Differential processing of osteopontin characterizes the proliferative vascular smooth muscle cell phenotype induced...

Repeated cycles of vascular injury by allylamine induce vascular lesions similar to those seen in atherosclerotic vessels, or following balloon catheterization. Vascular (aortic) smooth muscle cells harvested from allylamine-treated animals (i.e., allylamine cells) acquire a proliferative advantage relative to control counterparts that is associated with differential secretion and extracellular matrix sequestration of several proteins. In the present study, we have characterized two of these proteins (M(r) 52 and 36 kDa; pl 5.6 and 5.2, respectively) and their putative role in the expression of a proliferative phenotype. Because the physical properties of these proteins were comparable to those of osteopontin (OPN) and its thrombin-generated fragment(s), initial experiments were conducted to examine the expression and processing of OPN in this cell system. OPN mRNA expression was enhanced during early G1 cell cycle progression in allylamine cells relative to control counterparts. However, comparable amounts of OPN (M(r) 56, 52, and 50 kDa) were detected by Western analysis in media conditioned by both cell types using the OP-199 or B77-Rat1 antibodies to OPN. Allylamine cells, however, produced increased amounts of a 36 kDa protein recognized by the OP-199 antibody. Incubation of conditioned media from [35S]methionine-labeled allylamine cells with thrombin decreased the intensity of the 52 kDa protein, while increasing the intensity of a 36 kDa protein. RT-PCR analysis demonstrated expression of a 1.2 kb OPN band in both cell types consistent with the predicted size of OPN mRNA, suggesting that the 36 kDa fragment recognized by OP-199 in allylamine cells was likely not due to altered splicing of the OPN transcript. To determine if OPN and/or the 36 kDa fragment played a central role in the proliferative capacity of allylamine cells, the effect of an antibody to an alpha v integin subunit was examined. An antibody to the alpha v subunit, but not alpha 4, nullified the proliferative advantage of allylamine cells relative to control counterparts, suggesting that integrin-mediated signaling is a key feature of the proliferative phenotype of allylamine cells. We conclude that enhanced proteolytic cleavage of OPN may characterize the modulation of vascular SMCs to a more proliferative phenotype following chemical injury by allylamine.