The effect of pulsatile flow on co-cultured retinal endothelial & pericyte cells

Microvascular cell fate decisions are hallmarks of the microvascular cell response to injury and play a crucial role in the pathogenesis of retinal disease. Abnormalities in retinal blood flow play a critical role in remodeling of the retinal vasculature by altering microvascular endothelial and pericyte cell fate (proliferation, apoptosis and migration). Retinal blood flow is controlled locally by vasodilators such as nitric oxide, prostacyclin and the vasoconstrictor endothelin-1 , with considerable evidence linking retinal pathologies such as Normal Tension Glaucoma and Diabetic Retinopathy to altered retinal blood flow Shear stress has previously been shown to modulate EC production of these vasoactive agents in macrovascular cells. Therefore, using a perfused transcapillary coculture of bovine microvascular retinal endothelial cells (BRECs) and bovine retinal pericytes (BRPs), we examined the acute and chronic effect of pulsatile flow on the release of these vasoactive mediators and their subsequent role in modulating retinal vascular cell fate. Acute exposure to pulsatile flow increased BREC NO, PGI2 & ET-1 formation and release Similarly, chronic exposure to pulsatile flow enhanced NO and PGI2 release while concomitantly inhibiting ET-1 in these cells In parallel studies, there was an increase in BRP apoptosis following exposure to high pulsatile flow, whereas BREC apoptosis decreased. Furthermore, the pulsatile flow-induced increases in BRP apoptosis is dependent on increased PGI2 , whereas both ET-1 and NO mediate the protective effect of increased flow on BRECs survival. Notch receptor-hgand interactions and the Hedgehog signalling pathway have been strongly implicated in vascular morphogenesis and remodelling of the embryonic vasculature, with Hedgehog acting upstream of Notch signalling during development. We therefore tested the hypothesis that Hedgehog (Hh) and Notch pathway interact to promote changes in vascular cell fate in BRECs and BRPs in vitro in response to changes in pulsatile flow

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

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

The impact of a large-scale quality improvement programme on work engagement: preliminary results from a national cross-sectional-survey of the 'Productive Ward'

Background: Quality improvement (QI) Programmes, like the Productive Ward: Releasing-time-to-care initiative, aim to 'engage' and 'empower' ward teams to actively participate, innovate and lead quality improvement at the front line. However, little is known about the relationship and impact that QI work has on the 'engagement' of the clinical teams who participate and vice-versa. Objective: This paper explores and examines the impact of a large-scale QI programme, the Productive Ward, on the 'work engagement' of the nurses and ward teams involved. Design/methods: Using the Utrecht Work Engagement Scale (UWES), we surveyed, measured and analysed work engagement in a representative test group of hospital-based ward teams who had recently commenced the latest phase of the national 'Productive Ward' initiative in Ireland and compared them to a control group of similar size and matched (as far as is possible) on variables such as ward size, employment grade and clinical specialty area. Results: 338 individual datasets were recorded, n=. 180 (53.6) from the Productive Ward group, and n=. 158 (46.4) from the control group; the overall response rate was 67, and did not differ significantly between the Productive Ward and control groups. The work engagement mean score (±standard deviation) in the Productive group was 4.33(±0.88), and 4.07(±1.06) in the control group, representing a modest but statistically significant between-group difference (. p=. 0.013, independent samples t-test). Similarly modest differences were observed in all three dimensions of the work engagement construct. Employment grade and the clinical specialty area were also significantly related to the work engagement score (. p<. 0.001, general linear model) and (for the most part), to its components, with both clerical and nurse manager grades, and the elderly specialist areas, exhibiting substantially higher scores. Conclusions: The findings demonstrate how QI activities, like those integral to the Productive Ward programme, appear to positively impact on the work engagement (the vigour, absorption and dedication) of ward-based teams. The use and suitability of the UWES as an appropriate measure of 'engagement' in QI interventions was confirmed. The engagement of nurses and front-line clinical teams is a major component of creating, developing and sustaining a culture of improvement. © 2014 The Authors

Effect of remote ischaemic conditioning on clinical outcomes in patients with acute myocardial infarction (CONDI-2/ERIC-PPCI): a single-blind randomised controlled trial.

BACKGROUND: Remote ischaemic conditioning with transient ischaemia and reperfusion applied to the arm has been shown to reduce myocardial infarct size in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). We investigated whether remote ischaemic conditioning could reduce the incidence of cardiac death and hospitalisation for heart failure at 12 months. METHODS: We did an international investigator-initiated, prospective, single-blind, randomised controlled trial (CONDI-2/ERIC-PPCI) at 33 centres across the UK, Denmark, Spain, and Serbia. Patients (age >18 years) with suspected STEMI and who were eligible for PPCI were randomly allocated (1:1, stratified by centre with a permuted block method) to receive standard treatment (including a sham simulated remote ischaemic conditioning intervention at UK sites only) or remote ischaemic conditioning treatment (intermittent ischaemia and reperfusion applied to the arm through four cycles of 5-min inflation and 5-min deflation of an automated cuff device) before PPCI. Investigators responsible for data collection and outcome assessment were masked to treatment allocation. The primary combined endpoint was cardiac death or hospitalisation for heart failure at 12 months in the intention-to-treat population. This trial is registered with ClinicalTrials.gov (NCT02342522) and is completed. FINDINGS: Between Nov 6, 2013, and March 31, 2018, 5401 patients were randomly allocated to either the control group (n=2701) or the remote ischaemic conditioning group (n=2700). After exclusion of patients upon hospital arrival or loss to follow-up, 2569 patients in the control group and 2546 in the intervention group were included in the intention-to-treat analysis. At 12 months post-PPCI, the Kaplan-Meier-estimated frequencies of cardiac death or hospitalisation for heart failure (the primary endpoint) were 220 (8·6%) patients in the control group and 239 (9·4%) in the remote ischaemic conditioning group (hazard ratio 1·10 [95% CI 0·91-1·32], p=0·32 for intervention versus control). No important unexpected adverse events or side effects of remote ischaemic conditioning were observed. INTERPRETATION: Remote ischaemic conditioning does not improve clinical outcomes (cardiac death or hospitalisation for heart failure) at 12 months in patients with STEMI undergoing PPCI. FUNDING: British Heart Foundation, University College London Hospitals/University College London Biomedical Research Centre, Danish Innovation Foundation, Novo Nordisk Foundation, TrygFonden

The effect of pulsatile flow on co-cultured retinal endothelial & pericyte cells

Microvascular cell fate decisions are hallmarks of the microvascular cell response to injury and play a crucial role in the pathogenesis of retinal disease. Abnormalities in retinal blood flow play a critical role in remodeling of the retinal vasculature by altering microvascular endothelial and pericyte cell fate (proliferation, apoptosis and migration). Retinal blood flow is controlled locally by vasodilators such as nitric oxide, prostacyclin and the vasoconstrictor endothelin-1 , with considerable evidence linking retinal pathologies such as Normal Tension Glaucoma and Diabetic Retinopathy to altered retinal blood flow Shear stress has previously been shown to modulate EC production of these vasoactive agents in macrovascular cells. Therefore, using a perfused transcapillary coculture of bovine microvascular retinal endothelial cells (BRECs) and bovine retinal pericytes (BRPs), we examined the acute and chronic effect of pulsatile flow on the release of these vasoactive mediators and their subsequent role in modulating retinal vascular cell fate. Acute exposure to pulsatile flow increased BREC NO, PGI2 & ET-1 formation and release Similarly, chronic exposure to pulsatile flow enhanced NO and PGI2 release while concomitantly inhibiting ET-1 in these cells In parallel studies, there was an increase in BRP apoptosis following exposure to high pulsatile flow, whereas BREC apoptosis decreased. Furthermore, the pulsatile flow-induced increases in BRP apoptosis is dependent on increased PGI2 , whereas both ET-1 and NO mediate the protective effect of increased flow on BRECs survival. Notch receptor-hgand interactions and the Hedgehog signalling pathway have been strongly implicated in vascular morphogenesis and remodelling of the embryonic vasculature, with Hedgehog acting upstream of Notch signalling during development. We therefore tested the hypothesis that Hedgehog (Hh) and Notch pathway interact to promote changes in vascular cell fate in BRECs and BRPs in vitro in response to changes in pulsatile flow