Abstract
Abstract: :
Purpose: To determine if sympathetic denervation results in changes in gene expression of several known pro–angiogenic growth factors. Methods: Real–time polymerase chain reaction was carried out to measure steady–state mRNA expression for the following angiogenic factors: VEGF, VEGFR–2 (Flk–1/KDR), Ephrin–B2, EphB4, Angiopoietin–1, and TIE2. Western blotting was performed to measure protein expression of these genes. Results: Steady–state mRNA and protein expression was significantly decreased for both VEGF and VEGFR–2 at six weeks post–sympathectomy. Gene expression of ephrin–B2 was significantly upregulated, whereas that of EphB4 was unchanged at 6–weeks post–surgery. Protein expression of these genes followed the same trends. Both angiopoietin–1 and TIE–2 mRNA expression was significantly increased following sympathectomy. Densitometry showed that protein expression for angiopoietin–1 and TIE–2 were also increased. Conclusions: There are two possible explanations for the decrease in VEGF mRNA and protein expression. We have previously shown that sympathectomy causes increased capillary density in the outer nuclear layer of the retina . Since the increased capillary density has occurred by six–weeks post–sympathectomy, it is possible that VEGF levels have already peaked to elicit this response and have returned to basal levels at the six–week time point. Secondly, others have published that beta–adrenergic stimulation via norepinephrine induces VEGF gene expression (Fredriksson, Lindquist et al. 2000). Therefore, removing sympathetic nerve innervation via ganglionectomy should decrease VEGF gene expression due to denervation. The ephrin ligands and Eph receptor tyrosine kinases have been shown to play a role in neovascularization and other vascular events. The significant enhancement of ephrin–B2, a known ligand for EphB4, could play a role in the vascular remodeling event observed after sympathectomy, however, its regulation by sympathetic nerves is yet to be determined. Angiopoietin–1 and TIE–2 have been shown to elicit angiogenic effects later in the cascade than VEGF, regulating interaction between endothelial cells with surrounding mesenchymal cells and extracellular matrix . This correlates with our findings, suggesting a stabilizing role for angiopoietin–1 amidst areas of vascular remodeling. We propose that sympathetic nerves affect the expression and regulation of angiogenic growth factors in rat retina and that sympathetic nerves need to be an additional focus with respect to treatment of proliferative retinopathy.
Keywords: growth factors/growth factor receptors • innervation: neural regulation • diabetic retinopathy