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FUNDC1-dependent mitochondria-associated endoplasmic reticulum membranes are involved in angiogenesis and neoangiogenesis.

ABSTRACT: FUN14 domain-containing protein 1 (FUNDC1) is an integral mitochondrial outer-membrane protein, and mediates the formation of mitochondria-associated endoplasmic reticulum membranes (MAMs). This study aims to determine the contributions of FUNDC1-mediated MAMs to angiogenesis in vitro and in vivo. In cultured endothelial cells, VEGF significantly increases the formation of MAMs and MAM-related proteins, including FUNDC1. Endothelial cell-specific deletion of FUNDC1, which disrupts MAM formation in endothelial cells, lowers VEGFR2 expression and reduces tube formation, spheroid-sprouting, and functional blood vessel formation in vitro and in vivo. Conversely, increased MAM formation using MAM linkers mimics the effects of VEGF and promotes endothelial angiogenesis. Mechanistically, increased MAMs formation led to increased levels of Ca²⁺ in cytosol, promoted the phosphorylation of serum response factor (SRF) and enhanced the binding of SRF to VEGFR2 promoter, resulting in increased VEGFR2 production, with consequent angiogenesis. Moreover, blocking FUNDC1-related MAM formation with a cell-penetrating inhibitory peptide significantly suppresses the expressions of downstream angiogenic genes and inhibits tumor angiogenesis. We conclude that decreased MAMs formation by silencing FUNDC1 can inhibit angiogenesis by decreasing VEGFR2 expression, and targeting FUNDC1-dependent MAMs might be a promising approach for treating human disorders characterized by defective angiogenesis.

SUBMITTER: Wang C

PROVIDER: S-EPMC8110587 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:AimsCytokine storm is closely related to the initiation and progression of sepsis, and the level of IL-6 is positively correlated with mortality and organ dysfunction. Sepsis-induced myocardial dysfunction (SIMD) is one of the major complications. However, the role of the IL-6/STAT3 signaling in the SIMD remains unclear.Methods and resultsSeptic mice were induced by intraperitoneal injection of LPS (10 mg/kg). Echocardiography, cytokines detection, and histologic examination showed that sepsis mice developed cardiac systolic and diastolic dysfunction, increase of inflammatory cytokines in serum, activated STAT3 and TLR4/NFκB pathway in heart, and raised myocardial apoptosis, which were attenuated by IL-6/STAT3 inhibitor, Bazedoxifene. In vitro, we found that LPS decreased cell viability in a concentration-dependent manner and activated STAT3. Western blot and immunofluorescence results indicated that STAT3 phosphorylation induced by LPS was inhibited by Bazedoxifene. Bazedoxifene also suppressed LPS-induced IL-6 transcription. sIL-6R caused LPS-induced p-STAT3 firstly decreased and then significantly increased. More importantly, we found STAT3-knockdown suppressed LPS-induced expression of FUNDC1, a protein located in mitochondria-associated endoplasmic reticulum membranes (MAMs). Overexpression of STAT3 led to an increase in FUNDC1 expression. Dual-luciferase reporter assay was used to confirm that STAT3 was a potential transcription factor for FUNDC1. Moreover, we showed that LPS increased MAMs formation and intracellular Ca2+ levels, enhanced the expression of Cav1.2 and RyR2, decreased mitochondrial membrane potential and intracellular ATP levels, and promoted mitochondrial fragmentation, the expression of mitophagy proteins and ROS production in H9c2 cells, which were reversed by knockdown of FUNDC1 and IL-6/STAT3 inhibitor including Bazedoxifene and Stattic.ConclusionsIL-6/STAT3 pathway plays a key role in LPS-induced myocardial dysfunction, through regulating the FUNDC1-associated MAMs formation and interfering the function of ER and mitochondria. IL-6/STAT3/FUNDC1 signaling could be a new therapeutic target for SIMD.

Project description:Background: Neurotoxicity induced by the amyloid beta (Aβ) peptide is one of the most important pathological mechanisms of Alzheimer's disease (AD). Activation of the adaptive IRE1α-XBP1 pathway contributes to the pathogenesis of AD, making it a potential target for AD therapeutics. However, the mechanism of IRE1α-XBP1 pathway involvement in AD is unclear. We, therefore, investigated the effect of the IRE1α-XBP1 axis in an in vitro AD model and explored its potential mechanism. Methods: The human neuroblastoma cell line, SH-SY5Y, was used. Cells were treated with Aβ25-35, with or without 4μ8c, an inhibitor of IRE1α. Cells were collected and analyzed by Western blotting, quantitative real-time PCR, electron microscopy, fluorescence microscopy, calcium imaging, and other biochemical assays. Results: Aβ-exposed SH-SY5Y cells showed an increased expression of XBP1s and p-IRE1α. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and calcium imaging analysis showed that the IRE1α inhibitor, 4μ8c, reduced Aβ-induced cytotoxicity. Increased levels of ATP, restoration of mitochondrial membrane potential, and decreased production of mitochondrial reactive oxygen species after Aβ treatment in the presence of 4μ8c showed that inhibiting the IRE1α-XBP1 axis effectively mitigated Aβ-induced mitochondrial dysfunction in SH-SY5Y cells. Furthermore, Aβ treatment increased the expression and interaction of IP3R, Grp75, and vdac1 and led to an increased endoplasmic reticulum (ER)-mitochondria association, malfunction of mitochondria-associated ER-membranes (MAMs), and mitochondrial dysfunction. These deficits were rescued by inhibiting the IRE1α-XBP1 axis. Conclusion: These findings demonstrate that Aβ peptide induces the activation of the IRE1α-XBP1 axis, which may aggravate cytotoxicity and mitochondrial impairment in SH-SY5Y cells by targeting MAMs. Inhibition of the IRE1α-XBP1 axis provides the protection against Aβ-induced injury in SH-SY5Y cells and may, therefore, be a new treatment strategy.

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FUNDC1-dependent mitochondria-associated endoplasmic reticulum membranes are involved in angiogenesis and neoangiogenesis.

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