Xiu-Jie Wang

Thrombospondin-1 regulation of smooth muscle cell chemotaxis is extracellular signal-regulated protein kinases 1/2 dependent.

BACKGROUND Thrombospondin-1 (TSP-1), an extracellular matrix protein, induces vascular smooth muscle cell (VSMC) chemotaxis. We hypothesized that extracellular signal-regulated protein kinases 1/2 (ERK1/2), a pathway of the mitogen activated protein kinase (MAPK) family, is important in TSP-1-induced VSMC chemotaxis. METHODS A modified Boyden chamber was used to assess chemotaxis. First, a concentration curve was performed to determine the level for optimal TSP-1-induced chemotaxis. Then quiescent VSMCs were preincubated (30 minutes) in serum-free medium, dimethyl sulfoxide (the inhibitor vehicle), or PD98059 (10 mumol/L, an upstream inhibitor of ERK1/2). VSMCs (50,000 cells/well) with the appropriate preincubation were placed in the top chamber. The bottom chamber contained TSP-1 (20 micrograms/mL) or serum-free medium. Results were recorded as cells/5 fields (400x). Then quiescent VSMCs were exposed to TSP-1 (20 micrograms/mL) for 0, 1, 5, 10, 30, 120, or 300 minutes. Platelet-derived growth factor (10 ng/mL) was the positive control for ERK1/2 activation. Western blot analysis was performed for activated ERK1/2. All comparisons were made by a paired t test (n = 3). RESULTS TSP-1-induced chemotaxis peaks by a concentration of 20 micrograms/mL. PD98059 inhibited TSP-1-induced chemotaxis (P < .05). ERK1/2 was activated by TSP-1-stimulated VSMCs. CONCLUSIONS TSP-1-stimulated VSMCs activated ERK1/2. An ERK1/2 inhibitor abolished chemotaxis, suggesting the functional importance of MAPK in TSP-1-induced VSMC chemotaxis.

Thrombospondin-1 induces activation of focal adhesion kinase in vascular smooth muscle cells.

PURPOSE Vascular smooth muscle cell (VSMC) proliferation and migration are important events in the development of intimal hyperplasia. Thrombospondin-1 (TSP-1), an extracellular matrix protein present in intimal hyperplastic lesions, has been shown to stimulate VSMC proliferation and migration. We hypothesized that the focal adhesion plaque, specifically the focal adhesion kinase (FAK) protein, may be important in the signal transduction pathway for TSP-1-induced VSMC migration. METHODS Growth-arrested bovine aortic VSMCs were treated with TSP-1 (20 microg/mL) for set intervals (15, 30, and 120 minutes) and compared with VSMCs grown in serum-free medium (negative control) or in the presence of a known mitogen and chemotactic factor, platelet-derived growth factor (10 ng/mL; positive control). Crude cell lysates and anti-FAK immunoprecipitates were assayed for phosphotyrosine activity by means of antiphosphotyrosine immunoblotting. The blots were quantified by means of densitometric analysis. RESULTS TSP-1 increased tyrosine phosphorylation of three protein bands of molecular weights 68, 125 (consistent with FAK), and 180 kDa. Immunoprecipitation with FAK antibody, followed by antiphosphotyrosine immunoblotting, indicated that there was an increase in tyrosine phosphorylation of FAK at 15, 30, and 120 minutes in the TSP-1-treated groups (P <.05). CONCLUSION Tyrosine phosphorylation of FAK is induced by TSP-1 stimulated VSMCs. This suggests an outside-inside signaling pathway by which TSP-1 stimulates VSMC migration.

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

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.

Thrombospondin-1-Induced Migration Is Functionally Dependent Upon Focal Adhesion Kinase

Vascular smooth muscle cell migration is important in vascular disease. Previously, we showed thrombospondin-1 activates focal adhesion kinase in these cells. We hypothesized that focal adhesion kinase is important for thrombspondin-1-induced vascular smooth muscle cell migration. Bovine aortic smooth muscle cells were transfected with FAK397, FAK-wild type, pcDNA, or β-Gal plasmids. Migration was assessed with thrombospondin-1 or serum-free medium in quiescent transfected cells or quiescent cells pretreated with the focal adhesion kinase inhibitor, geldanamycin. Number of cells migrated per 5 fields (×400) were recorded. Antihemagglutinin immunoprecipitation and Western blot were used to examine thrombospondin-1-induced focal adhesion kinase phosphorylation in transfected cells. FAK397 transfection inhibited thrombospondin-1-induced focal adhesion kinase phosphorylation and migration (P < .05). Geldanamycin inhibited thrombospondin-1-induced smooth muscle cell migration (P < .05). In conclusion, vascular smooth muscle cells transfected with FAK397 inhibited thrombosponin-1-induced migration and tyrosine phosphorylation. Further, geldanamycin also inhibited migration. These results suggest focal adhesion kinase is involved in thrombospondin-1-induced vascular smooth muscle cell migration.

Photochemotherapy of vascular cells with 8-methoxypsoralen and visible light: differential effects on endothelial and smooth muscle cells

Background: The long‐term efficacy of percutaneous transluminal coronary angioplasty is limited by the restenosis which occurs in approximately 40% of patients, usually within 6 months of the procedure.