Both AT1 and AT2 receptors mediate proliferation and migration of porcine vascular smooth muscle cells

Angiotensin receptor antagonists have shown clinical promise in modulating vascular disease, in part by limiting smooth muscle cell proliferation and migration. The majority of studies examining the contribution of these receptors have been undertaken in cells derived from rat aorta, which primarily express the ANG II type 1 (AT1) receptor. This investigation studied the relative contribution of AT1 and ANG II type 2 (AT2) receptors to the mitogenic program of porcine smooth muscle cells. Smooth muscle cells were derived from porcine coronary artery explants. The presence of both AT1 and AT2 receptors was demonstrated through ligand binding and RT-PCR analysis. Biochemical and cellular markers of proliferation were monitored in the presence of selective receptor antagonists. Smooth muscle cell migration was measured using both wound healing and Boyden chamber migration assays. Visualization of the AT1 and AT2 receptors in growing and quiescent porcine smooth muscle cells with epifluorescence microscopy demonstrated that their subcellular distribution varied with growth state. An examination with several growth assays revealed that both AT1-specific losartan and AT2-specific PD-123319 receptor antagonists inhibited ANG II-stimulated RNA and DNA synthesis, PCNA expression, and hyperplasia. ANG II induced both directional and nondirectional cell migration. AT1 receptor antagonist treatment significantly decreased ANG II-induced directional migration only, whereas AT2 receptor antagonist treatment significantly reduced both modes of migration. Interestingly, the focal adhesion kinase inhibitor PF-573228 also blocked migration but not proliferation. Furthermore, focal adhesion kinase activation in response to ANG II was prevented only by PD-123319, indicating that this activation is downstream of the AT2 receptor. The observed role of the AT2 receptor in ANG II-induced migration was confirmed with smooth muscle cells depleted of the AT2 receptor with short hairpin RNA treatment.