Slug Is Increased in Vascular Remodeling and Induces a Smooth Muscle Cell Proliferative Phenotype.
ABSTRACT: Previous studies have confirmed Slug as a key player in regulating phenotypic changes in several cell models, however, its role in smooth muscle cells (SMC) has never been assessed. The purpose of this study was to evaluate the expression of Slug during the phenotypic switch of SMC in vitro and throughout the development of vascular remodeling.Slug expression was decreased during both cell-to-cell contact and TGF?1 induced SMC differentiation. Tumor necrosis factor-? (TNF?), a known inductor of a proliferative/dedifferentiated SMC phenotype, induces the expression of Slug in SMC. Slug knockdown blocked TNF?-induced SMC phenotypic change and significantly reduced both SMC proliferation and migration, while its overexpression blocked the TGF?1-induced SMC differentiation and induced proliferation and migration. Genome-wide transcriptomic analysis showed that in SMC, Slug knockdown induced changes mainly in genes related to proliferation and migration, indicating that Slug controls these processes in SMC. Notably, Slug expression was significantly up-regulated in lungs of mice using a model of pulmonary hypertension-related vascular remodeling. Highly remodeled human pulmonary arteries also showed an increase of Slug expression compared to less remodeled arteries.Slug emerges as a key transcription factor driving SMC towards a proliferative phenotype. The increased Slug expression observed in vivo in highly remodeled arteries of mice and human suggests a role of Slug in the pathogenesis of pulmonary vascular diseases.
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:Vascular smooth muscle cell (SMC) migration is an essential step involved in neointimal formation in restenosis and atherosclerosis. Lysophosphatidic acid (LPA) is a bioactive component of oxidized low-density lipoprotein and is produced by activated platelets, implying that LPA influences vascular remodeling. Our previous study revealed that matricellular protein CCN1, a prominent extracellular matrix (ECM) protein, mediates LPA-induced SMC migration in vitro. Here we examined the role of CCN1 in LPA-induced neointimal formation. By using LPA infusion of carotid artery in a mouse model, we demonstrated that LPA highly induced CCN1 expression (approximately six- to sevenfold) in neointimal lesions. Downregulation of CCN1 expression with the specific CCN1 siRNA in carotid arteries blocked LPA-induced neointimal formation, indicating that CCN1 is essential in LPA-induced neointimal formation. We then used LPA receptor knockout (LPA1-/-, LPA2-/-, and LPA3-/-) mice to examine LPA receptor function in CCN1 expression in vivo and in LPA-induced neointimal formation. Our data reveal that LPA1 deficiency, but not LPA2 or LPA3 deficiency, prevents LPA-induced CCN1 expression in vivo in mouse carotid arteries. We also observed that LPA1 deficiency blunted LPA infusion-induced neointimal formation, indicating that LPA1 is the major mediator for LPA-induced vascular remodeling. Our in vivo model of LPA-induced neointimal formation established a key role of the ECM protein CCN1 in mediating LPA-induced neointimal formation. Our data support the notion that the LPA1-CCN1 axis may be the central control for SMC migration and vascular remodeling. CCN1 may serve as an important vascular disease marker and potential target for vascular therapeutic intervention.
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:In response to various stimuli, vascular smooth muscle cells (SMCs) can de-differentiate, proliferate and migrate in a process known as phenotypic modulation. However, the phenotype of modulated SMCs in vivo during atherosclerosis and the influence of this process on coronary artery disease (CAD) risk have not been clearly established. Using single-cell RNA sequencing, we comprehensively characterized the transcriptomic phenotype of modulated SMCs in vivo in atherosclerotic lesions of both mouse and human arteries and found that these cells transform into unique fibroblast-like cells, termed 'fibromyocytes', rather than into a classical macrophage phenotype. SMC-specific knockout of TCF21-a causal CAD gene-markedly inhibited SMC phenotypic modulation in mice, leading to the presence of fewer fibromyocytes within lesions as well as within the protective fibrous cap of the lesions. Moreover, TCF21 expression was strongly associated with SMC phenotypic modulation in diseased human coronary arteries, and higher levels of TCF21 expression were associated with decreased CAD risk in human CAD-relevant tissues. These results establish a protective role for both TCF21 and SMC phenotypic modulation in this disease.
Project description:Despite modern therapies, pulmonary arterial hypertension (PAH) harbors a high mortality. Vascular remodeling is a hallmark of the disease. Recent clinical studies revealed that antiremodeling approaches with tyrosine-kinase inhibitors such as imatinib are effective, but its applicability is limited by significant side effects. Although imatinib has multiple targets, expression analyses support a role for platelet-derived growth factor (PDGF) in the pathobiology of the disease. However, its precise role and downstream signaling events have not been established.Patients with PAH exhibit enhanced expression and phosphorylation of ? PDGF receptor (?PDGFR) in remodeled pulmonary arterioles, particularly at the binding sites for phophatidyl-inositol-3-kinase and PLC? at tyrosine residues 751 and 1021, respectively. These signaling molecules were identified as critical downstream mediators of ?PDGFR-mediated proliferation and migration of pulmonary arterial smooth muscle cells. We, therefore, investigated mice expressing a mutated ?PDGFR that is unable to recruit phophatidyl-inositol-3-kinase and PLC? (?PDGFR(F3/F3)). PDGF-dependent Erk1/2 and Akt phosphorylation, cyclin D1 induction, and proliferation, migration, and protection against apoptosis were abolished in ?PDGFR(F3/F3) pulmonary arterial smooth muscle cells. On exposure to chronic hypoxia, vascular remodeling of pulmonary arteries was blunted in ?PDGFR(F3/F3) mice compared with wild-type littermates. These alterations led to protection from hypoxia-induced PAH and right ventricular hypertrophy.By means of a genetic approach, our data provide definite evidence that the activated ?PDGFR is a key contributor to pulmonary vascular remodeling and PAH. Selective disruption of PDGF-dependent phophatidyl-inositol-3-kinase and PLC? activity is sufficient to abolish these pathogenic responses in vivo, identifying these signaling events as valuable targets for antiremodeling strategies in PAH.
Project description:We hypothesized that redox-mediated apoptosis of medial smooth muscle cells (SMC) during the acute phase of vascular injury contributes to the pathophysiology of vascular disease.Apoptosis of medial SMC (1-14 days following balloon injury) was identified in rat carotid arteries by in situ DNA labeling. NADPH-derived superoxide and expression of Bcl-xL, Bax, caspase-3 and caspase-9 were assessed. The antioxidant tempol was administered in drinking water throughout the experimental period, and local adenoviral-mediated gene transfer of eNOS was performed prior to vascular injury.Balloon injury increased NADPH-dependent superoxide production, medial SMC apoptosis, Bax-positive medial SMC index, Bax/Bcl-xL ratio, and caspase-3 and caspase-9 expression in the injured arteries. Treatment with tempol or eNOS gene transfer decreased superoxide levels and medial SMC apoptosis, with a concomitant increase in medial SMC density. Inhibition of superoxide was associated with a decreased Bax/Bcl-xL ratio, and caspase-3 and -9 expression. Tempol treatment and eNOS gene therapy significantly reduced neointima formation.Vascular generation of reactive oxygen species participates in Bax activation and medial SMC apoptosis. These effects likely contribute to the shedding of cell-cell adhesion molecules and promote medial SMC migration and proliferation responsible for neointimal hyperplasia.
Project description:All forms of chronic pulmonary hypertension (PH) are characterized by structural remodeling of the pulmonary artery (PA) media, a process previously attributed solely to changes in the phenotype of resident smooth muscle cells (SMC). However, recent experimental evidence in both systemic and pulmonary circulations suggests that other cell types, including circulating and local progenitors, contribute significantly to this process. The goal of this study was to determine if hypoxia-induced remodeling of distal PA (dPA) media involves the emergence of cells with phenotypic and functional characteristics distinct from those of resident dPA SMC and fibroblasts. In vivo, in contrast to the phenotypically uniform SMC composition of dPA media in control calves, the remodeled dPA media of neonatal calves with severe hypoxia-induced PH comprised cells exhibiting a distinct phenotype, including the expression of hematopoetic (CD45), leukocytic/monocytic (CD11b, CD14), progenitor (cKit), and motility-associated (S100A4) cell markers. Consistent with these in vivo observations, primary cell cultures isolated from dPA media of hypertensive calves yielded not only differentiated SMC, but also smaller, morphologically rhomboidal (thus termed here "R") cells that transiently expressed CD11b, constitutively expressed the mesenchymal cell marker type I procollagen, expressed high mRNA levels of progenitor cell markers cKit, CD34, CD73, as well as for inflammatory mediators, IL-6 and MCP-1, and, with time in culture, gained expression of a myofibroblast marker, alpha-SM-actin. R cells exhibited highly augmented proliferative, migratory, invasive, and potent promitogenic capabilities, which were due, at least in part, to the production of PDGFs, SDF-1/CXCL12, and S100A4. These data suggest that the cellular mechanisms of dPA remodeling include the emergence of cells with phenotypic and functional characteristics markedly distinct from those of resident dPA cells.
Project description:Phenotypic switching of vascular smooth muscle cells (VSMCs) is known to play a critical role in the development of atherosclerosis. However, the factors present within lesions that mediate VSMC phenotypic switching are unclear. Oxidized phospholipids (OxPLs), including 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC), are active components of minimally modified low density lipoprotein and have been previously shown to induce multiple proatherogenic events in endothelial cells and macrophages, but their effects on VSMCs have been largely unexplored until recently. We previously showed that OxPLs induced phenotypic switching of VSMCs, including suppression of SMC differentiation marker genes. The goal of the present studies was to test the hypothesis that OxPLs alter extracellular matrix production and VSMC migration. Results showed that POVPC activated expression of several extracellular matrix proteins in VSMC. POVPC increased expression of type VIII collagen alpha1 chain (Col8a1) mRNA in cultured VSMCs and in vivo in rat carotid arteries by 9-fold and 4-fold, respectively. POVPC-induced activation of Col8a1 gene expression was reduced by small interfering RNA-mediated suppression of Krüppel-like factor 4 (Klf4) and Sp1, and was abolished in Klf4-knockout VSMCs. POVPC increased Klf4 binding to the Col8a1 gene promoter both in vivo in rat carotid arteries and in cultured VSMCs based on chromatin immunoprecipitation assays. Moreover, POVPC-induced VSMC migration was markedly reduced in Klf4- or type VIII collagen-knockout VSMCs. Given evidence that OxPLs are present within atherosclerotic lesions, it is interesting to suggest that OxPL-induced changes in VSMC phenotype may contribute to the pathogenesis of atherosclerosis at least in part through changes in extracellular matrix composition.
Project description:Autophagy is recently implicated in regulating vascular smooth muscle cell (SMC) homeostasis and in the pathogenesis of vascular remodeling. Transcription factor EB (TFEB) is a master regulator of autophagy signaling pathways. However, the molecular mechanisms and functional roles of TFEB in SMC homeostasis have not been elucidated. Here, we surveyed the ability of TFEB to regulate autophagy pathway in SMCs, and whether pharmacological activation of TFEB favors SMC homeostasis preventing dedifferentiation and pathogenic vascular remodeling. In primary cultured SMCs, TFEB activator trehalose induced nuclear translocation of TFEB and upregulation of TFEB-controlled autophagy genes leading to enhanced autophagy signaling. Moreover, trehalose suppressed serum-induced SMC dedifferentiation to synthetic phenotypes as characterized by inhibited proliferation and migration. These effects of trehalose were mimicked by ectopic upregulation of TFEB and inhibited by TFEB gene silencing. In animal experiments, partial ligation of carotid arteries induced downregulation of TFEB pathway in the media layer of these arteries. Such TFEB suppression was correlated with increased SMC dedifferentiation and aggravated high-fat diet (HFD)-induced neointima formation. Treatment of mice with trehalose reversed this TFEB pathway suppression, and prevented SMC dedifferentiation and HFD-induced neointima formation. In conclusion, our findings have identified TFEB as a novel positive regulator for autophagy pathway and cellular homeostasis in SMCs. Our data suggest that suppression of TFEB may be an initiating mechanism that promotes SMC dedifferentiation leading to accelerated neointima formation in vascular disorders associated with metabolic stress, whereas trehalose reverses these changes. These findings warrant further evaluation of trehalose in the clinical settings.