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Background: Thrombospondin-1 (TSP-1) is functionally important to intimal hyperplasia (IH) development. Statin drugs have beneficial pleiotropic effects, including reduced IH; however, the effect of statins on IH in a TSP-1-independent setting is unknown., Hypothesis: Statins will be less effective in attenuating IH after vascular injury in TSP-1-null (Thbs1 -/- ) mice compared with wild-type (WT) mice., Materials and Methods: Carotid artery ligation was performed on WT and Thbs1 -/- mice. Each strain was divided into two groups: no statin control or standard chow containing fluvastatin (10 or 40 mg/kg/d). After 28 d, analysis included morphometric analysis and real-time quantitative reverse transcription polymerase chain reaction on the arteries and enzyme-linked immunosorbent assay on plasma (TSP-1 WT, TSP-2 WT, and Thbs1 -/- ). Comparisons were made by analysis of variance, with P < 0.05 considered significant., Results: In no statin controls, WT mice had more IH than Thbs1 -/- mice (0.46 ± 0.09 versus 0.15 ± 0.04). Fluvastatin reduced IH in the WT (0.46 ± 0.09 versus 0.23 ± 0.06), but not in Thbs1 -/- groups (0.15 ± 0.04 versus 0.22 ± 0.07). No difference in IH existed between Thbs1 -/- no statin controls and fluvastatin WT and Thbs1 -/- groups. Statin dose did not affect IH. TSP-1 plasma levels were increased in fluvastatin WT. TSP-2 levels were decreased in fluvastatin WT and elevated in fluvastatin Thbs1 -/- . Fluvastatin had no effect on tissue Thbs1 or Thbs2 gene expression., Conclusions: TSP-1 is necessary for robust IH after arterial injury. Because fluvastatin had no effect on IH in Thbs1 -/- , the data suggest that the statin effect on IH may be largely TSP-1 dependent. Both statins and the presence of TSP-1 affect TSP-1 and TSP-2 plasma levels., (Published by Elsevier Inc.)

Thrombospondin-1 (TSP-1) is an important regulator of vascular smooth muscle cell (VSMC) physiology and gene expression. MicroRNAs (microRNA), small molecules that regulate protein translation, have emerged as potent regulators of cell function. MicroRNAs have been shown to be involved in intimal hyperplasia, atherosclerosis, and upregulated in the vasculature in diabetes. The purpose of this study was to identify microRNAs regulated by TSP-1 in vascular smooth muscle cells (VSMCs). Human VSMCs were treated for 6 h with basal media or TSP-1 both supplemented with 0.2% FBS. Cells were then snap frozen and RNA extracted. An Affymetrix GeneChip microRNA array analysis was performed in triplicate on three separate collections. Confirmatory qrtPCR was performed. Data were analyzed by ANOVA or t test, with significance set at p < 0.05. MicroRNAs identified were subjected to KEGG pathway analysis using the DIANA tools miRPath online tool. TSP-1 upregulated 22 microRNAs and downregulated 18 microRNAs in VSMCs (p < 0.05). The most upregulated microRNA was miR-512-3p (45.12 fold). The microRNA most downregulated by TSP-1 was miR-25-5p, which was decreased by 9.61. Of note, five members of the mir-17-92 cluster were downregulated. KEGG analysis revealed that thirty-three cellular signaling pathways were impacted by these microRNAs and that nine pathways were relevant to vascular disease. MicroRNAs regulate protein expression at the level of translation and may represent a significant mechanism by which TSP-1 regulates VSMC function. Several of the microRNAs identified have a role in vascular function. The miR-17-92 cluster family, which was found to exhibit reduced expression in this study, is known to be involved in angiogenesis and vascular function. TSP-1 regulates multiple microRNAs in VSMCs adding a new layer of complexity to TSP-1 regulation of VSMC function.

Unlabelled: 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., Hypothesis: 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.

Introduction: The 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., Methods: Human 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., Results: TSP-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 Relevance: The 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., (Published by Elsevier Inc.)

Background: Thrombospondin 1 (TSP-1) induces vascular smooth muscle cell (VSMC) migration and intimal hyperplasia. Statins and nitric oxide (NO) donors decrease intimal hyperplasia. We previously showed that statins (long-term exposure) and NO donors inhibit TSP-1-induced VSMC chemotaxis., Hypotheses: (1) Pretreatment with short-term statin will inhibit TSP-1-induced VSMC chemotaxis and (2) NO donors will enhance statin inhibition of TSP-1-induced or platelet-derived growth factor (PDGF)-induced VSMC chemotaxis., Methods: We examined these treatment effects on TSP-1-induced VSMC chemotaxis: (1) long-term (20 hours) versus short-term (20 minutes) pravastatin, (2) diethylenetriamine NONOate (DETA/NO) or S-nitroso-N-acetylpenicillamine (SNAP) in combination with pravastatin, and (3) comparison of TSP-1 to PDGF as a chemoattractant., Results: Pravastatin (long term or short term) inhibited TSP-1-induced chemotaxis. Diethylenetriamine NONOate and SNAP impeded statin inhibition of TSP-1-induced chemotaxis. Platelet-derived growth factor and TSP-1 had opposite effects on DETA/NO-pravastatin treatment., Conclusion: Short-term statin pretreatment inhibited TSP-1-induced VSMC chemotaxis, suggesting a pleiotropic effect. High-dose NO reversed statin inhibition of TSP-1-induced chemotaxis, suggesting NO and statin combination therapies warrant further study., (© The Author(s) 2014.)

Objectives: Thrombospondin-1 (TSP-1) is a matricellular glycoprotein released from platelets at sites of arterial injury and is important in neointima development after balloon angioplasty. MicroRNAs are small noncoding RNAs that function by binding target gene mRNA and inhibiting protein translation. MicroRNA-21 (miR-21) is up-regulated after angioplasty, and inhibition of miR-21 leads to decreased intimal hyperplasia. In this study, we examined the effects of miR-21 inhibition on vascular smooth muscle cell (VSMC) processes., Methods: VSMCs were exposed to TSP-1 and miR-21 inhibitor for 20 minutes. TSP-1-induced migration was assessed with a modified Boyden microchemotaxis chamber and proliferation with calcein-AM fluorescence. Phosphorylated extracellular signaling kinase (ERK) 1/2 expression was determined by Western Blot and densitometry. Quantitative real-time polymerase chain reaction for TSP-1, hyaluronic acid synthase 2 (HAS2), and transforming growth factor beta 2 (TGFβ2) was performed. Statistical analysis was performed with analysis of variance (P < .05)., Results: Inhibition of miR-21 blocked TSP-1-induced VSMC migration, proliferation, and ERK 1/2 phosphorylation (P < .05) and had no effect on TSP-1-stimulated expression of genes for TSP-1, HAS2, or TGFβ2 (P > .05)., Conclusion: Acute inhibition of miR-21 led to a decrease in VSMC migration and proliferation caused by TSP-1. The decrease in TSP-1's activation of ERK 1/2 after acute miR-21 inhibition indicates an active role for miR-21 in TSP-1's cell signaling cascade. No effect on TSP-1-induced expression of the pro-stenotic genes thbs1, tgfb2, or has2, occurred after acute miR-21 inhibition. These data indicate that miR-21 directly modulates cell function and signaling pathways in ways other than inhibition of protein translation., (Copyright © 2014 Mosby, Inc. All rights reserved.)

Angioplasty causes local vascular injury, leading to the release of thrombospondin-1 (TSP-1), which stimulates vascular smooth muscle cell (VSMC) migration and proliferation, important steps in the development of intimal hyperplasia. Transforming growth factor beta 2 (TGF-β2) and hyaluronic acid synthase (HAS) are two pro-stenotic genes upregulated in VSMCs by TSP-1. We hypothesized that inhibition of TGF-β2 or HAS would inhibit TSP-1-induced VSMC migration, proliferation, and TSP-1 signaling. Our data demonstrate that Inhibition of either TGF-β2 or HAS inhibited TSP-1-induced VSMC migration and proliferation. Activation of ERK 1 was decreased by TGF-β2 inhibition and unaffected by HAS inhibition. TGF-β2 and HAS are not implicated in TSP-1-induced thbs1 expression, while they are each implicated in TSP-1-induced expression of their own gene. In summary, TSP-1-induced VSMC migration and proliferation rely on intact TGF-β2 signaling and HAS function. TSP-1 activation of ERK 1 is dependent on TGF-β2. These data further expand our understanding of the complexity of TSP-1 cellular signaling and the involvement of TGF-β2 and HAS.

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., (Published by Elsevier Inc.)

Background: 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., Hypothesis: Lovastatin inhibits TSP-1-induced VSMC chemotaxis by inhibiting small G proteins via the mevalonate pathway., Methods: 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., Results: 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., Conclusion: 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., (Published by Elsevier Inc.)

Background: 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., Hypothesis: Ras, ERK1/2, and p38 differentially affect TSP-1-, Fn-, and Vn-induced VSMC chemotaxis., Methods: 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)., Results: 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., Conclusions: 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.

Background: Neointimal 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., Methods: VSMCs 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., Results: PDGF, 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., Conclusion: The complex interactions of these proteins in vivo will need to be considered when developing NO-dependent therapies for neointimal hyperplasia., (Published by Elsevier Inc.)

Background: Thrombospondin-1 (TSP-1) induces vascular smooth muscle cell (VSMC) migration after arterial injury. TSP-1 up-regulates hyaluronic acid (HyA)-inducing genes in VSMCs. HyA also induces VSMC migration. Our hypothesis was that TSP-1-induced VSMC migration is dependent on the CD44 receptor, and that HyA and TSP-1 share migratory signaling pathways., Methods: VSMC migration was assessed using TSP-1, HyA, or serum-free medium as chemoattractants. VSMCs were treated with inhibitors to CD44, Ras, phosphatidylinositol-3 kinase, Raf-1 kinase, or c-SRC. TSP-1- and HyA-induced epidermal growth factor receptor (EGFR) activity was determined by enzyme-linked immunosorbent assay. Comparisons were made by the Student t test and a P value less than .05 was considered significant., Results: Inhibiting CD44 reduced TSP-1- and HyA-induced migration. Phosphatidylinositol-3 kinase and c-SRC inhibitors prevented TSP-1- and HyA-induced migration, whereas Ras and Raf-1 kinase inhibitors only affected TSP-1. TSP-1 and HyA activate the EGFR., Conclusions: TSP-1- and HYA-induced migration share some of the same signaling pathways and the EGFR/CD44 receptors may be a common link.

Purpose: 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)., Methods: 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., Results: 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., Conclusion: 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.