Vascular Smooth Muscle Cells Differentiation Marker Research Articles

Loss of Smooth Muscle Tenascin-X Inhibits Vascular Remodeling Through Increased TGF-β Signaling.

Vascular smooth muscle cells (VSMCs) are highly plastic. Vessel injury induces a phenotypic transformation from differentiated to dedifferentiated VSMCs, which involves reduced expression of contractile proteins and increased production of extracellular matrix and inflammatory cytokines. This transition plays an important role in several cardiovascular diseases such as atherosclerosis, hypertension, and aortic aneurysm. TGF-β (transforming growth factor-β) is critical for VSMC differentiation and to counterbalance the effect of dedifferentiating factors. However, the mechanisms controlling TGF-β activity and VSMC phenotypic regulation under in vivo conditions are poorly understood. The extracellular matrix protein TN-X (tenascin-X) has recently been shown to bind TGF-β and to prevent it from activating its receptor. We studied the role of TN-X in VSMCs in various murine disease models using tamoxifen-inducible SMC-specific knockout and adeno-associated virus-mediated knockdown. In hypertensive and high-fat diet-fed mice, after carotid artery ligation as well as in human aneurysmal aortae, expression of Tnxb, the gene encoding TN-X, was increased in VSMCs. Mice with smooth muscle cell-specific loss of TN-X (SMC-Tnxb-KO) showed increased TGF-β signaling in VSMCs, as well as upregulated expression of VSMC differentiation marker genes during vascular remodeling compared with controls. SMC-specific TN-X deficiency decreased neointima formation after carotid artery ligation and reduced vessel wall thickening during Ang II (angiotensin II)-induced hypertension. SMC-Tnxb-KO mice lacking ApoE showed reduced atherosclerosis and Ang II-induced aneurysm formation under high-fat diet. Adeno-associated virus-mediated SMC-specific expression of short hairpin RNA against Tnxb showed similar beneficial effects. Treatment with an anti-TGF-β antibody or additional SMC-specific loss of the TGF-β receptor reverted the effects of SMC-specific TN-X deficiency. In summary, TN-X critically regulates VSMC plasticity during vascular injury by inhibiting TGF-β signaling. Our data indicate that inhibition of vascular smooth muscle TN-X may represent a strategy to prevent and treat pathological vascular remodeling.

The role of transforming growth factor- in hypertension-induced cerebrovascular remodeling

Background: Hypertension (HT) promotes structural and functional changes in the cerebral microcirculation that can provoke irreversible cerebrovascular injury, leading to neuronal loss and brain atrophy,1 cognitive impairment, vascular dementia,2 and Alzheimer’s disease.3 Currently, the mechanisms and consequences of such remodeling are not fully understood. Transforming growth factor (TGF) is a morphogen that regulates cellular differentiation, induces endothelial-to-mesenchymal transition (EndoMT), and works as a contractile-to-synthetic switch in vascular smooth muscle cells (VSMC) phenotype.4 Plasma levels of TGF are increased in HT patients, and in spontaneously hypertensive rats (SHR) that exhibit vascular fibrosis.5 Although coincidental, causal roles through which elevated TGF may drive cerebrovascular remodeling in the HT brain are suspected, but unproven. We hypothesize that HT-induced TGF drives cerebrovascular remodeling, which decreases blood-brain barrier and impairs cerebrovascular autoregulation.
 Methods: Brain cortices and penetrating cerebral microvessels (BMVs) were isolated from male and female SHR and Wistar-Kyoto rats (WKY, control), and subjected to western blotting and immunofluorescence labeling for endothelial cell (EC) and VSMC differentiation markers, TGF canonical pathway markers SMAD2, 3 and 4, as well as basement membrane protein expression, and glial fibrillary acidic protein (GFAP) as a marker of astrocyte inflammation. Additionally, TGF-treated rat retinal microvascular endothelial cells (RRMECs), and human brain microvascular endothelial cells (hCMEC/D3) ± the TGF inhibitor vactosertib, were also subjected to western blotting and immunofluorescence labeling to assess endothelial cell (EC) and VSMC differentiation markers, as well as basement membrane protein expression.
 Results: TGF was significantly increased in SHR BMVs. Moreover, GFAP levels were significantly increased in the SHR cortex. The EC junctional proteins platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial-cadherin (VE-cadherin), were significantly decreased in SHR. In contrast, CRBP-1, a marker for synthetic VSMC, and collagen IV and fibronectin levels significantly increased in SHR. Interestingly, female SHR rats had significantly increased levels of SMAD2/3, which was not evident in male SHRs, indicating a possible role for the non-canonical TGF pathway in male SHRs. Additionally, TGF significantly increased -smooth muscle actin (-SMA) and decreased VE-cadherin expression in RRMECs and D3 cells, consistent with EndoMT. TGF inhibition with vactosertib reversed these effects and significantly suppressed -SMA, and maintained VE-Cadherin similar to control RRMECs and D3s.
 Conclusions: We now have strong evidence for HT-induced cerebrovascular remodeling, where TGF mediates loss of both smooth muscle and endothelial differentiated phenotypes. The reversal of these effects using TGF blockers suggests that therapy to modulate TGF may represent a means to control HT-induced cerebrovascular remodeling.

Smooth Muscle Overexpression of PGC1α Attenuates Atherosclerosis in Rabbits.

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MiR-17 Knockdown Promotes Vascular Smooth Muscle Cell Phenotypic Modulation Through Upregulated Interferon Regulator Factor 9 Expression

MiR-17 is a small noncoding RNA that plays an important role in the development of tumorgenesis, which recently has emerged to be involved in regulation of inflammatory responses and angiogenesis. However, the effect and underlying mechanism of miR-17 on vascular smooth muscle cell (VSMC) phenotypic modulation have not been investigated. In the current study, we observed that miR-17 expression tested by real-time polymerase chain reaction (RT-PCR) was downregulated in VSMCs administrated with platelet-derived growth factor-BB stimulation and carotid arteries subjected to wire injury, which were accompanied with decreased VSMC differentiation markers. Loss-of-function strategy demonstrated that miR-17 knockdown promoted VSMC phenotypic modulation characterized as decreased VSMC differentiation marker genes, increased proliferated and migrated capability of VSMC examined by RT-PCR and western blot analysis. Mechanistically, the bioinformatics analysis and luciferase assay demonstrated that miR-17 directly targeted Interferon Regulator Factor 9 (IRF9) and the upregulated IRF9 expression was responsible for the promoted effect miR-17 knockdown on VSMC phenotypic modulation. Taken together, our results demonstrated that miR-17 knockdown accelerated VSMC phenotypic modulation partially through directly targeting to IRF9, which suggested that miR-17 may act as a novel therapeutic target for intimal hyperplasia management.

Vascular Smooth Muscle Cell Plasticity and Autophagy in Dissecting Aortic Aneurysms.

Objective- Recent studies suggested the occurrence of phenotypic switching of vascular smooth muscle cells (VSMCs) during the development of aortic aneurysm (AA). However, lineage-tracing studies are still lacking, and the behavior of VSMCs during the formation of dissecting AA is poorly understood. Approach and Results- We used multicolor lineage tracing of VSMCs to track their fate after injury in murine models of Ang II (angiotensin II)-induced dissecting AA. We also addressed the direct impact of autophagy on the response of VSMCs to AA dissection. Finally, we studied the relevance of these processes to human AAs. Here, we show that a subset of medial VSMCs undergoes clonal expansion and that VSMC outgrowths are observed in the adventitia and borders of the false channel during Ang II-induced development of dissecting AA. The clonally expanded VSMCs undergo phenotypic switching with downregulation of VSMC differentiation markers and upregulation of phagocytic markers, indicative of functional changes. In particular, autophagy and endoplasmic reticulum stress responses are activated in the injured VSMCs. Loss of autophagy in VSMCs through deletion of autophagy protein 5 gene ( Atg5) increases the susceptibility of VSMCs to death, enhances endoplasmic reticulum stress activation, and promotes IRE (inositol-requiring enzyme) 1α-dependent VSMC inflammation. These alterations culminate in increased severity of aortic disease and higher incidence of fatal AA dissection in mice with VSMC-restricted deletion of Atg5. We also report increased expression of autophagy and endoplasmic reticulum stress markers in VSMCs of human dissecting AAs. Conclusions- VSMCs undergo clonal expansion and phenotypic switching in Ang II-induced dissecting AAs in mice. We also identify a critical role for autophagy in regulating VSMC death and endoplasmic reticulum stress-dependent inflammation with important consequences for aortic wall homeostasis and repair.

TEAD1 (TEA Domain Transcription Factor 1) Promotes Smooth Muscle Cell Proliferation Through Upregulating SLC1A5 (Solute Carrier Family 1 Member 5)-Mediated Glutamine Uptake.

TEAD (TEA domain transcription factor) 1-a major effector of the Hippo signaling pathway-acts as an oncoprotein in a variety of tumors. However, the function of TEAD1 in vascular smooth muscle cells (VSMCs) remains unclear. To assess the role of TEAD1 in vascular injury-induced smooth muscle proliferation and delineate the mechanisms underlying its action. We found that TEAD1 expression is enhanced in mouse femoral artery after wire injury and correlates with the activation of mTORC1 (mechanistic target of rapamycin complex 1) signaling in vivo. Using an inducible smooth muscle-specific Tead1 KO (knockout) mouse model, we found that specific deletion of Tead1 in adult VSMCs is sufficient to attenuate arterial injury-induced neointima formation due to inhibition of mTORC1 activation and VSMC proliferation. Furthermore, we found that TEAD1 plays a unique role in VSMCs, where it not only downregulates VSMC differentiation markers but also activates mTORC1 signaling, leading to enhanced VSMC proliferation. Using whole-transcriptome sequencing analysis, we identified Slc1a5 (solute carrier family 1 member 5)-a key glutamine transporter-as a novel TEAD1 target gene. SLC1A5 overexpression mimicked TEAD1 in promoting mTORC1 activation and VSMC proliferation. Moreover, depletion of SLC1A5 by silencing RNA or blocking SLC1A5-mediated glutamine uptake attenuated TEAD1-dependent mTORC1 activation and VSMC proliferation. Our study unravels a novel mechanism by which TEAD1 promotes VSMC proliferation via transcriptional induction of SLC1A5, thereby activating mTORC1 signaling and promoting neointima formation.

Combined effects of hyperphosphatemia and hyperglycemia on the calcification of cultured human aortic smooth muscle cells.

Vascular calcification (VC) is common in patients with diabetes and/or chronic kidney disease (CKD). It is strongly associated with cardiovascular morbidity and mortality. Hyperphosphatemia caused by CKD induces the transformation of vascular smooth muscle cells (VSMCs) into chondrocytes or osteoblast-like cells. Hyperglycemia may also accelerate VC. However, the exact mechanisms of this remain unclear. The effects of simultaneous hyperphosphatemia and hyperglycemia require investigation. CKD rat models are typically used to study VC, which are far removed from the clinical situations of patients with CKD. The present study cultured human aortic smooth muscle cells (HASMCs) in normal, hyperphosphatemic and/or hyperglycemic conditions for 14 days. Alizarin red staining, calcification content, VSMC differentiation marker gene expression, phenotypic osteoblast gene expression and type III sodium-dependent phosphate cotransporter-1 (Pit-1) protein expression was examined. Hyperphosphatemia and hyperglycemia had combined effects in promoting calcification, phenotypic transition and Pit-1 expression in cultured HASMCs. In the present study, the combined effects of hyperphosphatemia and hyperglycemia on the calcification and phenotypic transition of HASMCs were demonstrated. Hyperphosphatemia combined with hyperglycemia medium should be considered an appropriate experimental model to study VC in diabetic kidney disease (DKD). Pit-1 should be considered as a promising index of VC.

MicroRNA-132 targeting PTEN contributes to cilostazol-promoted vascular smooth muscle cell differentiation

Background and aimsCilostazol, beyond its antiplatelet effect, is also capable of promoting vascular smooth muscle cell (VSMC) differentiation. The aim of this study was to explore the potential role of PTEN, known to associate with VSMC differentiation, and its related microRNA (miRNA) in cilostazol-dependent effects. Methods and resultsMicroarray analysis in balloon-injured rat carotid arteries comparing with and without balloon injury revealed that miR-132 was differentially expressed. Bioinformatic analysis predicts PTEN as a novel target of miR-132. Western blot and quantitative real-time reverse transcription-polymerase chain reaction along with in situ hybridization documented that cilostazol treatment enhanced PTEN and reduced miR-132 expression in the neointima of balloon-injured arteries. Treatment of cultured rat VSMCs with cilostazol resulted in the up-regulation of PTEN mRNA and the down-regulation of miR-132, supporting an in vitro relevance. Co-transfection experiments showed that transfection of miR-132 mimic into VSMCs suppressed PTEN 3′UTR activities, further reflecting that PTEN is the direct target of miR-132. Over-expression of miR-132 in VSMCs led to an attenuation of cilostazol-induced PTEN and its downstream VSMC differentiation marker (calponin) expression, confirming the critical role of miR-132 in VSMC differentiation. Transient transfection studies demonstrated that cilostazol reduced the activity of miR-132 promoter, which was mediated via cyclic AMP response element-binding protein. Notably, the use of lentivirus to over-express miR-132 in the neointima of balloon-injured arteries could reverse the effect of cilostazol in vivo. ConclusionsThese results suggest that miR-132 by targeting PTEN may be an important regulator in mediating cilostazol actions on VSMC differentiation.

Deletion of DGCR8 in VSMCs of adult mice results in loss of vascular reactivity, reduced blood pressure and neointima formation

DiGeorge syndrome chromosomal region 8 (DGCR8), a double-stranded-RNA-binding protein, participates in the miRNA biogenesis pathway and contributes to miRNA maturation by interacting with the RNAase III enzyme Drosha in cell nuclei. To investigate the role of DGCR8 in vascular smooth muscle cells (VSMCs) at the postnatal stages, we generated tamoxifen-inducible VSMC specific knockout (iKO) mice by crossing DGCR8loxp/loxp with VSMC specific tamoxifen-inducible Cre transgenic mice SMA-Cre-ERT2. DGCR8iKO mice display reduced body weight one month following tamoxifen treatment and died around 3 months. Blood pressure and vascular reactivity were significantly reduced in DGCR8iKO mice compared to control. Furthermore, loss of DGCR8 in VSMCs inhibited cell proliferation, migration and neointima formation. VSMC differentiation marker genes, including SMA and SM22, were downregulated in DGCR8 iKO mice. The majority of miRNAs were downregulated in DGCR8iKO mice. Disruption of the DGCR8-mediated miRNA biogenesis pathway attenuated multiple signaling pathways including ERK1/2 and AKT. Our results demonstrate that the DGCR8-mediated miRNA pathway is required for maintaining blood pressure, vascular reactivity and vascular wall remodeling at the postnatal stages.

MiR-26a contributes to the PDGF-BB-induced phenotypic switch of vascular smooth muscle cells by suppressing Smad1.

The phenotypic switch of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of various vascular diseases, such as atherosclerosis and post-angioplasty restenosis. Small non-coding microRNAs (miRNAs) have emerged as critical modulators of VSMC function. In the present study, miR-26a was significantly increased in cultured VSMCs stimulated by platelet-derived growth factor-BB (PDGF-BB) and in arteries with neointimal lesion formation. Moreover, we demonstrated that miR-26a regulates the expression of VSMC differentiation marker genes such as α-smooth muscle actin (α-SMA), calponin and smooth muscle myosin heavy chain (SM-MHC) in PDGF-BB-treated VSMCs. We further confirmed that the regulatory effect of miR-26a during the phenotypic transition occurs through its target gene Smad1, which is a critical mediator of the pro-contractile signal transmitted by bone morphogenetic protein (BMP) and transforming growth factor-beta (TGF-β). This discovery proposed a new channel for communication between PDGF and the BMP/TGF-β family. We concluded that miR-26a is an important regulator in the PDGF-BB-mediated VSMC phenotypic transition by targeting Smad1. Interventions aimed at miR-26a may be promising in treating numerous proliferative vascular disorders.

Macrophage migration inhibitory factor triggers vascular smooth muscle cell dedifferentiation by a p68-serum response factor axis.

Macrophage migration inhibitory factor (MIF) is an important proinflammatory mediator linked to arterial diseases. Although its inflammatory property such as macrophage recruitment is known for contributing to vascular pathogenesis, the direct effects of MIF on homeostasis and biological function of vascular smooth muscle cell (VSMC) that are crucial for development of arterial abnormalities, are poorly understood. We show that MIF is able to directly induce VSMC dedifferentiation, a pathophysiological process fundamental for progression of various arterial diseases. Mechanistically, MIF suppresses p68 protein, a crucial regulator of cell growth and organ differentiation, via activation of JNK and p38 MAPKs. siRNA targeting of p68 facilitated dedifferentiation state in VSMCs, whereas p68 overexpression blocked MIF-elicited transition. In addition, MIF decreased the expression of serum response factor (SRF) that governs VSMC differentiation marker genes transcription, through repression of p68 protein. Furthermore, we showed a previously uncharacterized molecular interaction between p68 and SRF by co-immunoprecipitation assay. p68 attenuated MIF-elicited suppression of SRF recruitment to VSMC-specific promoter. Finally, anti-MIF treatment could reverse VSMC dedifferentiation, preserve vascular function, and inhibit remodelling due to vascular injury. Our results demonstrate a novel mechanism for the regulation of VSMC differentiation by MIF involving p68 and SRF. Strategy for targeting of MIF could inhibit aberrant transition of VSMC in cardiovascular pathogenesis, and may be of therapeutic benefit in phenotype-related arterial remodelling.

A Lindera obtusiloba Extract Blocks Calcium-/Phosphate-Induced Transdifferentiation and Calcification of Vascular Smooth Muscle Cells and Interferes with Matrix Metalloproteinase-2 and Metalloproteinase-9 and NF-κB.

Vascular calcifications bear the risk for cardiovascular complications and have a high prevalence among patients with chronic kidney disease. Central mediators of vascular calcifications are vascular smooth muscle cells (VSMC). They transdifferentiate into a synthetic/osteoblast-like phenotype, which is induced, for example, by elevated levels of calcium and phosphate (Ca/P) due to a disturbed mineral balance. An aqueous extract from Lindera obtusiloba (LOE) is known to exert antifibrotic and antitumor effects or to interfere with the differentiation of preadipocytes. Using murine and rat VSMC cell lines, we here investigated whether LOE also protects VSMC from Ca/P-induced calcification. Indeed, LOE effectively blocked Ca/P-induced calcification of VSMC as shown by decreased VSMC mineralization and secretion of alkaline phosphatase. In parallel, mRNA expression of the calcification markers osterix and osteocalcin was reduced. Vice versa, the Ca/P-induced loss of the VSMC differentiation markers alpha smooth muscle actin and smooth muscle protein 22-alpha was rescued by LOE. Further, LOE blocked Ca/P-induced mRNA expressions and secretions of matrix metalloproteinases-2/-9 and activation of NF-κB, which are known contributors to vascular calcification. In conclusion, LOE interferes with the Ca/P-induced transdifferentiation/calcification of VSMC. Thus, LOE should be further analysed regarding a potential complementary treatment option for cardiovascular diseases including vascular calcifications.

Altered vascular smooth muscle cell differentiation in the endometrial vasculature in menorrhagia.

How does the smooth muscle content and differentiation stage of vascular smooth muscle cells (VSMCs) in endometrial blood vessels change according to the different phases of the menstrual cycle and is this altered in women with menorrhagia? The smooth muscle content (as a proportion of the vascular cross-sectional area) of endometrial blood vessels remained unchanged during the normal menstrual cycle and in menorrhagia; however, expression of the VSMC differentiation markers, smoothelin and calponin, was dysregulated in endometrial blood vessels in samples from women with menorrhagia compared with controls. Menorrhagia affects 30% of women of reproductive age and is the leading indication for hysterectomy. Previous studies have suggested important structural and functional roles for endometrial blood vessels, including impaired vascular contractility. Differentiation of VSMC from a synthetic to contractile state is associated with altered cellular phenotype that contributes to normal blood flow and pressure. This vascular maturation process has been little studied in endometrium both across the normal menstrual cycle and in menorrhagia. Endometrial biopsies were taken from hysterectomy specimens or by pipelle biopsy prior to hysterectomy in controls without endometrial pathology and in women with menorrhagia (n = 7 for each of proliferative, early-secretory, mid-secretory and late-secretory phases for both groups). Biopsies were formalin fixed and embedded in paraffin wax. Paraffin-embedded sections were immunostained for α smooth muscle actin (αSMA), myosin heavy chain (MyHC), H-caldesmon, desmin, smoothelin and calponin (h1 or basic). VSMC content was measured in 25 αSMA(+) vascular cross sections per sample and expressed as a ratio of the muscular area:gross vascular cross-sectional area. VSMC differentiation was analysed by the presence/absence of differentiation markers compared with αSMA expression. Smoothelin and calponin expression was also analysed in relation to total number of blood vessels by double immunostaining for endothelial cell markers. Study of VSMC differentiation markers revealed decreased expression of calponin both in αSMA(+) vessels (P = 0.008) and in relation to total number of vessels (P = 0.001) in late secretory phase endometrium in menorrhagia compared with controls. Smoothelin expression in αSMA(+) vessels was increased (P = 0.03) in menorrhagia, although this was not significant in relation to the total number of vessels. In normal endometrium, the proportion of blood vessels expressing αSMA increased from 63% in proliferative endometrium to 81% in the late secretory phase (P = 0.002). The overall arterial muscle content did not differ between control and menorrhagia at any phase of the menstrual cycle, occupying 78-81% of gross vascular cross-sectional area during the different menstrual cycle phases. This study included both straight and spiral arterioles and analysed only stratum functionalis. The VSMC differentiation with respect to αSMA expression is an observational study and the data are presented as presence or absence of the differentiation markers in each field of view, corresponding with the vascular cross sections included in the study of vascular muscle content. Smoothelin and calponin have been widely implicated as important regulators of vascular tone, vascular contractility and rate of blood flow. Our results have uncovered a disparate pattern of calponin expression, potentially indicating a dysfunctional contraction mechanism in the endometrial blood vessels in menorrhagia, thus implicating calponin as a potential therapeutic target. This study was funded by Wellbeing of Women (RG1342) and Newcastle University. There are no competing interests to declare. Not applicable.

Perivascular adipose tissue-derived leptin promotes vascular smooth muscle cell phenotypic switching via p38 mitogen-activated protein kinase in metabolic syndrome rats

Perivascular adipose tissue (PVAT)-derived leptin is a detrimental adipocytokine and plays a critical role in the development of cardiovascular diseases in metabolic syndrome (MetS). During vascular remodeling, vascular smooth muscle cells (VSMCs) undergo phenotypic switching into a synthetic phenotype characterized by decreased expression of differentiation markers (smooth muscle myosin heavy chain, α-smooth muscle actin, and calponin) and increased proliferation. We aimed to determine whether PVAT-derived leptin influences VSMC phenotypic switching and to explore the underlying mechanisms in MetS rats. Invivo, 32 Wistar rats were divided into two groups that received either a normal diet (control rat) or a high-fat diet (MetS rats). After 16 weeks, rat aortas were stained using hematoxylin-eosin and imaged. VSMC differentiation markers and proliferating cell nuclear antigen (PCNA), PVAT-derived leptin, aortic leptin receptor (ObR), and p38 mitogen-activated protein kinase (MAPK) expression were detected. Invitro, aortic VSMCs were incubated with MetS rat PVAT conditioned medium (PVAT-CM) to mimic invivo conditions and were pretreated with a p38 MAPK inhibitor (SB 203580) or leptin antagonist. Differentiation marker expression, including PCNA and p38 MAPK, was detected. MetS rats exhibited pronounced insulin resistance, hyperglycemia, hyperlipidemia, hypertension, obesity, and an associated increase in PVAT weight. VSMCs underwent phenotypic switching in MetS rat aorta and contributed to vascular remodeling. PVAT-derived leptin expression was higher in MetS rats than in control rats (P < 0.01). ObRa expression and p38 MAPK phosphorylation were upregulated in MetS rat aorta. Invitro, VSMCs incubated with MetS rat PVAT-CM underwent phenotypic switching, associated with increased p38 MAPK phosphorylation. This VSMC phenotypic switching was inhibited by pretreatment with SB 203580 or a leptin antagonist. These results suggest that in MetS rats, PVAT-derived leptin promotes VSMC phenotypic switching via a p38 MAPK-dependent pathway to exacerbate vascular remodeling.

The 10 Most Read Articles Published in Circulation Research in 2013

The 10 Most Read Articles Published in <i>Circulation Research</i> in 2013

MiR-18a-5p MicroRNA Increases Vascular Smooth Muscle Cell Differentiation by Downregulating Syndecan4.

Background and ObjectivesDifferentiation and de-differentiation of vascular smooth muscle cells (VSMCs) are important events in atherosclerosis and restenosis after angioplasty. MicroRNAs are considered a key regulator in cellular processes such as differentiation, proliferation, and apoptosis. Here, we report the role of new miR-18a-5p microRNA and its downstream target genes in VSMCs and in a carotid balloon injury model.Materials and MethodsExpression of miR-18a-5p and its candidate genes was examined in VSMCs and in a carotid artery injury model by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and microRNA microarray analysis. VSMC differentiation marker genes including smooth muscle (SM) α-actin and SM22α were determined by Western blot, qRT-PCR, and a SM22α promoter study. Gene overexpression or knockdown was performed in VSMCs.ResultsmiR-18a-5p was upregulated in the rat carotid artery at the early time after balloon injury. Transfection of the miR-18a-5p mimic promoted the VSMC differentiation markers SM α-actin and SM22α. In addition, miR-18a-5p expression was induced in differentiated VSMCs, whereas it decreased in de-differentiated VSMCs. We identified syndecan4 as a downstream target of miR-18-5p in VSMCs. Overexpression of syndecan4 decreased Smad2 expression, whereas knockdown of syndecan4 increased Smad2 expression in VSMCs. Finally, we showed that Smad2 induced the expression of VSMC differentiation marker genes in VSMCs.ConclusionThese results indicate that miR-18a-5p is involved in VSMC differentiation by targeting syndecan4.

MicroRNA-663 Regulates Human Vascular Smooth Muscle Cell Phenotypic Switch and Vascular Neointimal Formation

Abnormal phenotypic switch of vascular smooth muscle cell (VSMC) is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. MicroRNAs (miRNAs) have emerged as important regulators for VSMC function, and we recently identified miR-663 as critical for controlling human aortic smooth muscle cell proliferation. To investigate whether miR-663 plays a role in human VSMC phenotypic switch and the development of neointima formation. By using quantitative reverse-transcription polymerase chain reaction, we found that miR-663 was significantly downregulated in human aortic VSMCs on platelet-derived growth factor treatment, whereas expression was markedly increased during VSMC differentiation. Furthermore, we demonstrated that overexpression of miR-663 increased expression of VSMC differentiation marker genes, such as smooth muscle 22α, smooth muscle α-actin, calponin, and smooth muscle myosin heavy chain, and potently inhibited platelet-derived growth factor-induced VSMC proliferation and migration. We identified the transcription factor JunB and myosin light chain 9 as downstream targets of miR-663 in human VSMCs, because overexpression of miR-663 markedly inhibited expression of JunB and its downstream molecules, such as myosin light chain 9 and matrix metalloproteinase 9. Finally, we showed that adeno-miR-663 markedly suppressed the neointimal lesion formation by ≈50% in mice after vascular injury induced by carotid artery ligation, specifically via decreased JunB expression. These results indicate that miR-663 is a novel modulator of human VSMC phenotypic switch by targeting JunB/myosin light chain 9 expression. These findings suggest that targeting miR-663 or its specific downstream targets in human VSMCs may represent an attractive approach for the treatment of proliferative vascular diseases.

Abstract 212: MicroRNA-663 is a Novel Regulator of Human Vascular Smooth Muscle Cell Phenotypic Switch in vitro and Neointimal Formation in vivo

Background Abnormal phenotypic switch of vascular smooth muscle cell (VSMC) is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. Recently, microRNAs (miRNAs) emerge as critical regulators for vascular smooth muscle cell (VSMC) function. Our initial study identified miR-663 as one of the most sharply downregulated miRNAs in human proliferative aortic smooth muscle cells. Hypothesis MiR-663 is implicated in human VSMC phenotypic switch and the development of neointima formation. Methods and Results By using quantitative real-time PCR (qRT-PCR), we found that microRNA-663 (miR-663) was significantly downregulated in cultured human aortic VSMCs upon platelet-derived growth factor (PDGF) treatment, whereas its expression was markedly increased during VSMC differentiation as induced by either retinoid acid or SMC differentiation medium, a condition which induces SMC differentiation and inhibits cell proliferation. Furthermore, we demonstrated that overexpression of miR-663 significantly increased the expression of VSMC differentiation marker genes, such as SM22α, SM α-action, calponin, and SM myosin heavy chain, suggesting that miR-663 is a novel modulator implicated in human VSMC phenotypic switch. Moreover, miR-663 potently inhibited PDGF induced VSMC proliferation and migration. Mechanistically, we identified JunB as a downstream target of miR-663 in human VSMCs. Indeed, overexpression of miR-663 markedly inhibited the expression of the transcription factor JunB as well as its downstream molecules including matrix metallopeptidase-9 (MMP-9) and myosin light chain-9 (Myl9), thus inhibiting VSMC proliferation and migration. Finally, we showed that adeno-miR-663 markedly suppressed the neointimal lesion formation by approximately 50% in mice after vascular injury induced by carotid artery ligation, specifically via decreased JunB expression. Conclusion These results indicate that miR-663 is a novel modulator implicated in human VSMC phenotypic switch through targeting JunB expression and suggest that specific modulation of miR-663 in human VSMCs may represent a novel and attractive approach for the treatment of vascular proliferative diseases.

MiRNA Biogenesis Enzyme Drosha Is Required for Vascular Smooth Muscle Cell Survival

miRNA biogenesis enzyme Drosha cleaves double-stranded primary miRNA by interacting with double-stranded RNA binding protein DGCR8 and processes primary miRNA into precursor miRNA to participate in the miRNA biogenesis pathway. The role of Drosha in vascular smooth muscle cells (VSMCs) has not been well addressed. We generated Drosha conditional knockout (cKO) mice by crossing VSMC-specific Cre mice, SM22-Cre, with Drosha loxp/loxp mice. Disruption of Drosha in VSMCs resulted in embryonic lethality at E14.5 with severe liver hemorrhage in mutant embryos. No obvious developmental delay was observed in Drosha cKO embryos. The vascular structure was absent in the yolk sac of Drosha homozygotes at E14.5. Loss of Drosha reduced VSMC proliferation in vitro and in vivo. The VSMC differentiation marker genes, including αSMA, SM22, and CNN1, and endothelial cell marker CD31 were significantly downregulated in Drosha cKO mice compared to controls. ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated in VSMCs in vitro and in vivo. Disruption of Drosha in VSMCs of mice leads to the dysregulation of miRNA expression. Using bioinformatics approach, the interactions between dysregulated miRNAs and their target genes were analyzed. Our data demonstrated that Drosha is required for VSMC survival by targeting multiple signaling pathways.

MicroRNA-31 controls phenotypic modulation of human vascular smooth muscle cells by regulating its target gene cellular repressor of E1A-stimulated genes

Phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of a variety of proliferative vascular diseases. The cellular repressor of E1A-stimulated genes (CREG) has been shown to play an important role in phenotypic modulation of VSMCs. However, the mechanism regulating CREG upstream signaling remains unclear. MicroRNAs (miRNAs) have recently been found to play a critical role in cell differentiation via target-gene regulation. This study aimed to identify a miRNA that binds directly to CREG, and may thus be involved in CREG-mediated VSMC phenotypic modulation. Computational analysis indicated that miR-31 bound to the CREG mRNA 3′ untranslated region (3′-UTR). miR-31 was upregulated in quiescent differentiated VSMCs and downregulated in proliferative cells stimulated by platelet-derived growth factor and serum starvation, demonstrating a negative relationship with the VSMC differentiation marker genes, smooth muscle α-actin, calponin and CREG. Using gain-of-function and loss-of-function approaches, CREG and VSMC differentiation marker gene expression levels were shown to be suppressed by a miR-31 mimic, but increased by a miR-31 inhibitor at both protein and mRNA levels. Notably, miR-31 overexpression or inhibition affected luciferase expression driven by the CREG 3′-UTR containing the miR-31 binding site. Furthermore, miR-31-mediated VSMC phenotypic modulation was inhibited in CREG-knockdown human VSMCs. We also determined miR-31 levels in the serum of patients with coronary artery disease (CAD), with or without in stent restenosis and in healthy controls. miR-31 levels were higher in the serum of CAD patients with restenosis compared to CAD patients without restenosis and in healthy controls. In summary, these data demonstrate that miR-31 not only directly binds to its target gene CREG and modulates the VSMC phenotype through this interaction, but also can be an important biomarker in diseases involving VSMC phenotypic modulation. These novel findings may have extensive implications for the diagnosis and therapy of a variety of proliferative vascular diseases.