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

Physiological response of the retinal pigmented epithelium to 3-ns pulse laser application, in vitro and in vivo

BACKGROUND: To treat healthy retinal pigmented epithelium (RPE) with the 3-ns retinal rejuvenation therapy (2RT) laser and to investigate the subsequent wound-healing response of these cells. METHODS: Primary rat RPE cells were treated with the 2RT laser at a range of energy settings. Treated cells were fixed up to 7 days post-irradiation and assessed for expression of proteins associated with wound-healing. For in vivo treatments, eyes of Dark Agouti rats were exposed to laser and tissues collected up to 7 days post-irradiation. Isolated wholemount RPE preparations were examined for structural and protein expression changes. RESULTS: Cultured RPE cells were ablated by 2RT laser in an energy-dependent manner. In all cases, the RPE cell layer repopulated completely within 7 days. Replenishment of RPE cells was associated with expression of the heat shock protein, Hsp27, the intermediate filament proteins, vimentin and nestin, and the cell cycle-associated protein, cyclin D1. Cellular tight junctions were lost in lased regions but re-expressed when cell replenishment was complete. In vivo, 2RT treatment gave rise to both an energy-dependent localised denudation of the RPE and the subsequent repopulation of lesion sites. Cell replenishment was associated with the increased expression of cyclin D1, vimentin and the heat shock proteins Hsp27 and αB-crystallin. CONCLUSIONS: The 2RT laser was able to target the RPE both in vitro and in vivo, causing debridement of the cells and the consequent stimulation of a wound-healing response leading to layer reformation.John P. M. Wood, Marzieh Tahmasebi, Robert J. Casson, Malcolm Plunkett, Glyn Chidlo

Negative regulators of angiogenesis, ocular vascular homeostasis, and pathogenesis and treatment of exudative AMD

Angiogenesis, the formation of new blood vessels from pre-existing capillaries, is very tightly regulated and normally does not occur except during developmental and reparative processes. This tight regulation is maintained by a balanced production of positive and negative regulators, and alterations under pathological conditions such as retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration can lead to growth of new and abnormal blood vessels. Although the role of proangiogenic factors such as vascular endothelial growth factor has been extensively studied, little is known about the roles of negative regulators of angiogenesis in the pathogenesis of these diseases. Here, we will discuss the role of thrombospondin-1 (TSP1), one of the first known endogenous inhibitors of angiogenesis, in ocular vascular homeostasis, and how its alterations may contribute to the pathogenesis of age-related macular degeneration and choroidal neovascularization. We will also discuss its potential utility as a therapeutic target for treatment of ocular diseases with a neovascular component

Expression of thrombospondin-1 modulates the angioinflammatory phenotype of choroidal endothelial cells.

The choroidal circulation plays a central role in maintaining the health of outer retina and photoreceptor function. Alterations in this circulation contribute to pathogenesis of many eye diseases including exudative age-related macular degeneration. Unfortunately, very little is known about the choroidal circulation and its molecular and cellular regulation. This has been further hampered by the lack of methods for routine culturing of choroidal endothelial cells (ChEC), especially from wild type and transgenic mice. Here we describe a method for isolation and culturing of mouse ChEC. We show that expression of thrombospondin-1 (TSP1), an endogenous inhibitor of angiogenesis and inflammation, has a significant impact on phenotype of ChEC. ChEC from TSP1-deficient (TSP1-/-) mice were less proliferative and more apoptotic, less migratory and less adherent, and failed to undergo capillary morphogenesis in Matrigel. However, re-expression of TSP1 was sufficient to restore TSP1-/- ChEC migration and capillary morphogenesis. TSP1-/- ChEC expressed increased levels of TSP2, phosphorylated endothelial nitric oxide synthase (NOS) and inducible NOS (iNOS), a marker of inflammation, which was associated with significantly higher level of NO and oxidative stress in these cells. Wild type and TSP1-/- ChEC produced similar levels of VEGF, although TSP1-/- ChEC exhibited increased levels of VEGF-R1 and pSTAT3. Other signaling pathways including Src, Akt, and MAPKs were not dramatically affected by the lack of TSP1. Together our results demonstrate an important autocrine role for TSP1 in regulation of ChEC phenotype

Fingolimod (FTY720), a Sphinogosine-1-Phosphate Receptor Agonist, Mitigates Choroidal Endothelial Proangiogenic Properties and Choroidal Neovascularization

Neovascular or wet age-related macular degeneration (nAMD) causes vision loss due to inflammatory and vascular endothelial growth factor (VEGF)-driven neovascularization processes in the choroid. Due to the excess in VEGF levels associated with nAMD, anti-VEGF therapies are utilized for treatment. Unfortunately, not all patients have a sufficient response to such therapies, leaving few if any other treatment options for these patients. Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator found in endothelial cells that participates in modulating barrier function, angiogenesis, and inflammation. S1P, through its receptor (S1PR1) in endothelial cells, prevents illegitimate sprouting angiogenesis during vascular development. In the present paper, we show that, in choroidal endothelial cells, S1PR1 is the most abundantly expressed S1P receptor and agonism of S1PR1-prevented choroidal endothelial cell capillary morphogenesis in culture. Given that nAMD pathogenesis draws from enhanced inflammation and angiogenesis as well as a loss of barrier function, we assessed the impact of S1PR agonism on choroidal neovascularization in vivo. Using laser photocoagulation rupture of Bruch’s membrane to induce choroidal neovascularization, we show that S1PR non-selective (FTY720) and S1PR1 selective (CYM5442) agonists significantly inhibit choroidal neovascularization in this model. Thus, utilizing S1PR agonists to temper choroidal neovascularization presents an additional novel use for these agonists presently in clinical use for multiple sclerosis as well as other inflammatory diseases

Expression and phosphorylation of Src, Akt, and MAPKs signaling pathways in ChEC.

<p>Expression and phosphorylation of Src and Akt were analyzed by Western blotting (<i>A</i>). A similar level of phosphorylated and total Src and Akt was observed in TSP1+/+ and TSP1−/− choroidal EC. Expression and phosphorylation of ERKs, JNK and p38 MAP kinases were analyzed by Western blotting (<i>B</i>). Please note minimal impact of TSP1-deficincy on phosphorylation and expression of ERKs in ChEC. A significant increase in phosphorylation of STAT3 was observed in TSP1−/− ChEC, while total level of STAT3 was not affected. These experiments were repeated with two different isolations of cells with similar results.</p

Isolation and characterization of mouse choroidal endothelial cells (ChEC).

<p>Thrombospondin1 (TSP1)+/+ and TSP1−/− ChEC were prepared as described in MATERIALS AND METHODS and cultured on gelatin-coated plates in 60-mm dishes. A: cells were photographed in digital format at ×40 and ×100 magnification. Note TSP1−/− ChEC exhibited a similar elongated and spindly morphology compared with TSP1+/+ ChEC. B: The expression of vascular EC markers in ChEC. ChEC were examined for expression of PECAM-1, VE-cadherin (VE-cad), and B4 lectin by FACS analysis. Shaded areas show control IgG staining. Note the similar expression of these cellular markers in both cells. C: FACS analysis for expression of other cell surface markers. Please note expression of CD36, CD 47, ICAM-1, ICAM-2, and VCAM-1 expression in these cells. We also detected significant expression of VEGF-R1 in these cells whose level was increased in TSP1−/− ChEC. The VEGF-R2 expression was almost undetectable. D: FACS analysis of EC markers for fenestration, PV-1 and HTAR (stabilin-2). Please note minimal expression of these markers. These experiments were repeated at least twice with two different isolations of choroidal EC, with similar results.</p

Cellular localization and expression level of VE-cadherin, N-cadherin, β-catenin, and ZO-1.

<p>A: TSP1+/+ and TSP1−/− ChEC were grown on fibronectin-coated coverslips to confluence and stained as described in Methods with specific antibodies. No staining was observed when primary antibody was left out. Please note VE-cadherin showed no staining in both TSP1+/+ and TSP1−/− ChEC. N-cadherin, β-catenin had similar levels and junctional localization in TSP1+/+ and TSP1−/− choroidal EC. ZO-1 showed similar perinuclear localization and punctate junctional localization in both TSP1+/+ and TSP1−/− ChEC. B: Western blot analysis of junctional proteins. Consistent with immunofluorescence staining, no VE-cadherin protein was detectable in ChEC. Similar levels of N-cadherin, β-catenin, and ZO-1 were detected in ChEC. These experiments were repeated at least twice with two different isolations of choroidal EC, with similar results.</p

Altered expression of ECM proteins in TSP1−/− ChEC.

<p>TSP1+/+ and TSP1−/− ChEC were incubated for 2 days in serum-free medium. The collected conditioned medium (CM) and cell lysates were analyzed by Western blotting for TSP1, TSP2, fibronectin, tenascin-C, and osteopontin using specific antibodies. These experiments were repeated with two different isolations with similar results. Please note the lack of TSP1 expression but increased TSP2 expression in TSP1−/− ChEC. Similar expression of fibronectin, tenascin-C and osteopontin was observed in TSP1+/+ and TSP1−/− ChEC.</p