The Role of Shear-Induced Transforming Growth Factor-? Signaling in the EndotheliumSignificance

Objective Vascular endothelial cells (ECs) are continuously exposed to blood flow that contributes to the maintenance of vessel structure and function; however, the effect of hemodynamic forces on transforming growth factor-β (TGF-β) signaling in the endothelium is poorly described. We examined the potential role of TGF-β signaling in mediating the protective effects of shear stress on ECs. Approach and Results Human umbilical vein endothelial cells (HUVECs) exposed to shear stress were compared to cells grown under static conditions. Signaling through the TGF-β receptor ALK5 was inhibited with SB525334. Cells were examined for morphological changes and harvested for real-time PCR, western blot analysis, apoptosis, proliferation and immunocytochemistry. Shear stress resulted in ALK5-dependent alignment of HUVECs as well as attenuation of apoptosis and proliferation compared to static controls. Shear stress lead to an ALK5-dependent increase in TGF-β3 and Krüppel-like factor 2 (KLF2), phosphorylation of endothelial nitric oxide synthase (eNOS) and NO release. Addition of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) rescued the cells from apoptosis due to ALK5 inhibition under shear stress. Knockdown of TGF-β3, but not TGF-β1, disrupted the HUVEC monolayer and prevented the induction of KLF2 by shear. Conclusions Shear stress of HUVECs induces TGF-β3 signaling and subsequent activation of KLF2 and NO, and represents a novel role for TGF-β3 in the maintenance of HUVEC homeostasis in a hemodynamic environment

Expression and Role of VEGF-A in the Ciliary Body

Purpose.: The role of VEGF-A in the normal ciliary body is largely unexplored. The ciliary body is similar in many respects to the choroid plexus of the brain, and we demonstrated previously the importance of VEGF-A in maintenance of choroid plexus vasculature and ependymal cells. Therefore, the role of VEGF-A in ciliary body homeostasis was explored. Methods.: Swiss-Webster mice (VEGF-LacZ) were used to determine VEGF-A expression during ciliary body development and in the adult. VEGFR2 expression was determined in adult wild type C56BL/6J mice. Systemic VEGF-A neutralization in vivo was achieved with adenovirus-mediated overexpression of soluble VEGFR1 (sFlt1). Following VEGF-A neutralization, the ciliary epithelium was analyzed by light microscopy and transmission electron microscopy (TEM). The effect of VEGF-A blockade on ciliary body function also was assessed by measuring intraocular pressure. Results.: VEGF-A expression was detected at embryonic day 18.5 (E18.5), the onset of ciliary process formation. In the adult ciliary body, VEGF-A was expressed by the pigmented epithelium, whereas VEGFR2 was localized primarily to the capillary endothelium and nonpigmented epithelium. Systemic VEGF-A neutralization led to a thinning of the nonpigmented epithelium, vacuolization of the pigmented epithelium, loss of capillary fenestrations, and thrombosis. These changes were associated with impaired ciliary body function, as evidenced by decreased intraocular pressure in sFlt1-overexpressing animals (15.31 ± 2.06 mm Hg) relative to controls (18.69 ± 1.49 mm Hg). Conclusions.: VEGF-A has an important role in ciliary body homeostasis. Potential for undesired off-target effects should be considered with the chronic use of anti–VEGF-A therapies

VEGF and TGF-β are required for the maintenance of the choroid plexus and ependyma

Although the role of vascular endothelial growth factor (VEGF) in developmental and pathological angiogenesis is well established, its function in the adult is less clear. Similarly, although transforming growth factor (TGF) β is involved in angiogenesis, presumably by mediating capillary (endothelial cell [EC]) stability, its involvement in quiescent vasculature is virtually uninvestigated. Given the neurological findings in patients treated with VEGF-neutralizing therapy (bevacizumab) and in patients with severe preeclampsia, which is mediated by soluble VEGF receptor 1/soluble Fms-like tyrosine kinase receptor 1 and soluble endoglin, a TGF-β signaling inhibitor, we investigated the roles of VEGF and TGF-β in choroid plexus (CP) integrity and function in adult mice. Receptors for VEGF and TGF-β were detected in adult CP, as well as on ependymal cells. Inhibition of VEGF led to decreased CP vascular perfusion, which was associated with fibrin deposition. Simultaneous blockade of VEGF and TGF-β resulted in the loss of fenestrae on CP vasculature and thickening of the otherwise attenuated capillary endothelium, as well as the disappearance of ependymal cell microvilli and the development of periventricular edema. These results provide compelling evidence that both VEGF and TGF-β are involved in the regulation of EC stability, ependymal cell function, and periventricular permeability

TGF-β Is Required for Vascular Barrier Function, Endothelial Survival and Homeostasis of the Adult Microvasculature

Pericyte-endothelial cell (EC) interactions are critical to both vascular development and vessel stability. We have previously shown that TGF-β signaling between EC and mural cells participates in vessel stabilization in vitro. We therefore investigated the role of TGF-β signaling in maintaining microvessel structure and function in the adult mouse retinal microvasculature. TGF-β signaling was inhibited by systemic expression of soluble endoglin (sEng) and inhibition was demonstrated by reduced phospho-smad2 in the adult retina. Blockade of TGF-β signaling led to increased vascular and neural cell apoptosis in the retina, which was associated with decreased retinal function, as measured by electroretinogram (ERG). Perfusion of the inner retinal vasculature was impaired and was accompanied by defective autoregulation and loss of capillary integrity. Fundus angiography and Evans blue permeability assay revealed a breakdown of the blood-retinal-barrier that was characterized by decreased association between the tight junction proteins zo-1 and occludin. Inhibition of TGF-β signaling in cocultures of EC and 10T1/2 cells corroborated the in vivo findings, with impaired EC barrier function, dissociation of EC from 10T1/2 cells, and endothelial cell death, supporting the role of EC-mesenchymal interactions in TGF-β signaling. These results implicate constitutive TGF-β signaling in maintaining the integrity and function of the adult microvasculature and shed light on the potential role of TGF-β signaling in vasoproliferative and vascular degenerative retinal diseases

Endogenous VEGF Is Required for Visual Function: Evidence for a Survival Role on Müller Cells and Photoreceptors

Vascular endothelial growth factor (VEGF) is well known for its role in normal and pathologic neovascularization. However, a growing body of evidence indicates that VEGF also acts on non-vascular cells, both developmentally as well as in the adult. In light of the widespread use of systemic and intraocular anti-VEGF therapies for the treatment of angiogenesis associated with tumor growth and wet macular degeneration, systematic investigation of the role of VEGF in the adult retina is critical.Using immunohistochemistry and Lac-Z reporter mouse lines, we report that VEGF is produced by various cells in the adult mouse retina and that VEGFR2, the primary signaling receptor, is also widely expressed, with strong expression by Müller cells and photoreceptors. Systemic neutralization of VEGF was accomplished in mice by adenoviral expression of sFlt1. After 14 days of VEGF neutralization, there was no effect on the inner and outer retina vasculature, but a significant increase in apoptosis of cells in the inner and outer nuclear layers. By four weeks, the increase in neural cell death was associated with reduced thickness of the inner and outer nuclear layers and a decline in retinal function as measured by electroretinograms. siRNA-based suppression of VEGF expression in a Müller cell line in vitro supports the existence of an autocrine role for VEGF in Müller cell survival. Similarly, the addition of exogenous VEGF to freshly isolated photoreceptor cells and outer-nuclear-layer explants demonstrated VEGF to be highly neuroprotective.These results indicate an important role for endogenous VEGF in the maintenance and function of adult retina neuronal cells and indicate that anti-VEGF therapies should be administered with caution

A proteogenomic signature of age-related macular degeneration in blood

© 2022. The Author(s). Funding Information: The authors acknowledge the contribution of the Icelandic Heart Association (IHA) staff to the AGES-RS, as well as the involvement of all study participants. We thank the IAMDGC consortium for supplying us with their GWAS summary statistics data. National Institute on Aging (NIA) contracts N01-AG-12100 and HHSN271201200022C for V.G. financed the AGES study; retinal image collection and AMD readings were funded by the NIH Intramural Research Program (ZIAEY000401). V.G. received a funding from the NIA (1R01AG065596), and IHA received a support from Althingi (the Icelandic Parliament). The Icelandic Research Fund (IRF) funded V.E. and Va.G. with grants 195761-051, 184845-053, and 206692-051, while Va.G. received a postdoctoral research grant from the University of Iceland Research Fund Funding Information: The study was supported by the Novartis Institute for Biomedical Research. M.T., N.F., S.P., X.L., R.E., Y.Z., S.J., C.L.H., S.M.L., J.L., C.L.G., A.A.N., B.L., R.P., Z.L., L.L.J., T.E.W., Q.Z., Q.H., and J.R.L. are employees and stockholders of Novartis. All other authors have no conflict of interests to declare. Publisher Copyright: © 2022, The Author(s).Age-related macular degeneration (AMD) is one of the most common causes of visual impairment in the elderly, with a complex and still poorly understood etiology. Whole-genome association studies have discovered 34 genomic regions associated with AMD. However, the genes and cognate proteins that mediate the risk, are largely unknown. In the current study, we integrate levels of 4782 human serum proteins with all genetic risk loci for AMD in a large population-based study of the elderly, revealing many proteins and pathways linked to the disease. Serum proteins are also found to reflect AMD severity independent of genetics and predict progression from early to advanced AMD after five years in this population. A two-sample Mendelian randomization study identifies several proteins that are causally related to the disease and are directionally consistent with the observational estimates. In this work, we present a robust and unique framework for elucidating the pathobiology of AMD.Peer reviewe

Pulsatile flow increases the expression of eNOS, ET-1, and prostacyclin in a novel in vitro coculture model of the retinal vasculature. Invest Ophthalmol Vis Sci 46: 375–382

PURPOSE. By the development of a novel retinal microvascular endothelial and pericyte cell coculture system, this study determined the effects of pulsatile flow on the activation of the endothelial cell markers nitric oxide (NO), prostacyclin (PGI 2 ), and endothelin (ET)-1. METHODS. Monocultured bovine retinal endothelial cells (BRECs) and cocultured BRECs with bovine retinal pericytes (BRPs) were exposed to low flow (flow rate, 0.3 mL/min; pulse pressure, 6 mm Hg; shear stress, 0.5 dyne/cm 2 ) or high flow (flow rate, 25 mL/min; pulse pressure, 56 mm Hg; shear stress, 23 dynes/cm 2 ) for 24 hours, by using a novel perfused transcapillary culture system. The cells were characterized by immunohistochemistry and electron and confocal microscopy. Endothelial nitric oxide synthase (eNOS) and phosphorylatedeNOS Ser1179 (pp-eNOS) were determined by Western blot analysis. Nitrate, PGI 2 , and ET-1 levels were quantified in the medium perfusate by using fluorometric and enzyme-linked immunosorbent assays, respectively. Activation of cyclooxygenase (COX)-2 in BRECs was determined by measuring COX-2 promoter activity with a luciferase reporter assay. RESULTS. The presence of BRPs and BRECs was confirmed by Western blot, immunocytochemistry, and scanning electron microscopy. Phosphorylated eNOS (pp-eNOS) protein levels in BRECs were significantly increased from low to high flow in both mono-and cocultures, concomitant with a significant increase in nitrate levels in the conditioned medium after exposure to pulsatile flow. In parallel cultures, PGI 2 levels were also significantly enhanced concomitant with an increase in the transactivation of a COX-2 promoter BREC after exposure to pulsatile flow. ET-1 levels were also increased in both mono-and cocultured cells. CONCLUSIONS. In this study a novel, functioning, in vitro model of retinal microvascular endothelial and pericyte cells that respond to changes in pulsatile flow was established. (Invest Ophthalmol Vis Sci. 2005;46:375-382

Role of shear-stress-induced VEGF expression in endothelial cell survival

Vascular endothelial growth factor (VEGF) plays a crucial role in developmental and pathological angiogenesis. Expression of VEGF in quiescent adult tissue suggests a potential role in the maintenance of mature blood vessels. We demonstrate, using a Vegf–lacZ reporter mouse model, that VEGF is expressed by arterial but not by venous or capillary endothelial cells (ECs) in vivo. Using an in vitro model, we show that arterial shear stress of human umbilical vein ECs (HUVECs) decreases apoptosis and increases VEGF expression, which is mediated by the induction of Krüppel-like factor 2 (KLF2). Additionally, shear stress stimulates the expression of VEGF receptor 2 (VEGFR2) and is associated with its activation. Knockdown of VEGF in shear stressed HUVECs blocks the protective effect of shear stress, resulting in EC apoptosis equivalent to that in control ECs cultured under static conditions. Similarly, treatment of ECs subjected to arterial shear stress with the VEGF receptor tyrosine kinase inhibitor SU1498, or VEGFR2 neutralizing antiserum, led to increased apoptosis, demonstrating that the mechanoprotection from increased shear is mediated by VEGFR2. Taken together, these studies suggest that arterial flow induces VEGF–VEGFR2 autocrine–juxtacrine signaling, which is a previously unidentified mechanism for vascular EC survival in adult arterial blood vessels