Farnesoid X Receptor Protects Murine Lung against IL-6-promoted Ferroptosis Induced by Polyriboinosinic-Polyribocytidylic Acid.

Various infections trigger a storm of proinflammatory cytokines in which IL-6 acts as a major contributor and leads to diffuse alveolar damage in patients. However, the metabolic regulatory mechanisms of IL-6 in lung injury remain unclear. Polyriboinosinic-polyribocytidylic acid [poly(I:C)] activates pattern recognition receptors involved in viral sensing and is widely used in alternative animal models of RNA virus-infected lung injury. In this study, intratracheal instillation of poly(I:C) with or without an IL-6-neutralizing antibody model was combined with metabonomics, transcriptomics, and so forth to explore the underlying molecular mechanisms of IL-6-exacerbated lung injury. We found that poly(I:C) increased the IL-6 concentration, and the upregulated IL-6 further induced lung ferroptosis, especially in alveolar epithelial type II cells. Meanwhile, lung regeneration was impaired. Mechanistically, metabolomic analysis showed that poly(I:C) significantly decreased glycolytic metabolites and increased bile acid intermediate metabolites that inhibited the bile acid nuclear receptor farnesoid X receptor (FXR), which could be reversed by IL-6-neutralizing antibody. In the ferroptosis microenvironment, IL-6 receptor monoclonal antibody tocilizumab increased FXR expression and subsequently increased the Yes-associated protein (YAP) concentration by enhancing PKM2 in A549 cells. FXR agonist GW4064 and liquiritin, a potential natural herbal ingredient as an FXR regulator, significantly attenuated lung tissue inflammation and ferroptosis while promoting pulmonary regeneration. Together, the findings of the present study provide the evidence that IL-6 promotes ferroptosis and impairs regeneration of alveolar epithelial type II cells during poly(I:C)-induced murine lung injury by regulating the FXR-PKM2-YAP axis. Targeting FXR represents a promising therapeutic strategy for IL-6-associated inflammatory lung injury.

RNA N6-methyladenosine modulates endothelial atherogenic responses to disturbed flow in mice.

Atherosclerosis preferentially occurs in atheroprone vasculature where human umbilical vein endothelial cells are exposed to disturbed flow. Disturbed flow is associated with vascular inflammation and focal distribution. Recent studies have revealed the involvement of epigenetic regulation in atherosclerosis progression. N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic mRNA, but its function in endothelial atherogenic progression remains unclear. Here, we show that m6A mediates the epidermal growth factor receptor (EGFR) signaling pathway during EC activation to regulate the atherosclerotic process. Oscillatory stress (OS) reduced the expression of methyltransferase like 3 (METTL3), the primary m6A methyltransferase. Through m6A sequencing and functional studies, we determined that m6A mediates the mRNA decay of the vascular pathophysiology gene EGFR which leads to EC dysfunction. m6A modification of the EGFR 3' untranslated regions (3'UTR) accelerated its mRNA degradation. Double mutation of the EGFR 3'UTR abolished METTL3-induced luciferase activity. Adenovirus-mediated METTL3 overexpression significantly reduced EGFR activation and endothelial dysfunction in the presence of OS. Furthermore, thrombospondin-1 (TSP-1), an EGFR ligand, was specifically expressed in atheroprone regions without being affected by METTL3. Inhibition of the TSP-1/EGFR axis by using shRNA and AG1478 significantly ameliorated atherogenesis. Overall, our study revealed that METTL3 alleviates endothelial atherogenic progression through m6A-dependent stabilization of EGFR mRNA, highlighting the important role of RNA transcriptomics in atherosclerosis regulation.

Inhibition of Soluble Epoxide Hydrolase Attenuates Bosutinib-Induced Blood Pressure Elevation.

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c-Abl regulates YAPY357 phosphorylation to activate endothelial atherogenic responses to disturbed flow.

Local flow patterns determine the uneven distribution of atherosclerotic lesions. This research aims to elucidate the mechanism of regulation of nuclear translocation of Yes-associated protein (YAP) under oscillatory shear stress (OSS) in the atheroprone phenotype of endothelial cells (ECs). We report here that OSS led to tyrosine phosphorylation and strong, continuous nuclear translocation of YAP in ECs that is dependent on integrin α5ß1 activation. YAP overexpression in ECs blunted the anti-atheroprone effect of an integrin α5ß1-blocking peptide (ATN161) in Apoe-/- mice. Activation of integrin α5ß1 induced tyrosine, but not serine, phosphorylation of YAP in ECs. Blockage of integrin α5ß1 with ATN161 abolished the phosphorylation of YAP at Y357 induced by OSS. Mechanistic studies showed that c-Abl inhibitor attenuated the integrin α5ß1-induced YAP tyrosine phosphorylation. Furthermore, the phosphorylation of c-Abl and YAPY357 was significantly increased in ECs in atherosclerotic vessels of mice and in human plaques versus normal vessels. Finally, bosutinib, a tyrosine kinase inhibitor, markedly reduced the level of YAPY357 and the development of atherosclerosis in Apoe-/- mice. The c-Abl/YAPY357 pathway serves as a mechanism for the activation of integrin α5ß1 and the atherogenic phenotype of ECs in response to OSS, and provides a potential therapeutic strategy for atherogenesis.

Elevating ATP-binding cassette transporter G1 improves re-endothelialization function of endothelial progenitor cells via Lyn/Akt/eNOS in diabetic mice.

Endothelial progenitor cell (EPC) dysfunction contributes to diabetes-induced delay in endothelium repair after vessel injury, prominently associated with diabetic cardiovascular complications such as neointima formation. ATP-binding cassette transporter G1 (ABCG1) promotes cholesterol efflux to HDL, which may favorably affect EPC function. However, whether ABCG1 improves EPC function, especially in diabetes, remains unknown. Here we investigated the role of ABCG1 in EPCs by using Tie2-mediated ABCG1 transgenic (Tie2- ABCG1Tg) mice. Mice were injected with streptozotocin to induce diabetes mellitus. As compared with wild-type (WT) mice, in Tie2- ABCG1Tg mice, diabetes-impaired EPC migration and tube formation were reversed. In vitro gain-of-function and loss-of-function studies further revealed that ABCG1-overexpressing EPCs showed increased migration and tube formation and differentiation via the Lck/Yes-related novel protein tyrosine kinase /Akt/endothelial NO synthase pathway by enhancing cellular cholesterol efflux. Finally, type 1 and type 2 diabetic mouse models with arterial injury were intravenously injected with labeled EPCs from WT or Tie2- ABCG1Tg mice. Re-endothelialization in diabetic mice was improved to a greater extent by injection of ABCG1-overexpressing than WT EPCs. Our study demonstrated that ABCG1 in EPCs improved repair after vascular injury in diabetes by increasing EPC function such as migration, tube formation and differentiation, and subsequent re-endothelialization. ABCG1 might be a promising therapeutic target for diabetes-associated vascular diseases.-Shi, Y., Lv, X., Liu, Y., Li, B., Liu, M., Yan, M., Liu, Y., Li, Q., Zhang, X., He, S., Zhu, M., He, J., Zhu, Y., Zhu, Y., Ai, D. Elevating ATP-binding cassette transporter G1 improves re-endothelialization function of endothelial progenitor cells via Lyn/Akt/eNOS in diabetic mice.

Yes-Associated Protein Promotes Angiogenesis via Signal Transducer and Activator of Transcription 3 in Endothelial Cells.

RATIONALE: Angiogenesis is a complex process regulating endothelial cell (EC) functions. Emerging lines of evidence support that YAP (Yes-associated protein) plays an important role in regulating the angiogenic activity of ECs. OBJECTIVE: The objective of this study was to specify the effect of EC YAP on angiogenesis and its underlying mechanisms. METHOD AND RESULTS: In ECs, vascular endothelial growth factor reduced YAP phosphorylation time and dose dependently and increased its nuclear accumulation. Using Tie2Cre-mediated YAP transgenic mice, we found that YAP promoted angiogenesis in the postnatal retina and tumor tissues. Mass spectrometry revealed signal transducer and activator of transcription 3 (STAT3) as a potential binding partner of YAP in ECs. Western blot and immunoprecipitation assays indicated that binding with YAP prolonged interleukin 6-induced STAT3 nuclear accumulation by blocking chromosomal maintenance 1-mediated STAT3 nuclear export without affecting its phosphorylation. Moreover, angiopoietin-2 expression induced by STAT3 was enhanced by YAP overexpression in ECs. Finally, a selective STAT3 inhibitor or angiopoietin-2 blockage partly attenuated retinal angiogenesis in Tie2Cre-mediated YAP transgenic mice. CONCLUSIONS: YAP binding sustained STAT3 in the nucleus to enhance the latter's transcriptional activity and promote angiogenesis via regulation of angiopoietin-2.

Endothelial ATP-binding cassette G1 in mouse endothelium protects against hemodynamic-induced atherosclerosis.

Activated vascular endothelium inflammation under persistent hyperlipidemia is the initial step of atherogenesis. ATP-binding cassette G1 (ABCG1) is a crucial factor maintaining sterol and lipid homeostasis by transporting cholesterol efflux to high-density lipoprotein. In this study, we investigated the protective effects of ABCG1 in endothelial inflammation activation during early-stage atherogenesis in mice and the underlying mechanisms. Endothelial cell (EC)-specific ABCG1 transgenic (EC-ABCG1-Tg) mice were generated and cross-bred with low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. After a 4-week Western-type diet, the mice were sacrificed for assessing atherosclerosis. Human umbilical vein ECs were treated with different flows, and ABCG1 was adenovirally overexpressed to investigate the mechanism in vitro. Compared with Ldlr(-/-) mouse aortas, EC-ABCG1-Tg/Ldlr(-/-) aortas showed decreased early-stage lesions. Furthermore, the lesion area in the EC-ABCG1-Tg/Ldlr(-/-) mouse aortic arch but not thoracic aorta was significantly reduced, which suggests a protective role of ABCG1 under atheroprone flow. In vitro, overexpression of ABCG1 attenuated EC activation caused by oscillatory shear stress. Overexpression of ABCG1 blunted cholesterol-activated ECs in vitro. In exploring the mechanisms of ABCG1 attenuating endothelial inflammation, we found that ABCG1 inhibited oscillatory flow-activated nuclear factor kappa B and NLRP3 inflammasome in ECs. ABCG1 may play a protective role in early-stage atherosclerosis by reducing endothelial activation induced by oscillatory shear stress via suppressing the inflammatory response.