Thy-1 Regulates VEGF-Mediated Choroidal Endothelial Cell Activation and Migration: Implications in Neovascular Age-Related Macular Degeneration

Abstract

PurposeThis study addresses the hypothesis that age-related stresses upregulate Thy-1 in choroidal endothelial cells (CECs) and contribute to CEC activation and migration, processes important in choroidal neovascularization (CNV).MethodsMeasurements were made of Thy-1 protein (Western blot) in CECs and Thy-1 mRNA (real time quantitative PCR) in CECs treated with VEGF, CCL11, or PBS or in RPE/choroids from young or old donors or lasered or nonlasered mice. Immunolabeled Thy-1 in ocular sections was compared from young versus old human donor eyes or those with or without neovascular AMD or from lasered versus nonlasered mice. Choroidal endothelial cells transfected with Thy-1 or control siRNA or pretreated with Thy-1 blocking peptide or control were stimulated with VEGF or 7-ketocholesterol (7-KC). Choroidal endothelial cell migration, proliferation, cytoskeletal stress fibers, Rac1 activation, and phosphorylated VEGF receptor 2 (VEGFR2), integrin β3, and Src were measured. Statistics were performed using ANOVA.ResultsThy-1 was expressed in retinal ganglion cells and in vascular endothelial-cadherin–labeled choroid and localized to human or mouse laser-induced CNV lesions. Thy-1 protein and mRNA were significantly increased in CECs treated with VEGF or CCL11 and in RPE/choroids from aged versus young donor eyes or from lasered mice versus nonlasered controls. Knockdown or inhibition of Thy-1 in CECs significantly reduced VEGF-induced CEC migration and proliferation, stress fiber formation and VEGFR2, Src, integrin β3 and Rac1 activation, and 7-KC–induced Rac1 and Src activation.ConclusionsThy-1 in CECs regulates VEGF-induced CEC activation and migration and links extracellular 7-KC to intracellular signaling. Future studies elucidating Thy-1 mechanisms in neovascular AMD are warranted.

Serine racemase deficiency attenuates choroidal neovascularization and reduces nitric oxide and VEGF levels by retinal pigment epithelial cells

“…; Wang et al . ). Macrophages infiltration into the adjacent sites of laser injury begins on the first day and peaks on the third to fifth day post laser photocoagulation (Sakurai et al .…”

“…VEGF promotes the proliferation and migration of choroidal endothelial cells (CEC) (Wang et al . ) and disrupts the tight junction between RPE cells (Hartnett et al . ), thus facilitating growth of choroidal vessels into the subretinal space.…”

Computational Approach to Investigating Key GO Terms and KEGG Pathways Associated with CNV

“…The next GO term, called GO: 0005576 , describes a general term called extracellular region. Various extracellular substances participate in the pathogenesis of CNV, including LOX [ 63 ], LOXL2 [ 63 ], Thy-1 [ 64 ], and integrins [ 65 ]. Such specific substances may play irreplaceable roles during the initiation and progression of CNV; thus, this GO term that describes the extracellular regions of a certain focus is a potential CNV-associated biological process.…”

Memantine, Simvastatin, and Epicatechin Inhibit 7-Ketocholesterol-induced Apoptosis in Retinal Pigment Epithelial Cells But Not Neurosensory Retinal Cells In Vitro

“…[6,[8][9][10] 7kCh activates three distinct kinase signaling pathways via NFkB, MAPK, and ERK to upregulate proinflammatory cytokines: IL-6, IL-8, and vascular endothelial growth factor (VEGF) which induce neovascularization in the choroid. [7,8,[11][12][13] In previous studies, retinal pigment epithelial (RPE) cells and vascular endothelial cells had an 8-to 10-fold increase in VEGF levels, [14] and a 2-fold increase in endothelial smooth muscle cells after exposure to 7kCh. [15] Our previous studies showed that in both ARPE-19 and rat neuroretinal R28 cell cultures, 7kCh significantly increased the levels of pro-apoptotic caspase-3, -8, and -12 activities.…”