Microenvironment-Induced Endothelial Heterogeneity Stimulates Retinal Neovascularization in Sickle Mice.
Authors: Gupta Kalpna , Chen Chunsheng , Lutty Gerard A. , Hebbel Robert P. 
Published: April 2020

Abstract
Retinopathy is a serious complication of sickle cell disease (SCD) which is accompanied by endothelial activation and a pro-inflammatory microenvironment. We hypothesized that endogenous and exogenous factors modulate the endothelial microenvironment and endothelial specific alterations leading to retinal neovascularization. While VEGF receptor 2/Flk1 is a key player in the process of angiogenesis, opioids used to treat pain in SCD also promote angiogenesis. Therefore, we examined the role of opioids in the regulation of endothelial microenvironment, Flk1 expression and endothelial activity, using wild type (WT) and NY1DD sickle mice with transgenes for human α and βS globin. We injected 9–11 month old mice with morphine subcutaneously (50 mg/day/70 Kg with 10 mg increments every 2 weeks and/or equimolar amount of naloxone) for 10 to 16 months. NY1DD mouse retina exhibited appreciably higher expression of phospho-Flk1 vs WT which was further upregulated by morphine treatment for 10 and 16 months. Naloxone treatment antagonized morphine induced upregulation of phospho-Flk1, suggesting an opioid receptor mediated effect. Upregulation of phospho-Flk1 was accompanied by an increase in cell cycle regulatory protein cyclin D1 and stimulation of phospho-MAPK/ERK and phospho-Akt in morphine vs PBS treated mouse retina. Furthermore, morphine treatment led to a 5 and 20 fold increase in plasma IL6 and TNFα, respectively as compared to PBS treatment and promoted retinal neovascularization. Thus morphine treatment exaggerates the pro-inflammatory micro-environment and orchestrates a pro-angiogenic tone by upregulating Flk1 and activating the growth and survival promoting signaling in NY1DD mouse retina. To specifically delineate the role of opioids in endothelial activity leading to retinopathy, we isolated retinal endothelial cells (REC) from WT and NY1DD mice. Both VEGF and morphine stimulated REC proliferation in a dose-dependent fashion. Maximum proliferation was achieved with 1 μm morphine or 10 ng/ml VEGF in WT REC, but it was unsaturable upto 10 μm morphine or 1 μg/ml VEGF in NY1DD REC. Optimum morphine and VEGF-induced REC proliferation was 1.5 and 3 fold, respectively in NY1DD Vs WT REC, suggestive of a heterogeneity between WT and NY1DD REC. Saturation kinetics and opioid receptor binding studies showed that the Bmax for 3H-diprenorphine and the number of opioid receptor binding sites were 3-fold higher in NY1DD vs WT REC. RT-PCR analysis showed a higher expression of both Flk1 and μ opioid receptor (MOR) in NY1DD vs WT REC. Incubation of WT REC with IL6 as well as morphine for 48h upregulated Flk1 protein expression. Morphine-induced upregulation of Flk1 was abrogated by naloxone, anti-IL6 antibodies and STAT3 inhibitor Ac-PpYLKTK-OH. On the other hand, incubation of WT-REC with VEGF or IL6 for 48h upregulated MOR expression. Together, these data suggest that morphine stimulates Flk1 expression directly via opioid receptor(s) and also by upregulating IL6 via a STAT3 signaling pathway, while VEGF and IL6 also stimulate MOR expression. Therefore, a pro-inflammatory microenvironment in SCD upregulates endothelial Flk1 and opioid receptors, which is further exaggerated by addition of morphine leading to increased retinal neovascularization. It is likely that opioid use in SCD promotes retinopathy and that opioid receptor antagonists may play a preventive role.