Vascular endothelial growth factor and hepatocyte growth factor levels are differentially elevated in patients with advanced retinopathy of prematurity

Abstract

Although the roles of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in angiogenesis are well described, the putative roles of these factors in retinopathy of prematurity (ROP) remain unknown. We evaluated VEGF and HGF protein levels in subretinal fluid of eyes with ROP, and expression of their corresponding receptors in retrolental membranes associated with stage 5 ROP. We examined subretinal fluid samples from eyes using rhegmatogenous retinal detachment as a control. VEGF and HGF were differentially elevated in eyes with ROP. In Stage 5 ROP (n = 22), the mean VEGF and HGF levels were 14.77 +/- 14.01 ng/ml and 16.56 +/- 9.62 ng/ml, respectively. Interestingly, in patients with active stage 4 ROP, mean VEGF levels were highly elevated (44.16 +/- 18.72 ng/ml), whereas mean HGF levels remained very low (4.77 +/- 2.50 ng/ml). Next, we investigated in vivo expression of VEGF receptor-2 and HGF receptor in retrolental membranes from 16 patients with stage 5 ROP. Both VEGF receptor-2 and HGF receptor proteins were detected mainly in posterior portions of the membrane as well as in vessel walls and along the retinal interface where angiogenesis was active. These findings together suggest that VEGF and HGF play important roles in the pathogenesis of ROP.

Figure 1.

HGF-dependent tyrosine phosphorylation of the HGF receptor (HGFR) in response to stimulation with subretinal fluid from stage 5 retinopathy of prematurity (ROP) and rhegmatogenous retinal detachment (RRD). A549 cells were grown to 80% confluence and serum-starved overnight in DMEM containing 0.1% calf serum. Cells were stimulated for 10 minutes with either 50 ng/ml HGF (as control), or with subretinal fluid collected from 3 eyes with stage 5 ROP and 3 eyes with RRD. Cells were lysed and immunoprecipitated with anti-phosphotyrosine antibody and then subjected to Western blot analysis using anti-HGFR antibody. The blot shows that subretinal fluid from both stage 5 ROP and RRD were able to trigger tyrosine phosphorylation of HGFR, suggesting that HGF is secreted in an active form within the subretinal space. The arrow shows the HGFR (145 kd) protein.

Figure 2.

Immunohistochemical localization of vascular endothelial growth factor receptor-2 (VEGFR-2) and hepatocyte growth factor receptor (HGFR) in retrolental membranes (RLF) obtained from patients with stage 5 retinopathy of prematurity (ROP). Protein expression was detected using the DAB reagent (peroxidase; brown color) and tissue was counterstained with hematoxylin. Bar, 100 μm. A: Using an antibody that recognizes VEGFR-2, its expression is seen in population of cells and in the vascular compartment within the posterior segment of the RLF adjacent to the retinal interface (arrowhead). B: HGFR expression is seen in vessel walls and within stromal cells, and more concentrated along the retinal interface. C: Negative control using nonimmune polyclonal rabbit antibody. Trace pigment deposition is along the posterior segment of the RLF membrane.

Figure 3.

Expression of vascular endothelial growth factor receptor-2 (A) and hepatocyte growth factor receptor (B) by intrastromal spindle cells within the retrolental membrane from stage 5 retinopathy of prematurity. Proteins were detected using DAB reagent (peroxidase; brown color), and tissue was counterstained with hematoxylin. Bar, 50 μm. C: Negative control using nonimmune polyclonal rabbit antibody.