Issues with the Specificity of Immunological Reagents for NLRP3: Implications for Age-related Macular Degeneration.

Contradictory data have been presented regarding the implication of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in age-related macular degeneration (AMD), the leading cause of vision loss in the Western world. Recognizing that antibody specificity may explain this discrepancy and in line with recent National Institutes of Health (NIH) guidelines requiring authentication of key biological resources, the specificity of anti-NLRP3 antibodies was assessed to elucidate whether non-immune RPE cells express NLRP3. Using validated resources, NLRP3 was not detected in human primary or human established RPE cell lines under multiple inflammasome-priming conditions, including purported NLRP3 stimuli in RPE such as DICER1 deletion and Alu RNA transfection.

Anti-inflammatory activity of Barleria lupulina: Identification of active compounds that activate the Nrf2 cell defense pathway, organize cortical actin, reduce stress fibers, and improve cell junctions in microvascular endothelial cells.

Ethnopharmacological Relevance: Hot aqueous extracts of the plant Barleria lupulina (BL) are used for treating inflammatory conditions and diabetic vascular complications.

Aim Of The Study: The goal was to identify active compounds in hot aqueous extracts of BL (HAE-BL) that are consistent with a role in reducing inflammation and reducing the vascular pathology associated with diabetes. In particular, we examined activation of the Nrf2 cell defense pathway because our initial findings indicated that HAE-BL activates Nrf2, and because Nrf2 is known to suppress inflammation.

Moderation of calpain activity promotes neovascular integration and lumen formation during VEGF-induced pathological angiogenesis.

Background: Successful neovascularization requires that sprouting endothelial cells (ECs) integrate to form new vascular networks. However, architecturally defective, poorly integrated vessels with blind ends are typical of pathological angiogenesis induced by vascular endothelial growth factor-A (VEGF), thereby limiting the utility of VEGF for therapeutic angiogenesis and aggravating ischemia-related pathologies. Here we investigated the possibility that over-exuberant calpain activity is responsible for aberrant VEGF neovessel architecture and integration.

Active Rac1 improves pathologic VEGF neovessel architecture and reduces vascular leak: mechanistic similarities with angiopoietin-1.

Architecturally defective, leaky blood vessels typify pathologic angiogenesis induced by vascular endothelial growth factor-A (VEGF-A). Such neovascular defects aggravate disease pathology and seriously compromise the therapeutic utility of VEGF. Endothelial cell (EC) transduction with active L61Rac1 strongly improved VEGF-driven angiogenesis in vivo as measured by increased neovascular density, enhanced lumen formation, and reduced vessel leakiness.

Cdc42-mediated inhibition of GSK-3β improves angio-architecture and lumen formation during VEGF-driven pathological angiogenesis.

Vascular endothelial growth factor-A (VEGF) typically induces abnormal angiogenesis in the adult, thereby aggravating disease pathology and limiting utility of VEGF for therapeutic angiogenesis. To identify strategies for rectifying defects in pathological VEGF neovessels, we investigated consequences of modulating the Rho GTPase Cdc42. In a mouse skin model of VEGF-driven pathological angiogenesis, transduction with active Cdc42 (L28Cdc42) markedly improved VEGF neovessels, as measured by increased lumen formation, enlarged vessel diameter, and enhanced perfusion of macromolecular tracers.

Moderate GSK-3β inhibition improves neovascular architecture, reduces vascular leakage, and reduces retinal hypoxia in a model of ischemic retinopathy.

In ischemic retinopathies, unrelieved hypoxia induces the formation of architecturally abnormal, leaky blood vessels that damage retina and ultimately can cause blindness. Because these newly formed blood vessels are functionally defective, they fail to alleviate underlying hypoxia, resulting in more pathological neovascularization and more damage to retina. With an established model of ischemic retinopathy, we investigated inhibition of glycogen synthase kinase-3β (GSK-3β) as a means for improving the architecture and functionality of pathological blood vessels in retina.

Calpain inhibitors reduce retinal hypoxia in ischemic retinopathy by improving neovascular architecture and functional perfusion.

In ischemic retinopathies, underlying hypoxia drives abnormal neovascularization that damages retina and causes blindness. The abnormal neovasculature is tortuous and leaky and fails to alleviate hypoxia, resulting in more pathological neovascularization and retinal damage. With an established model of ischemic retinopathy we found that calpain inhibitors, when administered in moderation, reduced architectural abnormalities, reduced vascular leakage, and most importantly reduced retinal hypoxia.

Down syndrome candidate region 1 isoform 1 mediates angiogenesis through the calcineurin-NFAT pathway.

Down syndrome candidate region 1 (DSCR1) is one of more than 50 genes located in a region of chromosome 21 that has been implicated in Down syndrome. DSCR1 can be expressed as four isoforms, one of which, isoform 4 (DSCR1-4), has recently been found to be strongly induced by vascular endothelial growth factor A (VEGF-A(165)) and to provide a negative feedback loop that inhibits VEGF-A(165)-induced endothelial cell proliferation in vitro and angiogenesis in vivo. We report here that another DSCR1 isoform, DSCR1-1L, is also up-regulated by VEGF-A(165) in cultured endothelial cells and is strongly expressed in several types of pathologic angiogenesis in vivo.

In vivo and in vitro models of Mammalian angiogenesis.

Angiogenesis is a complex process involving the organization of proliferating endothelial cells into new blood vessels. Both in vivo models and in vitro models are important for investigating angiogenesis and for defining the involvement of specific molecules. This chapter describes a basic mouse model of vascular endothelial growth factor-driven angiogenesis in mouse skin together with a modified version of this model in which retrovirus-packaging cells are included as a means to efficiently achieve retroviral transduction in vivo.

Rho activity critically and selectively regulates endothelial cell organization during angiogenesis.

The mechanisms that control organization of endothelial cells (ECs) into new blood vessels are poorly understood. We hypothesized that the GTPase Rho, which regulates cytoskeletal architecture, is important for EC organization during neovascularization. To test this hypothesis, we designed a highly versatile mouse skin model that used vascular endothelial growth factor-expressing cells together with packaging cells producing retroviruses encoding RhoA GTPase mutants.