Vascular Smooth Muscle Cell Chemotaxis Research Articles

Intraluminal Delivery of Simvastatin Attenuates Intimal Hyperplasia After Arterial Injury

Oral statins reduce intimal hyperplasia (IH) after arterial injury by only ∼25%. Alternative drug delivery systems have gained attention as carriers for hydrophobic drugs. We studied the effects of simvastatin (free vs hyaluronic acid-tagged polysialic acid-polycaprolactone micelles) on vascular smooth muscle cell (VSMC) migration, VSMC proliferation and intimal hyperplasia. We hypothesized both free and micelle containing simvastatin would inhibit VSMC chemotaxis and proliferation, and local statin treatment would be more effective than oral in reducing IH in rats following carotid balloon injury. VSMCs pretreated with free simvastatin (20 minutes or 20 hours) or simvastatin-loaded micelles underwent chemotaxis and proliferation to platelet-derived growth factor. Next, rats that underwent balloon injury of the common carotid artery received statin therapy-intraluminal simvastatin-loaded micelles prior to injury, periadventitial pluronic gel following injury, or combinations of gel, micelle, and oral simvastatin. After 14 days, morphometric analysis determined the -intimal to medial ratio. Findings were compared to controls receiving oral simvastatin or no statin therapy. Statistical analysis was by analysis of variance for the in vitro experiments and a factorial general linear model for the in vivo experiments. The simvastatin-loaded micelles and free simvastatin inhibited VSMC chemotaxis (54%-60%). IH was induced in all injured vessels. Simvastatin in pluronic gel or micelles reduced IH compared to untreated controls (0.208 ± 0.04 or 0.160 ± 0.03 vs 0.350 ± 0.03, respectively); however, neither gel nor simvastatin-loaded micelles were superior to oral statins (0.261 ± 0.03). Addition of oral statins or combining both local therapies did not provide additional benefit. Micelles were the single greatest contributing factor in IH attenuation. Intraluminally or topically delivered statins reduced IH. The efficacy of single-dose, locally delivered statin alone may lead to novel treatments to prevent IH. The different routes of administration may allow for treatment during endovascular procedures, without the need for systemic therapy.

Dyslipidemia regulates thrombospondin-1-induced vascular smooth muscle cell chemotaxis.

Dyslipidemia is a risk factor for intimal hyperplasia (IH). Key to IH is vascular smooth muscle cell (VSMC) migration. Thrombospondin-1 (TSP-1) is a matricellular protein that stimulates VSMC migration. HDL will inhibit and LDL will augment TSP-1-induced VSMC chemotaxis. VSMC chemotaxis will be inhibited by the HDL moiety, S1P, through the S1PR1 receptor, and augmented by the LDL component, LPA, through the LPAR1 receptor. The goal of this study was to determine the effect of HDL and LDL and their receptors on TSP-1-induced VSMC chemotaxis. For VSMC chemotaxis to TSP-1 cells received the following pretreatments: low (25 µg/ml) or optimal (75 µg/ml) concentration of HDL, S1P, optimal (75 µg/ml) or high (175 µg/ml) concentration of LDL, or LPA. For the receptor studies, VSMCs were transfected with siRNA to S1PR1, S1PR3, LPAR1, LPAR2, LPAR3, or a S1PR2 receptor antagonist. The TSP-1-induced chemotaxis results were (1) HDL (25 µg/ml) or LDL (75 µg/ml) exhibited no effect on chemotaxis; (2) HDL (75 µg/ml) inhibited chemotaxis by 50.9 ± 8 % and S1P by 43.4 ± 11.6 %; (3) LDL (175 µg/ml) augmented chemotaxis by 30 ± 10.4 % and LPA by 25.6 ± 12.3 %; (4) S1PR1 and S1PR3 knockdown and S1PR2 antagonist-treated cells augmented chemotaxis; and (5) LPAR1 and LPAR2 knockdown inhibited and LPAR3 knockdown had no effect on chemotaxis. In conclusion, HDL/S1P inhibits, while LDL/LPA stimulates TSP-1-induced VSMC chemotaxis. The HDL/S1P effect is mediated by the S1PR1-3 receptors. The LDL/LPA effects are mediated by the LPAR1 and LPAR2 receptors, but not LPAR3. Therefore, lipids have significant effects on TSP-1-induced VSMC chemotaxis.

Thrombospondin-1, -2 and -5 have differential effects on vascular smooth muscle cell physiology

IntroductionThe thrombospondins (TSPs) are matricellular proteins that exert multifunctional effects by binding cytokines, cell-surface receptors and other proteins. TSPs play important roles in vascular pathobiology and are all expressed in arterial lesions. The differential effects of TSP-1, -2, and -5 represent a gap in knowledge in vascular smooth muscle cell (VSMC) physiology. Our objective is to determine if structural differences of the TSPs imparted different effects on VSMC functions critical to the formation of neointimal hyperplasia. We hypothesize that TSP-1 and -2 induce similar patterns of migration, proliferation and gene expression, while the effects of TSP-5 are different. MethodsHuman aortic VSMC chemotaxis was tested for TSP-2 and TSP-5 (1–40 μg/mL), and compared to TSP-1 and serum-free media (SFM) using a modified Boyden chamber. Next, VSMCs were exposed to TSP-1, TSP-2 or TSP-5 (0.2–40 μg/mL). Proliferation was assessed by MTS assay. Finally, VSMCs were exposed to TSP-1, TSP-2, TSP-5 or SFM for 3, 6 or 24 h. Quantitative real-time PCR was performed on 96 genes using a microfluidic card. Statistical analysis was performed by ANOVA or t-test, with p < 0.05 being significant. ResultsTSP-1, TSP-2 and TSP-5 at 20 μg/mL all induce chemotaxis 3.1 fold compared to serum-free media. TSP-1 and TSP-2 induced proliferation 53% and 54% respectively, whereas TSP-5 did not. In the gene analysis, overall, cardiovascular system development and function is the canonical pathway most influenced by TSP treatment, and includes multiple growth factors, cytokines and proteases implicated in cellular migration, proliferation, vasculogenesis, apoptosis and inflammation pathways. Conclusions and relevanceThe results of this study indicate TSP-1, -2, and -5 play active roles in VSMC physiology and gene expression. Similarly to TSP-1, VSMC chemotaxis to TSP-2 and -5 is dose-dependent. TSP-1 and -2 induces VSMC proliferation, but TSP-5 does not, likely due conservation of N-terminal domains in TSP-1 and -2. In addition, TSP-1, -2 and -5 significantly affect VSMC gene expression; however, little overlap exists in the specific genes altered. This study further delineates TSP-1, -2 and -5's contributions to processes related to VSMC physiology.

Abstract 187: Regulation of Vascular Smooth Muscle Cell (VSMC) Inflammation and Migration by the Pro-resolving Lipid Mediator Maresin-1 (mar1)

Introduction Resolution of acute inflammation is regulated by endogenous lipid mediators derived from polyunsaturated fatty acids such as docosahexaenoic acid (DHA), however little is known about mechanisms of resolution in vascular injury. We investigated the effects of the DHA-derived mediator Mar1 on VSMC phenotype responses. Methods Primary human VSMCs were obtained from saphenous vein. VSMC were pretreated with Mar1 (10-100nM) then exposed to TNF-α (10ng/ml), and inflammatory responses assessed using a monocyte adhesion (U937) assay, expression of cell adhesion molecules and pro-inflammatory molecules (qPCR, western blot, ELISA), and production of superoxide (DHE). VSMC migration was measured in a transwell assay with PDGF-AB as the agonist, and cyotskeletal changes were assessed by actin-phalloidin staining. Results Mar-1 (100 nM) reduced U937 adhesion to TNF-stimulated VSMC, VCAM-1, and pro-inflammatory cytokine (IL-6, IL-8) expression. Superoxide production measured by DHE fluorescence was reduced by 57% (p=0.002) and Nox4 expression was markedly attenuated (43%, p=0.01). Mar-1 (0.01-100nM) induced rapid cytoskeletal changes with increased cell area, and reduced VSMC migration (76%, p=0.004) to PDGF-AB (50ng/ml; Figure). Conclusions Mar-1 attenuates TNF-α inflammatory activation of VSMC, with reduction in pro-inflammatory gene expression, oxidant stress, and monocyte adhesion. Mar-1 reduces actin polymerization and inhibits VSMC chemotaxis to PDGF. Pro-resolving mediators may represent a new class of endogenous vascular therapeutics.

Activation of PDK1 mediates VSMC migration and may contribute to vascular remodeling following injury

We hypothesized that increased 3‐phosphoinositide‐dependent kinase 1 (PDK1) activation contributes to arterial wall thickening following vascular injury. Vascular smooth muscle cells (VSMCs) were pretreated with BX‐912, a small molecule inhibitor of PDK1, prior to stimulation with PDGF. Western analysis confirmed inhibition of PDK1 activity in the presence of BX‐912 by decreased phosphorylation of PDK1 (ser241) by ~60% and Akt (thr308) by ~ 70%. Conversely, Akt phosphorylation at ser473 was only mildly affected. To examine VSMC migration, both scratch wound assays and modified Boyden chamber studies were completed. In both instances, wound‐ and PDGF‐induced chemotaxis was attenuated following BX‐912 treatment. Following wire injury of the carotid artery in C57BL6/J mice, in vivo PDK1 phosphorylation (ser 241) was assessed by immunoblotting in both injured and contralateral control carotids at 3, 7, and 14 days post injury. PDK1 phosphorylation was markedly increased at day 3 (~5 fold vs. non‐injured), remained slightly elevated at day 7 (~2 fold) and returned close to baseline by later timepoints following injury. Our findings indicate that BX‐912 is effective in attenuating PDK1 activity and chemotaxis in VSMCs and that increased PDK1 activity occurs following vascular injury. Thus, PDK1 inhibition, via BX‐912 treatment, could potentially be utilized to attenuate wall remodeling in response to injury. Support: NIH HL084159

Abstract 488: Acute Fluvastatin Pleiotropy Is Mevalonate Independent

Objectives Thrombospondin 1 (TSP1) is a matricellular glycoprotein present in injured arteries, and acts as a chemoattractant for vascular smooth muscle cells (VSMC). Migration of VSMCs to an area of injury plays a role in the development of intimal hyperplasia. We have recently shown that acute treatment of VSMCs with statins leads to a decrease in TSP1-induced VSMC chemotaxis. The purpose of this study was to determine whether the acute statin effect is dependent on the mevalonate pathway or is a pleiotropic effect. Methods Quiescent VSMCs were treated for 20 hours (standard time) or 20 minutes (acute treatment) with 100μM mevalonolactone, 0.5 μM fluvastatin, or mevalonolactone and fluvastatin combined. Migration of VSMCs towards 20 μg/ml TSP1 was assessed using a modified Boyden microchemotaxis chamber. Data was recorded as cells migrated per 5 high power fields and converted to percent positive control. Extracellular signal-regulated kinase 1/2 (ERK 1/2) activation was determined by Western blot. Densitometry is presented as percent positive control. Results TSP1 induced VSMC migration compared to SFM, 100% + 7.6 vs. 35.3% + 4.5. Treatment with fluvastatin for 20 hours (34.3% + 6.9) or 20 minutes (38.4% + 9) inhibited TSP1-induced VSMC migration. 20 hour mevalonate treatment together with fluvastatin restored the migratory effect of TSP1, 81% +9.9, p=0.11. Addition of mevalonate to acute fluvastatin treatment, did not reverse the anti-migratory effect of fluvastatin compared to TSP1 alone, 35.6% + 9.9, p&lt;0.0001. TSP1 significantly activated ERK 1/2 compared to basal medium, p42: 100% + 0 vs. 62.3% + 12.6; p44: 100% + 0 vs. 56.9% + 8.7. Acute fluvastatin did not inhibit ERK1/2 activation compared to TSP1, p42: 86.7% + 12.7; p44: 81.7% + 9.9. Conclusion These data indicate long-term fluvastatin inhibition of TSP1 stimulated migration is at least partially dependent on the mevalonate pathway. In contrast, acute fluvastatin inhibition of TSP1-induced chemotaxis involves an alternate pathway resulting in pleiotropic statin effects. Previous studies found that standard treatment time with statins inhibited ERK 1/2 activation. Our current study found that acute fluvastatin inhibition of TSP1-induced chemotaxis does not appear to be ERK1/2 dependent.

The effects of nicotine on vascular smooth muscle cell chemotaxis induced by thrombospondin-1 and fibronectin

Background Vascular smooth muscle cell (VSMC) migration is an important process in many vascular disorders. Nicotine, thrombospondin-1 (TSP-1) and fibronectin (Fn) separately induce VSMC migration. The hypothesis of this study was that nicotine treatment of vascular cells would augment TSP-1-induced and Fn-induced VSMC migration. Methods VSMCs or endothelial cells (ECs) were treated with serum-free medium or nicotine. Migration of VSMCs was assessed using a modified Boyden chemotaxis chamber to serum-free medium, TSP-1, Fn, EC basal medium, and conditioned EC medium or nicotine-treated conditioned EC medium alone or with supplemented TSP-1 or Fn. Results Nicotine treatment increased VSMC chemotaxis to serum-free medium, but TSP-1 or Fn had no further effect on chemotaxis. Conditioned EC and nicotine-treated conditioned EC enhanced VSMC chemotaxis, which was further augmented by Fn supplementation. Conclusions Nicotine-stimulated EC derived factors induce VSMC migration, which is augmented by the addition of Fn.

Vascular smooth muscle cell migration induced by domains of thrombospondin-1 is differentially regulated

Background Thrombospondin-1 (TSP-1) stimulates vascular smooth muscle cell (VSMC) migration via defined intracellular signaling pathways. The aim of this study was to examine the signaling pathways whereby TSP-1 folded domains (amino-terminal [NH 2], procollagen homology [PCH], all 3 type 1 repeats [3TSR], and a single recombinant protein containing the 3rd type 2 repeat, the type 3 repeats, and the carboxyl-terminal [E3T3C1]) induce VSMC migration. Methods Quiescent VSMCs were pretreated with serum-free media or inhibitors: PP2 (c-Src), LY294002 (phosphatidylinositol 3-kinase), FPT (Ras), Y27632 (Rho kinase), SB202190 (p38 kinase), and PD98059 (extracellular signal-regulated kinase). Migration induced by serum-free media, TSP-1, NH 2, PCH, 3TSR, and E3T3C1 was assessed using a modified Boyden chamber. Results TSP-1, NH 2, 3TSR, and E3T3C1 induced VSMC chemotaxis ( P < .05), but PCH did not ( P > .05). PP2, FPT, SB202190, and PD98059 attenuated chemotaxis stimulated by TSP-1, NH 2, 3TSR, and E3T3C1 ( P < .05). LY294002 inhibited TSP-1-induced and E3T3C1-induced ( P < .05) but not NH 2-induced or 3TSR-induced ( P > .05) chemotaxis. Y27632 inhibited NH 2-induced, 3TSR-induced, and E3T3C1-induced ( P < .05) but not TSP-1-induced ( P > .05) induced chemotaxis. Conclusions TSP-1 folded domains are differentially dependent on intracellular signaling pathways to induce migration.

Chrysin restores PDGF-induced inhibition on protein tyrosine phosphatase and reduces PDGF signaling in cultured VSMCs

Previous studies have shown that an increased intake of dietary flavonoids is associated with a decreased risk of cardiovascular diseases (CVDs). PDGF is a major mitogen for vascular smooth muscle cell (VSMC) and participates in the pathogenesis of many CVDs. The study investigated whether the flavone chrysin affected PDGF functions in VSMCs and neointma formation in rat artery. We found that chrysin concentration-dependently inhibited PDGF-induced proliferation and chemotaxis and reduced PDGF signaling in VSMCs. Chrysin attenuated H2O2 signaling and PDGF-induced reactive oxygen species production and NADPH oxidase activation but did not interfere with PDGF binding to VSMCs. The further analyses revealed that chrysin relieved PDGF-induced inhibition on activity of protein tyrosine phosphatase (PTP) and reduced PDGF-induced oxidation of PTP cysteinyl active site. Moreover, it inhibited PDGF receptor autophosphorylation induced by low-dose vanadate (an inhibitor for PTP). The effect of chrysin, but not of the flavonoid (-)-epigallocatechin-3-gallate and antioxidant N-acetylcysteine, on PDGF signaling and PTP activity was reversed by depletion of intracellular glutathione (GSH), suggesting an involvement of chrysin on GSH/glutaredoxin system for PTP reactivation. Finally, to demonstrate the effectiveness of chrysin in vivo, we showed that oral administration of chrysin before and after angioplasty could reduce neointima formation in balloon-injured carotid artery in rats. In conclusion, we provide here evidence that chrysin can regulate intracellular PTP activity during PDGF signaling, inhibits PDGF-induced VSMC proliferation and chemotaxis, and reduces arterial intima hyperplasia in vivo.

Anti-inflammatory cytokine interleukin-19 inhibits smooth muscle cell migration and activation of cytoskeletal regulators of VSMC motility

Vascular smooth muscle cell (VSMC) migration is an important cellular event in multiple vascular diseases, including atherosclerosis, restenosis, and transplant vasculopathy. Little is known regarding the effects of anti-inflammatory interleukins on VSMC migration. This study tested the hypothesis that an anti-inflammatory Th2 interleukin, interleukin-19 (IL-19), could decrease VSMC motility. IL-19 significantly decreased platelet-derived growth factor (PDGF)-stimulated VSMC chemotaxis in Boyden chambers and migration in scratch wound assays. IL-19 significantly decreased VSMC spreading in response to PDGF. To determine the molecular mechanism(s) for these cellular effects, we examined the effect of IL-19 on activation of proteins that regulate VSMC cytoskeletal dynamics and locomotion. IL-19 decreased PDGF-driven activation of several cytoskeletal regulatory proteins that play an important role in smooth muscle cell motility, including heat shock protein-27 (HSP27), myosin light chain (MLC), and cofilin. IL-19 decreased PDGF activation of the Rac1 and RhoA GTPases, important integrators of migratory signals. IL-19 was unable to inhibit VSMC migration nor was able to inhibit activation of cytoskeletal regulatory proteins in VSMC transduced with a constitutively active Rac1 mutant (RacV14), suggesting that IL-19 inhibits events proximal to Rac1 activation. Together, these data are the first to indicate that IL-19 can have important inhibitory effects on VSMC motility and activation of cytoskeletal regulatory proteins. This has important implications for the use of anti-inflammatory cytokines in the treatment of vascular occlusive disease.

Thrombospondin 1, Fibronectin, and Vitronectin are Differentially Dependent Upon RAS, ERK1/2, and p38 for Induction of Vascular Smooth Muscle Cell Chemotaxis

Thrombospondin 1 (TSP-1), fibronectin (Fn), and vitronectin (Vn) promote vascular smooth muscle cell (VSMC) chemotaxis through a variety of second messenger systems, including Ras, ERK1/2, and p38. Ras, ERK1/2, and p38 differentially affect TSP-1-, Fn-, and Vn-induced VSMC chemotaxis. Bovine VSMCs were transfected with Ras N17 or treated with the following inhibitors: a farnesyl protein transferase (FPT) inhibitor, PD098059 (ERK1/2 inhibitor), or SB202190 (p38 inhibitor). Thrombospondin 1, Fn, and Vn were used as chemoattractants. Results were analyzed by analysis of variance (ANOVA) with post hoc testing (P < .05). Ras N17 transfection or FPT inhibitor treatment inhibited TSP-1-, Fn-, and Vn-induced chemotaxis. PD098059 or SB202190 resulted in more inhibition of VSMC migration to TSP-1 than to Fn or Vn. Ras appears equally relevant in the signal transduction pathways of TSP-1-, Fn-, and Vn-induced VSMC chemotaxis. Thrombospondin 1-induced migration is more dependent upon ERK1/2 and p38 than Fn- or Vn-included migration.

Differential effect of nitric oxide on thrombospondin-1-, PDGF- and fibronectin-induced migration of vascular smooth muscle cells

BackgroundNeointimal hyperplasia involves the migration of medial vascular smooth muscle cells (VSMCs) in response to arterial injury. Thrombospondin-1 (TSP1), platelet-derived growth factor (PDGF), and fibronectin (Fn) induce VSMC migration. Nitric oxide (NO) limits VSMC migration. The hypothesis of this study is that NO would dose dependently inhibit TSP1-induced, PDGF-induced, and Fn-induced VSMC chemotaxis. MethodsVSMCs were pretreated with serum free media or the NO donors diethylenetriamine NONOate or S-nitroso-N-acetyl-D,L-penicillamine. Chemotaxis to TSP1, PDGF, or Fn was determined. Analysis of variance with post hoc testing was done. P values < .05 were considered significant. ResultsPDGF, TSP1, and Fn induced VSMC chemotaxis. NO donors inhibited chemotaxis of VSMCs to PDGF in a concentration-dependent manner. NO donors had a variable effect on TSP1-induced chemotaxis. NO donors did not inhibit Fn-induced chemotaxis. ConclusionThe complex interactions of these proteins in vivo will need to be considered when developing NO-dependent therapies for neointimal hyperplasia.

SDF‐1α/CXCR4 axis is involved in glucose‐potentiated proliferation and chemotaxis in rat vascular smooth muscle cells

Excessive proliferation of vascular smooth muscle cells (VSMCs), which migrate from the tunica media to the subendothelial region, is one of the primary lesions involved in atherogenesis in diabetes. Here, we investigated whether high glucose potentiated the proliferation and chemotaxis of VSMCs by activating SDF-1α/CXCR4/PI-3K/Akt signalling. The expression of SDF-1α, CXCR4 and PCNA was up-regulated in tunica media of thoracic aortas by streptozotocin-induced hyperglycaemic Sprague-Dawley rats. Exposure of primary VSMCs to high glucose (25 mM) led to the up-regulated expression of SDF-1α and CXCR4, activated PI-3K/Akt signalling, and consequently promoted the proliferation and chemotaxis of VSMCs. Interestingly, the administration of SDF-1 siRNA or neutralizing antibody against SDF-1α abolished high glucose-induced up-regulation of CXCR4. Moreover, pretreatment with SDF-1α neutralizing antibody, CXCR4 specific inhibitor (AMD3100) or PI-3K inhibitor (LY294002) attenuated the high glucose-potentiated proliferation and chemotaxis in VSMCs. These results suggested that high glucose activated the SDF-1α/CXCR4/PI-3K/Akt signalling pathway in VSMCs in an autocrine manner, which enhanced the proliferation and chemotaxis of VSMCs.

Lovastatin Inhibits Thrombospondin-1-Induced Smooth Muscle Cell Chemotaxis

Thrombospondin-1 (TSP-1) induces vascular smooth muscle cell (VSMC) migration and is important in the development of intimal hyperplasia. HMG-CoA reductase inhibitors, such as lovastatin, reduce the incidence of vascular restenosis after angioplasty by both cholesterol lowering and pleiotropic effects. Inhibition of the mevalonate pathway is largely responsible for these pleiotropic properties. This inhibition prevents isoprenylation of the small G proteins, Rho and Ras, by geranylgeranyl and farnesyl pyrophosphate, respectively. Isoprenylation is required for Ras and Rho activation, which is relevant for cell migration. Lovastatin inhibits TSP-1-induced VSMC chemotaxis by inhibiting small G proteins via the mevalonate pathway. Chemotaxis was assessed using a modified Boyden chamber. Quiescent VSMCs were pretreated with serum free media (SFM), lovastatin with or without mevalonate farnesyl (FTI), geranylgeranyl transferase inhibitors (GGTI), farnesyl transferase inhibitor (FPT), or the Rho kinase inhibitor (Y-27632). Chemoattractants were SFM or TSP-1. Comparisons were made by ANOVA followed by post-hoc testing (P<0.05). The effect of lovastatin on Ras activation was evaluated using cells pretreated with SFM or lovastatin, with or without mevalonate prior to TSP-1 exposure. Western blot for Ras activation was performed. Lovastatin dose-dependently inhibited TSP-1-induced chemotaxis, which was reversed by mevalonate. Mevalonate did not induce chemotaxis independently. FTI and FPT, but not GGTI or Y-27632, inhibited TSP-1-induced Ras activation and TSP-1-induced chemotaxis. Lovastatin inhibition of Ras activation was reversed with mevalonate. Ras, not Rho, is relevant for TSP-1-induced VSMC chemotaxis. These data suggest that lovastatin suppresses TSP-1-induced chemotaxis by inhibition of Ras.

The role of G proteins in thromospondin-1–induced vascular smooth muscle cell migration

Thrombospondin-1 (TSP-1), which is a matricellular glycoprotein associated with chemotaxis of vascular smooth muscle cells (VSMCs), is relevant to the development of arterial lesions. Evidence suggests that TSP-1 receptors are linked to guanosine triphosphate-binding proteins (G proteins). The purpose of this study was to determine the role of G proteins in TSP-1-induced VSMC chemotaxis and whether this pathway was associated with extracellular signal-regulated kinase 1/2 (ERK) or p38 kinase activation (downstream pathways associated with VSMC chemotaxis). In all studies, quiescent VSMCs were preincubated either with serum-free medium, cholera toxin, pertussis toxin, forskolin, or 3-isobutyl-1-methylxanthine. Using a microchemotaxis chamber, preincubated VSMCs were exposed to TSP-1 or serum-free medium. Migrated VSMCs were recorded as cells/5 fields (400x) and analyzed by paired t-test. To evaluate the effect of G proteins on TSP-1-induced ERK or p38 activation, preincubated VSMCs were exposed to serum-free medium or TSP-1 and analyzed by Western immunoblotting. For measurement of intracellular cyclic adenosine monophosphate (cAMP) levels, enzyme-linked immunosorbant assay was performed on preincubated VSMCs exposed to serum-free medium or TSP-1. Although pertussis toxin attenuated TSP-1-induced chemotaxis, cholera toxin abolished TSP-1-induced chemotaxis. Cholera toxin, but not pertussis toxin, inhibited both ERK and p38 activation. The cAMP stimulators forskolin and IBMX abolished TSP-1-induced chemotaxis and ERK and p38 activation. Although no changes were observed in cAMP levels in VSMCs treated with serum-free medium, TSP-1, or pertussis toxin, cholera toxin alone significantly increased cAMP levels. G(s) protein signaling inhibits TSP-1-induced VSMC chemotaxis by increasing the levels of cAMP. G(i) signaling is involved in the mechanism of TSP-1 stimulated chemotaxis and warrants additional study. Agents that increase cAMP levels may be beneficial in reducing TSP-1-induced chemotaxis in response to vascular injury.

Internal mammary artery smooth muscle cells resist migration and possess high antioxidant capacity

This study investigated whether differences exist in atherogen-induced migratory behaviors and basal antioxidant enzyme capacity of vascular smooth muscle cells (VSMC) from human coronary (CA) and internal mammary (IMA) arteries. Migration experiments were performed using the Dunn chemotaxis chamber. The prooxidant [NAD(P)H oxidase] and antioxidant [NOS, superoxide dismutase, catalase and glutathione peroxidase] enzyme activities were determined by specific assays. Chemotaxis experiments revealed that while both sets of VSMC migrated towards platelet-derived growth factor-BB (1-50 ng/ml) and angiotensin II (1-50 nM), neither oxidized-LDL (ox-LDL, 25-100 microg/ml) nor native LDL (100 microg/ml) affected chemotaxis in IMA VSMC. However, high dose ox-LDL produced significant chemotaxis in CA VSMC that was inhibited by pravastatin (100 nM), mevastatin (10 nM), losartan (10 nM), enalapril (1 microM), and MnTBAP (a free radical scavenger, 50 microM). Microinjection experiments with isoprenoids i.e. geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) showed distinct involvement of small GTPases in atherogen-induced VSMC migration. Significant increases in antioxidant enzyme activities and nitrite production along with marked decreases in NAD(P)H oxidase activity and O2- levels were determined in IMA versus CA VSMC. Enhanced intrinsic antioxidant capacity may confer on IMA VSMC resistance to migration against atherogenic agents. Drugs that regulate ox-LDL or angiotensin II levels also exert antimigratory effects.

YKL-40, a new inflammatory marker with relation to insulin resistance and with a role in endothelial dysfunction and atherosclerosis

Substantial evidence supports a role of chronic subclinical inflammation and activation of the innate immune system in the pathogenesis of insulin resistance and endothelial dysfunction and the development of type 2 diabetes (T2D) and atherosclerosis. Several proinflammatory cytokines, acute phase-reactants and cell adhesion molecules play a pivotal role in this chronic subclinical inflammation but a comprehensive understanding of the interrelations of these molecules is still needed. YKL-40 is a new inflammatory marker with relation to acute and chronic inflammation as well as cancer. It is secreted in vitro from a variety of human cells, including vascular smooth muscle cells (VSMCs), activated macrophages and macrophages during late stages of differentiation and is found in vivo in subpopulations of macrophages in tissues with inflammation and extracellular tissue remodelling, such as macrophages in atherosclerotic plaques. YKL-40 promotes chemotaxis, cell attachment and migration of VSMCs and the formation of branching tubules suggesting that YKL-40 plays a role in angiogenesis. Latest studies reveal that YKL-40 is elevated in patients with T2D and is related to insulin resistance. This article reviews the studies of YKL-40 with focus on a possible role of YKL-40 in insulin resistance, endothelial dysfunction and atherosclerosis.

Thrombospondin-1 antagonizes nitric oxide-stimulated vascular smooth muscle cell responses

Endothelial-derived nitric oxide (NO), by increasing cGMP, is a major physiological regulator of vascular tone and of vascular smooth muscle cell (VSMC) adhesion, chemotaxis, and proliferation. Thrombospondin-1 is a potent antagonist of NO/cGMP signaling in endothelial cells. Because endothelial and VSMC typically exhibit opposing responses to thrombospondin-1, we examined thrombospondin-1 effects on NO signaling in VSMC. Effects of exogenous thrombospondin-1 on human VSMC adhesion, chemotaxis, proliferation, and cGMP signaling were examined. Endogenous thrombospondin-1 function was characterized by comparing NO signaling in VSMC from wild type and thrombospondin-1 null mice. Picomolar concentrations of exogenous thrombospondin-1 prevented adhesive, chemotactic, and proliferative responses of human aortic VSMC stimulated by low dose NO. A recombinant CD36-binding domain of thrombospondin-1 or antibody ligation of CD36 similarly inhibited NO-stimulated VSMC responses. Thrombospondin-1 and CD36 ligation inhibited NO responses in VSMC by preventing cGMP accumulation. Thrombospondin-1 null VSMC responses to NO and cGMP signaling were enhanced relative to wild type murine VSMC. In the presence of NO, thrombospondin-1 is converted from a weak stimulator to a potent inhibitor of VSMC responses. Both exogenous and endogenous thrombospondin-1 inhibit NO signaling in VSMC. This activity is mediated by the type 1 repeats and utilizes the same CD36-dependent cGMP signaling pathway in endothelial and VSMC.

Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not proliferation☆

Reactive oxygen species (ROS) produced by NAD(P)H oxidases (Nox) play a significant role in the pathophysiology of cardiovascular diseases. Expression and activity of NAD(P)H oxidases are regulated by growth factors such as angiotensin II and platelet-derived growth factor (PDGF). We characterized the effects of the novel Nox inhibitor VAS2870 on PDGF-dependent ROS liberation and cellular events in vascular smooth muscle cells (VSMC). PDGF-BB increased NAD(P)H oxidase activity (lucigenin-enhanced chemiluminescence) and intracellular ROS levels (detected by confocal laserscanning microscopy using 2,7-DCF) to 229+/-9% and 362+/-54% at 1 and 2 h, respectively. Preincubation with VAS2870 (10 and 20 microM) completely abolished PDGF-mediated NAD(P)H oxidase activation and ROS production. Since ROS are involved in various growth factor-induced cellular functions, the influence of VAS2870 on PDGF-induced DNA synthesis and chemotaxis was determined. PDGF promoted a 4.2+/-0.2-fold increase of VSMC migration (modified Boyden chamber, p<0.01) and increased DNA synthesis by maximally 3.2+/-0.4-fold (BrdU incorporation, p<0.01) in a concentration-dependent manner. Preincubation with VAS2870 (0.1-20 microM) did not affect PDGF-induced cell cycle progression. However, it abolished PDGF-dependent chemotaxis of VSMC in a concentration-dependent manner (100% inhibition at 10 microM). These findings were related to PDGF-dependent signaling events. Western blot analyses using phospho-specific antibodies revealed that the downstream signaling molecules Akt, Erk, and Src were activated by PDGF. However, VAS2870 blocked PDGF-dependent activation of Src, but not of Akt and Erk, in a concentration-dependent manner. VAS2870 effectively suppresses growth factor-mediated ROS liberation in VSMC. Furthermore, it completely inhibits PDGF-dependent VSMC migration, whereas it does not affect DNA synthesis. These divergent effects reflect the critical role of Src activity, which-in contrast to Akt and Erk-appears to be redox-sensitive and is absolutely required for PDGF-induced chemotaxis, but not cell cycle progression.

Regulation of vascular smooth muscle cell proliferation, migration and death by heparan sulfate 6- O-endosulfatase1

Migration, proliferation and death of vascular smooth muscle cells (VSMC) are important events in vascular pathology regulated by heparan sulfate proteoglycans and hence potentially by cell surface HS 6- O-endosulfatase1 (sulf1). Sulf1 mRNA expression was increased in cultured VSMC compared to rat aorta. Furthermore, adenovirus mediated overexpression of quail sulf1 decreased adhesion, and increased proliferation and apoptosis of VSMC. Overexpression of a dominant negative variant also decreased adhesion of VSMC and increased proliferation, apoptosis, migration and chemotaxis of VSMC. Our results imply that only normal levels of 6- O-sulfation maintained by sulf1 are optimal for several functions of VSMC.