Vascular Smooth Muscle Cell Growth Research Articles

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit vascular smooth muscle cell proliferation via differential effects on the cell cycle.

Abnormal vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of both atherosclerosis and restenosis. Recent studies suggest that high-dose salicylates, in addition to inhibiting cyclooxygenase activity, exert an antiproliferative effect on VSMC growth both in-vitro and in-vivo. However, whether all non-steroidal anti-inflammatory drugs (NSAIDs) exert similar antiproliferative effects on VSMCs, and do so via a common mechanism of action, remains to be shown. In this study, we demonstrate that the NSAIDs aspirin, sodium salicylate, diclofenac, ibuprofen, indometacin and sulindac induce a dose-dependent inhibition of proliferation in rat A10 VSMCs in the absence of significant cytotoxicity. Flow cytometric analyses showed that exposure of A10 cells to diclofenac, indometacin, ibuprofen and sulindac, in the presence of the mitotic inhibitor, nocodazole, led to a significant G0/G1 arrest. In contrast, the salicylates failed to induce a significant G1 arrest since flow cytometry profiles were not significantly different from control cells. Cyclin A levels were elevated, and hyperphosphorylated p107 was present at significant levels, in salicylate-treated A10 cells, consistent with a post-G1/S block, whereas cyclin A levels were low, and hypophosphorylated p107 was the dominant form, in cells treated with other NSAIDs consistent with a G1 arrest. The ubiquitously expressed cyclin-dependent kinase (CDK) inhibitors, p21 and p27, were increased in all NSAID-treated cells. Our results suggest that diclofenac, indometacin, ibuprofen and sulindac inhibit VSMC proliferation by arresting the cell cycle in the G1 phase, whereas the growth inhibitory effect of salicylates probably affects the late S and/or G2/M phases. Irrespective of mechanism, our results suggest that NSAIDs might be of benefit in the treatment of certain vasculoproliferative disorders.

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.

Nitric Oxide–Dependent Bone Marrow Progenitor Mobilization by Carbon Monoxide Enhances Endothelial Repair After Vascular Injury

Carbon monoxide (CO) has emerged as a vascular homeostatic molecule that prevents balloon angioplasty-induced stenosis via antiproliferative effects on vascular smooth muscle cells. The effects of CO on reendothelialization have not been evaluated. Exposure to CO has diametrically opposite effects on endothelial cell (EC) and vascular smooth muscle cell proliferation in rodent models of carotid injury. In contrast to its effect of blocking vascular smooth muscle cell growth, CO administered as a gas or as a CO-releasing molecule enhances proliferation and motility of ECs in vitro by >50% versus air controls, and in vivo, it accelerates reendothelialization of the denuded artery by day 4 after injury versus day 6 in air-treated animals. CO enhanced EC proliferation via rapid activation of RhoA (Ras homolog gene family, member A), followed by downstream phosphorylation of Akt, endothelial nitric oxide (NO) synthase phosphorylation, and a 60% increase in NO generation by ECs. CO drives cell cycle progression through phosphorylation of retinoblastoma, which is dependent in part on endothelial NO synthase-generated NO. Similarly, endothelial repair in vivo requires NO-dependent mobilization of bone marrow-derived EC progenitors, and CO yielded a 4-fold increase in the number of mobilized green fluorescent protein-Tie2-positive endothelial progenitor cells versus controls, with a corresponding accelerated deposition of differentiated green fluorescent protein-Tie2-positive ECs at the site of injury. CO was ineffective in augmenting EC repair and the ensuing development of intimal hyperplasia in eNOS(-/-) mice. Collectively, the present data demonstrate that CO accelerates EC proliferation and vessel repair in a manner dependent on NO generation and enhanced recruitment of bone marrow-derived endothelial progenitor cells.

Evidence for prostacyclin and cAMP upregulation by bradykinin and insulin-like growth factor 1 in vascular smooth muscle cells

Although bradykinin (BK) and insulin like growth factor-1 (IGF-1) have been shown to modulate the functional and structural integrity of the arterial wall, the cellular mechanisms through which this regulation occurs is still undefined. The present study examined the role of second messenger molecules generated by BK and IGF-1 that could ultimately result in proliferative or antiproliferative signals in vascular smooth muscle cells (VSMC).Activation of BK or IGF-1 receptors stimulated the synthesis and release of prostacyclin (PGI2) leading to increased production of cAMP in VSMC. Inhibition of p42/p44mapk or src kinases prevented the increase in PGI2 and cAMP observed in response to BK or IGF-1, indicating a role for these kinases in the regulation of cPLA2 activity in the VSMC. Inhibition of PKC failed to alter production of PGI2 in response to BK, but further increased both p42/p44mapk activation and the synthesis of PGI2 produced in response to IGF-1. In addition, both BK and IGF-1 significantly induced the expression of c-fos mRNA levels in VSMC, and this effect of BK was accentuated in the presence a cPLA2 inhibitor. Finally, inhibition of cPLA2 activity and/or cyclooxygenase activity enhanced the expression of collagen I mRNA levels in response to BK and IGF-1 stimulation.These findings indicate that the effect of BK or IGF-1 to stimulate VSMC growth is an integrated response to the activation of multiple signaling pathways. Thus, the excessive cell growth that occurs in certain forms of vascular disease could reflect dysfunction in one or more of these pathways.

Abstract 5279: GIT1 is Required for Neointima Formation by Regulating Notch1 Signaling Pathway

Notch1 is a critical regulator of cell fate, angiogenesis and cardiovascular development. Upon ligand (Delta and Serrate/Jagged) binding, Notch1 is proteolytically cleaved by γ -secretase to generate the Notch intracellular domain (N1-ICD). N1-ICD translocates to the nucleus to stimulate target gene expression. Recently it was shown that Notch1 mediates vascular smooth muscle cell (VSMC) proliferation and neointimal formation after vascular injury. G-protein-coupled receptor-kinase-2 interacting protein 1 (GIT1), is a multi-domain scaffold protein, that plays an important role in angiotensin II (AngII) induced Ca(2+)-calmodulin (CaM)-dependent kinase II (CaMKII) activation. It has been shown that AngII stimulates VSMC growth through CamKII-Akt pathway. Activation of Akt increases γ -secretese activity and subsequently increases Notch1 cleavage. Our hypothesis is that knockdown of GIT1 will impair the activation of CamKII-Akt pathway, which will decrease Notch1 cleavage, and subsequently decrease neointima formation. Stimulation of VSMC with AngII time- dependently caused Notch1 cleavage, N1-ICD formation, and Hey1 expression. VSMC treated with AngII and GIT1 siRNA showed dramatic decreases in Notch1 cleavage and Akt activation. Since Notch 1 is important for cell proliferation and survival, knockdown of GIT1 may inhibit cell proliferation and increase cell apoptosis. Serum-stimulated growth was decreased by 60% in GIT1 KO VSMC compared to WT VSMC. Moreover, apoptosis induced by serum derivation in GIT1 KO VSMC increased 2-fold compared with WT VSMC. Cyclin D1, which is a key regulator of cell cycle, was significantly decreased in GIT1 KO VSMC. Finally to confirm the physiological role of GIT1 in VSMCs in vivo, complete carotid ligation was performed in GIT1 WT and KO mice. After carotid ligation, GIT1 expression in vessels of WT mice increased. Intimal and medial hyperplasia after 4 weeks was markedly decreased in GIT1 knockout mice. In conclusion, GIT1 plays a crucial role in VSMC proliferation and survival by regulating Notch1 activation, thereby regulating flow-mediated vascular remodeling and intima formation. This research has received full or partial funding support from the American Heart Association, Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).

Endothelial and Smooth Muscle-derived Neuropilin-like Protein Regulates Platelet-derived Growth Factor Signaling in Human Vascular Smooth Muscle Cells by Modulating Receptor Ubiquitination

Endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is up-regulated in the neointima of remodeling arteries and modulates vascular smooth muscle cell (VSMC) growth. Platelet-derived growth factor (PDGF) is the prototypic growth factor for VSMCs and plays a key role in vascular remodeling. Here, we sought to further define ESDN function in primary human VSMCs. ESDN down-regulation by RNA interference significantly enhanced PDGF-induced VSMC DNA synthesis and migration. This was associated with increased ERK1/2, Src, and PDGF receptor (PDGFR)beta phosphorylation, without altering total PDGFRbeta expression levels. In binding assays, ESDN down-regulation significantly increased (125)I-PDGF maximum binding (B(max)) to PDGF receptors on VSMCs without altering the binding constant (K(d)), raising the possibility that ESDN regulates PDGFR processing. ESDN down-regulation significantly reduced ligand-induced PDGFRbeta ubiquitination. This was associated with a significant reduction in the expression level of c-Cbl, an E3 ubiquitin ligase that ubiquitinylates PDGFRbeta. Thus, ESDN modulates PDGF signaling in VSMCs via regulation of PDGFR surface levels. The ESDN effect is mediated, at least in part, through effects on PDGFRbeta ubiquitination. ESDN may serve as a target for regulating PDGFRbeta signaling in VSMCs.

17β-Estradiol inhibits oleic acid-induced rat VSMC Proliferation and migration by restoring PGC-1α expression

Estrogen shows a vasoprotective role through inhibiting the proliferation and migration of vascular smooth muscle cells (VSMCs). The mechanism underlying the effect of estrogen, however, is not completely understood. Here, we explored the role of peroxisome proliferator-activated receptor-gamma (PPARgamma) coactivator-1alpha (PGC-1alpha) in estrogen-mediated vasoprotection. Firstly, we showed that oleic acid (OA) decreased PGC-1alpha expression while stimulating VSMC proliferation and migration. In contrast, administration of VSMCs with 17beta-estradiol (E(2), 1 or 10nM) significantly restored OA-decreased PGC-1alpha expression, treatment with 10nM E(2) almost completely abolished OA-induced VSMC proliferation and migration. Secondly, by using PGC-1alpha siRNA, the inhibitory effect of E(2) on VSMC growth is strongly reduced via suppressing PGC-1alpha expression, indicating that E(2) may exert its role through restoring PGC-1alpha. Finally, E(2) (10nM) treatment inhibits OA-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, however, suppression of PGC-1alpha expression abolishes this inhibitory effect of E(2). Our findings demonstrate for the first time that in OA-stimulated rat VSMCs, treatment with E(2) (1 or 10nM) diminishes VSMC proliferation and migration via restoring OA-decreased PGC-1alpha expression. This observation offers a novel molecular basis of the vasoprotective effect of estrogen.

Differential regulation of vascular smooth muscle and endothelial cell proliferation in vitro and in vivo by cAMP/PKA-activated p85αPI3K

cAMP inhibits proliferation in most cell types, triggering different and sometimes opposing molecular pathways. p85alpha (phosphatidylinositol 3-kinase regulatory subunit) is phosphorylated by cAMP/PKA in certain cell lineages, but its effects on vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) are unknown. In the present study, we evaluated 1) the role of p85alpha in the integration of cAMP/PKA-dependent signaling on the regulation of VSMC and EC growth in vitro; and 2) the effects of PKA-modified p85alpha on neointimal hyperplasia and endothelial healing after balloon injury in vivo. Plasmid constructs carrying wild-type and PKA-modified p85alpha were employed in VSMCs and ECs in vitro and after balloon injury in rat carotid arteries in vivo. cAMP/PKA reduced VSMC proliferation through p85alpha phosphorylation. Transfected PKA-activated p85alpha binds p21ras, reducing ERK1/2 activation and VSMC proliferation in vitro. In contrast, EC proliferation inhibition by cAMP is independent from PKA modification of p85alpha and ERK1/2 inhibition; indeed, PKA-activated p85alpha did not inhibit per se ERK1/2 activation and proliferation in ECs in vitro. Interestingly, cAMP reduced both VSMC and EC apoptotic death through p85alpha phosphorylation. Accordingly, PKA-activated p85alpha triggered Akt activation, reducing both VSMC and EC apoptosis in vitro. Finally, compared with controls, vascular gene transfer of PKA-activated p85alpha significantly reduced neointimal formation after balloon injury in rats, without inhibiting endothelial regeneration of the injured arterial segment. In conclusions, PKA-activated p85alpha integrates cAMP/PKA signaling differently in VSMCs and ECs. By reducing neointimal hyperplasia without inhibiting endothelial regeneration, it exerts a protective effect against restenosis after balloon injury.

Negative Action of Hepatocyte Growth Factor/c-Met System on Angiotensin II Signaling via Ligand-Dependent Epithelial Growth Factor Receptor Degradation Mechanism in Vascular Smooth Muscle Cells

Neointimal hyperplasia contributes to atherosclerosis and restenosis after percutaneous coronary intervention. Vascular injury in each of these conditions results in the release of mitogenic growth factors and hormones that contribute to pathological vascular smooth muscle cell growth and inflammation. Hepatocyte growth factor (HGF) is known as an antiinflammatory growth factor, although it is downregulated in injured tissue. However, the precise mechanism how HGF reduces inflammation is unclear. To elucidate the mechanism how HGF and its receptor c-Met reduces angiotensin II (Ang II)-induced inflammation. HGF reduced Ang II-induced vascular smooth muscle cell growth and inflammation by controlling translocation of SHIP2 (Src homology domain 2-containing inositol 5'-phosphatase 2), which led to Ang II-dependent degradation of epithelial growth factor receptor. Moreover, the present study also revealed a preventive effect of HGF on atherosclerotic change in an Ang II infusion and cuff HGF transgenic mouse model. These data suggest that the HGF/c-Met system might regulate extrinsic factor signaling that maintains the homeostasis of organs.

Nobiletin, a dietary phytochemical, inhibits vascular smooth muscle cells proliferation via calcium-mediated c-Jun N-terminal kinases pathway

Dietary flavonoids have been shown to reduce risk of cardiovascular disease, but the underlying molecular mechanisms are not known. The objective of this study was to investigate the effect of nobiletin, a dietary phytochemical belonging to polymethoxy flavonoid from the peel of Citrus fruit, on vascular smooth muscle cells (VSMCs) proliferation and its mechanisms. VSMCs proliferation was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) and [ 3H]thymidine incorporation assay. The activity of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinases (MAPK) were determined by western blotting. [Ca 2+] i was measured by laser scanning confocal microscopy. Our results showed that angiotensin II-induced VSMCs proliferation was inhibited by nobiletin. While no effect on ERK1/2 and p38 MAPK, nobiletin markedly inhibited angiotensin II-induced activation of JNK. Anthra[1-9-cd]pyrazol-6(2H)-one (SP600125), an inhibitor of JNK, decreased the [ 3H]thymidine incorporation induced by angiotensin II. Nobiletin also attenuated both the intracellular Ca 2+ mobilization and the extracellular Ca 2+ influx induced by angiotensin II. Furthermore, intracellular Ca 2+ chelation by BAPTA-AM, extracellular Ca 2+ chelation by EGTA or blockade of L-type Ca 2+ channel with verapamil inhibited angiotensin II-induced JNK activation. These findings suggest that the preventing effect of nobiletin on angiotensin II-induced VSMCs proliferation is attributed, in part, to its inhibitory effect on Ca 2+-dependent JNK activation in VSMCs. Thus, inhibition of JNK by nobiletin may imply its usefulness for the treatment of cardiovascular diseases relevant to VSMCs growth.

Growth of fibroblast and vascular smooth muscle cells in fibroin/collagen scaffold

In our recent study, a novel fibroin/collagen scaffold with improved mechanical properties and controllable porous structure was prepared through freeze–drying method. In this research, the cyto-compatibility was further studied, using fibroblast and vascular smooth muscle cells (VSMC) as the model cells. MTT results indicated that the growth of fibroblast and VSMC both further improved in the fibroin/collagen scaffold than in pure fibroin scaffolds. The confocal and SEM results showed that fibroblast cells and VSMCs had better adhesion and spreading properties in the fibroin/collagen scaffolds. Although further studies, such as the extracellular matrix production and the functional gene expression, are necessary to clarify the biocompatibility of the fibroin/collagen scaffolds, the present results indicate that the fibroin/collagen scaffold is a new scaffold material suitable for tissue engineering. On the other hand, the mild and all-aqueous preparation processes also make it possible to embed different growth factors inside the scaffolds to maximize cell functions and the formation of specific tissues.

Sonic Hedgehog Induces Notch Target Gene Expression in Vascular Smooth Muscle Cells via VEGF-A

Notch, VEGF, and components of the Hedgehog (Hh) signaling pathway have been implicated in vascular morphogenesis. The role of Notch in mediating hedgehog control of adult vascular smooth muscle cell (SMC) growth and survival remains unexplored. In cultured SMCs, activation of Hh signaling with recombinant rShh (3.5 mug/mL) or plasmid encoded Shh increased Ptc1 expression, enhanced SMC growth and survival and promoted Hairy-related transcription factor (Hrt) expression while concomitantly increasing VEGF-A levels. These effects were significantly reversed after Hh inhibition with cyclopamine. Shh-induced stimulation of Hrt-3 mRNA and SMC growth and survival was attenuated after inhibition of Notch-mediated CBF-1/RBP-Jk-dependent signaling with RPMS-1 while siRNA knockdown of Hrt-3 inhibited SMC growth and survival. Recombinant VEGF-A increased Hrt-3 mRNA levels while siRNA knockdown abolished rShh stimulated VEGF-A expression while concomitantly inhibiting Shh-induced increases in Hrt-3 mRNA levels, proliferating cell nuclear antigen (PCNA), and Notch 1 IC expression, respectively. Hedgehog components were expressed within intimal SMCs of murine carotid arteries after vascular injury concomitant with a significant increase in mRNA for Ptc1, Gli(2), VEGF-A, Notch 1, and Hrts. Hedgehog promotes a coordinate regulation of Notch target genes in adult SMCs via VEGF-A.

The tight junction protein ZO-2 mediates proliferation of vascular smooth muscle cells via regulation of Stat1

Recent evidence suggests that the zonula occludens protein 2 (ZO-2) might have additional cellular functions, beyond regulation of paracellular permeability of epithelial and endothelial cells. Deregulation of ZO-2 in response to ischaemia, hypertensive stress, and vascular injury implies its involvement in cardiovascular disorders, most likely via regulating the functional behaviour of vascular smooth muscle cells (VSMC). However, a role of ZO-2 in VSMC biology has yet to be established. Our study was designed to understand the specific functions of ZO-2 in human VSMC. The expression of ZO-2 and Stat1 upon vascular injury was studied using ex vivo organ culture of coronary arteries combined with immunohistochemistry. ZO-2 silencing in human primary VSMC was achieved by means of lentiviral gene transfer. Cell proliferation was assessed by analysing DNA synthesis and by cell counting. Stat1 expression was examined using immunoblotting, immunocytochemistry, TaqMan, and fluorescence activated cell sorting (FACS) analysis. Functional relevance of Stat1 up-regulation was studied using a Stat1 promoter-luciferase reporter assay and intracellular microinjections of a Stat1 specific antibody. ZO-2 was highly expressed in the media and neointima of dilated but not of control arteries, whereas expression of the transcription factor Stat1 was inversely regulated upon injury. Analysis of VSMC with down-regulated ZO-2 revealed increased expression of Stat1 in these cells, whereas Stat1 phosphorylation was not affected. Stat1 up-regulation in VSMC with ZO-2 silencing resulted in a coordinate activation of Stat1-specific genes and consequently led to inhibition of cell proliferation. This effect was restored by microinjection of a Stat1 neutralising antibody. Our data suggest that the tight junction protein ZO-2 is involved in regulation of VSMC growth control upon vascular injury that is mediated by the transcription factor Stat1. Our findings point to a novel function of ZO-2 in VSMC and implicate ZO-2 as a novel important molecular target in pathological states of vascular remodelling in cardiovascular diseases.

Calcineurin-NFAT signaling is involved in phenylephrine-induced vascular smooth muscle cell proliferation

Catecholamine-induced vascular smooth muscle cell (VSMC) proliferation is one of the major events in the pathogenesis of atherosclerosis and vascular remodeling. The calcineurin-NFAT pathway plays a role in regulating growth and differentiation in various cell types. We investigated whether the calcineurin-NFAT pathway was involved in the regulation of phenylephrine-induced VSMC proliferation. Proliferation of VSMC was measured using an MTT assay and cell counts. Localization of NFATc1 was detected by immunofluorescence staining. NFATc1-DNA binding was determined by EMSA and luciferase activity analyses. NFATc1 and calcineurin levels were assayed by immunoprecipitation. Phenylephrine (PE, an alpha(1)-adrenoceptor agonist) increased VSMC proliferation and cell number. Prazosin (an alpha(1)-adrenoceptor antagonist), cyclosporin A (CsA, an inhibitor of calcineurin) and chelerythrine (an inhibitor of PKC) decreased PE-induced proliferation and cell number. Additional treatment of VSMC with CsA or chelerythrine further inhibited proliferation and cell number in the chelerythrine-pretreatment group and the CsA-pretreatment group. CsA and chelerythrine alone had no effect on either absorbance or cell number. CsA decreased PE-induced calcineurin levels and activity. NFATc1 was translocated from the cytoplasm to the nucleus upon treatment with PE. This translocation was reversed by CsA. CsA decreased the PE-induced NFATc1 level in the nucleus. PE increased NFAT's DNA binding activity and NFAT-dependent reporter gene expression. CsA blocked these effects. CsA partially suppresses PE-induced VSMC proliferation by inhibiting calcineurin activity and NFATc1 nuclear translocation. The calcineurin-NFATc1 pathway is involved in the hyperplastic growth of VSMC induced by phenylephrine.

CREB-Mediated IL-6 Expression Is Required for 15(S)-Hydroxyeicosatetraenoic Acid–Induced Vascular Smooth Muscle Cell Migration

Migration of vascular smooth muscle cells (VSMCs) from media to intima is a key event in the pathophysiology of atherosclerosis and restenosis. The lipoxygenase products of polyunsaturated fatty acids (PUFA) were shown to play a role in these diseases. cAMP response element binding protein (CREB) has been implicated in the regulation of VSMC growth and motility in response to thrombin and angiotensin II. The aim of the present study was to test the role of CREB in an oxidized lipid molecule, 15(S)-HETE-induced VSMC migration and neointima formation. 15(S)-HETE stimulated VSMC migration in CREB-dependent manner, as measured by the modified Boyden chamber method. Blockade of MEK1, JNK1, or p38MAPK inhibited 15(S)-HETE-induced CREB phosphorylation and VSMC migration. 15(S)-HETE induced expression and secretion of interleukin-6 (IL-6), as analyzed by RT-PCR and ELISA, respectively. Neutralizing anti-IL-6 antibodies blocked 15(S)-HETE-induced VSMC migration. Dominant-negative mutant-mediated blockade of ERK1/2, JNK1, p38MAPK, or CREB suppressed 15(S)-HETE-induced IL-6 expression in VSMCs. Serial 5' deletions and site-directed mutagenesis of IL-6 promoter along with chromatin immunoprecipitation using anti-CREB antibodies showed that cAMP response element is essential for 15(S)-HETE-induced IL-6 expression. Dominant-negative CREB also suppressed balloon injury-induced IL-6 expression, SMC migration from media to intimal region, and neointima formation. Adenovirus-mediated transduction of 15-lipoxygenase 2 (15-LOX2) caused increased production of 15-HETE in VSMCs and enhanced IL-6 expression, SMC migration from media to intimal region, and neointima formation in response to arterial injury. The above results suggest a role for 15-LOX2-15-HETE in the regulation of VSMC migration and neointima formation involving CREB-mediated IL-6 expression.

Physiologic Stimulation of Bladder Smooth Muscle Cell Growth is Mediated by Mammalial Target of Rapamycin (Mtor)

Purpose Maladaptive bladder muscle overgrowth in human bladder dysfunction is instigated by coordinate responses to mechanical strain, tissue hypoxia, and extracellular matrix (ECM) remodeling. Though mTOR's role in vascular smooth muscle cell (SMC) growth and cardiomyocyte hypertrophy is known, mTOR's role in bladder SMC (BSMC) hypertrophy and hyperplasia is unknown. Rapamycin, a specific inhibitor of mTOR, induces differentiation of vascular SMC, and reverses overgrowth of SMC in vitro and in vivo. Our purpose was to study rapamycin's ability to reverse the de-differentiated phenotype of BSMC during injury. Material and Methods BSMCs were slowly ramped up to 5% mechanical strain on flexible collagen membranes to mimic bladder distension in vivo. A humidified hypoxia chamber was used for BSMC culture at 3%O2 to simulate local tissue hypoxia. To simulate altered ECM induced by stretch and hypoxia, denatured collagen (DNC) was used as substrate and stimulus and compared to native fibrillar collagen. Proliferation was assessed by 3H-thymidine incorporation or cell counting. Real-time PCR and immunofluorescent staining for smooth muscle actin (SMA) assessed BSMC differentiation. Transfections were performed with HA-S6 K wildtype and constutively active mutants. Westerns for proteins of the mTOR pathway were also performed. Results Rapamycin significantly inhibited BSMC proliferation in response to hypoxia (p = 0.0001), DNC, and strain (p = 0.0001) while maintaining basal growth. Strain induced a significant decrease in SMA mRNA, which was restored by rapamycin. Similarly, rapamycin restored a spindle-like contractile morphology in BSMC on DNC. Many components of the mTOR pathway increased in activity post-strain, including S6 K and S6. Forced expression of S6 K resulted in decreased SMA immunofluorescence, as compared to untransfected neighbouring cells. Conclusions Rapamycin inhibition of BSMC growth induced by three coordinate stimuli, which define hypertrophic bladder dysfunction (stretch, hypoxia and altered ECM), demonstrates the potential of mTOR and S6 K as targets of therapy for maladaptive BSMC hyperplasia and hypertrophy.

CREB‐MEDIATED IL‐6 EXPRESSION IS REQUIRED FOR 15(S)‐HYDROXYEICOSATETRAENOIC ACID‐INDUCED VASCULAR SMOOTH MUSCLE CELL MIGRATION

Migration of vascular smooth muscle cells (VSMC) from media to intima is a key event in the pathophysiology of atherosclerosis and restenosis. Cyclic AMP response element binding protein (CREB) has been implicated in the regulation of VSMC growth and motility in response to thrombin and angiotensin II. In this study, we tested the role of CREB in an oxidized lipid molecule, 15(S)‐HETE‐induced VSMC migration and injury‐induced restenosis. 15(S)‐HETE stimulated VSMC migration in a CREB‐dependent manner, as measured by the modified Boyden chamber method. Blockade of MEK1, JNK1 or p38MAPK inhibited 15(S)‐HETE‐induced CREB phosphorylation and VSMC migration. 15(S)‐HETE induced expression and secretion of interleukin‐6 (IL‐6), as analyzed by RT‐PCR and ELISA, respectively. Neutralizing anti‐IL‐6 antibodies blocked 15(S)‐HETE‐induced VSMC migration. Adenoviral mediated expression of dominant negative MEK1, JNK1, p38MAPK or CREB suppressed 15(S)‐HETE induced IL‐6 expression in VSMC. Serial 5′‐deletions and site‐directed mutagenesis analysis in an IL‐6 promoter‐dependent luciferase reporter system showed that cAMP response element is essential for 15(S)‐HETE‐induced IL‐6 expression. Dominant negative CREB also suppressed balloon injury‐induced IL‐6 expression and neointima formation. Thus, these results suggest a role for MAPK‐dependent CREB‐mediated IL‐6 expression in 15(S)‐HETE‐induced VSMC migration. These findings also suggest a role for CREB in balloon injury‐induced IL‐6 expression and neointima formation.

Cytocompatibility of Ar+ plasma treated and Au nanoparticle-grafted PE

Polyethylene (PE) was irradiated with inert Ar plasma, and the chemically active PE surface was grafted with Au nanoparticles. The composition and the structure of the modified PE surface were studied using X-ray photoelectron spectroscopy (XPS) and Rutherford backscattering spectroscopy (RBS). Changes in the surface wettability were determined from the contact angle measured in a reflection goniometer. The changes in the surface roughness and morphology were followed by atomic force microscopy (AFM). The modified PE samples were seeded with rat vascular smooth muscle cells (VSMC) or mouse NIH 3T3 fibroblasts, and their adhesion and proliferation were studied. We found that plasma discharge and Au grafting lead to dramatic changes in the surface morphology and roughness of PE. The Au nanoparticles were found not only on the sample surface, but also in the sample interior up to the depth of about 100nm. In addition, plasma modification of the PE surface, followed with grafting Au-nanoparticles, significantly increased the attractiveness of the PE surface for the adhesion and growth of VSMC, and particularly for mouse embryonic 3T3 fibroblasts.

Atrophin Proteins Interact with the Fat1 Cadherin and Regulate Migration and Orientation in Vascular Smooth Muscle Cells

Fat1, an atypical cadherin induced robustly after arterial injury, has significant effects on mammalian vascular smooth muscle cell (VSMC) growth and migration. The related Drosophila protein Fat interacts genetically and physically with Atrophin, a protein essential for development and control of cell polarity. We hypothesized that interactions between Fat1 and mammalian Atrophin (Atr) proteins might contribute to Fat1 effects on VSMCs. Like Fat1, mammalian Atr expression increased after arterial injury and in VSMCs stimulated with growth and chemotactic factors including angiotensin II, basic fibroblast growth factor, and platelet-derived growth factor BB. Two distinct Atr2 transcripts, atr2L and atr2S, were identified by Northern analysis; in VSMCs, atr2S mRNA expression was more responsive to stimuli. By immunocytochemistry, Fat1 and Atrs colocalized at cell-cell junctions, in the perinuclear area, and in the nucleus. In coimmunoprecipitation studies, Fat1 interacted with both Atr1 and Atr2; these interactions required Fat1 amino acids 4300-4400 and an intact Atro-box in the Atrs. Knock-down of Atrs by small interfering RNA did not affect VSMC growth but had complex effects on migration, which was impaired by Atr1 knockdown, enhanced by Atr2L knockdown, and unchanged when both Atr2S and Atr2L were depleted. Enhanced migration caused by Atr2L knockdown required Fat1 expression. Similarly, orientation of cells after monolayer denudation was impaired in cells with Atr1 knockdown but enhanced in cells selectively depleted of Atr2L. Together these findings suggest that Fat1 and Atrs act in concert after vascular injury but show further that distinct Atr isoforms have disparate effects on VSMC directional migration.

Sulfasalazine induces haem oxygenase-1 via ROS-dependent Nrf2 signalling, leading to control of neointimal hyperplasia

Inflammation, and the subsequent proliferative activity of vascular smooth muscle cells (VSMCs), is one of the major pathophysiological mechanisms associated with neointimal hyperplasia following vascular injury. Although sulfasalazine (SSZ) has been used as an anti-inflammatory and immune-modulatory agent in various inflammatory diseases, its primary targets and therapeutic effects on vascular disease have not yet been determined. We investigated whether SSZ could suppress VSMC growth and prevent neointimal hyperplasia. SSZ was found to have pro-apoptotic and anti-proliferative activity in cultured VSMCs. Unexpectedly, these effects were not mediated by nuclear factor kappa B (NF-kappaB) inhibition, which has been suggested to be the anti-inflammatory mechanism associated with the effects of SSZ. Instead, cell-cycle arrest of the VSMCs was observed, which was mediated by induction of haem oxygenase-1 (HO-1) followed by an increased expression of p21(waf1/Cip1). The underlying mechanism for SSZ-induced HO-1 expression was by reactive oxygen species (ROS)-dependent nuclear translocation and activation of nuclear factor erythroid-2-related factor 2 (Nrf2). In a rat carotid artery balloon injury model, administration of SSZ significantly suppressed neointimal growth. In a series of reverse experiments, inhibition of HO-1 by shRNA, ROS by N-acetylcysteine (NAC) or Nrf2 by dominant-negative Nrf2 abrogated the beneficial effects of SSZ. Our data demonstrate that SSZ inhibits VSMC proliferation in vitro and in vivo through a novel signalling pathway and may be a promising therapeutic option for the treatment of proliferative vascular disease.