Urokinase and Tissue-type Plasminogen Activator Are Required for the Mitogenic and Chemotactic Effects of Bovine Fibroblast Growth Factor and Platelet-derived Growth Factor-BB for Vascular Smooth Muscle Cells

Abstract

The present study was undertaken to evaluate in vitro the relative importance of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) in the mitogenic and chemotactic potential of bovine fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF)-BB for smooth muscle cells (SMC). Aortic SMC were isolated from transgenic mice showing single inactivations of the t-PA, u-PA, plasminogen activator inhibitor-1, or urokinase-type plasminogen activator receptor (u-PAR) genes. With regard to serum-induced proliferation, all cell types showed similar responses. However, SMC isolated from t-PA-deficient mice did not proliferate or migrate in response to PDGF, whereas SMC isolated from u-PA-deficient animals appeared to be much less sensitive to bFGF than the cells isolated from the other animals. Supplementation of cells from deficient animals with exogenous murine t-PA or u-PA restored the normal response of the growth factors with regard to both migration and proliferation. The mitogenic and chemotactic responses of bFGF were specifically inhibited in u-PAR-deficient cells or in wild-type SMC, cultured in the presence of antibodies to u-PAR. The role of u-PA and t-PA in bFGF and PDGF-induced growth and migration of SMC was not dependent on plasmin generation and activity as demonstrated by the inactivity of ⑀-aminocaproic acid and aprotinin. A 4 -5-fold increase in the steady-state levels of u-PA and t-PA mRNA and proteins were observed after 24 h of incubation of the cell cultures with bFGF and PDGF-BB, respectively. These results therefore indicate that, at least in vitro, t-PA is an important element of the activity of PDGF-BB with regard to the proliferation and migration of SMC whereas u-PA is a key factor in the effect of bFGF on SMC.The accumulation of neointimal SMC 1 resulting from media smooth muscle proliferation and migration in response to vascular injury is believed to be one of the main events involved in the initiation of atherosclerosis or during restenosis following angioplasty (1, 2). Although the general contribution of thrombosis to the development of atherosclerosis has been acknowledged for a long time, recent investigations suggested that the fibrinolytic system may also play an important role in the process of cell proliferation or migration (3, 4). Indeed, several authors described both a mitogenic and a chemotactic effect of two types of plasminogen activators: t-PA and u-PA for several cell types including vascular smooth muscle cells (5-8) 2 and a recent study (10) showed that both u-PA and t-PA produced by endothelial cells were sequestered in an active form by the subendothelial extracellular matrix suggesting that they may participate in sequential matrix degradation during cell invasion but also function in the release of extracellular matrixbound growth factor-like bFGF, that will stimulate SMC growth, a crucial step in atherogenesis or post-percutaneous transluminal coronary angioplasty restenosis.Most interesting were the works of Clow...

Urokinase-type plasminogen activator increases hepatocyte growth factor activity required for skeletal muscle regeneration

“…26,39 Studies on vascular smooth muscle cells produced similar findings that uPA induces proliferation of these cells and may mediate growth factor-induced proliferation. [40][41][42] Our data demonstrated that (1) intact uPA but not the aminoterminal fragment, which lacks the proteolytic domain, stimulated myoblast proliferation; (2) the specific proteolytic inhibitor PAI-1 blocked uPA-mediated myoblast proliferation; (3) the HGFblocking antibody reduced uPA stimulation of myoblast proliferation; and (4) both the PI3K inhibitor LY and the MEK1 inhibitor PD98059 reduced uPA-mediated myoblast proliferation. Taken together, these data indicate that uPA stimulates myoblast proliferation through proteolytic activation of HGF and via signaling through PI3K and MEK1.…”

Mechanisms of Vascular Smooth Muscle Cell Migration

“…Transforming growth factor-␤ 1 (TGF␤ 1 ) 183,184 Tumor necrosis factor-␣ (TNF␣) 185,161 Thrombin 186 Urokinase plasminogen activator 187,188 Vascular endothelial growth factor (VEGF) 189,190 Extracellular matrix components Collagen I, IV 37 Collagen VIII 191,192 Sphingosine-1 phosphate (S1P) 75,76 formins (mDia1 and mDia2), which act on the plus end in concert with profilin. The formin mDia1 is activated by RhoA, and mDia2 is activated by Cdc42.…”

Characterization of SIS3, a Novel Specific Inhibitor of Smad3, and Its Effect on Transforming Growth Factor-β1-Induced Extracellular Matrix Expression

“…Normal human dermal fibroblasts were plated at a density of 10 5 cells/well in six-well culture plates and grown until subconfluence in MEM containing 10% FCS. Cells were quiesced by 24-h incubation in serum-free MEM, followed by incubation in serum-free medium in the presence or absence of SIS3 before the collection of cells for 72 h. Then, the cells were detached from the wells by trypsin treatment and counted using a Coulter counter (Beckman Coulter, Fullerton, CA) (Herbert et al, 1997 3 H]proline} (GE Healthcare) was added to the medium and incubated overnight. Medium was harvested from each well, and the incorporated radioactivity was counted in a liquid scintillation counter (Ziyadeh et al, 1994;Isono et al, 2000).…”