Abstract
Purpose :
In addition to the well-known pathological consequences of elevated total VEGFA, reports of isoform switching of VEGFA165b to VEGFA165a in the vitreous of patients with proliferative eye diseases led us to ask whether this isoform shift is a significant contributor to the hyperactivation of primary human retinal microvascular endothelial cells (HRMECs). As part of this investigation, we compared the contributions of MAPK, AKT, and p38-MAPK to proliferation and migration in primary HRMECs using in situ immunofluorescence assays.
Methods :
VEGFA165 isoforms were compared for their effects on cell proliferation, measured with infrared-fluorescence Cell-Tag assays. Cell migration was compared using a fluorescent transmembrane migration assay in the multiwell plate format. Relative contribution of the MAPK, AKT, and p38-MAPK pathway to cell proliferation and migration were assessed using pharmacological inhibition of MAPK, AKT, and p38-MAPK activation.
Results :
At a saturating activation concentration (5,000pM), VEGFA165b was as good or slightly more potent than VEGFA165a as a stimulator of cell proliferation but was not significantly different between both isoforms. The p38-MAPK inhibitor SB203580 increased HRMEC proliferation but was also found to be a potent activator of MAPK, while blocking p38-MAPK activation. VEGFA165a stimulated cell migration more than 6-fold compared to VEGFA165b at 1000 pM. Inhibition of MAPK with U0126 (MEK inhibitor), did not inhibit VEGFA165-induced migration. Inhibition of AKT activation with MK2206 did not inhibit a VEGFA165-induced cell migration. Inhibition of p38-MAPK with BIRB796 completely blocked VEGFA165-induced cell migration.
Conclusions :
VEGFA165b was a poor activator of HRMEC migration at lower, physiological, concentrations where VEGFA165a induced substantial cell migration. This was consistent with our dose-response analysis of their relative activation of intracellular signaling pathways (MAPK, AKT, and p38-MAPK). VEGFA165 driven cell migration was mostly mediated via activation of the p38-MAPK pathway. VEGFA165-induced proliferation of primary HRMECs did not require activation of p38-MAPK in addition to MAPK.
This is a 2021 ARVO Annual Meeting abstract.