Vascular Smooth Muscle Cell Survival Research Articles

Role of EZH2-mediated epigenetic modification on vascular smooth muscle in cardiovascular diseases: A mini-review.

Vascular smooth muscle cells (VSMCs) are integral to the pathophysiology of cardiovascular diseases (CVDs). Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, plays a crucial role in epigenetic regulation of VSMCs gene expression. Emerging researches suggest that EZH2 has a dual role in VSMCs, contingent on the pathological context of specific CVDs. This mini-review synthesizes the current knowledge on the mechanisms by which EZH2 regulates VSMC proliferation, migration and survival in the context of CVDs. The goal is to underscore the potential of EZH2 as a therapeutic target for CVDs treatment. Modulating EZH2 and its associated epigenetic pathways in VSMCs could potentially ameliorate vascular remodeling, a key factor in the progression of many CVDs. Despite the promising outlook, further investigation is warranted to elucidate the epigenetic mechanisms mediated by EZH2 in VSMCs, which may pave the way for novel epigenetic therapies for conditions such as atherosclerosis and hypertension.

Carrageenan maintains the contractile phenotype of vascular smooth muscle cells by increasing macromolecular crowding in vitro

BackgroundThe contractile phenotype of vascular smooth muscle cells (VSMCs) results in good diastolic and contractile capacities, and its altered function is the main pathophysiological basis for diseases such as hypertension. VSMCs exist as a synthetic phenotype in vitro, making it challenging to maintain a contractile phenotype for research. It is widely recognized that the common medium in vitro is significantly less crowded than in the in vivo environment. Additionally, VSMCs have a heightened sense for detecting changes in medium crowding. However, it is unclear whether macromolecular crowding (MMC) helps maintain the VSMCs contractile phenotype.PurposeThis study aimed to explore the phenotypic, behavioral and gene expression changes of VSMCs after increasing the crowding degree by adding carrageenan (CR).MethodsThe degree of medium crowding was examined by a dynamic light scattering assay; VSMCs survival and activity were examined by calcein/PI cell activity and toxicity and CCK-8 assays; VSMCs phenotypes and migration were examined by WB and wound healing assays; and gene expression was examined by transcriptomic analysis and RT-qPCR.ResultsNotably, 225 μg/mL CR significantly increased the crowding degree of the medium and did not affect cell survival. Simultaneously, CR significantly promoted the contraction phenotypic marker expression in VSMCs, shortened cell length, decreased cell proliferation, and inhibited cell migration. CR significantly altered gene expression in VSMCs. Specifically, 856 genes were upregulated and 1207 genes were downregulated. These alterations primarily affect the cellular ion channel transport, microtubule movement, respiratory metabolism, amino acid transport, and extracellular matrix synthesis. The upregulated genes were primarily involved in the cytoskeleton and contraction processes of VSMCs, whereas the downregulated genes were mainly involved in extracellular matrix synthesis.ConclusionsThe in vitro study showed that VSMCs can maintain the contractile phenotype by sensing changes in the crowding of the culture environment, which can be maintained by adding CR.Graphical

G6PD maintains the VSMC synthetic phenotype and accelerates vascular neointimal hyperplasia by inhibiting the VDAC1-Bax-mediated mitochondrial apoptosis pathway.

Glucose-6-phosphate dehydrogenase (G6PD) plays an important role in vascular smooth muscle cell (VSMC) phenotypic switching, which is an early pathogenic event in various vascular remodeling diseases (VRDs). However, the underlying mechanism is not fully understood. An IP‒LC‒MS/MS assay was conducted to identify new binding partners of G6PD involved in the regulation of VSMC phenotypic switching under platelet-derived growth factor-BB (PDGF-BB) stimulation. Co-IP, GST pull-down, and immunofluorescence colocalization were employed to clarify the interaction between G6PD and voltage-dependent anion-selective channel protein 1 (VDAC1). The molecular mechanisms involved were elucidated by examining the interaction between VDAC1 and apoptosis-related biomarkers, as well as the oligomerization state of VDAC1. The G6PD level was significantly elevated and positively correlated with the synthetic characteristics of VSMCs induced by PDGF-BB. We identified VDAC1 as a novel G6PD-interacting molecule essential for apoptosis. Specifically, the G6PD-NTD region was found to predominantly contribute to this interaction. G6PD promotes VSMC survival and accelerates vascular neointimal hyperplasia by inhibiting VSMC apoptosis. Mechanistically, G6PD interacts with VDAC1 upon stimulation with PDGF-BB. By competing with Bax for VDAC1 binding, G6PD reduces VDAC1 oligomerization and counteracts VDAC1-Bax-mediated apoptosis, thereby accelerating neointimal hyperplasia. Our study showed that the G6PD-VDAC1-Bax axis is a vital switch in VSMC apoptosis and is essential for VSMC phenotypic switching and neointimal hyperplasia, providing mechanistic insight into early VRDs.

Role of FOXM1 in vascular smooth muscle cell survival and neointima formation following vascular injury

BackgroundAccelerated smooth muscle cell (SMC) proliferation is the primary cause of intimal hyperplasia (IH) following vascular interventions. Forkhead Box M1 (FOXM1) is considered a proliferation-associated transcription factor. However, the presence and role of FOXM1 in IH following vascular injury have not been determined. ObjectiveWe examined the expression of FOXM1 in balloon-injured rat carotid arteries and investigated the effect of FOXM1 inhibition in SMCs and on the development of IH. Methods and resultsFOXM1 was detected by immunofluorescent staining in balloon-injured rat carotid arteries where we observed an upregulation at day 7, 14, and 28 compared to uninjured controls. Immunofluorescence staining revealed FOXM1 coincided with proliferating cell nuclear antigen (PCNA). FOXM1 was also detectable in human carotid plaque samples. Western blot showed an upregulation of FOXM1 protein in serum-stimulated SMCs. Inhibition of FOXM1 using siRNA or chemical inhibition led to the induction of apoptosis as measured by flow cytometry and western blot for cleaved caspase 3. Perturbations in survival signaling were measured by western blot following FOXM1 inhibition, which showed a decrease in phosphorylated AKT and β-catenin. The chemical inhibitor thiostrepton was delivered by intraperitoneal injection in rats that underwent balloon injury and led to reduced intimal thickening compared to DMSO controls. ConclusionsFOXM1 is an important molecular mediator of IH that contributes to the proliferation and survival of SMCs following vascular injury.

Cyclodextrin Prevents Abdominal Aortic Aneurysm via Activation of Vascular Smooth Muscle Cell Transcription Factor EB.

Abdominal aortic aneurysm (AAA) is a severe aortic disease with a high mortality rate in the event of rupture. Pharmacological therapy is needed to inhibit AAA expansion and prevent aneurysm rupture. Transcription factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis, is critical to maintain cell homeostasis. In this study, we aim to investigate the role of vascular smooth muscle cell (VSMC) TFEB in the development of AAA and establish TFEB as a novel target to treat AAA. The expression of TFEB was measured in human and mouse aortic aneurysm samples. We used loss/gain-of-function approaches to understand the role of TFEB in VSMC survival and explored the underlying mechanisms through transcriptome and functional studies. Using VSMC-selective Tfeb knockout mice and different mouse AAA models, we determined the role of VSMC TFEB and a TFEB activator in AAA in vivo. We found that TFEB is downregulated in both human and mouse aortic aneurysm lesions. TFEB potently inhibits apoptosis in VSMCs, and transcriptome analysis revealed that TFEB regulates apoptotic signaling pathways, especially apoptosis inhibitor B-cell lymphoma 2. B-cell lymphoma 2 is significantly upregulated by TFEB and is required for TFEB to inhibit VSMC apoptosis. We consistently observed that TFEB deficiency increases VSMC apoptosis and promotes AAA formation in different mouse AAA models. Furthermore, we demonstrated that 2-hydroxypropyl-β-cyclodextrin, a clinical agent used to enhance the solubility of drugs, activates TFEB and inhibits AAA formation and progression in mice. Last, we found that 2-hydroxypropyl-β-cyclodextrin inhibits AAA in a VSMC TFEB-dependent manner in mouse models. Our study demonstrated that TFEB protects against VSMC apoptosis and AAA. TFEB activation by 2-hydroxypropyl-β-cyclodextrin may be a promising therapeutic strategy for the prevention and treatment of AAA.

Altered mitochondrial quality control in autophagy-deficient vascular smooth muscle cells is linked to high-risk atherosclerotic plaque phenotype

Atherosclerosis is one of the most common causes of stroke worldwide. Intracranial or extracranial atherosclerosis may occur concomitantly with systemic atherosclerosis involving other arterial beds, such as coronary, or peripheral arteries. A major mechanism of stroke, involves unstable plaque as described in the coronary model. Vascular smooth muscle cells (VSMCs) regulate various aspects of vessel homeostasis, including contraction, dilation, and vessel remodeling. Consequently, VSMCs are one of the main cellular determinants in arterial pathology and several evidence indicates that apoptosis of VSMCs is associated with features of high-risk/vulnerable atherosclerotic lesions. Mitochondrial turnover is an essential aspect of the mitochondrial quality control in which dysfunctional mitochondria are selectively eliminated through autophagy. Even though successful autophagy promotes VSMC survival, it is unclear whether reduced autophagic flux affects mitochondrial quality control of VSMCs in atherosclerotic plaques. By using a mouse model of atherosclerosis carrying a VSMC-specific deletion of the essential autophagy gene Atg7, we observed that impaired VSMC autophagy promotes an unstable plaque phenotype as well as the accumulation of fragmented mitochondria with reduced bioenergetic efficiency and more oxidative stress. Furthermore, we demonstrate that disrupted autophagic flux is linked to defective mitophagy and biogenesis of mitochondria, which exacerbate the apoptosis of VSMCs and plaque vulnerability. Overall, our results indicate that mitochondrial quality control could be a promising therapeutic target to stabilize atherosclerotic plaques and to decrease the risk of stroke.

The Multifunctional Sorting Protein PACS-2 Controls Mitophagosome Formation in Human Vascular Smooth Muscle Cells through Mitochondria-ER Contact Sites.

Mitochondria-associated ER membranes (MAMs) are crucial for lipid transport and synthesis, calcium exchange, and mitochondrial functions, and they also act as signaling platforms. These contact sites also play a critical role in the decision between autophagy and apoptosis with far reaching implications for cell fate. Vascular smooth muscle cell (VSMC) apoptosis accelerates atherogenesis and the progression of advanced lesions, leading to atherosclerotic plaque vulnerability and medial degeneration. Though the successful autophagy of damaged mitochondria promotes VSMC survival against pro-apoptotic atherogenic stressors, it is unknown whether MAMs are involved in VSMC mitophagy processes. Here, we investigated the role of the multifunctional MAM protein phosphofurin acidic cluster sorting protein 2 (PACS-2) in regulating VSMC survival following a challenge by atherogenic lipids. Using high-resolution confocal microscopy and proximity ligation assays, we found an increase in MAM contacts as in PACS-2-associated MAMs upon stimulation with atherogenic lipids. Correspondingly, the disruption of MAM contacts by PACS-2 knockdown impaired mitophagosome formation and mitophagy, thus potentiating VSMC apoptosis. In conclusion, our data shed new light on the significance of the MAM modulatory protein PACS-2 in vascular cell physiopathology and suggest MAMs may be a new target to modulate VSMC fate and favor atherosclerotic plaque stability.

LncRNA GAS5 regulates vascular smooth muscle cell cycle arrest and apoptosis via p53 pathway

Vascular remodeling is a pathological process following cardiovascular intervention. Vascular smooth muscle cells (VSMC) play a critical role in the vascular remodeling. Long noncoding RNAs (lncRNA) are a class of gene regulators functioning through various mechanisms in physiological and pathological conditions. By using cultured VSMC and rat carotid artery balloon injury model, we found that lncRNA growth arrest specific 5 (GAS5) serves as a negative regulator for VSMC survival in vascular remodeling. By manipulating GAS5 expression via adenoviral overexpression or short hairpin RNA knockdown, we found that GAS5 suppresses VSMC proliferation while promoting cell cycle arrest and inducing cell apoptosis. Mechanistically, GAS5 directly binds to p53 and p300, stabilizes p53-p300 interaction, and thus regulates VSMC cell survival via induction of p53-downstream target genes. Importantly, local delivery of GAS5 via adenoviral vector suppresses balloon injury-induced neointima formation along with an increased expression of p53 and apoptosis in neointimal SMCs. Our study demonstrated for the first time that GAS5 negatively impacts VSMC survival via activation the p53 pathway during vascular remodeling.

UNK cell-derived TGF-β1 regulates the long noncoding RNA MEG3 to control vascular smooth muscle cell migration and apoptosis in spiral artery remodeling.

Successful pregnancy depends on correct spiral artery (SpA) remodeling, and thus, on normal patterns of the vascular smooth muscle cell (VSMC) apoptosis and migration. Uterine natural killer (uNK) cells-derived transforming growth factor β1 (TGF-β1) is known to mediate the separation of VSMC layers via as yet unknown mechanisms. Likewise, the long noncoding RNA maternally expressed gene 3 (MEG3) is a tumor suppressor that has been shown to regulate cancer cell apoptosis and migration; however, its role in VSMC loss is unclear. Thus, the aim of the present study was to assess the effects of uNK-derived TGF-β1 and MEG3 on VSMC function during SpA. Analyses were conducted to assess the effects of downregulating MEG3 expression, and/or administering treatments to increase or block TGF-β1 signaling on VSMC survival and behavior. The results of these analyses showed that treating the VSMC with uNK cell-derived supernatant or recombinant human TGF-β1 promoted MEG3 and matrix metalloprotease 2 expression and VSMC apoptosis and migration, and suppressed VSMC proliferation. Conversely, MEG3 silencing promoted VSMC proliferation and inhibited VSMC apoptosis and migration. Notably, TGF-β1 signaling induction had no significant effect on the proliferation, apoptosis, nor migration of the MEG3-silenced VSMC. Together, these findings suggest that MEG3 is regulated by uNK-derived TGF-β1, and itself mediates VSMC apoptosis and migration; thus, it may be an important positive regulator of VSMCs separation during maternal SpA remodeling.

Promotion of PTEN on apoptosis through PI3K/Akt signal in vascular smooth muscle cells of mice model of coronary heart disease.

Previous studies have shown that phosphatase and tensin homolog (PTEN) are key regulators of the development of many malignant tumors and other diseases. However, its regulatory effect on coronary heart disease (CHD) has rarely been reported. Therefore, the regulatory effect of PTEN on the survival and cell death of vascular smooth muscle cells (VSMCs) in CHD mice was elucidated in this study. It was found that the protein and messenger RNA expressions of PTEN in VSMCs of 10 CHD mice were lower than those of normal mice. Then PTEN was overexpressed in VSMCs. It was suggested that the upregulation of PTEN was not conducive to the proliferation and survival of VSMCs in the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assay. The flow cytometry (Annexin V-Fluorescein isothiocyanate (FITC)/propidium iodide) and the terminal deoxynucleotidyl transferase dUTP nick end labeling assay were used to detect the apoptotic rate of overexpressing PTEN cells. Some data showed that the expression of PTEN could lead to increased apoptotic rate. It was shown that antiapoptotic Bcl-2 levels were decreased, but cleaved caspase-3 and proapoptotic Bax levels were promoted by SIRT6 overexpression in Western blot analysis. Moreover, PI3K/Akt expression and phosphorylation were significantly decreased in cells expressing PTEN. Recovery of PI3K expression inhibited the suppressive influence of PTEN on VSMC survival, as evidenced by the activated PI3K/Akt pathway, increased cell proliferative rate, reduced the apoptotic level, and reversed expression patterns of Bcl-2 and Bax. Therefore, the findings in this study provide a new idea on the occurrence and development mechanism of CHD and may promote the discovery of innovative therapies.

Optimization of Electrospun Poly(caprolactone) Fiber Diameter for Vascular Scaffolds to Maximize Smooth Muscle Cell Infiltration and Phenotype Modulation.

Due to the morphological resemblance between the electrospun nanofibers and extracellular matrix (ECM), electrospun fibers have been widely used to fabricate scaffolds for tissue regeneration. Relationships between scaffold morphologies and cells are cell type dependent. In this study, we sought to determine an optimum electrospun fiber diameter for human vascular smooth muscle cell (VSMC) regeneration in vascular scaffolds. Scaffolds were produced using poly(caprolactone) (PCL) electrospun fiber diameters of 0.5, 0.7, 1, 2, 2.5, 5, 7 or 10 μm, and VSMC survivals, proliferations, infiltrations, and phenotypes were recorded after culturing cells on these scaffolds for one, four, seven, or 10 days. VSMC phenotypes and macrophage infiltrations into scaffolds were evaluated by implanting scaffolds subcutaneously in a mouse for seven, 14, or 28 days. We found that human VSMC survival was not dependent on the electrospun fiber diameter. In summary, increasing fiber diameter reduced VSMC proliferation, increased VSMC infiltration and increased macrophage infiltration and activation. Our results indicate that electrospun PCL fiber diameters of 7 or 10 µm are optimum in terms of VSMC infiltration and macrophage infiltration and activation, albeit at the expense of VSMC proliferation.

Altered mitochondrial quality control in Atg7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype

Vascular smooth muscle cells (VSMCs) are one of the main cellular determinants in arterial pathology. A large body of evidence indicates that death of VSMCs is associated with features of high-risk/vulnerable atherosclerotic plaques. Mitochondrial turnover is an essential aspect of the mitochondrial quality control in which dysfunctional mitochondria are selectively eliminated through autophagy and replaced through expansion of preexisting mitochondria. Even though successful autophagy promotes VSMC survival, it is unclear whether reduced autophagic flux affects mitochondrial quality control of VSMCs in atherosclerotic plaques. By using apolipoprotein E-deficient (ApoE−/−) mice carrying a VSMC-specific deletion of the essential autophagy gene Atg7, we show in the present study that impaired VSMC autophagy promotes an unstable plaque phenotype, as well as the accumulation of fragmented mitochondria with reduced bioenergetic efficiency and more oxidative stress. Furthermore, we demonstrate that disrupted autophagic flux is linked to defective mitophagy and biogenesis of mitochondria, which exacerbate VSMC apoptosis and in turn plaque vulnerability. Overall, our data indicate that mitochondrial quality control is a promising therapeutic target to stabilize atherosclerotic plaques.

Aging differentially modulates the Wnt pro-survival signalling pathways in vascular smooth muscle cells.

We previously reported pro‐survival effects of Wnt3a and Wnt5a proteins in vascular smooth muscle cells (VSMCs). Wnt5a achieved this through induction of Wnt1‐inducible signalling pathway protein‐1 (WISP‐1) consequent to β‐catenin/CREB‐dependent, TCF‐independent, signalling. However, we found that as atherosclerosis advances, although Wnt5a protein was increased, WISP‐1 was reduced. We hypothesized this disconnect could be due to aging. In this study, we elucidate the mechanism underlying Wnt3a pro‐survival signalling and demonstrate the differential effect of age on Wnt3a‐ and Wnt5a‐mediated survival. We show Wnt3a protein was expressed in human atherosclerotic coronary arteries and co‐located with macrophages and VSMCs. Meanwhile, Wnt3a stimulation of primary mouse VSMCs increased β‐catenin nuclear translocation and TCF, but not CREB, activation. Wnt3a increased mRNA expression of the pro‐survival factor WISP‐2 in a TCF‐dependent manner. Functionally, β‐catenin/TCF inhibition or WISP‐2 neutralization significantly impaired Wnt3a‐mediated VSMC survival. WISP‐2 was upregulated in human atherosclerosis and partly co‐localized with Wnt3a. The pro‐survival action of Wnt3a was effective in VSMCs from young (2 month) and old (18–20 month) mice, whereas Wnt5a‐mediated rescue was impaired with age. Further investigation revealed that although Wnt5a induced β‐catenin nuclear translocation in VSMCs from both ages, CREB phosphorylation and WISP‐1 upregulation did not occur in old VSMCs. Unlike Wnt5a, pro‐survival Wnt3a signalling involves β‐catenin/TCF and WISP‐2. While Wnt3a‐mediated survival was unchanged with age, Wnt5a‐mediated survival was lost due to impaired CREB activation and WISP‐1 regulation. Greater understanding of the effect of age on Wnt signalling may identify targets to promote VSMC survival in elderly patients with atherosclerosis.

Abstract 505: Targeting FOXM1 in Vascular Smooth Muscle Cells Induces Apoptotic Cell Death

Objectives: Vascular smooth muscle cell (VSMC) proliferation and dedifferentiation are key contributors to the initiation and progression of vascular diseases including restenosis. Forkhead Box M1 (FOXM1) is a proliferation-associated transcription factor shown to play a role in a variety of biological processes including cell cycle progression, cell survival, and apoptosis in many cell types. However, the role of FOXM1 in VSMC phenotypic transformation as a result of vascular injury following balloon angioplasty has not been studied. We hypothesize that FOXM1 modulates VSMC response by enhancing pro-proliferative and pro-survival signaling following vascular injury. Methods & Results: The rat carotid artery balloon injury was used to model vascular injury. Immunofluorescence staining was carried out on sections from injured carotid arteries or uninjured controls collected at 3, 7, or 14 days post injury (dpi). We observed that FOXM1 expression was upregulated in injured arteries at 7 and 14 dpi. Expression of FOXM1 in injured arteries co-localized with PCNA, a marker of proliferation. The upregulation of FOXM1 protein was replicated in cultured rat VSMCs by serum stimulation. Using thymidine synchronization, we observed that FOXM1 expression followed a cell cycle specific pattern in vitro . Chemical inhibition of FOXM1 using thiostrepton or FDI-6, or via siRNA resulted in decreased VSMC viability as measured by CCK-8, induction of apoptosis as measured by flow cytometry, and cleaved caspase 3 induction. More detailed immunocytochemistry analysis revealed that FDI-6 treated VSMCs exhibit disorganized microtubule networks. Furthermore, FDI-6 treatment decreased expression of Eg5, a motor protein essential for microtubule orientation and critical for mitosis execution. Additionally, FOXM1 inhibition decreased levels of β-catenin, a known activator of cell cycle-related genes and proliferation. Conclusions: In conclusion, our data suggest FOXM1 is critical to VSMC viability and may modulate VSMC proliferation and survival via regulation of important cell cycle and pro-proliferative proteins, and therefore may serve as a novel therapeutic target to prevent VSMC pathophysiology in the context of restenosis.

Vascular smooth muscle cell death, autophagy and senescence in atherosclerosis.

In the present review, we describe the causes and consequences of loss of vascular smooth muscle cells (VSMCs) or their function in advanced atherosclerotic plaques and discuss possible mechanisms such as cell death or senescence, and induction of autophagy to promote cell survival. We also highlight the potential use of pharmacological modulators of these processes to limit plaque progression and/or improve plaque stability. VSMCs play a pivotal role in atherogenesis. Loss of VSMCs via initiation of cell death leads to fibrous cap thinning and promotes necrotic core formation and calcification. VSMC apoptosis is induced by pro-inflammatory cytokines, oxidized low density lipoprotein, high levels of nitric oxide and mechanical injury. Apoptotic VSMCs are characterized by a thickened basal lamina surrounding the cytoplasmic remnants of the VSMC. Inefficient clearance of apoptotic VSMCs results in secondary necrosis and subsequent inflammation. A critical determinant in the VSMC stress response and phenotypic switching is autophagy, which is activated by various stimuli, including reactive oxygen and lipid species, cytokines, growth factors and metabolic stress. Successful autophagy stimulates VSMC survival, whereas reduced autophagy promotes age-related changes in the vasculature. Recently, an interesting link between autophagy and VSMC senescence has been uncovered. Defective VSMC autophagy accelerates not only the development of stress-induced premature senescence but also atherogenesis, albeit without worsening plaque stability. VSMC senescence in atherosclerosis is likely a result of replicative senescence and/or stress-induced premature senescence in response to DNA damaging and/or oxidative stress-inducing stimuli. The finding that VSMC senescence can promote atherosclerosis further illustrates that normal, adequate VSMC function is crucial in protecting the vessel wall against atherosclerosis.

Loss of SPRR3 in ApoE-/- mice leads to atheroma vulnerability through Akt dependent and independent effects in VSMCs.

Vascular smooth muscle cells (VSMCs) represent important modulators of plaque stability in advanced lesions. We previously reported that loss of small proline-rich repeat protein 3 (Sprr3), leads to VSMC apoptosis in a PI3K/Akt-dependent manner and accelerates lesion progression. Here, we investigated the role of Sprr3 in modulating plaque stability in hyperlipidemic ApoE-/- mice. We show that loss of Sprr3 increased necrotic core size and reduced cap collagen content of atheromas in brachiocephalic arteries with evidence of plaque rupture and development of intraluminal thrombi. Moreover, Sprr3-/-ApoE-/- mice developed advanced coronary artery lesions accompanied by intraplaque hemorrhage and left ventricle microinfarcts. SPRR3 is known to reduce VSMC survival in lesions by promoting their apoptosis. In addition, we demonstrated that Sprr3-/- VSMCs displayed reduced expression of procollagen in a PI3K/Akt dependent manner. SPRR3 loss also increased MMP gelatinase activity in lesions, and increased MMP2 expression, migration and contraction of VSMCs independently of PI3K/Akt. Consequently, Sprr3 represents the first described VSMC modulator of each of the critical features of cap stability, including VSMC numbers, collagen type I synthesis, and protease activity through Akt dependent and independent pathways.

Abstract 95: MiRNA-210 Enhances Fibrous Cap Stability in Advanced Atherosclerotic Lesions

In the search for markers and modulators of vascular disease, miRNAs have emerged as potent therapeutic targets. We investigated miRNAs of clinical interest in patients with unstable carotid stenosis at risk of stroke. Utilizing patient material from the Biobank of Karolinska Endarterectomies (BiKE), we profiled miRNA expression in symptomatic versus asymptomatic patients with high-grade carotid artery stenosis. A PCR-based miRNA of plasma, sampled at the carotid lesion site, identified eight deregulated miRNAs (miR-15b, -29c, -30c/d, -150, -191, -210 and -500). miR-210 was the most significantly downregulated miRNA in local plasma material. Laser-capture microdissection as well as in situ hybridization revealed a distinct localization of miR-210 in the fibrous caps of atherosclerotic lesions and showed reduced miR-210 expression in the unstable fibrous cap. We confirmed that miR-210 directly targets the tumor suppressor gene adenomatous polyposis coli (APC), thereby affecting Wnt signaling and regulating vascular smooth muscle cell survival, as well as differentiation, in advanced atherosclerotic lesions. Substantial changes in arterial miR-210 were detectable in two rodent models of vascular remodeling and plaque rupture. Modulating miR-210 in vitro and in vivo improved fibrous cap stability with implications for vascular disease. We discovered that an unstable carotid plaque at risk of stroke is characterized by low expression of miR-210. miR-210 contributes to stabilizing carotid plaques through inhibition of APC, ensuring vascular smooth muscle cell survival. We present local delivery of miR-210 as a therapeutic approach for prevention of atherothrombotic disease.

Vitamin K2 inhibits rat vascular smooth muscle cell calcification by restoring the Gas6/Axl/Akt anti-apoptotic pathway

Vascular calcification is associated with cardiovascular disease as a complication of hypertension, hyperlipidemia, diabetes mellitus, and chronic kidney disease. Vitamin K2 (VK2) delays vascular calcification by an unclear mechanism. Moreover, apoptosis modulates vascular smooth muscle cell (VSMC) calcification. This paper aimed to study VK2-modified VSMC calcification and survival cell signaling mediated by growth arrest-specific gene 6 (Gas6) and its tyrosine kinase receptor Axl. Primary-cultured VSMCs were dose-dependently treated with VK2 in the presence of calcification medium for 8days, or pre-treated for 1h with/without the Axl inhibitor R428 (2μmol/L) or the caspase inhibitor Z-VAD-fmk (20μmol/L) followed by treatment with VK2 (10μmol/L) or rmGas6 (200nmol/L) in calcification medium for 8days. Calcium deposition was determined by the o-cresolphthalein complexone assay and Alizarin Red S staining. Apoptosis was determined by TUNEL and flow cytometry using Annexin V-FITC and propidium iodide staining. Western blotting detected the expressions of Axl, Gas6, p-Akt, Akt, and Bcl2. VK2 significantly inhibited CaCl2- and β-sodium glycerophosphate (β-GP)-induced VSMC calcification and apoptosis, which was dependent on restored Gas6 expression and activated downstream signaling by Axl, p-Akt, and Bcl2. Z-VAD-fmk significantly inhibited CaCl2- and β-GP-induced VSMC calcification and apoptosis. Augmented recombinant mouse Gas6 protein (rmGas6) expression significantly reduced VSMC calcification and apoptosis. Furthermore, the Gas6/Axl interaction was inhibited by R428, which abolished the preventive effect of VK2 on CaCl2- and β-GP-induced apoptosis and calcification. These results suggest that Gas6 is critical in VK2-mediated functions that attenuate CaCl2- and β-GP-induced VSMC calcification by blocking apoptosis.

The microRNA miR‐124 inhibits vascular smooth muscle cell proliferation by targeting S100 calcium‐binding protein A4 (S100A4)

S100 calcium-binding protein A4 (S100A4) induces proliferation and migration of vascular smooth muscle cells (VSMCs). We aimed to find the microRNA regulating S100A4 expression. S100A4 transcripts are abruptly increased in the acute phase of carotid arterial injury 1 day later (at day 1) but gradually decreases at days 7 and 14. Bioinformatics analysis reveals that miR-124 targets S100A4. VSMC survival is attenuated by miR-124 mimic but increased by miR-124 inhibitor. miR-124 decreases immediately after carotid arterial injury but dramatically increases at days 7 and 14. miR-124 inhibitor-induced cell proliferation is blocked by S100A4 siRNA, whereas miR-124-induced cell death is recovered by S100A4. Our findings suggest that miR-124 is a novel regulator of VSMC proliferation and may play a role in the development of neointimal proliferation.

A highly versatile adaptor protein for the tethering of growth factors to gelatin-based biomaterials.

In an effort to functionalize collagen/gelatin-based biomaterials with growth factors, we have designed an adaptor protein corresponding to a collagen-binding domain fused to a coil peptide. In our strategy, this adaptor protein captures growth factors being tagged with the partner coil peptide in a specific, stable and oriented manner. We have found that the tethering of the Epidermal Growth Factor preserved its mitogenic and anti-apoptotic activity. In the case of the basic Fibroblast Growth Factor, the captured growth factor remained bioactive although its tethering via this adaptor protein modified its natural mode of interaction with gelatin. Altogether this strategy is easily adaptable to the simultaneous tethering of various growth factors.