Cytosolic 85-kDa phospholipase A2-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 A2 (PLA2) hydrolysis. The purpose of this study was to examine the roles of the distinct 14-kDa and 85-kDa PLA2 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 PLA2 activity nor mass changed over a 10-day culture period. In contrast, 85-kDa PLA2 protein activity and mRNA decreased as time in culture progressed. This reduction in 85-kDa PLA2 correlated with reductions in DNA synthesis and suggested a possible association between 85-kDa PLA2 and proliferation. To directly evaluate the role of the 85-kDa PLA2 in proliferation we examined the effects of an 85-kDa PLA2 inhibitor (AACOCF3) and 85-kDa PLA2 antisense oligonucleotides on proliferation. Both reagents dose dependently inhibited proliferation, whereas a 14-kDa PLA2 inhibitor (SB203347), a calcium-independent PLA2 inhibitor (HELSS), an 85-kDa sense oligonucleotide, and a nonrelevant scrambled control oligonucleotide had no effect. The mechanism by which 85-kDa PLA2 influences cellular proliferation remains unclear. Inhibition of 85-kDa PLA2 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 AACOCF3 and 85-kDa antisense oligonucleotides implicating AA as a key mediator in cellular proliferation. However, although prostaglandin E2 (PGE2) 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 PGE2. These data provide the first direct evidence that PLA2 is involved in control of VSMC proliferation and indicate that 85-kDa PLA2-mediated liberation of AA is critical for cellular proliferation.
Anderson
K M
KM
Departments of Cardiovascular Pharmacology and Immunopharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
Roshak
A
A
Winkler
J D
JD
McCord
M
M
Marshall
L A
LA
D016718:Arachidonic Acid / Q000378:metabolism; D001095:Arachidonic Acids / Q000494:pharmacology; D002453:Cell Cycle; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2
D001095:Arachidonic Acids; D004791:Enzyme Inhibitors; C081565:arachidonyltrifluoromethane; D016718:Arachidonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2