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 beta-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 (x400) 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.

Factor Xa-inhibitor (DX-9065a) modulates the leukocyte-endothelial cell interaction in endotoxemic rat.

Abnormalities of vascular endothelial function and coagulation play important roles in the development of septic organ dysfunction. DX-9065a is a novel Factor Xa inhibitor that is expected to modulate both coagulation and endothelial function. The purpose of this study is to examine the effect of DX-9065a on leukocyte-endothelial interaction. Rats were injected with 1.0 mg/kg of endotoxin simultaneously with saline, (placebo group), 0.3 mg/kg DX-9065a (low-dose group), or 3.0 mg/kg DX-9065a (high-dose group; n = 6 in each group). At 1 and 3 h after injection, the mesenteric microcirculation was observed under intravital microscopy. In addition, TNF, IL-6, alanine aminotransferase (ALT), blood urea nitrogen (BUN), and lactate levels were measured. The number of leukocytes adhering to the endothelium was significantly reduced in both the high-dose and low-dose groups (P < 0.05 for both, compared to the control group). A comparison of the cytokine levels showed that the peak levels in the treatment groups tended to be lower. Markers of organ damage also showed less increase in the treatment groups (P < 0.05 for both treatment groups compared to the control group). In summary, the Factor Xa inhibitor DX-9065a showed a protective effect on the microcirculation of endotoxemic rats by attenuating leukocyte-endothelial interaction. Although the mechanism for this effect could not be fully elucidated, suppression of both excessive coagulation and cytokine production appear to play a role.

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.

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

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.