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Joy Sarkar, Yuncin Luo, Qiang Zhou, Evguenia Ivakhnitskaia, Eitan Katz, Michael Sun, Daniel Lara, Victor H Guaiquil, Mark Rosenblatt; Differential Distribution of VEGF Receptor Dimers in Neuronal and Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2021;62(8):711.
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© ARVO (1962-2015); The Authors (2016-present)
VEGF Receptor dimer activation is critical for regulating downstream signaling cascades in angiogenesis. However, not much is known about these processes in corneal nerve repair. Previous studies have shown that the non-angiogenic ligand VEGF-B has potent roles in the peripheral nervous system (PNS). The molecular mechanism of VEGF-B-mediated corneal nerve growth is unclear and may be due to differences in VEGFR1 homo- and heterodimer presence. The purpose of this study was to investigate the presence of VEGFR1 and R2 homo- and heterodimers and characterize similarities and differences in their distribution compared to endothelial cells.
Rat neuronal (PC12), mouse aortic endothelial (MAE), mouse venous endothelial (MVE) and human umbilical venous endothelial (HUVEC) cell lines were used. Cells were acutely stimulated either with VEGF-A (50 ng/µL; VEGFR1 and VEGFR2 binding) or VEGF-B (50 ng/µL; VEGFR1 binding) or “vehicle” (PBS; control group). We also isolated mouse trigeminal ganglion cells from thy1-YFP neurofluorescent mice. After treatment, cells were used as follows: (i) One group was fixed in 4% paraformaldehyde and processed for VEGFR1 and VEGFR2 immunostaining and visualized using confocal fluorescence microscopy and Total Internal Reflection (TIRF) microscopy; (ii) the second group was 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) a third group of cells were also processed for Proximity Ligation Assay (PLA; Sigma) to assess the presence and distribution of VEGF Receptor homo- and heterodimers.
TIRF and fluorescence confocal microscopy showed presence of VEGFR1 co-localized with VEGFR2 in endothelial and neuronal cells. Cell lysates immunoprecipitated with anti-VEGFR1 further validated the existence of VEGFR1-R2 heterodimers in neuronal cells. Neuronal cells showed higher levels of VEGFR1-R2 heterodimers compared to endothelial cells whereas endothelial cells showed higher levels of VEGFR2-R2 homodimers as shown by PLA studies.
Differences in VEGF Receptor homo- and heterodimer distribution may explain the differential role of VEGF ligands in neuronal vs endothelial cell types.
This is a 2021 ARVO Annual Meeting abstract.
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