Abstract
Purpose :
We have previously reported that VEGF-B is more potent than VEGF-A in mediating corneal nerve growth in vitro and in vivo. This stimulation of nerve growth is different from stimulation of angiogenesis by the same ligands. In angiogenesis, VEGR1/R2 activation after ligand binding and subsequent phosphorylation alters the activation of downstream signaling cascades. However, our understanding of these processes in corneal nerve repair remains unclear. The purpose of this study was characterization of VEGF receptor activation and ligand interactions in neuronal cells to elucidate their role in corneal nerve repair.
Methods :
PC12 (rat neuronal cell line), MAEC (mouse aortic endothelial cell line), MVEC (mouse venous endothelial cell line) and HUVEC (human umbilical venous endothelial cell line; control group) were used. Cells were acutely stimulated either with VEGF-A (50 ng/µL; VEGFR1 and VEGFR2 binding) or VEGF-B (50 ng/µL; ostensible VEGFR1 binding) or “vehicle” (PBS; control group). After treatment, cells were used as follows: (i) 4% paraformaldehyde fixation and processed for VEGFR1 and VEGFR2 immunostaining and confocal fluorescence microscopy; (ii) Harvested in cell lysis buffer (containing anti-protease / anti-phosphatase cocktail), lysed and processed for immunoprecipitation (IP; Thermo Fisher IP kit) and immunoblotting (IB; LI-COR® Systems). Immunoprecipitated proteins were probed either with anti-VEGFR1 or anti-VEGFR2 IgG antibodies to evaluate VEGFR1-R2-heterodimerization; (iii) Phosphorylation at specific intracellular tyrosine residues was evaluated by immunoblotting using anti-phospho-antibodies directed to VEGFR1 residues (Y1213, Y1333) and VEGFR2 residues (Y1054, Y1175, Y1214, Y951).
Results :
Confocal Microscopy revealed VEGFR1-R2 colocalization in endothelial and neuronal cells. Cell lysates immunoprecipitated with anti-VEGFR1 showed the presence of VEGFR1-R2 heterodimers in both cell types. VEGF-A and B treatment induced ~ 2.5-fold increase in pTy1333 (VEGFR1) and pTy951 (VEGFR2) and ~ 2-fold increase in pTy1175 (VEGFR2) in PC12 cells as compared to MAEC and MVEC respectively whereas pTy1213 (VEGFR1) levels remained unchanged.
Conclusions :
Neuronal and endothelial cells exhibit differences in VEGF receptor tyrosine phosphorylation at specific residues after ligand-activation and VEGFR1-R2 heterodimers may influence VEGF activity and regulation of corneal neuronal cell homeostasis.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.