Vascular endothelial growth factor stimulates a novel calcium-signaling pathway in vascular smooth muscle cells.
BACKGROUND
Vascular endothelial growth factor (VEGF) is a potent vascular mitogen that selectively stimulates vascular smooth muscle cell (VSMC) migration through an unknown mechanism while having no effect on VSMC proliferation. It is known that VSMC migration and proliferation are dependent on the second messenger Ca2+ and, in particular, mitogen-stimulated Ca2+ influx. We hypothesized that the selective effect of VEGF on VSMC migration versus proliferation was a result of differential VEGF-stimulated Ca2+ signaling pathways.


METHODS
Primary cultured human aortic smooth muscle cells (VSMCs) were grown to subconfluency and assigned to the following experimental groups: no stimulation, stimulation with platelet-derived growth factor-BB (PDGF-BB) (20 ng/mL) as positive control, and stimulation with VEGF165 (40 ng/mL). Total increase in [Ca2+]cyt and intracellular calcium release was quantified with the use of a fura-2 fluorescence assay. Assays for the following receptors VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and PDGFR-beta were performed by immunoprecipitation, while PLCgamma1, Akt 1/2, and phospholamban B phosphorylation were assessed with Western immunoblotting.


RESULTS
VSMCs stimulated with VEGF165 exhibited no intracellular Ca2+ release, compared with a 75 +/- 30 nmol/L intracellular calcium release after PDGF-BB stimulation, (P < .02) VEGF165-stimulated VSMCs in Ca2+-containing media exhibited 192 +/- 26 nmol/L increase in [Ca2+]cyt, compared with 354 +/- 54 nmol/L increase after PDGF-BB stimulation (P < .02). VEGF165 did not phosphorylate PLCgamma1 after 1, 5, or 10 minutes of treatment. VEGF165 treatment did not result in PI3-K/Akt activation at 1-, 5-, or 10-minute time points. Calmodulin-dependent kinase II (CaMKII) was activated by both VEGF165 and PDGF-BB after 1 and 5 minutes of stimulation. The presence of the receptors VEGFR-1, VEGFR-2, and PDGFR-beta was confirmed in all experimental groups.


CONCLUSIONS
VEGF induces extracellular calcium influx but no intracellular calcium release in VSMCs. This lack of intracellular Ca2+ release stems from the inability of VEGF165 to activate the PLCgamma1 cascade and IP3 receptor-mediated Ca2+ release. The lack of PI3-K/Akt activation at these time points indicates a novel extracellular Ca2+ influx pathway sufficient to activate CaMKII. A paradigm relating extracellular Ca2+ influx to CaMKII activation and migration is suggested and may account for the selective effects of VEGF on VSMC migration.
Chandra
Ankur
A
UCSD Section of Vascular and Endovascular Surgery, UCSD Medical Center, San Diego, California 92103-8402, USA.
Angle
Niren
N
D001011:Aorta / Q000166:cytology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D045604:Extracellular Fluid / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051966:Phospholipase C gamma / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000187:drug effects / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism* / Q000494:pharmacology; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D019869:Phosphatidylinositol 3-Kinases; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D051057:Proto-Oncogene Proteins c-akt; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D051966:Phospholipase C gamma; D002118:Calcium