Response Of Vascular Smooth Muscle Cells Research Articles

C-Reactive Protein Induces TNF-α Secretion by p38 MAPK–TLR4 Signal Pathway in Rat Vascular Smooth Muscle Cells

Atherosclerosis is a chronic inflammatory disease. C-reactive protein (CRP) not only is an inflammatory marker but also regulates the expressions of other inflammatory cytokines associated with the pathogenesis of atherosclerosis. Toll-like receptor 4 (TLR4) also contributes to atherogenesis via transducting inflammatory signals. Herein, our studies focused on characterizing the effect of CRP on tumor necrosis factor α (TNF-α) production and TLR4-related molecular mechanisms in rat vascular smooth muscle cells (VSMCs). The results showed that CRP stimulated VSMCs to secrete TNF-α and enhanced TLR4 expression in a time-concentration-dependent manner. TLR4 knockdown significantly inhibited CRP-induced TNF-α generation, and p38 mitogen-activated protein kinase (MAPK) blocker SB203580 depressed TLR4 expression and TNF-α production initiated by CRP in VSMCs. The data demonstrate that CRP triggers an inflammatory response in rat VSMCs by inducing TNF-α secretion, which is mediated by p38 MAPK-TLR4 signaling pathway.

Underlying mechanism for suppression of vascular smooth muscle cells by green tea polyphenol EGCG released from biodegradable polymers for stent application

Epigallocatechin-3-O-gallate (EGCG), the predominant catechin from tea, is known to exert a variety of cardiovascular beneficial effects by affecting the activity of receptor and signal transduction kinases. In this study, we investigated the suppressive effects of EGCG released from biodegradable poly(L-lactide-co-ε-caprolactone, PLCL) films on the proliferation, cell cycle progression and matrix metalloproteinase-2 (MMP-2) expression of vascular smooth muscle cells (VSMCs). The involvement of phosphorylated Akt (pAkt) and nuclear factor-κB (pNF-κB) as well as the internalization of EGCG into VSMCs was also examined as underlying mechanisms for EGCG-mediated VSMC inhibition. The proliferation of canine aortic SMCs (CASMCs) on EGCG-releasing PLCL (E-PLCL) was significantly inhibited. The culture of CASMCs on E-PLCL resulted in induction of cell cycle arrest at G(0)/G(1) phase and inactivation of pAkt, leading to subsequent apoptosis. Active MMP-2 expression was directly lowered by EGCG released from E-PLCL and indirectly inhibited by the EGCG-mediated suppression of pNF-κB. We also observed the incorporation of fluorescein isothiocyanate-conjugated EGCG into the cytoplasm of CASMCs and its further nuclear translocation, which could lead to the interruption of the exogenous signals directed to genes responsible for cellular responses of CASMCs. Taken together, the attenuated responses of VSMCs to E-PLCL were shown to be mediated through the suppression of pNF-κB, pAkt and each subsequent target genes or proteins by EGCG incorporated into the cells.

IFN-γ Primes Intact Human Coronary Arteries and Cultured Coronary Smooth Muscle Cells to Double-Stranded RNA- and Self-RNA–Induced Inflammatory Responses by Upregulating TLR3 and Melanoma Differentiation-Associated Gene 5

Atherosclerosis of native coronary arteries and graft arteriosclerosis in transplanted hearts are characterized by activation of innate and adaptive immune responses. Nucleic acids generated by infections or cell death have been detected within arteriosclerotic lesions, and it is known that microbial and synthetic nucleic acids evoke inflammatory responses in cultured vascular cells. In this study, we report that model RNA, but not DNA, instigated robust cytokine and chemokine production from intact human coronary arteries containing both intrinsic vascular cells and resident/infiltrating leukocytes. An ssRNA analog induced TNF-alpha and IFN-gamma-induced protein of 10 kDa secretion by isolated human PBMCs, but not vascular cells. Conversely, synthetic dsRNA induced these inflammatory mediators by vascular cells, but not PBMCs. IFN-gamma, a cytokine linked to atherosclerosis and graft arteriosclerosis, potentiated the inflammatory responses of intact arteries and cultured vascular smooth muscle cells (VSMCs) to polyinosinic:polycytidylic acid [poly(I:C)] and was necessary for inflammatory responses of VSMC to self-RNA derived from autologous cells. IFN-gamma also induced the expression of TLR3, melanoma differentiation-associated gene 5, and retinoic acid-inducible gene I dsRNA receptors. Small interfering RNA knockdown revealed that TLR3 mediated VSMC activation by poly(I:C), whereas melanoma differentiation-associated gene 5 was more important for VSMC stimulation by self-RNA. IFN-gamma-mediated induction of dsRNA receptors and priming for inflammatory responses to poly(I:C) was confirmed in vivo using immunodeficient mice bearing human coronary artery grafts. These findings suggest that IFN-gamma, and by inference adaptive immunity, sensitizes the vasculature to innate immune activators, such as RNA, and activation of IFN-gamma-primed vascular cells by exogenous or endogenous sources of RNA may contribute to the inflammatory milieu of arteriosclerosis.

Extracellular matrix effect on RhoA signaling modulation in vascular smooth muscle cells

Morphological adaptations of vascular smooth muscle cells (VSMC) to the mechanically active environment in which they reside, are mediated by direct interactions with the extracellular matrix (ECM) which induces physiological changes at the intracellular level. This study aimed to analyze the effects of the ECM on RhoA-induced mechanical signaling that controls actin organization and focal adhesion formation. VSMC were transfected with RhoA constructs (wild type, dominant negative or constitutively active) and plated on different ECM proteins used as substrate (fibronectin, collagen IV, collagen I, and laminin) or poly- l-lysine as control. Morphological changes of the VSMC were detected by fluorescence confocal microscopy and total internal reflection fluorescence (TIRF) microscopy, and were independently verified using adhesion assays and Western blot analysis. Our results showed that the ECM has an important role in cell spreading, adhesion and morphology with a direct effect on modulating RhoA signaling. RhoA activity significantly affected the stress fibers and focal adhesions reorganization, but in a context imposed by the ECM. Thus, RhoA activity modulation in VSMC induced an increased activation of stress fibers and FA formation at 5 h, while a significant inhibition was recorded at 24 h after plating on the different ECM. Our findings provide biophysical evidence that ECM modulates VSMC response to mechanical stimuli inducing intracellular biochemical signaling involved in cellular adaptation to the local microenvironment.

FRNK overexpression limits the depth and frequency of vascular smooth muscle cell invasion in a three‐dimensional fibrin matrix

Pathological vascular smooth muscle cell (VSMC) behavior after vascular interventions such as angioplasty or bypass is initiated within the 3D environment of the vessel media. Here VSMCs proliferate, invade the surrounding matrix, migrate adluminally, and deposit substantial amounts of matrix, leading to myointimal hyperplasia and decreased blood flow to critical organs and tissue. Since focal adhesion kinase (FAK) mediates many of the VSMC responses to these pathologic events, it provides a reasonable pharmacologic target to limit this invasive VSMC behavior and to better understand the cellular pathophysiology of this disease. Here we quantified the effectiveness of disabling FAK in VSMCs with its dominant-negative inhibitor, FAK-related nonkinase (FRNK), in a clinically relevant 3D assay. We found that FRNK overexpression decreased VSMC invasion (both the length and frequency) in this matrix. These effects were demonstrated in the presence and absence of chemical mitotic inhibition, suggesting that FAK's effect on cellular matrix invasion, migration, and proliferation utilize separate and/or redundant signaling cascades. Mechanistically, FAK inhibition decreased its localization to focal adhesions which led to a significant decrease in FAK autophosphorylation and the phosphorylation of the serine/threonine kinase, AKT. Together these findings suggest that disruption of FAK signaling may provide a pharmaceutical tool that limits pathological VSMC cell behavior.

The mechanism by which RhoA regulates vascular reactivity after hemorrhagic shock in rats

RhoA, an important member of the Rho family of GTPases, has been implicated in many cellular processes. Our pilot study found that RhoA participated in the regulation of vascular reactivity after shock, but the mechanism was incompletely understood. Whether RhoA regulates vascular reactivity through the Rho kinase-myosin light-chain phosphatase (MLCP) and Rac1-p21-activated kinase (PAK)-myosin light-chain kinase (MLCK) signaling pathway needs investigation. With isolated, superior mesenteric arteries from hemorrhagic-shock rats and hypoxia-treated vascular smooth muscle cells (VSMCs), the effects of U-46619 (RhoA agonist) and C3 transferase (RhoA antagonist) on vascular reactivity, and the relationship to the Rho kinase-MLCP and Rac1-PAK-MLCK signaling pathways were observed. The vascular reactivity of the superior mesenteric artery and the contractile response of VSMCs to norepinephrine after prolonged hemorrhagic shock and hypoxia (2 h) were significantly decreased. Activation of RhoA with U-46619 significantly increased shock or hypoxia-induced decreased vascular reactivity. These effects of U-46619 were abolished by Y-27632 (Rho kinase inhibitor) and PDGF (Rac1 stimulator). Y-27632 had a stronger antagonistic effect than PDGF. U-46619 increased the activity of Rho kinase and MLCK, enhanced the phosphorylation of 20-kDa myosin light chain, and decreased the activity of Rac1, PAK, and MLCP in VSMCs after hypoxia. Y-27632-antagonized U-46619 induced the decrease of MLCP activity and the increase of 20-kDa myosin light chain phosphorylation. PDGF-antagonized U-46619 induced decrease of PAK activity and increase of MLCK activity. RhoA has an important role in the regulation of vascular reactivity after hemorrhagic shock. The Rho kinase-MLCP and Rac1-PAK-MLCK signal pathways participate in the regulatory process of RhoA. Rho kinase-MLCP may be the main signaling pathway by which RhoA regulates vascular reactivity.

Il-19 reduces VSMC activation by regulation of mRNA regulatory factor HuR and reduction of mRNA stability

While much is known about the deleterious effects of pro-inflammatory cytokines on development of vascular disease, little is reported on the direct effects of anti-inflammatory cytokines on the vascular smooth muscle cell (VSMC) response to injury. Interleukin-19 (IL-19) is a recently described Th2, anti-inflammatory interleukin. We have previously reported that IL-19 is absent in normal VSMC, but induced in VSMC by inflammatory cytokines and in arteries by injury. IL-19 is anti-proliferative for VSMC. The purpose of this study is to determine the molecular mechanism of these effects. In cultured, primary human VSMC, IL-19 reduces abundance of proliferative and inflammatory gene proteins and mRNA, including Cyclin D1, IL-1β, IL-8, and COX2. IL-19 does not inhibit NF-κB, but does transiently reduce cytoplasmic abundance of the mRNA stability factor HuR. The mRNA stabilizing function of HuR is linked to its phosphorylation and cytoplasmic translocation. IL-19 reduces serine phosphorylation of HuR, and activation of PKCα, a known regulator of HuR translocation. Actinomycin D transcription blockade demonstrates that IL-19 treatment significantly reduces stability of proliferative and inflammatory mRNAs. Knock down of HuR with siRNA also reduces stability of these inflammatory mRNA transcripts. These data indicate that IL-19 has direct effects on VSMC mRNA stability. One potential mechanism whereby IL-19 reduces the VSMC response to injury is by regulation of HuR abundance and cytoplasmic translocation, with a subsequent decrease in mRNA half-life of proliferative and inflammatory mRNA transcripts.

Thrombin promotes proinflammatory phenotype in human vascular smooth muscle cell

Expression of PAR, the thrombin receptor, is elevated in smooth muscle cell-rich areas in atherosclerotic plaques, where the cells change to proinflammatory or synthetic phenotype. In this study we investigated whether thrombin promotes a proinflammatory phenotype in vascular smooth muscle cell (VSMC), characterized by increased cytokine and chemokine synthesis. Thrombin not only elevated transcripts for IL-6, CXCL8, and CCL11 genes but also enhanced release of IL-6 and CXCL8 protein from human aortic smooth muscle cell (HAoSMC). Thrombin activated Akt, PKC and MAPK in HAoSMC, and thrombin-mediated expression of IL-6 and CXCL8 was significantly inhibited by LY294002, AKT IV, RO318220, and GF109203X as well as by diphenyleneiodium at the messenger RNA and the protein levels. SB202129 and U0126 also significantly attenuated thrombin-mediated release of IL-6 and CXCL8 proteins from HAoSMC. These results indicate that thrombin promotes proinflammatory phenotype in human VSMC and that PI3K, Akt, PKC, NADPH oxidase, and MAPK are involved in that process. We propose that activation VSMC in response to thrombin after endothelial injury and/or thrombus formation will enhance inflammation in vasculature.

Insulin-like Growth Factor-I-stimulated Insulin Receptor Substrate-1 Negatively Regulates Src Homology 2 Domain-containing Protein-tyrosine Phosphatase Substrate-1 Function in Vascular Smooth Muscle Cells

Vascular smooth muscle cells maintained in normal (5.6 mm) glucose respond to insulin-like growth factor-I (IGF-I) with increased protein synthesis but do not proliferate. In contrast, hyperglycemia alters responsiveness to IGF-I, resulting in increased SHPS-1 phosphorylation and assembly of a signaling complex that enhances MAPK and phosphatidylinositol 3-kinase pathways. Hyperglycemia also reduces the basal IRS-1 concentration and IGF-I-stimulated IRS-1-linked signaling. To determine if failure to down-regulate IRS-1 alters vascular smooth muscle cell (VSMC) responses to IGF-I, we overexpressed IRS-1 in VSMCs maintained in high glucose. These cultures showed reduced SHPS-1 phosphorylation, transfer of SHP-2 to SHPS-1, and impaired Shc and MAPK phosphorylation and cell proliferation in response to IGF-I. In vitro studies demonstrated that SHPS-1 was a substrate for type I IGF receptor (IGF-IR) and that IRS-1 competitively inhibited SHPS-1 phosphorylation. Exposure of VSMC cultures to a peptide that inhibited IRS-1/IGF-IR interaction showed that IRS-1 binding to IGF-IR impairs SHPS-1 phosphorylation in vivo. IRS-1 also sequestered SHP-2. Expression of an IRS-1 mutant (Y1179F/Y1229F) reduced IRS-1/SHP-2 association, and exposure of cells expressing the mutant to the inhibitory peptide enhanced SHPS-1 phosphorylation and SHP-2 transfer. This result was confirmed by expressing an IRS-1 mutant that had both impaired binding to IGF-IR and to SHP-2 IGF-I increased SHPS-1 phosphorylation, SHP-2 association with SHPS-1, Shc MAPK phosphorylation, and proliferation in cells expressing the mutant. We conclude that IRS-1 is an important factor for maintaining VSMCs in the non-proliferative state and that its down-regulation is a component of the VSMC response to hyperglycemic stress that results in an enhanced response to IGF-I.

Nucleotide oligomerization domain 1 is a dominant pathway for NOS2 induction in vascular smooth muscle cells: comparison with Toll‐like receptor 4 responses in macrophages

Background and purpose:Gram-negative bacteria contain ligands for Toll-like receptor (TLR) 4 and nucleotide oligomerization domain (NOD) 1 receptors. Lipopolysaccharide (LPS) activates TLR4, while peptidoglycan products activate NOD1. Activation of NOD1 by the specific agonist FK565 results in a profound vascular dysfunction and experimental shock in vivo.Experimental approach:Here, we have analysed a number of pharmacological inhibitors to characterize the role of key signalling pathways in the induction of NOS2 following TLR4 or NOD1 activation.Key results:Vascular smooth muscle (VSM) cells expressed NOD1 mRNA and protein, and, after challenge with Escherichia coli or FK565, NOS2 protein and activity were induced. Macrophages had negligible levels of NOD1 and were unaffected by FK565, but responded to E. coli and LPS by releasing increased NO and expression of NOS2 protein. Classic pharmacological inhibitors for NF-κB (SC-514) and mitogen-activated protein kinase (SB203580, PD98059) signalling pathways inhibited responses in both cell types regardless of agonist. While TLR4-mediated responses in macrophages were specifically inhibited by the pan-caspase inhibitor z-VAD-fmk and the PKC inhibitor Gö6976, NOD1-mediated responses in VSM cells were inhibited by the Rip2 inhibitor PP2.Conclusions and implications:Our findings suggest a selective role for NOD1 in VSM cells, and highlight NOD1 as a potential novel therapeutic target for the treatment of vascular inflammation.

Implication of adenylyl cyclase 8 in pathological smooth muscle cell migration occurring in rat and human vascular remodelling

Recently, we discovered on primary cell cultures that adenylyl cyclase type 8 (AC8) was involved in the transition of rat vascular smooth muscle cells (VSMCs) to an inflammatory phenotype. Here we demonstrate, in human vessels displaying early or advanced atherosclerotic lesions, that: (a) only intimal VSMCs strongly express AC8; and (b) very few AC8-positive VSMCs were detected in the medial layer, either in atherosclerotic or healthy arteries. Furthermore, over-expressing AC8 in primary rat VSMC cultures triggered the recolonization of a wounded zone and similar results were obtained in the presence of mitomycin, a potent inhibitor of proliferation. This phenomenon was prevented by silencing AC8. Indeed, in IL-1 beta-treated cells, AC8 silencing halted migration and decreased the matrix-metalloproteinases 2/9 secretion, known to be involved in VSMC migration. In vivo, we showed: (a) a pronounced up-regulation of AC8 expression in highly migrating VSMCs of the injured rat carotid artery; (b) an undetectable AC8 labelling in re-endothelized vessels where neo-intimal thickening had stopped. From our data, we conclude that AC8 expression appears closely linked to the properties developed by VSMCs in atherosclerosis and post-angioplasty neo-intima formation leading to restenosis. In addition, it reinforces the idea that VSMC responses to their cell environment greatly depend on the AC isoforms expressed and attributes a new role for AC8 in these pathological vascular processes.

Cellular signals associated with integrin activation in afferent arteriole

By pulling fibronectin‐coated magnetic beads to activate integrins in renal vascular smooth muscle cells (VSMCs) grown on flexible substratum, it was found that the traction force exerted on the substratum by VSMC remained elevated after the termination of pulling force. These observations showed that activation of α5β1 integrins (receptors for fibronectin in VSMCs) with mechanical force triggered myogenic‐like behavior in VSMCs. We sought to determine whether antibodies known to activate α5β1 integrins can elicit cellular signals related to myogenic response in VSMCs of perfused afferent arterioles. We monitored VSMC [Ca2+]i with fluo‐4 and ROS (reactive oxygen species) production with CM‐H2DCFD using confocal microscopy. ROS has been implicated in signaling events of myogenic response in other vascular beds. α5‐integrin antibody ( HMα5‐1, 10 μg/ml) induced an initial increase of [Ca2+]i ( 124 ± 8 % of baseline, 35 cells/5 vessels), which was followed by multiple Ca2+ spikes. The increase of [Ca2+]i was associated with reduction of luminal diameter. α5‐integrin antibody also increased ROS production, which reached the peak ( 110 ± 3% of baseline, 7 vessels) at about 30 s after the application. Control antibodies did not trigger Ca2+ mobilization and ROS production. Our observations suggested Ca2+ mobilization is the major response when α5β1 integrins were activated in renal VSMCs. Supported by AHA.

Intracellular Ca2+ regulating proteins in vascular smooth muscle cells are altered with type 1 diabetes due to the direct effects of hyperglycemia

BackgroundDiminished calcium (Ca2+) transients in response to physiological agonists have been reported in vascular smooth muscle cells (VSMCs) from diabetic animals. However, the mechanism responsible was unclear.Methodology/Principal FindingsVSMCs from autoimmune type 1 Diabetes Resistant Bio-Breeding (DR-BB) rats and streptozotocin-induced rats were examined for levels and distribution of inositol trisphosphate receptors (IP3R) and the SR Ca2+ pumps (SERCA 2 and 3). Generally, a decrease in IP3R levels and dramatic increase in ryanodine receptor (RyR) levels were noted in the aortic samples from diabetic animals. Redistribution of the specific IP3R subtypes was dependent on the rat model. SERCA 2 was redistributed to a peri-nuclear pattern that was more prominent in the DR-BB diabetic rat aorta than the STZ diabetic rat. The free intracellular Ca2+ in freshly dispersed VSMCs from control and diabetic animals was monitored using ratiometric Ca2+ sensitive fluorophores viewed by confocal microscopy. In control VSMCs, basal fluorescence levels were significantly higher in the nucleus relative to the cytoplasm, while in diabetic VSMCs they were essentially the same. Vasopressin induced a predictable increase in free intracellular Ca2+ in the VSMCs from control rats with a prolonged and significantly blunted response in the diabetic VSMCs. A slow rise in free intracellular Ca2+ in response to thapsigargin, a specific blocker of SERCA was seen in the control VSMCs but was significantly delayed and prolonged in cells from diabetic rats. To determine whether the changes were due to the direct effects of hyperglycemica, experiments were repeated using cultured rat aortic smooth muscle cells (A7r5) grown in hyperglycemic and control conditions. In general, they demonstrated the same changes in protein levels and distribution as well as the blunted Ca2+ responses to vasopressin and thapsigargin as noted in the cells from diabetic animals.Conclusions/SignificanceThis work demonstrates that the previously-reported reduced Ca2+ signaling in VSMCs from diabetic animals is related to decreases and/or redistribution in the IP3R Ca2+ channels and SERCA proteins. These changes can be duplicated in culture with high glucose levels.

Modulation of LPS-mediated Inflammation by Fenofibrate via the TRIF-dependent TLR4 Signaling Pathway in Vascular Smooth Muscle Cells

Lipopolysaccharide (LPS) induced-vascular inflammation plays a central role in vasculitis and atherosclerosis. The stimulation of toll-like receptor 4 (TLR4) by LPS elicits the release of major proinflammatory cytokines that aggravates cardiovascular disorders. Peroxisome proliferator- activated receptor Α (PPARΑ) agonists have been shown to reduce cardiovascular events by controlling lipid metabolism as well as inflammation. However, the role of PPARΑ agonist fenofibrate in modulating LPS-mediated inflammatory responses in vascular smooth muscle cells (VSMCs) remains elusive. The present study demonstrated that fenofibrate exerted a potent anti-inflammatory action through reducing interleckin-1(IL-18), tissue inhibitor of metalloproteinase-1(TIMP-1), TLR4 and enhancing PPARΑ in LPS-stimulated VSMCs. Additionally, treatment of VSMCs with the TLR4 inhibition or TLR4 small-interfering RNA illustrated that the modulatory effects of fenofibrate on LPS-mediated inflammatory responses in VSMCs were reliant on TLR4. Especially, the results suggested that beneficial effects of fenofibrate on LPS-stimulated inflammatory responses in VSMCs were mediated through interference of TLR4 and its downstream signaling components such as Toll-interleckin-1(IL-1) receptor domain- containing adaptor inducing interferon-Β (TRIF), interferon regulatory factor 3 (IRF3) and interferon-gamma inducible protein 10 (IP-10). In conclusion, PPARΑ agonist fenofibrate exerts anti-inflammatory property by antagonizing LPS-mediated inflammatory responses in VSMCs. More importantly, the modulation of the TRIF-dependent signaling pathway (TLR4/TRIF/IRF3/IP-10) might be a useful and novel anti-inflammatory strategy of fenofibrate.

Leptin Inhibits the Proliferation of Vascular Smooth Muscle Cells Induced by Angiotensin II through Nitric Oxide-Dependent Mechanisms

Objective. This study was designed to investigate whether leptin modifies angiotensin (Ang) II-induced proliferation of aortic vascular smooth muscle cells (VSMCs) from 10-week-old male Wistar and spontaneously hypertensive rats (SHR), and the possible role of nitric oxide (NO). Methods. NO and NO synthase (NOS) activity were assessed by the Griess and 3H-arginine/citrulline conversion assays, respectively. Inducible NOS (iNOS) and NADPH oxidase subutnit Nox2 expression was determined by Western-blot. The proliferative responses to Ang II were evaluated through enzymatic methods. Results. Leptin inhibited the Ang II-induced proliferative response of VSMCs from control rats. This inhibitory effect of leptin was abolished by NOS inhibitor, NMMA, and iNOS selective inhibitor, L-NIL, and was not observed in leptin receptor-deficient fa/fa rats. SHR showed increased serum leptin concentrations and lipid peroxidation. Despite a similar leptin-induced iNOS up-regulation, VSMCs from SHR showed an impaired NOS activity and NO production induced by leptin, and an increased basal Nox2 expression. The inhibitory effect of leptin on Ang II-induced VSMC proliferation was attenuated. Conclusion. Leptin blocks the proliferative response to Ang II through NO-dependent mechanisms. The attenuation of this inhibitory effect of leptin in spontaneous hypertension appears to be due to a reduced NO bioavailability in VSMCs.

Abstract 5463: Cyclophilin A Mediates Endothelial Damage and Promotes Recruitment of Inflammatory Cells and Atherosclerosis

Background- Cyclophilin A (CyPA) is a proinflammatory cytokine that is highly expressed in vascular smooth muscle cells (VSMC) in mice and humans and is also secreted from VSMC in response to reactive oxygen species (ROS). Development of atherosclerosis involves endothelial cell (EC) dysfunction and activation, vascular inflammatory cytokine release, macrophage migration/activation, and differentiation into foam cells. Based on involvement of CyPA in ROS signaling, we hypothesized that CyPA expression would be proatherogenic. Methods and Results- Using the ApoE −/− mouse model, we compared atherosclerosis development following genetic deletion of CyPA (ApoE −/− CyPA −/− ) in mice fed a high fat diet for 16 wks. Atherosclerosis was assessed by oil-red O (ORO) staining and was significantly greater in ApoE −/− mice compared with ApoE −/− CyPA −/− mice (% ORO, en face aorta; 19.3±7.5 vs. 8.2±2.0, P <0.01). Immunostaining of aortic cusp revealed significant reduction of CD45 + cells, CD3 + cells, and Mac3 + cells in ApoE −/− CyPA −/− compared with ApoE −/− mice, supporting the role of CyPA in inflammatory cell recruitment. En face aortic TUNEL staining revealed significant reduction of EC apoptosis in ApoE −/− CyPA −/− compared with ApoE −/− mice (count/area; 23.1±5.5 vs. 6.8±3.2, P <0.01). Moreover, ApoE −/− CyPA −/− mice had reduced expression of vascular cellular adhesion molecule 1 (VCAM-1) and increased expression of eNOS compared to ApoE −/− mice, suggesting a key role for CyPA in EC inflammation. These features were not altered by reconstitution of bone marrow cells from ApoE −/− CyPA −/− mice, suggesting that vascular-derived CyPA plays a crucial role in progression of atherosclerosis. Mechanistic experiments revealed that CyPA may be important for MCP-1 secretion from VSMC. These data are consistent with findings in humans where expression of CyPA, migration of inflammatory cells, and adhesion molecule expression are significantly augmented in ruptured atherosclerotic plaque areas of patients with acute coronary syndromes. Conclusion- CyPA is a novel mediator of atherogenesis by promoting EC damage, adhesion molecule expression, and inflammatory cell migration.

Aging increases p16INK4a expression in vascular smooth-muscle cells

Alteration of VSMC (vascular smooth-muscle cell) physiology is associated with the development of atherosclerosis and restenosis. We hypothesize that aging up-regulates the expression of p16INK4a in VSMCs, which may increase the susceptibility of blood vessels to vascular occlusive diseases. Aortic VSMCs were obtained from young and aged mice. Cells from aged mice grew more slowly than those from their younger counterparts. Progression of cell cycle in response to serum stimulation was significantly inhibited in those cells with aging, as determined by FACS after propidium iodide staining. A significant up-regulation of p16INK4a (2.5-fold, P=0.0012) was found in VSMC from aged animals using gene arrays. The up-regulation of this gene was further confirmed by quantitative RT–PCR (reverse transcription–PCR) and Western-blot experiments. Immunostaining for p16INK4a confirmed that aortas from aged mice contained more p16INK4a+ SMA (smooth-muscle cell actin)+ cells than aortas from young animals (26.79±2.45 versus 7.06±1.44, P=0.00027, n=4). In conclusion, we have shown that aging up-regulates the expression of p16INK4a in VSMC in both cultures and arteries. The increase in p16INK4a in the vasculature with aging may modify VSMC's response to post-injury stress and therefore accelerate the development of age-related cardiovascular diseases.

Effects of Cyclic Strain and Growth Factors on Vascular Smooth Muscle Cell Responses

Under physiological and pathological conditions, vascular smooth muscle cells (SMC) are exposed to different biochemical factors and biomechanical forces. Previous studies pertaining to SMC responses have not investigated the effects of both factors on SMCs. Thus, in our research we investigated the combined effects of growth factors like Bfgf (basic fibroblast growth factor), TGF-β (transforming growth factor β) and PDGF (platelet-derived growth factor) along with physiological cyclic strain on SMC responses. Physiological cyclic strain (10% strain) significantly reduced SMC proliferation compared to static controls while addition of growth factors bFGF, TGF-β or PDGF-AB had a positive influence on SMC growth compared to strain alone. Microarray analysis of SMCs exposed to these growth factors and cyclic strain showed that several bioactive genes (vascular endothelial growth factor, epidermal growth factor receptor, etc.) were altered upon exposure. Further work involving biochemical and pathological cyclic strain stimulation will help us better understand the role of cyclic strain and growth factors in vascular functions and development of vascular disorders.

Transcriptional regulation of Pim-1 kinase in vascular smooth muscle cells and its role for proliferation

The Ser/Thr-protein kinase Pim-1 has been discovered as a novel transducer of survival- and cell cycle promoting signals in the hematopoietic cell system. Although its significance for proliferation of vascular smooth muscle cells (VSMC) in vitro and neointima formation in vivo has been suggested recently, the mechanism has barely been characterized. This study aimed to foster the understanding of Pim-1 expression and regulation in murine VSMC in response to factors typically present within the atherosclerotic plaque. While oxidative stress, VEGF-A165 and angiotensin II did not have any effect on Pim-1 expression, VSMC strongly increased (3-fold) Pim-1 mRNA upon stimulation with PDGF(bb), followed by its protein upregulation. Half life of Pim-1 RNA and protein were determined to be 25 min and 6 h, respectively. PDGF(bb) induced a strong, 10-fold increase in BrdU-uptake, a marker of proliferation. This was effectively blocked by either Pim-1-specific inhibitor quercetagetin or adenovirally introduced Pim-1 shRNA. We further identified the signaling pathways linking PDGF(bb) to Pim-1 in VSMC: as expected, we determined transcriptional stimulation of Pim-1 via Janus-activated kinase (Jak), but also an additional pathway involving protein kinase C (PKC) and the mitogen-activated protein kinase Mek1/2. Blockade of Akt signaling did, however, not interfere with Pim-1 upregulation, suggesting an independence of either survival system. PDGF(bb)-induced proliferation of VSMC is partly attributed to transcriptionally upregulated Pim-1 and was assigned to distinct cell signaling. Our findings help to understand the fundamental processes of vasculoproliferative diseases thus opening avenues for its prevention and treatment.

Cell–cell bond modulates vascular smooth muscle cell responsiveness to Angiotensin II

Cell attachment is provided by cell–matrix and cell–cell bonds, and acts as a regulator of vascular smooth muscle cell (VSMC) survival, activity and homeostasis, as well as of VSMCs response to pathogenic stimuli. In this work we elicited an exclusive cell–cell contact by culturing A7r5 VSMCs on agarose-coated wells to form floating cell clusters, and we demonstrated that a steady state with a reduced response to the vasoactive peptide Angiotensin II (ATII) was induced. We found that clustered VSMCs showed subcortical stabilization of β-catenin and Caveolin 1 (Cav1), unlike adherent confluent counterparts. We demonstrated that β-catenin and Cav1 stabilization at the membrane level hampers the molecular cross-talk induced by ATII-activated AT1 receptor (AT1R), thereby impeding the phosphorylation of Cav1 and IGF1R, the NADPH oxidase activity, and counteracting ATII-dependent hypertrophy. Thus, elective cell–cell bond might modulate the proatherogenic activity of ATII, reducing the adverse vascular remodelling associated with AT1R activation.