Abstract
Release of endothelial cells from contact-inhibition and cell cycle re-entry is required for the induction of new blood vessel formation by angiogenesis. Using a combination of chemical inhibition, loss of function, and gain of function approaches, we demonstrate that endothelial cell cycle re-entry, S phase progression, and subsequent angiogenic tubule formation are dependent upon the activity of cytosolic phospholipase A(2)-alpha (cPLA(2)alpha). Inhibition of cPLA(2)alpha activity and small interfering RNA (siRNA)-mediated knockdown of endogenous cPLA(2)alpha reduced endothelial cell proliferation. In the absence of cPLA(2)alpha activity, endothelial cells exhibited retarded progression from G(1) through S phase, displayed reduced cyclin A/cdk2 expression, and generated less arachidonic acid. In quiescent endothelial cells, cPLA(2)alpha is inactivated upon its sequestration at the Golgi apparatus. Upon the stimulation of endothelial cell proliferation, activation of cPLA(2)alpha by release from the Golgi apparatus was critical to the induction of cyclin A expression and efficient cell cycle progression. Consequently, inhibition of cPLA(2)alpha was sufficient to block angiogenic tubule formation in vitro. Furthermore, the siRNA-mediated retardation of endothelial cell cycle re-entry and proliferation was reversed upon overexpression of an siRNA-resistant form of cPLA(2)alpha. Thus, activation of cPLA(2)alpha acts as a novel mechanism for the regulation of endothelial cell cycle re-entry, cell cycle progression, and angiogenesis.
Highlights
(AA) at the sn-2 position to liberate free AA [3]
Phase Progression—We have previously demonstrated that cPLA2␣ activity is reduced in confluent monolayers of endothelial cells that are in the G0 phase of quiescence [11], and we proposed that cPLA2␣ is inactivated in confluent endothelial cells upon its sequestration at the Golgi apparatus and exclusion from its intracellular membrane substrate [12]
This study shows that cPLA2␣ activation helps to regulate endothelial cell S phase progression and cell cycle entry
Summary
Cell Culture and Materials—Human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical cords as described previously [16, 17]. Subconfluent, confluent, or wounded cells were washed with PBS and incubated with cPLA2␣ inhibitors for 15 min or overnight as indicated. For measurement of passive AA release, media were collected following overnight incubation (18 h), cleared by centrifugation, and assayed for radioactivity by liquid scintillation counting. Cleared supernatant containing released AA was assayed for radioactivity by liquid scintillation as were total cell lysates prepared by lysis of cells in 0.5% Triton X-100. Cells were either chased in fresh media containing inhibitors for various time points prior to processing as above, or siRNA-treated cells were pulsed with media supplemented with 10 M BrdUrd for 30 min prior to harvesting, fixation, and analysis for BrdUrd incorporation. 20-min fixative step in 100% ethanol on ice. Cells were incubated with 2 N HCl, 0.5% Triton X-100 for 20 min at room temperature followed by a wash in 100 mM sodium tetraborohydrate. Cells were processed as above with the indicated antibodies
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