shRNA) (shMeox1), neointima formation was significantly inhibited. Elastin staining also confirmed the reduction of neointima in Meox1 shRNA-transduced arteries. Moreover, knockdown of Meox1 decreased the collagen production/deposition that was significantly increased in neointima induced by balloon injury.
Project description:Intima formation involves smooth muscle cell (SMC) proliferation and migration that ultimately drives arterial stenosis, thrombosis, and ischemia in atherosclerosis, hypertension, and arterial revascularization. Receptor for advanced glycation endproducts (RAGE) is a transmembrane signaling receptor implicated in diabetic renal and vascular complications, and post-injury intima formation, partly via Signal transducer and activator of transcription 3 (STAT3) activation. The metabolic super-regulator Adenosine monophosphate kinase (AMPK) inhibits SMC proliferation and intima formation. AMPK activation is promoted by liver kinase B1 (LKB1), and LKB1 inhibits STAT3 activation. Here, we tested the hypothesis that RAGE promotes arterial intima formation by modulating both LKB1 and AMPK.RAGE ligands (the calgranulin S100A11, and glycated albumin) suppressed AMPK activation in conjunction with increased proliferation and migration of cultured SMCs. These effects were inhibited both by RAGE deficiency and by prior AMPK activation. In SMCs, RAGE ligands decreased LKB1 activity. Moreover, knockdown of both LKB1 and AMPK were associated with increased STAT3 phosphorylation levels. In response to murine carotid artery ligation, expression of RAGE and S100A11 increased, whereas AMPK and LKB1 activity decreased in situ. Conversely, LKB1 and AMPK activity increased in situ, and neointima formation was attenuated in Rage(-/-) mice.The linkage of decreased LKB1 and AMPK activity with increased STAT3 in SMCs mediates the capacity of RAGE ligand-induced signaling to promote neointima formation in response to arterial injury.