Α-Melanocyte-Stimulating Hormone Protects Early Diabetic Retina from Blood-Retinal Barrier Breakdown and Vascular Leakage via MC4R

Abstract

Background/Aims: Blood-retinal barrier (BRB) breakdown and vascular leakage is the leading cause of blindness of diabetic retinopathy (DR). Hyperglycemia-induced oxidative stress and inflammation are primary pathogenic factors of this severe DR complication. An effective interventional modality against the pathogenic factors during early DR is needed to curb BRB breakdown and vascular leakage. This study sought to examine the protective effects of α-Melanocyte-stimulating hormone (α-MSH) on early diabetic retina against vascular hyperpermeability, electrophysiological dysfunction, and morphological deterioration in a rat model of diabetes and probe the mechanisms underlying the α-MSH’s anti-hyperpermeability in both rodent retinas and simian retinal vascular endothelial cells (RF6A). Methods: Sprague Dawley rats were injected through tail vein with streptozotocin to induce diabetes. The rats were intravitreally injected with α-MSH or saline at Week 1 and 3 after hyperglycemia. In another 2 weeks, Evans blue assay, transmission electron microscopy, electroretinogram (ERG), and hematoxylin and eosin (H&E) staining were performed to examine the protective effects of α-MSH in diabetic retinas. The expression of pro-inflammatory factors and tight junction at mRNA and protein levels in retinas was analyzed. Finally, the α-MSH’s anti-hyperpermeability was confirmed in a high glucose (HG)-treated RF6A cell monolayer transwell culture by transendothelial electrical resistance (TEER) measurement and a fluorescein isothiocyanate-Dextran assay. Universal or specific melanocortin receptor (MCR) blockers were also employed to elucidate the MCR subtype mediating α-MSH’s protection. Results: Evans blue assay showed that BRB breakdown and vascular leakage was detected, and rescued by α-MSH both qualitatively and quantitatively in early diabetic retinas; electron microscopy revealed substantially improved retinal and choroidal vessel ultrastructures in α-MSH-treated diabetic retinas; scotopic ERG suggested partial rescue of functional defects by α-MSH in diabetic retinas; and H&E staining revealed significantly increased thickness of all layers in α-MSH-treated diabetic retinas. Mechanistically, α-MSH corrected aberrant transcript and protein expression of pro-inflammatory factor and tight junction genes in the diseased retinas; moreover, it prevented abnormal changes in TEER and permeability in HG-stimulated RF6A cells, and this anti-hyperpermeability was abolished by a universal MCR blocker or an antagonist specific to MC4R. Conclusions: This study showed previously undescribed protective effects of α-MSH on inhibiting BRB breakdown and vascular leakage, improving electrophysiological functions and morphology in early diabetic retinas, which may be due to its down-regulating pro-inflammatory factors and augmenting tight junctions. α-MSH acts predominantly on MC4R to antagonize hyperpermeability in retinal microvessel endothelial cells.

Ultrastructural studies of the neurovascular unit reveal enhanced endothelial transcytosis in hyperglycemia‐enhanced hemorrhagic transformation after stroke

“…To assess BBB integrity after mild I/R (20 min / 4 h) injury, we monitored the extravasation of Evans blue (EB) dye into the brain parenchyma after injection into the mouse tail vein. Similar to previous reports, 34‐36 we observed more severe brain leakage of EB after ischemic stroke in the ipsilateral brain tissues of HG mice, compared to NG controls (Figure 1A,B). To further quantitatively evaluate the extent of EB leakage, fluorescence intensity was used to measure the EB absorbance at 620 nm.…”

Probing the Role of Melanocortin Type 1 Receptor Agonists in Diverse Immunological Diseases

“…Other studies have demonstrated protective, anti-inflammatory, and anti-apoptotic effects of α-MSH in the retina in rat models of diabetic retinopathy (Zhang et al, 2014; Cai et al, 2018). Potential protective mechanisms of α-MSH against diabetic retinopathy suggested by these studies include inhibition of Forkhead box O genes induced by high glucose concentrations (Zhang et al, 2014) and the correction of aberrant expression of inflammatory factors and tight junction genes, and inhibition of hyperpermeability, in diseased retinas (Cai et al, 2018). Investigation of whether PL-8177 and/or PL-8331 possess similar mechanisms and protective effects in preclinical models of diabetic retinopathy is of interest for future studies.…”

Connexin Hemichannel Block Using Orally Delivered Tonabersat Improves Outcomes in Animal Models of Retinal Disease

“…To investigate whether micro-and macroaneurysms in the hyperglycemic rat retinas (observed using OCT) reflect sites of vessel leak, rats were perfused at the age of 3 months with Evans Blue dye as previously described [33]. Briefly, Evans Blue dye (30 mg/ml; Sigma-Aldrich, USA) was dissolved in normal saline and filtered.…”