Proliferation, migration, and apoptosis of vascular smooth muscle cells (VSMC) are pivotal determinants of the pathogenesis of vascular diseases, which are mainly controlled by growth factor dependent activation of PI 3-Kinase (PI3K). Growth factors like platelet-derived growth factor (PDGF) activate class IA PI3Ks containing one of three p110 catalytic subunits (p110alpha, p110beta, and p110delta). We investigated the specific function of these isoforms for PDGF-controlled proliferation, migration, and apoptosis of VSMC using novel isoform-specific inhibitors. PDGF-dependent proliferation and migration solely depended on p110alpha. Stimulation of VSMC with PDGF-BB (50 ng/ml) mediated a 2.5±0.4 increase (
p
<0.05) of DNA-synthesis (BrdU incorporation assay) and induced a 3.4+/−0.7 fold increase (
p
<0.05) of VSMC migration (modified Boyden-chamber). Inhibition of p110alpha with PIK075 (1
μ
M, Ki=100 nM) completely abrogated PDGF-dependent DNA-synthesis and migration (
p
<0,05), whereas inhibitors against p110beta (TGX 221, 1
μ
M) or p110delta (IC87114 1
μ
M) had no influence. Consistently, PDGF-induced DNA-synthesis and migration were suppressed by siRNA-dependent downregulation of p110alpha (
p
<0,05) whereas p110beta or p110delta knockdown had no effect. Interestingly, stimulation of VSMC with PDGF-BB (50 ng/ml) induced anti- or proapoptotic effects depending on the duration of PDGFR activation. Incubation of VSMC with H
2
O
2
(50
μ
M, 16h) led to a 2.8±0.7 fold increase (
p
>0.05) of apoptosis (Cell Death Detection ELISA). Simultanous addition of PDGF-BB (50 ng/ml) significantly diminished the H
2
O
2
-induced apoptosis (52±7%,
p
>0.05). In contrast, prestimulation with PDGF-BB 24h prior to the addition of H
2
O
2
led to an increase of H
2
O
2
-induced apoptosis (7.8±1.3,
p
>0.05). The anti- as well as the proapoptotic effect depended strictly on p110alpha as PIK075 (1
μ
M,
p
<0,05) or p110alpha specific siRNA completely abrogated PDGF-BB-mediated pro- as well as antiapoptotic effects. Our results demonstrate that only the catalytical PI3K subunit p110alpha mediates the growth factor-induced atherogenic responses. Therefore, p110alpha represents an interesting therapeutic target for prevention of atherosclerosis and restenosis formation.
Tyrphostin inhibition of ATP-stimulated DNA synthesis, cell proliferation and Fos-protein expression in vascular smooth muscle cells
