Nuclei Of Vascular Smooth Muscle Cells Research Articles

Knockdown of LINC01128 Downregulates FUT8 to Inhibit OxLDL-Induced Vascular Smooth Muscle Cell Apoptosis in Atherosclerosis.

The apoptosis of vascular smooth muscle cells (VSMCs) contributes to the progression of atherosclerosis (AS). Long intergenic non-protein coding RNA 1128 (LINC01128) has been implicated in AS, and this study aims to uncover the role and mechanism of LINC01128 in regulating oxidized low-density lipoprotein (oxLDL)-induced VSMCs. The position of LINC01128 in cells and its target genes were predicted using bioinformatics. The localization of LINC01128 in human VSMCs was determined through fluorescence in situ hybridization. VSMCs were transfected, and the interaction between LINC01128 and fucosyltransferase 8 (FUT8) was validated by chromatin immunoprecipitation assay. The apoptotic VSMC model was established using oxLDL. LINC01128 expression in VSMCs was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), and FUT8 expression was detected by qRT-PCR and western blot. VSMC viability, migration, invasion abilities, and apoptosis were assessed using cell counting kit-8, transwell assay, and flow cytometry, respectively. OxLDL (200 μg/mL) upregulated the expression of LINC01128 and FUT8 mRNA, as well as FUT8 protein, in VSMCs. LINC01128 was expressed in the nucleus of VSMCs and bound to FUT8. Knockdown of LINC01128 alleviated the inhibitory effects of oxLDL (200 μg/mL) on viability, migration, and invasion, and mitigated the promotion of apoptosis and FUT8 expression in VSMCs. On the other hand, FUT8 overexpression enhanced the suppressive effects of oxLDL (200 μg/mL) on viability, migration, and invasion activities, and amplified the facilitating effect of oxLDL on apoptosis in VSMCs. Moreover, FUT8 overexpression reversed the impact of LINC01128 silencing on viability, migration, invasion, and apoptosis in oxLDL-stimulated VSMCs. The knockdown of LINC01128 downregulates FUT8, inhibiting the progression of VSMCs in AS.

Thoracic aortopathy in Marfan syndrome overlaps with mechanisms seen in bicuspid aortic valve disease.

Thoracic aortopathy is a serious complication which is more often seen in patients with Marfan syndrome (MFS) and patients with a bicuspid aortic valve (BAV) than in individuals with a tricuspid aortic valve (TAV). The identification of common pathological mechanisms leading to aortic complications in non-syndromic and syndromic diseases would significantly improve the field of personalized medicine. This study sought to compare thoracic aortopathy between MFS, BAV, and TAV individuals. Bicuspid aortic valve (BAV; n = 36), TAV (n = 23), and MFS (n = 8) patients were included. Ascending aortic wall specimen were studied for general histologic features, apoptosis, markers of cardiovascular ageing, expression of synthetic and contractile vascular smooth muscle cells (VSMC), and fibrillin-1 expression. The MFS group showed many similarities with the dilated BAV. Both patient groups showed a thinner intima (p < 0.0005), a lower expression of contractile VSMCs (p < 0.05), more elastic fiber thinning (p < 0.001), lack of inflammation (p < 0.001), and a decreased progerin expression (p < 0.05) as compared to the TAV. Other features of cardiovascular ageing differed between the BAV and MFS. Dilated BAV patients demonstrated less medial degeneration (p < 0.0001), VSMC nuclei loss (p < 0.0001), apoptosis of the vessel wall (p < 0.03), and elastic fiber fragmentation and disorganization (p < 0.001), as compared to the MFS and dilated TAV. This study showed important similarities in the pathogenesis of thoracic aortic aneurysms in BAV and MFS. These common mechanisms can be further investigated to personalize treatment strategies in non-syndromic and syndromic conditions.

Abstract 12715: Role of Novel Long Non-Coding RNA BAZ1A-AS1 in Neointima Formation in Human Saphenous Vein Graft Disease

Introduction: Saphenous veins (SVs) are frequently used in CABG surgery, but over 50% of the SV grafts fail in the first 10 years after surgery due to neointima formation. Long non-coding RNAs (lncRNAs) have emerged as attractive therapeutic targets and biomarkers for cardiovascular disease. However, studies on their role and mechanisms in humans are limited. This study aimed to unravel epigenetic mechanisms of neointima formation in human SVs and investigate the role of candidate lncRNAs in key vascular cell functions pertinent to neointima formation. Methods and Results: By using an ex vivo model of human SV graft disease wherein segments of human SV are incubated in tissue culture, we performed RNA-sequencing on human SV with and without neointima formation to interrogate the global transcriptomic changes, and determined 699 differentially expressed novel lncRNAs during neointimal formation. We then identified BAZ1A-antisense (AS)1 as a novel lncRNA highly upregulated during neointima formation. BAZ1A-AS1 overlaps antisense to its cis- regulatory gene, BAZ1A , which was reported to play a role in oxidative stress and DNA damage repair response. We found that one isoform of BAZ1A-AS1 primarily localizes to the nucleus of VSMCs. Interestingly, UV exposure of vascular smooth muscle cells (VSMCs) isolated from human SV induced significant upregulation of BAZ1A and BAZ1A-AS1, suggesting that BAZ1A-AS1 could be associated with chromatin repair processes activated during neointima formation. To investigate whether BAZ1A-AS1 can regulate VSMC functions pertinent to neointima formation, VSMCs were transfected with BAZ1A-AS1 antisense gapmers. Knockdown of BAZ1A-AS1 showed reduced cell proliferation and migration, and increased cell death and DNA damage in VSMCs. Furthermore, role of BAZ1A during neointima formation in vivo is currently under investigation in a carotid artery ligation model using BAZ1A knockout mice. Conclusions: We identified BAZ1A-AS1 as a novel lncRNA associated with neointima formation in human SV, and targeted inhibition of BAZ1A-AS1 or its cis-regulator gene BAZ1A could be a promising strategy for SV graft disease.

IL-37 Expression in Patients with Abdominal Aortic Aneurysm and Its Role in the Necroptosis of Vascular Smooth Muscle Cells.

Background Our previous studies have shown that interleukin- (IL-) 37 plays a protective role in patients and animal models with coronary artery disease. However, the role of IL-37 in patients with abdominal aortic aneurysm (AAA), another artery disease, is yet to be elucidated. Methods and Results AAA tissues and plasma samples were obtained from patients with or without surgical intervention. Normal renal aortic tissues were collected from kidney transplant donors. Our findings established that in AAA, IL-37 was distributed in endothelial cells, macrophages, and vascular smooth muscle cells (VSMCs) and that it was chiefly concentrated in VSMCs. Furthermore, the expression was found to be downregulated compared with that in normal artery tissues. Immunofluorescence showed that, unlike normal arteries, IL-37 was translocated to the nucleus of VSMCs in AAA. Moreover, in patients with AAA, the expressions of IL-37, IL-6, and tumor necrosis factor- (TNF-) α were increased in the plasma in comparison with the healthy controls. Correlation analysis revealed that IL-37 was positively correlated with IL-6, TNF-α, age, aneurysm diameter, and blood pressure. Furthermore, human aortic vascular smooth muscle cells (HASMCs) were stimulated with angiotensin II (AngII) in vitro to simulate smooth muscle cell (SMC) damage in AAA. A decrease in IL-37 expression and an increase in receptor-interacting serine/threonine-protein kinase 3 (RIPK3) expression were observed in HASMCs stimulated with AngII. On this basis, inhibition of RIPK3 with GSK'872 significantly attenuated necroptosis. Moreover, the necroptosis rates were significantly lowered in HASMCs treated with recombinant IL-37, whereas the rates were enhanced when the cells were depleted of the interleukin. Immunoblotting results showed that both exogenous and endogenous IL-37 could affect the expressions of RIPK3, NLRP3, and IL-1β. Also, the phosphorylation of RIPK3 and p65 was affected. Meanwhile, IL-37 promoted the transition of SMC from proliferative type to contractile type. Conclusions The expression of IL-37 in VSMCs decreases in patients with AAA, whereas IL-37 supplementation suppresses RIPK3-mediated necroptosis and promotes the transition of VSMCs from proliferative to contractile type.

Lin11-Isl1-Mec3 Domain Proteins as Mechanotransducers in Endothelial and Vascular Smooth Muscle Cells

Arterial hypertension is the leading risk factor for cardiovascular morbidity and mortality worldwide. However, little is known about the cellular mechanisms underlying it. In small arteries and arterioles, a chronic increase in blood pressure raises wall tension and hence stretches, namely, the medial vascular smooth muscle cells (VSMC) but also endothelial cell (EC) to cell contacts. Initially compensated by an increase in vascular tone, the continuous biomechanical strain causes a prominent change in gene expression in both cell types, frequently driving an arterial inward remodeling process that ultimately results in a reduction in lumen diameter, stiffening of the vessel wall, and fixation of blood pressure, namely, diastolic blood pressure, at the elevated level. Sensing and propagation of this supraphysiological stretch into the nucleus of VSMC and EC therefore seems to be a crucial step in the initiation and advancement of hypertension-induced arterial remodeling. Focal adhesions (FA) represent an important interface between the extracellular matrix and Lin11-Isl1-Mec3 (LIM) domain-containing proteins, which can translocate from the FA into the nucleus where they affect gene expression. The varying biomechanical cues to which vascular cells are exposed can thus be rapidly and specifically propagated to the nucleus. Zyxin was the first protein described with such mechanotransducing properties. It comprises 3 C-terminal LIM domains, a leucine-rich nuclear export signal, and N-terminal features that support its association with the actin cytoskeleton. In the cytoplasm, zyxin promotes actin assembly and organization as well as cell motility. In EC, zyxin acts as a transcription factor, whereas in VSMC, it has a less direct effect on mechanosensitive gene expression. In terms of homology and structural features, lipoma preferred partner is the nearest relative of zyxin among the LIM domain proteins. It is almost exclusively expressed by smooth muscle cells in the adult, resides like zyxin at FA but seems to affect mechanosensitive gene expression indirectly, possibly via altering cortical actin dynamics. Here, we highlight what is currently known about the role of these LIM domain proteins in mechanosensing and transduction in vascular cells.

A Loss of Nuclear-Cytoskeletal Interactions in Vascular Smooth Muscle Cell Differentiation Induced by a Micro-Grooved Collagen Substrate Enabling the Modeling of an In Vivo Cell Arrangement.

Vascular smooth muscle cells (VSMCs) remodel vascular walls actively owing to mechanical cues and dedifferentiate to the synthetic phenotype from contractile phenotype in pathological conditions. It is crucial to clarify the mechanisms behind the VSMC phenotypic transition for elucidating their role in the vascular adaptation and repair and for designing engineered tissues. We recently developed novel micro-grooved collagen substrates with “wavy wrinkle” grooves to induce cell–substrate adhesion, morphological polarization, and a tissue-like cell arrangement with cytoskeletal rearrangements similar to those in vascular tissue in vivo. We found that cultivation with this micro-grooved collagen significantly induced VSMC contractile differentiation. Nonetheless, the detailed mechanism underlying the promotion of such VSMC differentiation by micro-grooved collagen has not been clarified yet. Here, we investigated the detailed mechanism of the cell arrangement into a tissue and contractile-differentiation improvement by our micro-grooved collagen substrates in terms of nuclear–cytoskeletal interactions that possibly affect the nuclear mechanotransduction involved in the activation of transcription factors. We found that VSMCs on micro-grooved collagen manifested significant cell arrangement into a tissue and nucleus slimming with a volume reduction in response to the remodeling of the actin cytoskeleton, with consequent inhibition of nuclear shuttling of a transcriptional coactivator, Yes-associated protein (YAP), and improved contractile differentiation. Furthermore, VSMC nuclei rarely deformed during macroscopic cell stretching and featured a loss of nesprin-1–mediated nuclear–cytoskeletal interactions. These results indicate that our micro-grooved collagen induces a cell alignment mimicking in vivo VSMC tissue and promotes contractile differentiation. In such processes of contractile differentiation, mechanical interaction between the nucleus and actin cytoskeleton may diminish to prevent a nuclear disturbance from the excess mechanical stress that might be essential for maintaining vascular functions.

OCT4 regulated neointimal formation in injured mouse arteries by matrix metalloproteinase 2-mediated smooth muscle cells proliferation and migration.

The excessive proliferation and migration of vascular smooth muscle cells (VSMCs) play vital roles in neointimal hyperplasia and vascular restenosis. In the present study, we aimed to investigate the function and mechanism of octamer-binding transcription factor 4 (OCT4, a key transcription factor for maintaining stem cells in de-differentiated state) on neointima formation in response to vascular injury. Quantitative reverse-transcription polymerase chain reaction and western blot results displayed a significant increase of OCT4 levels in injured carotid arteries. Immunohistochemistry and immunofluorescence assays confirmed that the increased OCT4 expression was primarily localized in α-SMA-positive VSMCs from neointima, and colocalized with PCNA in the nuclei of VSMCs. Adenovirus-mediated OCT4 overexpression in injured carotid arteries exacerbated intimal thickening, while OCT4 knockdown significantly inhibited intimal thickening. In-vitro experiments confirmed that the increased OCT4 expression in VMSCs could be induced by platelet-derived growth factor-BB (PDGF-BB) in a time-dependent manner. Overexpression of OCT4 greatly promoted VSMCs proliferation and migration, while OCT4 knockdown significantly retarded the PDGF-BB-induced excessive proliferation and migration of VSMCs. Bioinformatics analysis, dual-luciferase reporter assay, and chromatin immunoprecipitation assay confirmed that OCT4 could upregulate matrix metalloproteinases 2 (MMP2) expression through promoting its transcription. Moreover, knockdown of MMP2 significantly attenuated OCT4-mediated VSMCs proliferation and migration. These results indicated that OCT4 facilitated neointimal formation in response to vascular injury by MMP2-mediated VSMCs proliferation and migration, and targeting OCT4 in VSMCs might be a novel therapeutic strategy for vascular restenosis.

LncRNA HOTAIR regulates the proliferation and apoptosis of vascular smooth muscle cells through targeting miRNA-130b-3p/PPARα axis.

To investigate the role of long non-coding RNA (lncRNA) HOTAIR in influencing the proliferative and apoptotic abilities of vascular smooth muscle cells (VSMCs) by regulating microRNA-130b-3p (miRNA-130b-3p)/peroxisome proliferator-activated receptor alpha (PPARα) axis. The expression levels of HOTAIR, miRNA-130b-3p, and PPARα in VSMCs treated with different doses of ox-LDL for different time points were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The subcellular distribution of HOTAIR in VSMCs was examined. The regulatory effects of HOTAIR, miRNA-130b-3p, and PPARα on the viability and apoptosis of ox-LDL-treated with VSMCs were assessed. The interaction among HOTAIR, miRNA-130b-3p, and PPARα was evaluated through Dual-luciferase reporter gene assay and Western blot. After treatment of different doses of ox-LDL in VSMCs, the levels of HOTAIR and PPARα were gradually downregulated, whereas miRNA-130b-3p was upregulated. The overexpression of HOTAIR reversed the enhanced viability and suppressed apoptosis in ox-LDL-treated VSMCs. HOTAIR was mainly distributed in nucleus of VSMCs. MiRNA-130b-3p was the direct target of HOTAIR and its level was negatively regulated by HOTAIR. Moreover, miRNA-130b-3p could bind to PPARα and could negatively regulate its level. The knockdown of HOTAIR could reverse the regulatory effect of PPARα on the proliferative and apoptotic abilities of VSMCs CONCLUSIONS: HOTAIR reduces the proliferative ability and stimulates the apoptosis in ox-LDL-treated VSMCs by targeting miRNA-130b-3p/PPARα axis.

Nuclear abnormalities in vascular myocytes in cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL).

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a stroke and dementia syndrome with degeneration and loss of vascular smooth muscle cells (VSMCs). The disease is due to mutations in NOTCH3 playing an important role in VSMC differentiation, proliferation and apoptosis. Searching for a possible cause of VSMC dysfunction in CADASIL, we investigated morphology and proliferative activity the affected myocytes. In material from autopsy brains and skin-muscle biopsies of patients with CADASIL diagnosis, assessment of VSMCs in arterial vessels at the level of light and electron microscopy was performed. Proliferative activity of VSMCs was evaluated in immune reactions to proliferative markers: proliferating cell nuclear antigen, and cyclins B1 and D. In CADASIL, abnormal morphology of VSMC nuclei was observed in 18.1%, 11.5%, and 6.9% of the cerebral, skin, and skeletal muscle vessels, respectively. The affected myocytes showed variability in nuclear size, irregularity in nuclear shape, and abnormal chromatin appearance. Frequently, double nuclei of equal size or micronuclei were observed. Sometimes, even multinuclear myocytes were found. In some of the nuclei immune reactions to the examined proliferative markers were positive. Aberrant structure and number of VSCM nuclei, and their immunoreactivity to proliferative markers suggest mitotic instability of vascular myocytes in CADASIL. We speculate that mutant NOTCH3 which is unable to control properly VSMC proliferation, and may be responsible for their premature or inappropriate entry into mitosis, irreversible arrest of the cell cycle, senescence or degeneration and loss.

Characterization of Monocyte Chemoattractant Proteins and CC Chemokine Receptor 2 Expression During Atherogenesis in Apolipoprotein E-null Mice

We aimed to investigate the expression of monocyte chemoattractant proteins (MCPs) and their cognate receptor CC chemokine receptor 2 (CCR2) in aortas of apolipoprotein E-null (apoE(-/-)) mice during atherogenesis as well as the possible transcription pathway involved in the early induction of MCP-1 in vascular smooth muscle cells (VSMCs) in vivo. Atherosclerotic lesion development, aortic MCPs and CCR2 mRNA expression as well as the cellular localization of MCP-1, CCR2 and MCP-1 related transcription factors in atherosclerotic lesions were analyzed in apoE(-/-) mice fed a high fat and cholesterol diet. MCP-1 and CCR2 mRNA expression was significantly induced during early atherogenesis and peaked after 10 and 12 weeks of diet, respectively, whereas MCP-2 and MCP-3 mRNA expression elevated in the late phases of lesion development. Immunostaining revealed that early MCP-1 expression was localized to VSMCs and that, in advanced lesions, both neointimal VSMCs and intimal macrophages expressed high levels of MCP-1. During the early (0 and 4 weeks of diet) induction of MCP-1 in VSMCs, the regulatory activator protein-1 (AP-1) proteins c-Jun and c-Fos were highly expressed and observed within the VSMCs nuclei, whereas nuclear factor-κB (NF-κB) protein p65 was only observed within the nuclei of VSMCs after 4 weeks of diet. CCR2 was also identified on intimal macrophages, endothelial cells and VSMCs in advanced lesions. This study provides fundamental information on the expression kinetics of MCPs and CCR2 during atherogenesis and indicates that the earliest induction of MCP-1 in VSMCs of apoE(-/-)mice appears to correlate with AP-1 but not NF-κB regulatory pathways.

Loss of Clusterin Limits Atherosclerosis in Apolipoprotein E-deficient Mice via Reduced Expression of Egr-1 and TNF-α

Whether clusterin/apolipoprotein J is antiatherogenic or proatherogenic is controversial. We reported that clusterin was markedly induced in media and neointima after vascular injury and that reduced clusterin expression reduced the proliferation of vascular smooth muscle cells (VSMCs), which induced G1 arrest via p53 and p21. The purpose of this study was to investigate the physiological function of clusterin in atherosclerosis using double-knockout mice (D-KO) of apolipoprotein E-deficient mice (apoE-KO) and clusterin-deficient mice (CLU-KO). Atherosclerotic lesions in the aortic root were quantitated at 20 weeks of age. Atherosclerotic lesions of D-KO were significantly smaller than those of apoE-KO (D-KO: 0.176±0.078 mm(2) vs. apoE-KO: 0.365±0.164 mm(2), p< 0.001). To identify underlying atherosclerotic mechanisms that were blocked by loss of clusterin, we performed immunohistochemical analysis of Egr-1. Egr-1 immunoreactivity in the nuclei of VSMCs in atherosclerotic lesions of apoE-KO was upregulated, whereas it was not in D-KO lesions. Western blotting demonstrated that the expression levels of Egr-1 and TNF-α in the D-KO were significantly lower than those in the apoE-KO. When VSMCs and macrophages were obtained from D-KO and apoE-KO, Western blotting showed that the expression levels of Egr-1 and TNF-α in VSMCs and macrophages of D-KO were significantly lower than those of apoE-KO. Loss of clusterin strongly suppressed apoE-KO-induced atherosclerotic lesions at a step prior to the expression of Egr-1 and TNF-α, suggesting that clusterin is a candidate for an antiatherogenic target.

Modulation of Myocardin Function by the Ubiquitin E3 Ligase UBR5

Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of smooth muscle-specific proteins. Although previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, the mechanisms regulating myocardin activity are still poorly understood. We used a yeast two-hybrid screen with myocardin as bait to search for factors that may regulate the transcriptional activity of the myocardin. From this screen we identified a HECT domain-containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin-binding protein. Previous studies have shown that HECT domain-containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has, however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of SMCs and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented the ability of myocardin to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by small interfering RNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in SMCs resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin protein.

Androgen Receptor-dependent Transactivation of Growth Arrest-specific Gene 6 Mediates Inhibitory Effects of Testosterone on Vascular Calcification

Recent epidemiological studies have found that androgen deficiency is associated with a higher incidence of cardiovascular disease in men. However, little is known about the mechanism underlying the cardioprotective effects of androgens. Here we show the inhibitory effects of testosterone on vascular calcification and a critical role of androgen receptor (AR)-dependent transactivation of growth arrest-specific gene 6 (Gas6), a key regulator of inorganic phosphate (P(i))-induced calcification of vascular smooth muscle cells (VSMC). Testosterone and nonaromatizable androgen dihydrotestosterone inhibited P(i)-induced calcification of human aortic VSMC in a concentration-dependent manner. Androgen inhibited P(i)-induced VSMC apoptosis, an essential process for VSMC calcification. The effects on VSMC calcification were mediated by restoration of P(i)-induced down-regulation of Gas6 expression and a subsequent reduction of Akt phosphorylation. These effects of androgen were blocked by an AR antagonist, flutamide, but not by an estrogen receptor antagonist, ICI 182,780. We then explored the mechanistic role of the AR in Gas6 expression and found an abundant expression of AR predominantly in the nucleus of VSMC and two consensus ARE sequences in the Gas6 promoter region. Dihydrotestosterone stimulated Gas6 promoter activity, and this effect was abrogated by flutamide and by AR siRNA. Site-specific mutation revealed that the proximal ARE was essential for androgen-dependent transactivation of Gas6. Furthermore, chromatin immunoprecipitation assays demonstrated ligand-dependent binding of the AR to the proximal ARE of Gas6. These results indicate that AR signaling directly regulates Gas6 transcription, which leads to inhibition of vascular calcification, and provides a mechanistic insight into the cardioprotective action of androgens.

Fibroblast growth factor-2 transiently activates the p53 oncosuppressor protein in human primary vascular smooth muscle cells: Implications for atherogenesis

ObjectiveThe development of atherosclerotic lesions associates with the proliferation of vascular smooth muscle cells (VSMC), and their migration from arterial tunica media to the intima. Fibroblast growth factor (FGF)-2 can trigger either phenomena, which are accompanied by the functional impairment of the p53 transcription factor. However, FGF-2 impact on p53 function in VSMC is largely unknown. Methods and resultsRT-PCR and Western blot analyses assayed FGF-2 effect on human primary VSMC expression of p53-induced molecules with a role in atherogenesis. Confocal microscopy evaluated whether FGF-2 could affect p53 distribution inside VSMC. Results indicate that VSMC exposure to FGF-2 at amounts stimulating the proliferation and migration of these cells promotes p53 phosphorylation and transient accumulation in VSMC nuclei. This is followed by an increase in the expression of the p53-induced thrombospondin (TSP)-1, a VSMC growth and motility factor, and human double minute 2 (HDM2), an antagonist of p53 transcriptional and growth suppressive activity. At later time points, in agreement with the increase of HDM2 and with the capability of this protein to export nuclear p53 to the cytoplasm, the content of p53 in VSMC nuclei is reduced, and the expression of the p53-targeted TSP-l and HDM2 is diminished. ConclusionsSince FGF-2, p53, TSP-1, and HDM2 are expressed in human atherosclerotic lesions, the in vitro effects of FGF-2 described herein may be operative in vivo, providing a molecular mechanism for FGF-2 pro-atherogenic activity.

Matrix metalloproteinase-2 and -9 are associated with high stresses predicted using a nonlinear heterogeneous model of arteries.

Arteries adapt to their mechanical environment by undergoing remodeling of the structural scaffold via the action of matrix metalloproteinases (MMPs). Cell culture studies have shown that stretching vascular smooth muscle cells (VSMCs) positively correlates to the production of MMP-2 and -9. In tissue level studies, the expressions and activations of MMP-2 and -9 are generally higher in the outer media. However, homogeneous mechanical models of arteries predict lower stress and strain in the outer media, which appear inconsistent with experimental findings. The effects of heterogeneity may be important to our understanding of VSMC function since arteries exhibit structural heterogeneity across the wall. We hypothesized that local stresses, computed using a heterogeneous mechanical model of arteries, positively correlate to the levels of MMP-2 and -9 in situ. We developed a model of the arterial wall accounting for nonlinearity, residual strain, anisotropy, and structural heterogeneity. The distributions of elastin and collagen fibers in situ, measured in the media of porcine carotid arteries, showed significant nonuniformities. Anisotropy was represented by the direction of collagen fibers measured by the helical angle of VSMC nuclei. The points at which the collagen fibers became load bearing were computed, assuming a uniform fiber strain and orientation under physiological loading conditions, an assumption motivated by morphological measurements. The distributions of circumferential stresses, computed using both heterogeneous and homogeneous models, were correlated to the distributions of expressions and activations of MMP-2 and -9 in porcine common carotid arteries incubated in an ex vivo perfusion organ culture system under physiological conditions for 48 h. While strains computed using incompressibility were identical in both models, the heterogeneous model, unlike the homogeneous model, predicted higher circumferential stresses in the outer layer correlated to the expressions and activations of MMP-2 and -9. This implies that localized remodeling occurs in the areas of high stress and agrees with results from cell culture studies. The results support the role of mechanical stress in vascular remodeling and the importance of structural heterogeneity in understanding mechanobiological responses.

A novel constitutively active isoform of cGMP‐dependent protein kinase I (PKGI) is localized in the nucleus of vascular smooth muscle cells (VSMC)

Objective: Pediatric pulmonary vascular disease (PVD) is associated with excessive VSMC proliferation. Because recent studies suggest that nitric oxide attenuates PVD and VSMC proliferation by stimulating PKGI activity, the mechanisms affecting PKGI's nuclear localization were investigated.Methods: PKGI was mapped to intracellular compartments using immunologic and biochemical methods and its catalytic activity was determined using a PKGI‐specific peptide substrate. The domain structure of nuclear PKGI was analyzed using antibodies and detailed using Edman‐based protein sequencing.Results: PKGI immunoreactivity was detected in the nuclei of VSMC, where its activity was cGMP‐independent. Immunoblotting revealed that the nuclear PKGI isoform (PKGIγ) is 18 kDa smaller than the cytosolic one and lacks a NH2‐terminal dimerization domain, which tethers it to cytosolic proteins. The PKGI dimerization domain was observed in the perinuclear region where it colocalized primarily with the endoplasmic reticulum. Sequencing of immunopurified endogenous nuclear PKGIγ confirmed the absence of a dimerization domain and revealed that its putative scissile amino acids are not encoded by DNA located near typical RNA splice donor‐acceptor guidance nucleotides.Conclusions: A novel constitutively active isoform of PKGI that appears to result from PKGI proteolytic processing is localized in the nuclei of VSMC.

526 細胞種間における単離細胞核の力学的特性の比較(T10-2 マイクロ・ナノバイオテクノロジー(2):細胞内部の現象を捉える,大会テーマセッション,21世紀地球環境革命の機械工学:人・マイクロナノ・エネルギー・環境)

Local stiffness of cell nuclei isolated from vascular smooth muscle cells (VSMCs) and fibroblasts (FBs) were determined using an atomic force microscope (AFM). Nuclei were chemically isolated and attached to a cover glass in Hanks' balanced salt solution of room temperature. Then, the surface of a nucleus was observed using AFM, and force curves were obtained from the central region (8×8 μm) of the nucleus at the interval of 0.5 μm. From force-indentation curves, the distribution of local stiffness was determined in the central portion (4×4 μm) of the measurement area. Initial stiffness of the nuclei obtained from VSMCs and FBs were 0.43 nN/μm and 0.36 nN/μm, respectively; there was a tendency that the nucleus of VSMCs was stiffer than that of FBs, but the difference was not statistically significant. These results suggest that the difference in cell types does not affect much to the local surface mechanical properties of cell nucleus.

Blockade of Nuclear Factor of Activated T Cells Activation Signaling Suppresses Balloon Injury-induced Neointima Formation in a Rat Carotid Artery Model

We have previously reported that nuclear factor of activated T cells (NFATs) play an important role in the regulation of vascular smooth muscle cell migration and proliferation by receptor tyrosine kinase and G protein-coupled receptor agonists, platelet-derived growth factor-BB and thrombin, respectively. To understand the role of NFATs in vascular disease, we have now studied the involvement of these transcription factors in neointima formation in a rat carotid artery balloon injury model. The levels of NFATc1 in injured right common carotid arteries were increased at 72 h, 1 week, and 2 weeks after balloon injury compared with its levels in uninjured left common carotid arteries. Intraperitoneal injection of cyclosporine A (CsA), a pharmacological inhibitor of the calcineurin-NFAT activation pathway, suppressed balloon injury-induced neointima formation by 40%. Similarly, adenoviral-mediated expression of GFPVIVIT, a competent peptide inhibitor of the calcineurin-NFAT activation pathway, in injured arteries also reduced neointima formation by about 40%. Furthermore, CsA and GFPVIVIT attenuated balloon injury-induced neointimal smooth muscle cell proliferation as determined by bromodeoxyuridine staining. Platelet-derived growth factor-BB induced the expression of COX-2 in cultured VSMC in a time- and NFAT-dependent manner. COX-2 expression was also increased in the right common carotid artery in a time-dependent manner after balloon injury as compared with its levels in uninjured left common carotid artery and both CsA and GFPVIVIT negated this response. Together these results for the first time demonstrate that NFATs play a critical role in neointima formation via induction of expression of COX-2.

Focal adhesion protein zyxin is a mechanosensitive modulator of gene expression in vascular smooth muscle cells.

Excessive deformation of vascular smooth muscle cells (SMCs) caused by a prolonged increase in blood pressure (eg, in hypertension) results in an adaptive remodeling of the vessel wall that is characterized by SMC hypertrophy or hyperplasia and contributes to fixation of the increase in blood pressure. The onset of this process is characterized by a unique change in gene expression in the SMC. However, thus far, no transcription factor has been identified that specifically mediates mechanosensitive gene expression in these cells. Therefore, the role of a putative mechanotransducer, the cytoskeletal protein zyxin, was investigated in rat aortic cultured SMCs. Immunofluorescence and Western blot analysis revealed that on exposure to cyclic stretch, but not to osmotic stress or treatment with proinflammatory cytokines, zyxin dissociates from focal adhesions and accumulates in the nucleus. Unlike zyxin, vinculin, another focal adhesion-associated protein, did not translocate. Moreover, antisense oligonucleotide downregulation of zyxin protein abundance suggested that zyxin accumulation in the nucleus is a prerequisite for mechanosensitive gene expression in these cells. Thus, stretch-induced endothelin B receptor expression, for example, was attenuated, whereas that of tenascin-C was augmented after zyxin suppression. The data are consistent with a role for zyxin in transducing mechanical stimuli from the cell membrane to the nucleus in vascular SMCs and in controlling the expression of mechanosensitive genes that have been implicated in hypertension-induced arterial remodeling.

SHIP-2 and PTEN Are Expressed and Active in Vascular Smooth Muscle Cell Nuclei, but Only SHIP-2 Is Associated with Nuclear Speckles

Recently, the control of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3)-dependant signaling by phosphatases has emerged, but there is a shortage of information on intranuclear PtdIns(3,4,5)P3 phosphatases. Therefore, we investigated the dephosphorylation of [32P]PtdIns(3,4,5)P3 specifically labeled on the D-3 position of the inositol ring in membrane-free nuclei isolated from pig aorta vascular smooth muscle cells (VSMCs). In vitro PtdIns(3,4,5)P3 phosphatase assays revealed the production of both [32P]PtdIns(3,4)P2 and inorganic phosphate, demonstrating the presence of PtdIns(3,4,5)P3 5- and 3-phosphatase activities inside the VSMC nucleus, respectively. Both activities presented the same potency in cellular lysates, whereas the nuclear PtdIns(3,4,5)P3 5-phosphatase activity appeared to be the most efficient. Immunoblot experiments showed for the first time the expression of the 5-phosphatase SHIP-2 (src homology 2 domain-containing inositol phosphatase) as well as the 3-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) in VSMC nuclei. In addition, immunoprecipitations from nuclear fractions indicated a [32P]PtdIns(3,4,5)P3 dephosphorylation by both SHIP-2 and PTEN. Moreover, confocal microscopy analyses demonstrated that SHIP-2 but not PTEN colocalized with a speckle-specific component, the SC35 splicing factor. These results suggest that SHIP-2 may be the primary enzyme for metabolizing PtdIns(3,4,5)P3 into PtdIns(3,4)P2 within the nucleus, thus producing another second messenger, whereas PTEN could down-regulate nuclear phosphoinositide 3-kinase signaling. Finally, intranuclear PtdIns(3,4,5)P3 phosphatases might be involved in the control of VSMC proliferation and the pathogenesis of vascular proliferative disorders.