Abstract
Purpose :
Cigarette smoke is a known risk factor for AMD. Mitochondrial dysfunction of RPE contributes to AMD progression. Humanin (HN) fused to elastin-like polypeptide (ELP) can protect RPE cells from mitochondrial damage from acute oxidative stress (PMID:31655204). We wished to investigate the effect of prolonged exposure of hRPE cells and mouse retina to cigarette smoke extract (CSE) and to determine whether HN-ELP (V96-HN) protects RPE/retina from injury.
Methods :
Primary cultured human fetal RPE cells were treated with varying doses (1–1000 µg/ml) of CSE for 24 h to evaluate the concentration-dependent cell viability. hRPE cells were treated 3 times a week with 1, 5, and 10 μg/ml CSE alone or CSE + HN-ELP (5 µM) for 21 days and mitochondrial bioenergetics were assessed. The regulation of mitochondrial Stat3 (mitoStat3) was studied after treatment of RPE with 200 μg/ml CSE alone or HN-ELP alone, or CSE + HN-ELP (5 µM) for 1 h. The therapeutic potential of HN-ELP was tested in vivo in 10-month-old male mice (C57BL/6J) intravitreally injected with CSE (1µl from 200 µg/ml) on Day 0 with appropriate DMSO controls. On day 3, one group received HN-ELP intravitreally (1µl from 400 µM stock). ERG, OCT, and fundus were analyzed on day 10, and eyes were processed for H&E and TUNEL staining.
Results :
Acute CSE doses >50 µg/ml caused significant cell death and a dose range of 1-10 µg/ml with < 10% cell death was chosen for chronic studies. We measured mitochondrial respiration (OXPHOS) to study CSE effect on mitochondrial function. Chronic CSE at three doses (1, 5, and 10 µg/ml) caused a dose-dependent decrease in basal respiration, ATP production, and maximal respiration. HN-ELP-cotreated RPE showed a significant improvement in mitochondrial bioenergetics. With HN-ELP, Stat3 was colocalized with RPE mitochondria while Stat3 was not detected in mitochondria of CSE-treated cells. Increased Stat3 was found in the mitochondria of RPE co-treated with CSE and HN-ELP. Studies in vivo demonstrated that a single intravitreal injection of HN-ELP significantly augmented photoreceptor and RPE function and protected retinal structure in CSE-injected mice. HN-ELP also protected the retina against CSE-induced apoptosis.
Conclusions :
HN-ELP protects the retina from damage and mitochondrial dysfunction in a model of CSE-induced early AMD, which may involve mitoStat3 activation in RPE.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.