Biochemistry and Molecular Biology b2-Adrenergic Receptor Antagonism Attenuates CNV Through Inhibition of VEGF and IL-6 Expression

Citation: Lavine JA, Farnoodian M, Wang S, et al. b2-adrenergic receptor antagonism attenuates CNV through inhibition of VEGF and IL-6 expression. Invest Ophthalmol Vis Sci. 2017;58:299-308. DOI:10.1167/ iovs.16-20204 PURPOSE. The role of b-adrenergic receptor (AR) signaling in neovascular ocular diseases has recently emerged. We have previously reported that intraperitoneal propranolol inhibits choroidal neovascularization (CNV) in vivo and b2-AR blockade reduces vascular endothelial growth factor (VEGF) expression in mouse retinal pigment epithelium and choroidal endothelial cells in culture. Here we tested the hypothesis that the b2-AR regulates CNV through modulation of VEGF and inflammatory cytokine expression. METHODS. Mice were subjected to laser burns, inducing CNV, and were treated with an intravitreal b2-AR antagonist. After 3 and 5 days, total eye interleukin-6 (IL-6) and VEGF protein levels were measured, respectively. After 14 days, CNV was measured on choroidalscleral flatmounts. The effects of b-AR signaling on VEGF and IL-6 expression were investigated in various mouse retinal and human RPE cells by using specific b-AR agonists and antagonists. RESULTS. b2-Adrenergic receptor signaling increased Vegf mRNA expression by approximately 3-to 4-fold in mouse retinal microglia and pericytes in culture. b2-Adrenergic receptor signaling upregulated IL-6 mRNA expression between 10-and 60-fold in mouse retinal microglia, pericytes, RPE, and choroidal endothelial cells in culture. Intravitreal injection of b2-AR antagonist ICI 118,551 reduced CNV by 35% and decreased IL-6 protein levels by approximately 50%. In primary human RPE cells, b2-AR activation also stimulated VEGF and IL-6 mRNA expression by 2-and 10-fold, respectively. CONCLUSIONS. Anti-VEGF therapy for CNV is highly effective; however, some patients are resistant to therapy while others undergo repeated, frequent treatments. b2-Adrenergic receptor signaling is a potential therapeutic target because of its angiogenic and inflammatory properties

Vitamin D receptor expression is essential during retinal vascular development and attenuation of neovascularization by 1, 25(OH)2D3.

Vitamin D provides a significant benefit to human health, and its deficiency has been linked to a variety of diseases including cancer. Vitamin D exhibits anticancer effects perhaps through inhibition of angiogenesis. We previously showed that the active form of vitamin D (1, 25(OH)2D3; calcitriol) is a potent inhibitor of angiogenesis in mouse model of oxygen-induced ischemic retinopathy (OIR). Many of vitamin D's actions are mediated through vitamin D receptor (VDR). However, the role VDR expression plays in vascular development and inhibition of neovascularization by 1, 25(OH)2D3 remains unknown. Here using wild type (Vdr +/+) and Vdr-deficient (Vdr -/-) mice, we determined the impact of Vdr expression on postnatal development of retinal vasculature and retinal neovascularization during OIR. We observed no significant effect on postnatal retinal vascular development in Vdr -/- mice up to postnatal day 21 (P21) compared with Vdr +/+ mice. However, we observed an increase in density of pericytes (PC) and a decrease in density of endothelial cells (EC) in P42 Vdr -/- mice compared with Vdr +/+ mice, resulting in a significant decrease in the EC/PC ratio. Although we observed no significant impact on vessel obliteration and retinal neovascularization in Vdr -/- mice compared with Vdr +/+ mice during OIR, the VDR expression was essential for inhibition of retinal neovascularization by 1, 25(OH)2D3. In addition, the adverse impact of 1, 25(OH)2D3 treatment on the mouse bodyweight was also dependent on VDR expression. Thus, VDR expression plays a significant role during retinal vascular development, especially during maturation of retinal vasculature by promoting PC quiescence and EC survival, and inhibition of ischemia-mediated retinal neovascularization by 1, 25(OH)2D3

Bim Expression Promotes the Clearance of Mononuclear Phagocytes during Choroidal Neovascularization, Mitigating Scar Formation in Mice

Inflammation is increasingly recognized as an important modulator in the pathogenesis of neovascular age-related macular degeneration (nAMD). Although significant progress has been made in delineating the pathways that contribute to the recruitment of inflammatory cells and their contribution to nAMD, we know little about what drives the resolution of these inflammatory responses. Gaining a better understanding of how immune cells are cleared in the choroid will give a novel insight into how sustained inflammation could influence the pathogenesis of nAMD. The pro-apoptotic Bcl-2 family member Bim is a master regulator of immune cell homeostasis. In its absence, immune cell lifespan and numbers increase. Most therapeutic regimes that squelch inflammation do so by enhancing immune cell apoptosis through enhanced Bim expression and activity. To test the hypothesis that Bim expression tempers inflammation during the pathogenesis of nAMD, we used the mouse laser-induced choroidal neovascularization (CNV) model in which inflammation acts as a facilitator of CNV. Here, we showed minimal to no change in the recruitment of F4/80-, CD80-, CD11b-, and Iba1-positive myeloid-derived mononuclear phagocytes to the site of laser photocoagulation in the absence of Bim expression. However, the resolution of these cells from the choroid of Bim-deficient (Bim -/-) mice was significantly diminished following laser photocoagulation. With time, we noted increased scar formation, demonstrated by collagen I staining, in Bim -/- mice with no change in the resolution of neovascularization compared to wild-type littermates. We also noted that mice lacking Bim expression in mononuclear phagocytes (BimFlox/Flox; Lyz2-Cre (BimMP) mice) had delayed resolution of F4/80-, CD80-, CD11b-, and Iba1-positive cells, while those lacking Bim expression in endothelial cells (BimFlox/Flox; Cad5-Cre (BimEC) mice) had delayed resolution of only CD11b- and Iba1-positive cells. Both BimMP and BimEC mice demonstrated increased scar formation, albeit to differing degrees. Thus, our studies show that resolving inflammation plays an important role in moderating scar formation in nAMD, and it is impacted by Bim expression in both the endothelium and mononuclear phagocyte lineages

Cytochrome P450 1B1 Expression Regulates Intracellular Iron Levels and Oxidative Stress in the Retinal Endothelium

Cytochrome P450 (CYP) 1B1 is a heme-containing monooxygenase found mainly in extrahepatic tissues, including the retina. CYP1B1 substrates include exogenous aromatic hydrocarbons, such as dioxins, and endogenous bioactive compounds, including 17β-estradiol (E2) and arachidonic acid. The endogenous compounds and their metabolites are mediators of various cellular and physiological processes, suggesting that CYP1B1 activity is likely important in maintaining proper cellular and tissue functions. We previously demonstrated that lack of CYP1B1 expression and activity are associated with increased levels of reactive oxygen species and oxidative stress in the retinal vasculature and vascular cells, including retinal endothelial cells (ECs). However, the detailed mechanism(s) of how CYP1B1 activity modulates redox homeostasis remained unknown. We hypothesized that CYP1B1 metabolism of E2 affects bone morphogenic protein 6 (BMP6)-hepcidin-mediated iron homeostasis and lipid peroxidation impacting cellular redox state. Here, we demonstrate retinal EC prepared from Cyp1b1-deficient (Cyp1b1−/−) mice exhibits increased estrogen receptor-α (ERα) activity and expresses higher levels of BMP6. BMP6 is an inducer of the iron-regulatory hormone hepcidin in the endothelium. Increased hepcidin expression in Cyp1b1−/− retinal EC resulted in decreased levels of the iron exporter protein ferroportin and, as a result, increased intracellular iron accumulation. Removal of excess iron or antagonism of ERα in Cyp1b1−/− retinal EC was sufficient to mitigate increased lipid peroxidation and reduce oxidative stress. Suppression of lipid peroxidation and antagonism of ERα also restored ischemia-mediated retinal neovascularization in Cyp1b1−/− mice. Thus, CYP1B1 expression in retinal EC is important in the regulation of intracellular iron levels, with a significant impact on ocular redox homeostasis and oxidative stress through modulation of the ERα/BMP6/hepcidin axis

Altered vascular cell density and EC/PC ratios in Vdr -/- mice.

<p>Retinas from P21 and P42 mice were prepared by trypsin digest and H&E/PAS staining. Slides were then scanned and images captured at x400. (A) Representative images are shown; Scale bar = 50 μm. Number of EC (red arrow head) and PC (green arrow head) were counted for at least 6 images per mice, and EC/PC ratio calculated for P21 (B) and for P42 (C) mice; (***P = 0.0008). (D) The quantitative assessment of this data, and the number of EC and PC along with the number of retinas counted in each group in parentheses, are shown. (n≥ 5; **P = 0.0043).</p

Similar degree of ischemia-driven retinal neovascularization in Vdr+/+ and Vdr-/- during OIR.

<p>(A) Representative images (x20) of wholemount retinal neovascularization isolated from P17 mice exposed to a cycle of hyperoxia and room air (OIR) and stained with collagen IV. Retinas from Vdr +/+, Vdr +/-, and Vdr -/- littermates were wholemount stained with anti-collagen IV to visualize the vasculature. Scale bar = 2,000 μm. Quantitative assessment of the neovascularization (histological evaluation and quantitative analysis of images) and area of vessel obliteration are shown in (B), (C), and (D) respectively. (n≥ 7; each point represents one mice).</p

<i>Vdr</i> expression is required for significant inhibition of retinal neovascularization by1, 25(OH)₂D₃.

<p>(A) Representative images (x20) of collagen IV stained wholemount retinal neovascularization from P17 OIR with and without 1, 25(OH)₂D₃ treatment from <i>Vdr</i> +/+, <i>Vdr</i> +/-, and <i>Vdr</i> -/- mice. Scale bar = 2,000 μm. Quantitative assessment of the neovascularization (histological evaluation) and area of vessel obliteration from these groups are shown in (B) and (C), respectively; (***P = 0.0006, *P = 0.0121). (D) The evaluated mice bodyweight (gr) comparison from the above groups is shown; (****P<0.0001). (n≥ 7; each point represents one mouse).</p

The development of superficial layer of retinal vasculature is independent of Vdr expression.

<p>(A) Demonstrates GFAP and Col IV stained retinal vessels prepared from postnatal day 5 (P5) Vdr +/+ and Vdr -/- mice. Please note similar expansion of astrocytes (green, GFAP) and progression of expanding vessels (red, Col IV). Scale bar = 200 μm for x25 and Scale bar = 50 μm for x100 images. (B) The mean number of angiogenic sprouts at the angiogenic fronts were quantified per field (x100) in each retina. (C) Coverage of retinal vasculature relative to total retina area were measured for each retina and is shown as a percentage. (n≥ 5; each point represents one mouse).</p