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Endothelial FAM3A positively regulates post-ischaemic angiogenesis.

ABSTRACT: BACKGROUND:Angiogenesis improves reperfusion to the ischaemic tissue after vascular obstruction. The underlying molecular mechanisms of post-ischaemic angiogenesis are not clear. FAM3A belongs to the family with sequence similarity 3 (FAM3) genes, but its biological function in endothelial cells in regards to vascular diseases is not well understood. METHODS:Gain- and loss-of-function methods by adenovirus or associated-adenovirus (AAV) in different models were applied to investigate the effects of FAM3A on endothelial angiogenesis. Endothelial angiogenesis was analysed by tube formation, migration and proliferation in vitro, and the blood flow and capillary density in a hind limb ischaemic model in vivo. FINDINGS:Endothelial FAM3A expression is downregulated under hypoxic conditions. Overexpression of FAM3A promotes, but depletion of FAM3A suppresses, endothelial tube formation, proliferation and migration. Utilizing the mouse hind limb ischaemia model, we also observe that FAM3A overexpression can improve blood perfusion and increase capillary density, whereas FAM3A knockdown has the opposite effects. Mechanistically, mitochondrial FAM3A increases adenosine triphosphate (ATP) production and secretion; ATP binds to P2 receptors and then upregulates cytosolic free Ca2+ levels. Increased intracellular Ca2+ levels enhance phosphorylation of the transcriptional factor cAMP response element binding protein (CREB) and its recruitment to the VEGFA promoter, thus activating VEGFA transcription and the final endothelial angiogenesis. INTERPRETATION:In summary, our data demonstrate that FAM3A positively regulates angiogenesis through activation of VEGFA transcription, suggesting that FAM3A may constitute a novel molecular therapeutic target for ischaemic vascular disease.

SUBMITTER: Xu W

PROVIDER: S-EPMC6562148 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Endothelial FAM3A positively regulates post-ischaemic angiogenesis.

Xu Wenjing W Liang Minglu M Zhang Yanqing Y Huang Kai K Wang Cheng C

EBioMedicine 20190416

<h4>Background</h4>Angiogenesis improves reperfusion to the ischaemic tissue after vascular obstruction. The underlying molecular mechanisms of post-ischaemic angiogenesis are not clear. FAM3A belongs to the family with sequence similarity 3 (FAM3) genes, but its biological function in endothelial cells in regards to vascular diseases is not well understood.<h4>Methods</h4>Gain- and loss-of-function methods by adenovirus or associated-adenovirus (AAV) in different models were applied to investig ...[more]

PMID: 31000420

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Project description:RATIONALE:Postischemic angiogenesis is critical to limit the ischemic tissue damage and improve the blood flow recovery. The regulation and the underlying molecular mechanisms of postischemic angiogenesis are not fully unraveled. TFEB (transcription factor EB) is emerging as a master gene for autophagy and lysosome biogenesis. However, the role of TFEB in vascular disease is less understood. OBJECTIVE:We aimed to determine the role of endothelial TFEB in postischemic angiogenesis and its underlying molecular mechanism. METHODS AND RESULTS:In primary human endothelial cells (ECs), serum starvation induced TFEB nuclear translocation. VEGF (vascular endothelial growth factor) increased TFEB expression level and nuclear translocation. Utilizing genetically engineered EC-specific TFEB transgenic and KO (knockout) mice, we investigated the role of TFEB in postischemic angiogenesis in the mouse hindlimb ischemia model. We observed improved blood perfusion and increased capillary density in the EC-specific TFEB transgenic mice compared with the wild-type littermates. Furthermore, blood flow recovery was attenuated in EC-TFEB KO mice compared with control mice. In aortic ring cultures, the TFEB transgene significantly increased vessel sprouting, whereas TFEB deficiency impaired the vessel sprouting. In vitro, adenovirus-mediated TFEB overexpression promoted EC tube formation, migration, and survival, whereas siRNA-mediated TFEB knockdown had the opposite effect. Mechanistically, TFEB activated AMPK (AMP-activated protein kinase)-? signaling and upregulated autophagy. Through inactivation of AMPK? or inhibition of autophagy, we demonstrated that the AMPK? and autophagy are necessary for TFEB to regulate angiogenesis in ECs. Finally, the positive effect of TFEB on AMPK? activation and EC tube formation was mediated by TFEB-dependent transcriptional upregulation of MCOLN1 (mucolipin-1). CONCLUSIONS:In summary, our data demonstrate that TFEB is a positive regulator of angiogenesis through activation of AMPK? and autophagy, suggesting that TFEB constitutes a novel molecular target for ischemic vascular disease.

Project description:Purpose: Angiogenesis post-ischemia plays an essential role in preventing ischemic damage to tissue by improving the blood recovery. Determining the regulatory mechanism of ischemic angiogenesis, therefore, could provide effective therapeutics for ischemic injury. Materials and Methods: The RNA sequencing (RNA-seq) database was used to predict the association of gamma-aminobutyric acid type B receptor subunit 2 (GABBR2) with endothelial-specific expression. The role of GABBR2 in angiogenesis was verified in vitro by downregulating GABBR2 in human umbilical vein endothelial cells (HUVECs) with lentiviral vectors. Besides, the in vivo effect of GABBR2 on the blood recovery of an ischemic hindlimb was demonstrated by establishing a hindlimb ischemia model in normal and GABBR2 adenoviral vector-infected mice. Then, the mobilization of endothelial progenitor cells (EPCs) in peripheral blood post-ischemia was determined by flow cytometry. Finally, the XF analyzer and Western blot were used to determine the effect of GABBR2 on endothelial metabolism. Results: The RNA-seq results indicated a strong association between GABBR2 and endothelial revascularization, and the upregulation of GABBR2 was detected in both hypoxia-treated HUVECs and ischemic mouse hindlimb. Hypoxia treatment for 6 h increased the proliferation, migration, and tube formation of HUVECs, which were inhibited by GABBR2 knockdown. Additionally, GABBR2 downregulation significantly decreased the blood flow recovery of mouse ischemic hindlimb. The expressions of the EPC markers CD34+ and CD133+ significantly decreased in the peripheral blood in hindlimb post-ischemia. Mechanically, glycolysis-dominated metabolism of HUVECs was compromised by GABBR2 knockdown. Evidences of the decreased expressions of HKII, PFKFB3, and PKM1 also supported the compromised glycolysis induced by GABBR2 downregulation. Conclusion: Our study demonstrated that GABBR2 regulated angiogenesis post-ischemia by inhibiting the glycolysis pathway.

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Project description:BackgroundAngiogenesis is critical for forming new blood vessels from antedating vascular vessels. The endothelium is essential for angiogenesis, vascular remodelling and minimisation of functional deficits following ischaemia. The insulin-like growth factor (IGF) family is crucial for angiogenesis. Insulin-like growth factor-binding protein 5 (IGFBP5), a binding protein of the IGF family, may have places in angiogenesis, but the mechanisms are not yet completely understood. We sought to probe whether IGFBP5 is involved in pathological angiogenesis and uncover the molecular mechanisms behind it.Methods and resultsIGFBP5 expression was elevated in the vascular endothelium of gastrocnemius muscle from critical limb ischaemia patients and hindlimb ischaemic (HLI) mice and hypoxic human umbilical vein endothelial cells (HUVECs). In vivo, loss of endothelial IGFBP5 (IGFBP5EKO) facilitated the recovery of blood vessel function and limb necrosis in HLI mice. Moreover, skin damage healing and aortic ring sprouting were faster in IGFBP5EKO mice than in control mice. In vitro, the genetic inhibition of IGFBP5 in HUVECs significantly promoted tube formation, cell proliferation and migration by mediating the phosphorylation of IGF1R, Erk1/2 and Akt. Intriguingly, pharmacological treatment of HUVECs with recombinant human IGFBP5 ensued a contrasting effect on angiogenesis by inhibiting the IGF1 or IGF2 function. Genetic inhibition of IGFBP5 promoted cellular oxygen consumption and extracellular acidification rates via IGF1R-mediated glycolytic adenosine triphosphate (ATP) metabolism. Mechanistically, IGFBP5 exerted its role via E3 ubiquitin ligase Von Hippel-Lindau (VHL)-regulated HIF1α stability. Furthermore, the knockdown of the endothelial IGF1R partially abolished the reformative effect of IGFBP5EKO mice post-HLI.ConclusionOur findings demonstrate that IGFBP5 ablation enhances angiogenesis by promoting ATP metabolism and stabilising HIF1α, implying IGFBP5 is a novel therapeutic target for treating abnormal angiogenesis-related conditions.

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Endothelial FAM3A positively regulates post-ischaemic angiogenesis.

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Endothelial FAM3A positively regulates post-ischaemic angiogenesis.

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