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Ocular levels of IL-1β, TNFα, IL-8, and IL-6 correlate with progression of diabetic retinopathy (DR). Müller cells (MC), which are crucial to maintaining retinal homeostasis, are targets and sources of these cytokines. We explored the relative capacities of these four DR-associated cytokines to amplify inflammatory signal expression both in and between human MC (hMC) and retinal microvascular endothelial cells (hRMEC) and in the mouse retina. Of the four cytokines, IL-1β was the most potent stimulus of transcriptomic alterations in hMC and hRMEC in vitro, as well as in the mouse retina after intravitreal injection in vivo. Stimulation with IL-1β significantly induced expression of all four transcripts in hMC and hRMEC. TNFα significantly induced expression of some, but not all, of the four transcripts in each cell, while neither IL-8 nor IL-6 showed significant induction in either cell. Similarly, conditioned media (CM) derived from hMC or hRMEC treated with IL-1β, but not TNFα, upregulated inflammatory cytokine transcripts in the reciprocal cell type. hRMEC responses to hMC-derived CM were dependent on IL-1R activation. In addition, we observed a correlation between cytokine expression changes following direct and CM stimulation and NFκB-p65 nuclear translocation in both hMC and hRMEC. Finally, in mice, intravitreal injections of IL-1β, but not TNFα, induced retinal expression of Il1b and CXCL8 homologues Cxcl1, Cxcl2, Cxcl3, and Cxcl5, encoding pro-angiogenic chemokines. Our results suggest that expression of IL-1β, TNFα, IL-8, and IL-6 may be initiated, propagated, and sustained by autocrine and paracrine signals in hRMEC and hMC through a process involving IL-1β and NFκB. Targeting these signals may help thwart inflammatory amplification, preventing progression to vision-threatening stages and preserving sight., Competing Interests: Declaration of competing interest There are no financial or non-financial competing interests declared by any author., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: The peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is a transcription factor with roles in metabolism, angiogenesis, and inflammation. It has yet undefined roles in retinal inflammation and diabetic retinopathy (DR). We used RNA-seq to better understand the role of the antagonist and inverse agonist of PPARβ/δ, GSK0660, in TNFα-induced inflammation. Understanding the underlying mechanisms of vascular inflammation could lead to new treatments for DR., Methods: RNA was isolated from human retinal microvascular endothelial cells treated with a vehicle, TNFα, or TNFα plus GSK0660. RNA-seq was performed with a 50 bp single read protocol. The differential expression was determined using edgeR and gene ontology, and a pathway analysis was performed using DAVID. RNA-seq validation was performed using qRT-PCR using the primers for ANGPTL4, CCL8, NOV, CXCL10, and PDPK1., Results: TNFα differentially regulated 1,830 transcripts, many of which are involved in the cytokine-cytokine receptor interaction, chemokine signaling, and inflammatory response. Additionally, TNFα highly upregulated genes involved in leukocyte recruitment, including CCL5, CX3CL1, and CXCL10. GSK0660 differentially regulated 273 transcripts in TNFα-treated cells compared to TNFα alone. A pathway analysis revealed the enrichment of cytokine-cytokine receptor signaling. In particular, GSK0660 blocks the TNFα-induced upregulation of CCL8, a chemokine involved in leukocyte recruitment., Conclusions: TNFα regulates several genes related to retinal leukostasis in retinal endothelial cells. GSK0660 blocks the effect of TNFα on the expressions of cytokines involved in leukocyte recruitment, including CCL8, CCL17, and CXCL10 and it may therefore block TNFα-induced retinal leukostasis.

Purpose: We investigated endoglin expression in hypoxic microvascular endothelial cells and retinal endoglin expression in rats that develop experimental oxygen-induced retinopathy (OIR). We also tested neutralizing antibodies (Abs) against endoglin (anti-CD105 Ab) and VEGF (anti-VEGF Ab) either alone or in combination for efficacy against serum-induced retinal microvascular endothelial cell proliferation and retinal neovascularization (NV) in OIR rats. To our knowledge, this marks the first time that a biologic agent has been used to target retinal endoglin and modulate retinal neovascularization., Methods: Induction of endoglin by hypoxia was measured by immunohistochemical analysis and ELISA. Proliferation was quantified using a colorimetric 5-bromo-2-deoxyuridine ELISA. Western blots were used to measure endoglin levels in retinas of OIR rats. Immunohistochemical staining was also preformed in OIR rats using anti-CD105 and fluorescein isothiocyanate-conjugated isolectin B4 antibodies., Results: Anti-CD105 Ab and Anti-VEGF Ab, administered alone or in combination, reduced serum-induced retinal microvascular endothelial cell proliferation. Additionally, in a rat model of oxygen-induced retinopathy, retinal endoglin was significantly increased at 14(2), 14(3), 14(4) and 14(6) compared with retinal levels in control rats. At 14(2), immunohistochemical analysis demonstrated that endoglin was elevated in newly developed vessels at the peripheral extent of major veins, precisely where NV is expected to develop in OIR rats. Neutralizing anti-CD105 reduced retinal NV in OIR rats., Conclusions: Our data support other studies showing that reduction of endoglin expression inhibits retinal NV. Our findings demonstrate that retinal endoglin immunolocalization overlaps with nascent neovascular structures in OIR rats. Therefore, endoglin may serve as a useful predictor of incipient neovascular disease., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: To develop new therapies against ocular neovascularization (NV), we tested the effect of peroxisome proliferator-activated receptor-β/δ (PPAR-β/δ) agonism and antagonism on angiogenic behaviors and in human retinal microvascular endothelial cells (HRMEC) and on preretinal NV in rat oxygen-induced retinopathy (OIR)., Methods: HRMECs were treated with the PPAR-β/δ agonist GW0742 and the antagonist GSK0660. Messenger RNA levels of a PPAR-β/δ target gene, angiopoietin-like-4 (angptl4) were assayed by qRT-PCR. HRMEC proliferation and tube formation were assayed according to standard protocols. OIR was induced in newborn rats by exposing them to alternating 24-hour episodes of 50% and 10% oxygen for 14 days. OIR rats were treated with GW0742 or GSK0660. Angptl4 protein levels were assessed by ELISA and preretinal NV was quantified by adenosine diphosphatase staining., Results: GW0742 significantly increased angptl4 mRNA, and GSK0660 significantly decreased angptl4 mRNA. GW0742 had no effect on HRMEC proliferation, but caused a significant and dose-responsive increase in tube formation. GSK0660 significantly reduced serum-induced HRMEC proliferation and tube formation in a dose-dependent manner. Intravitreal injection of GW0742 significantly increased total retinal Angptl4 protein, but intravitreal injection of GSK0660 had no effect. Intravitreal injection of GW0742 significantly increased retinal NV, as did GW0742 administered by oral gavage. Conversely, both intravitreal injection and intraperitoneal injection of GSK0660 significantly reduced retinal NV., Conclusions: PPAR-β/δ activation exacerbates, and its inhibition reduces, preretinal NV. PPAR-β/δ may regulate preretinal NV through a prodifferentiation/maturation mechanism that depends on Angptl4. Pharmacologic inhibition of PPAR-β/δ may provide a rational basis for therapeutic targeting of ocular NV.