lnvolvement of N-formyl peptide receptors in modulating the pro-angiogenic pro-inflammatory activity of human vitreous in proliferative diabetic retinopathy (POR)

Purpose: POR is characterized by neovascularization and a persistent grade of infiammati an. N-formyl peptide receptors (FPRs) belong to a transmembrane G protein-coupled receptor superfamily ab le to modulate angiogenic and inflammatory responses. However, their involvement in POR remains to be elucidated. H ere, we examined FPR expression in human umbilical vein endothelial cells (HUVECs). We further assessed the capacity ofvitreous fluid obtained from POR patients after pars plana vitrectomy to induce pro-angiogenic/pro-inflammatory responses in endothelium an d the contribution of FPR inhibitors to abrogate these responses. Methods : Expression of FPRs {FPR1-FPR3} was examined in HUVECs at mRNA and protein levels. The capacity of POR vitreous samples to induce pro-angiogenic and pro-inflammatory responses in HUVECs was assessed by celi proliferati o n, motility and sprouting assays and by evaluating the activation of pro-intlammatory transcription factors, disruption of junctional protein and RT-PCR analysis of upregulation leukocyte adhesion molecules, respectively./n vivo, the pro-angiogenic/pro-intlammatory activity of PDR vitreous was tested in the chick embryo chorioallantoic membrane (CAM) assay. Finally, FPR1 inhibitor cyclosporin (CsH), FPR2 inhibitorWRW4 (Trp-Arg-Trp-Trp-Trp-Trp), pan-FPR inhibitor BOC-Phe-Leu-Phe-Leu-Phe (BOC-FLFLF), and the novel FPR inhibitor Ac-L-Arg-Aib-L Arg-L-Ca(Me)Phe-NH tetrapeptide (UPARANn were tested for their capacity to inhibit the biologica! responses elicited by PDR vitreous in vitro and in vivo. Results: Data from semi-quantitative RT-PCR, FACS an d Western blot analyses indicate that primary HUVEC cultures express FPR3 but not FPR1 and FPR2. PDR vitreous activates a pro-angiogenic/pro-inflammatory phenotype in endothelial cells. Accordingly, PDR vitreous triggers a potent angiogenic/intlammatory response in vivo. The pan-FPR inhibitor BOC-FLFLF and the novel FPR antagonist UPARANT inhibited the capacity of PDR vitreous to stimulate the sprouting of HUVEC spheroids embedded in 30-fibrin gel and neovessel formation in the CAM assay. Conclusions: Together, our data point to the involvement of FPRs in modulating the pro-angiogenic/pro-inflammatory response of PDR vitreous. FPRs may represent a target for the development of novel anti-intlammatory/anti-angiogenic approaches for PDR therapy.