Pulmonary artery smooth muscle cells from chronically hypoxic neonatal calves retain fetal-like and acquire new growth properties

Abstract

Growth properties retained and acquired by immature pulmonary artery (PA) smooth muscle cells (SMC) in vivo after chronic exposure to hypoxia and the mechanisms that regulate hypoxia-induced change in proliferative phenotype are not known. We tested the hypothesis that PA SMC from neonatal calves exposed to hypoxia after birth would both retain fetal-like and acquire new growth characteristics and that these changes would be at least partially dependent on protein kinase C (PKC), a key proproliferative signal transduction pathway. Like fetal cells, PA SMC from hypoxic calves grew faster in the presence and absence of serum and were more responsive to insulin-like growth factor I and platelet-derived growth factor-BB than control neonatal and adult cells. PA SMC from hypoxic calves also acquired other growth properties (i.e., including increased hypoxic growth after PKC activation) that were new compared with those observed for fetal cells. The proliferative response to hypoxia was first detectable in the neonatal period and was further increased in cells from hypoxic calves. SMC from fetuses and hypoxic calves were more susceptible to the growth-inhibiting effects of PKC antagonists (dihydrosphingosine and calphostin C) than control neonatal and adult cells. To test if the Ca(2+)-dependent isozymes of PKC were uniquely important in the developmental and acquired growth changes observed, the antagonistic effect of the specific, but isozyme nonselective, PKC inhibitor Ro-81-8220 was then compared with GF-109203X, a structural analog with relative specificity for the Ca(2+)-dependent isozymes of PKC (alpha and beta in PA SMC). The faster growing PA SMC from bovine fetuses and hypoxia-exposed calves again demonstrated greater growth inhibition in response to both inhibitors. GF-109203X was equipotent to Ro-31-8220, and its antiproliferative effects were shown to not be due to an increase in apoptosis. Phorbol ester-induced PKC downregulation, another inhibitor strategy that selectively depletes bovine PA SMC of PKC-alpha, but not -beta, mimicked the antiproliferative effects of GF-109203X. Whole cellular PKC catalytic activity paralleled the pattern of peptide-induced growth and susceptibility to PKC inhibition. These results suggest that PA SMC from hypoxia-exposed neonatal calves retain enhanced fetal-like proliferative capacity and acquire new growth properties that are at least partially dependent on the Ca(2+)-regulated isozymes of PKC and in particular PKC-alpha.

Mechanisms regulating cAMP-mediated growth of bovine neonatal pulmonary artery smooth muscle cells

“…Identical results were achieved with the PKC blocker Ro 31-8220 (5 µM; data not shown). A previous report from our laboratory (30) has shown that PKC inhibitors at concentrations presently reported do not cause significant cell death, confirming that inhibition of PKC decreased proliferation rather than induced apoptosis or necrosis. We next tested the role of adenylyl cyclase and cAMP in increased proliferation by blocking cAMPdependent protein kinase activity with Rp-cAMPS (1 mM).…”

“…3). Cell counts after forskolin and 8-bromo-cAMP application were higher in neonatal but not in adult PASMCs (data not shown), consistent with a proproliferative effect of cAMP in these neonatal cells (30).…”

“…Dempsey et al (6) previously (30). Interestingly, the ␣-isozyme of PKC activates type II adenylyl cyclase in Sf9 cells (34).…”

“…Vascular SMCs derived from neonatal PAs exhibit enhanced growth capacities to growth-promoting stimuli compared with SMCs derived from adult pulmonary arteries (6,30). The reason for this unique phenomenon is unclear, although enhanced growth capacity may contribute to normal adaptive mechanisms after birth as well as the need for continued pulmonary vascular growth.…”

“…Several studies (15,26) have demonstrated that when the normal transition to postnatal life is interrupted by hypoxia or increased pulmonary blood flow, marked proliferative changes in pulmonary artery (PA) SMCs (PASMCs) are observed that exceed those observed when adult animals are exposed to these stimuli. Similarly, SMCs derived from neonatal pulmonary arteries are less differentiated and exhibit enhanced growth responses to mitogenic stimuli compared with the relatively differentiated and quiescent SMCs derived from the adult pulmonary artery (6,30). Thus the increased growth capacity of neonatal PASMCs likely contributes to both normal pulmonary vascular development and the predisposition to develop exorbitant pulmonary vascular remodeling in response to injury in the neonatal period.…”