Flt-1 and KDR knockdown affects receptor tyrosine phosphorylation and VEGF-mediated neurite growth in PC12 neuronal cells

Abstract

Purpose : In angiogenesis, activation of Flt-1/KDR by ligand binding leads to phosphorylation at intracellular tyrosine residues and activation of downstream signaling cascades. Nevertheless, details of these processes in corneal nerve repair are unclear. VEGF-B has previously been shown to have potent effects in the peripheral nervous system (PNS) possibly due to the existence of receptor heterodimers. The purpose of this study was to investigate Flt-1/KDR tyrosine phosphorylation at specific residues in neuronal cells and characterize similarities and differences with endothelial cells as well as investigate the effects of Flt-1/KDR knockdown on phosphorylation and VEGF-mediated neuronal growth.

Methods : Rat neuronal (PC12), mouse aortic endothelial (MVEC), mouse venous endothelial (MVEC) and human umbilical venous endothelial (HUVEC) cell lines were used. “Receptor Control Group” of cells were acutely stimulated either with VEGF-A or VEGF-B (50 ng/µL) or “vehicle” (PBS; control group). “Receptor Knockdown Group” of cells were treated with siRNA specifically directed against Flt-1 or KDR as per manufacturer’s instructions and then acutely stimulated with VEGF-A or B as described earlier. After treatment, cells were used as follows: (i) One group was fixed in 4% paraformaldehyde and processed for TUBB3 immunostaining and neurite length was visualized using fluorescence microscopy (ii) the second group was harvested in cell lysis buffer (containing anti-protease / anti-phosphatase cocktail), lysed and processed for immunoblotting (IB; LI-COR® Systems) with anti-Flt-1, anti-KDR, anti-phospho-Flt-1 (Tyr1213, Tyr1333), anti-phospho-KDR (Tyr951, Tyr1054, Tyr1175, Tyr1214) antibodies to evaluate receptor tyrosine phosphorylation at specific residues.

Results : Differences in tyrosine phosphorylation at specific residues were observed for Flt1 and KDR in neuronal versus endothelial cells. siRNA-mediated knockdown of Flt1 and KDR caused decreased tyrosine phosphorylation at specific residues in neuronal and endothelial cells. VEGF-A and -B treatment increased neurite outgrowth and branching in PC12 neuronal cells. siRNA knockdown of Flt1 or KDR caused reduction in neurite length and number.

Conclusions : Differences in Flt-1/KDR tyrosine phosphorylation at specific residues may regulate downstream signaling events and influence corneal nerve regeneration.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.