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.