Cytosolic 85-kDa phospholipase A(2)-mediated release of arachidonic acid is critical for proliferation of vascular smooth muscle cells

Recent evidence suggests that arachidonic acid (AA) may be involved in regulating cellular proliferation, The predominant mechanism of AA release from cellular phospholipids is via phospholipase A(2) (PLA(2)) hydrolysis, The purpose of this study was to examine the roles of the distinct 14-kDa and 85-kDa PLA(2) enzymes in human coronary artery vascular smooth muscle cell (hCAVSMC) proliferation, Cultured hCAVSMCs proliferate in the presence of growth medium with a typical doubling time of 30-40 h, grow at a slower proliferative rate upon reaching confluency (day 8), and eventually undergo contact inhibition of growth (day 10), Neither Type II 14-kDa PLA(2) activity nor mass changed over a 10-day culture period, In contrast, 85-kDa PLA(2) protein activity and mRNA decreased as time in culture progressed. This reduction in 85-kDa PLA(2) correlated with reductions in DNA synthesis and suggested a possible association between 85-kDa PLA(2) and proliferation, To directly evaluate the role of the 85-kDa PLA(2) in proliferation we examined the effects of an 85-kDa PLA(2) inhibitor (AACOCF(3)) and 85-kDa PLA(2) antisense oligonucleotides on proliferation. Both reagents dose dependently inhibited proliferation, whereas a 14-kDa PLA(2) inhibitor (SB203347), a calcium-independent PLA(2) inhibitor (HELSS), all 85-kDa sense oligonucleotide, and a nonrelevant scrambled control oligonucleotide had no effect, The mechanism by which 85-kDa PLA(2) influences cellular proliferation remains unclear. Inhibition of 85-kDa PLA(2) activity produced neither phase-specific cell cycle arrest nor apoptosis (fluorescence-activated cell sorter analysis). Addition of AA (20 mu M) attenuated the effects of both AACOCF(3) and 85-kDa antisense oligonucleotides implicating AA as a key mediator in cellular proliferation However, although prostaglandin E-2 (PGE(2)) was present in the culture medium, it peaked early (day 3) in culture, and indomethacin had no effect on cellular proliferation indicating that hCAVSMC proliferation was not mediated through PGE(2). These data provide the first direct evidence that PLA(2) is involved in control of VSMC proliferation and indicate that 85-kDa PLA(2)-mediated liberation of AA is critical for cellular proliferation.