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Purpose: To investigate the roles of sphingosine kinases (SphKs) and sphingosine-1-phosphate receptors (S1PRs) in endotoxin-induced uveitis (EIU) mice. Methods: EIU model was induced using an intraperitoneal injection of lipopolysaccharide (LPS). The expression of SphKs and S1PRs in the retina was assessed using quantitative polymerase chain reaction (qPCR) and immunofluorescence. The effects of S1PR antagonists on the expression of inflammatory cytokines in the retina were evaluated using qPCR and western blotting. Effects of leukocyte infiltration of the retinal vessels were evaluated to determine the effects of the S1PR2 antagonist and genetic deletion of S1PR2 on retinal inflammation. Results: Retinal SphK1 expression was significantly upregulated in EIU. SphK1 was expressed in the GCL, IPL, and OPL and S1PR2 was expressed in the GCL, INL, and OPL. Positive cells in IPL and OPL of EIU retina were identified as endothelial cells. S1PR2 antagonist and genetic deletion of S1PR2 significantly suppressed the expression of IL-1α, IL-6, TNF-α, and ICAM-1, whereas S1PR1/3 antagonist did not. Use of S1PR2 antagonist and S1PR2 knockout in mice significantly ameliorated leukocyte adhesion induced by LPS. Conclusion: SphK1/S1P/S1PR2 signaling was upregulated in EIU and S1PR2 inhibition suppressed inflammatory response. Targeting this signaling pathway has potential for treating retinal inflammatory diseases. [ABSTRACT FROM AUTHOR]

Introduction Lysophosphatidic acid (LPA; 1- or 2-acyl-sn-glycerol-3-phosphate) is a bioactive lipid mediator that regulates cell migration, proliferation, morphological change, and the production of cytokines (1, 2). LPA is mainly produced [...], Lysophosphatidic acid (LPA) is a potent lipid mediator with various biological functions mediated through six G proteincoupled receptors (GPCRs), LPA1-LPA6. Previous studies have demonstrated that LPA-G[alpha]12/G[alpha]13 signaling plays an important role in embryonic vascular development. However, the responsible LPA receptors and underlying mechanisms are poorly understood. Here, we show a critical role of LPA4 and LPA6 in developmental angiogenesis. In mice, Lpa4; Lpa6 double-knockout (DKO) embryos were lethal due to global vascular deficiencies, and endothelial cell-specific (EC-specific) Lpa4; Lpa6-DKO retinas had impaired sprouting angiogenesis. Mechanistically, LPA activated the transcriptional regulators YAP and TAZ through LPA4/LPA6-mediated G[alpha]12/G[alpha]13-Rho-ROCK signaling in ECs. YAP/TAZ knockdown increased endothelial expression of the Notch ligand delta-like ligand 4 (DLL4) that was mediated by [beta]-catenin and Notch intracellular domain (NICD). Fibrin gel sprouting assay revealed that LPA4/LPA6, G[alpha]12/G[alpha]13, or YAP/TAZ knockdown consistently blocked EC sprouting, which was rescued by a Notch inhibitor. Notably, the inhibition of Notch signaling also ameliorated impaired retinal angiogenesis in EC-specific Lpa4; Lpa6-DKO mice. Overall, these results suggest that the G[alpha]12/G[alpha]13coupled receptors LPA4 and LPA6 synergistically regulate endothelial Dll4 expression through YAP/TAZ activation. This could in part account for the mechanism of YAP/TAZ-mediated developmental angiogenesis. Our findings provide insight into the biology of GPCR- activated YAP/TAZ.

Pulmonary fibrosis, especially idiopathic pulmonary fibrosis (IPF), portends significant morbidity and mortality, and current therapeutic options are suboptimal. We have previously shown that type I collagen signaling through discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase expressed by fibroblasts, is critical for the regulation of fibroblast apoptosis and progressive fibrosis. However, the downstream signaling pathways for DDR2 remain poorly defined and could also be attractive potential targets for therapy. A recent phosphoproteomic approach indicated that PIK3C2α, a poorly studied member of the PI3 kinase family, could be a downstream mediator of DDR2 signaling. We hypothesized that collagen I/DDR2 signaling through PIK3C2α regulates fibroblast activity during progressive fibrosis. To test this hypothesis, we found that primary murine fibroblasts and IPF‐derived fibroblasts stimulated with endogenous or exogenous type I collagen led to the formation of a DDR2/PIK3C2α complex, resulting in phosphorylation of PIK3C2α. Fibroblasts treated with an inhibitor of PIK3C2α or with deletion of PIK3C2α had fewer markers of activation after stimulation with TGFβ and more apoptosis after stimulation with a Fas‐activating antibody. Finally, mice with fibroblast‐specific deletion of PIK3C2α had less fibrosis after bleomycin treatment than did littermate control mice with intact expression of PIK3Cα. Collectively, these data support the notion that collagen/DDR2/PIK3C2α signaling is critical for fibroblast function during progressive fibrosis, making this pathway a potential target for antifibrotic therapy. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Formation of the vascular network by vasculogenesis and angiogenesis is essential for embryonic development, repair and remodeling of tissues in adults, and tumor growth. The angiogenic response to vascular endothelial [...], The class II α-isoform of phosphatidylinositol 3-kinase (PI3K-C2α) is localized in endosomes, the trans-Golgi network and clathrin-coated vesicles; however, its functional role is not well understood. Global or endothelial-cell-specific deficiency of PI3K-C2α resulted in embryonic lethality caused by defects in sprouting angiogenesis and vascular maturation. PI3K-C2α knockdown in endothelial cells resulted in a decrease in the number of PI3-phosphate-enriched endosomes, impaired endosomal trafficking, defective delivery of VE-cadherin to endothelial cell junctions and defective junction assembly. PI3K-C2α knockdown also impaired endothelial cell signaling, including vascular endothelial growth factor receptor internalization and endosomal RhoA activation. Together, the effects of PI3K-C2α knockdown led to defective endothelial cell migration, proliferation, tube formation and barrier integrity. Endothelial PI3K-C2α deficiency in vivo suppressed postischemic and tumor angiogenesis and diminished vascular barrier function with a greatly augmented susceptibility to anaphylaxis and a higher incidence of dissecting aortic aneurysm formation in response to angiotensin II infusion. Thus, PI3K-C2α has a crucial role in vascular formation and barrier integrity and represents a new therapeutic target for vascular disease.

Introduction Atherosclerosis is a chronic inflammatory process involving complex interactions of modified lipoproteins, monocyte-derived macrophages or foam cells, T lymphocytes, ECs, and SMCs (1, 2). Oxidized LDL (oxLDL) and other [...], Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that has pleiotropic effects in a variety of cell types including ECs, SMCs, and macrophages, all of which are central to the development of atherosclerosis. It may therefore exert stimulatory and inhibitory effects on atherosclerosis. Here, we investigated the role of the S1P receptor S1PR2 in atherosclerosis by analyzing [S1pr2.sup.-/-] mice with an [Apoe.sup.-/-] background. S1PR2 was expressed in macrophages, ECs, and SMCs in atherosclerotic aortas. In [S1pr2.sup.-/-][Apoe.sup.-/-] mice fed a high-cholesterol diet for 4 months, the area of the atherosclerotic plaque was markedly decreased, with reduced macrophage density, increased SMC density, increased eNOS phosphorylation, and downregulation of proinflammatory cytokines compared with [S1pr2.sup.+/+][Apoe.sup.-/-] mice. Bone marrow chimera experiments indicated a major role for macrophage S1PR2 in atherogenesis. [S1pr2.sup.-/-][Apoe.sup.-/-] macrophages showed diminished Rho/Rho kinase/NF-κB (ROCK/NF-κB) activity. Consequently, they also displayed reduced cytokine expression, reduced oxidized LDL uptake, and stimulated cholesterol efflux associated with decreased scavenger receptor expression and increased cholesterol efflux transporter expression. [S1pr2.sup.-/-][Apoe.sup.-/-] ECs also showed reduced ROCK and NF-κB activities, with decreased MCP-1 expression and elevated eNOS phosphorylation. Pharmacologic S1PR2 blockade in [S1pr2.sup.+/+][Apoe.sup.-/-] mice diminished the atherosclerotic plaque area in aortas and modified LDL accumulation in macrophages.We conclude therefore that S1PR2 plays a critical role in atherogenesis and may serve as a novel therapeutic target for atherosclerosis.