AhR agonists prevent both atRAL and 4-HNE-induced ARPE-19 cell death.

(A) Measurement of CYP1A1 transcription in ARPE-19 after dosed treatment with AhR agonists DF 203, FICZ and Kynurenic Acid. CYP1A1 gene expression levels were normalized to β-actin. (B-C) atRAL and 4-HNE mediated cell death inhibition after pre-treatment with AhR agonists, measured by CytoTox-Glo (B) or PI (C). Each data point represents biological replicates (n = 3–4) and indicated as mean±S.D. Non-parametric Kruskal-Wallis test was applied for statistical analysis. * p<0.05 and ** p<0.01 compared to vehicle control.</p

atRAL and 4-HNE cause ARPE-19 cell death in dose- and time-dependent manners.

atRAL and 4-HNE-induced ARPE-19 cell death was visualized and analyzed using PI staining and IncuCyte. (A) The represented pictures of atRAL or 4-HNE-induced ARPE-19 cell death with indicated concentrations at 24 h post-treatment. The dose and time responses of atRAL (B) and 4-HNE (C) induced-cell death were quantified by the number of PI positive cells. Each data point represents biological replicates (n = 3–4), and indicated as mean±S.D. (TIF)</p

iPS-RPE has lower expression of apoptotic related genes compared to ARPE-19.

mRNA from both ARPE-19 and iPS-RPE was collected and the expression levels of apoptotic related genes, such as CASP8, BAX, and FAS, were evaluated using qPCR. The relative expression levels were normalized to the relevant gene expression in ARPE-19. Each data point represents biological replicates (n = 3–4) and is indicated as mean±S.D. Statistical analysis was performed using the non-parametric Mann-Whitney test. ** p (TIF)</p

DF 203 identified as target inhibiting atRAL- and 4-HNE-induced ARPE-19 cell death.

(A) Percentage inhibition of compounds on atRAL-induced ARPE-19 cell death. atRAL-treated group-only set as 0% inhibition, and untreated group set as 100% inhibition. Dotted line indicates 2xS.D. cutoff limit. The yellow line linked 2-dots replicates. (B) Dose-dependent inhibition of atRAL-mediated cell death of ARPE-19 by DF 203 measured by CytoTox-Glo. (C) Dose-dependent inhibition of 4-HNE mediated cell death of ARPE-19 by DF203 measured by PI. Each data point (B-C) represents biological replicates (n = 3–4), and indicated as mean±S.D.. Non-parametric Kruskal-Wallis test was applied for statistical analysis. * p<0.05, ** p<0.01 and *** p<0.001 compared to vehicle control.</p

Compound screen identification for prevention of both atRAL and 4-HNE-induced ARPE-19 cell death.

(A) Screening flowchart for the study, showing experiment treatment conditions, hit selection criteria and number of compounds passing each step. (B) Dose-dependent atRAL induction of ARPE-19 cell death, detection using CytoTox-Glo (EC50 = 12.0 μM, R2 = 0.9343). (C) Z’ factor for all assay plates was calculated to confirm screening quality based on vehicle control and 18 μM atRAL treatments. Outliers (Z’<0.7) were excluded from further analysis.</p

Schematic mechanism of AhR agonism preventing toxin-induced RPE death.

Various toxins can induce different types of cell death. In ARPE-19 cells, all-trans retinal (atRAL) induces apoptosis, while 4-hydroxynonenal (4-HNE) induces necrotic cell death. Activation of the aryl hydrocarbon receptor (AhR) leads to the upregulation of several downstream signaling pathways, which may include enzymes and other factors that can prevent cell death caused by these toxins. Figure created with BioRender.com.</p

atRAL induces apoptosis while 4-HNE induces necroptosis in ARPE-19 cells.

(A-B) Analysis of general cell death (PI) and caspase3/7 mediated apoptosis (CellEvent) following atRAL (A) and 4-HNE (B) treatments. (C-D) Quantification of atRAL- or 4-HNE-mediated ARPE-19 cell death using PI following pretreatment with increasing doses of Z-VAD (C) or Nec-1 (D) before challenge. Each data point represents biological replicates (n = 3–4) with five images captured per replicate and indicated as mean±S.D. Non-parametric Kruskal-Wallis test was applied for statistical analysis. * p<0.05, ** p<0.01 and *** p<0.001 compared to vehicle control.</p

AhR agonists could prevent 4-HNE but not atRAL induced iPS-RPE cell death.

(A-B) Analysis of general cell death (PI) following atRAL (A) and 4-HNE (B) treatments. (C-D) AhR agonist effects on atRAL (C) and 4-HNE (D) mediated iPS-RPE cell death, measured by PI staining. Each data point represents biological replicates (n = 3–4) and indicated as mean±S.D. Non-parametric Kruskal-Wallis test was applied for statistical analysis. * p<0.05 and *** p<0.001 compared to vehicle control.</p

Inhibition of TGF-β decreases retinal perfusion and vascular autoregulation.

<p>After fourteen days of Ad-sEng and Ad-null expression, mice were injected with h.m.w. FITC-dextran through the left ventricle to localize perfused vessels. (A) Confocal analysis of retinal flat-mounts revealed reduced perfusion of the retina in the sEng-expressing mice compared to the control (representative photo of n = 12 mice). Scale bar = 200 µm. (B) The perfused vessels were visualized on retinal flat mounts by comparing the co-localization of type IV collagen (Cy3-red) and FITC and quantified on cryosections by comparing the number of vessels in the innermost vascular plexus (arrowheads) positive for both type IV collagen- and FITC to the number of vessels positive for type IV collagen but negative for FITC (C). (D) The retinas of sEng expressing mice show a marked reduction in the number of perfused vessels (n = 5, ** p<0.01). (E) After seven days of adenoviral expression, blood flow rates in the tail were measured non-invasively in response to intravascular injection of ACh. Measurements were made over 5 cycles pre-injection of ACh, normalized to 1 for each animal, and averaged at 5-cycle intervals post ACh injection. In Ad-null control mice, ACh increased tail vein blood flow rates 6–10 cycles post-injection, whereas blood flow rates were unchanged in Ad-sEng expressing mice (Ad-null: 1.619 µl/cycle, n = 5; Ad-sEng: 0.960 µl/cycle, n = 4, * p<0.01). Injection of 100 µl of saline in Ad-null or Ad-sEng mice elicited no response.</p

Inhibition of TGF-β decreases endothelial barrier function in vitro.

<p>(A) SEM analysis demonstrated a characteristic endothelial cobblestone morphology in EC monocultures. Co-culture enhanced the association between adjacent cells, which was reversed with addition of SB-431542. Scale bars = 100 µm (left panel); 10 µm (right panel). Cell size was calculated by tracing individual ECs using ImageJ. Morphology of 10T1/2 cells from cocultures with ECs was not visibly altered when compared to 10T1/2 monoculture or with the addition of SB431542. (B) Analysis of EC size in monoculture or coculture with 10T1/2 cells (+/−SB431542). Coculture did not significantly alter average cell size, however, EC size were more uniform when compared to EC mono-cultures. Addition of SB431542 led to larger and more heterogenous cell size.</p