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Background: The apoptosis and inflammation in cardiac microvascular endothelial cells (CMECs) promote the development of coronary microvascular dysfunction (CMD). The present study aimed to explore the role of E3 ubiquitin ligase mind bomb 1 (MIB1) in the apoptosis and inflammation in CMECs during CMD., Methods: In vivo, CMD in rats was induced by sodium laurate injection. In vitro, rat primary CMECs were stimulated by homocysteine (Hcy). The apoptosis of CMECs was measured using flow cytometry. The inflammation of CMECs was evaluated by the level of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β). The interplay between MIB1 and mitogen-activated protein kinase kinase kinase 5 (map3k5, also called ASK1) was measured using Co-immunoprecipitation., Results: MIB1 expression was decreased and ASK1 expression was increased in the heart tissues of CMD rats and Hcy-treated CMECs. MIB1 overexpression decreased fibrinogen-like protein 2 (FGL2) secretion, inflammation, and apoptosis induced by Hcy in CMECs. Meanwhile, MIB1 overexpression decreased the protein levels of ASK1 and p38, while not affected ASK1 mRNA levels. The following mechanism experiments revealed that MIB1 downregulated ASK1 expression by increasing its ubiquitination. ASK1 overexpression reversed the inhibitory effect of MIB1 on FGL2 secretion, apoptosis, inflammation, and p38 activation in Hcy-treated CMECs. In CMD rats, MIB1 overexpression partly retarded CMD progression, manifesting as increased coronary capillary density and decreased microthrombi formation., Conclusion: MIB1 overexpression relieved apoptosis and inflammation of CMECs during CMD by targeting the ASK1/p38 pathway., (Copyright © 2021 Elsevier Inc. All rights reserved.)

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. An Expression of Concern for this article was previously published while an investigation was conducted (see related editorial: https://doi.org/10.1016/j.biopha.2022.113812). This retraction notice supersedes the Expression of Concern published earlier. Concerns were raised about the provenance of the flow cytometry data shown in Figure 5A, as detailed here: https://pubpeer.com/publications/46C8B5439C5C617A60494BA4C15479; and here: https://docs.google.com/spreadsheets/d/1r0MyIYpagBc58BRF9c3luWNlCX8VUvUuPyYYXzxWvgY/edit#gid=262337249. Independent analysis also identified additional suspected image duplications between the Bax and Bcl-2 Western blots in Figure 6A. The journal requested the corresponding author comment on these concerns and provide the associated raw data. The authors did not respond to this request and therefore the Editor-in-Chief decided to retract the article., (Copyright © 2018. Published by Elsevier Masson SAS.)

Cardiac microvascular endothelial cells (CMECs) dysfunction is an important pathophysiological event in the cardiovascular complications induced by diabetes. However, the underlying mechanism is not fully clarified. Autophagy is involved in programmed cell death. Here we investigated the potential role of autophagy on the CMECs injury induced by high glucose. CMECs were cultured in normal or high glucose medium for 6, 12 and 24 h respectively. The autophagy of CMECs was measured by green fluorescence protein (GFP)-LC3 plasmid transfection. Moreover, the apoptosis of CMEC was determined by flow cytometry. Furthermore, 3-Methyladenine (3MA), ATG7 siRNA and rapamycin were administrated to regulate the autophagy state. Moreover, Western blotting assay was performed to measure the expressions of Akt, mTOR, LC3 and p62. High glucose stress decreased the autophagy, whereas increased the apoptosis in CMECs time dependently. Meanwhile, high glucose stress activated the Akt/mTOR signal pathway. Furthermore, autophagy inhibitor, 3-MA and ATG7 siRNA impaired the autophagy and increased the apoptosis in CMECs induced by high glucose stress. Conversely, rapamycin up-regulated the autophagy and decreased the apoptosis in CMECs under high glucose condition. Our data provide evidence that high glucose directly inhibits autophagy, as a beneficial adaptive response to protect CMECs against apoptosis. Furthermore, the autophagy was mediated, at least in part, by mTOR signaling.

We aimed to explore the effects of myeloid-derived growth factor (Mydgf) on the regulation of hypoxia/reoxygenation (HR)–induced apoptosis of cardiac microvascular endothelial cells (CMECs). CMECs were exposed to hypoxia for 24 h and reoxygenation for 6 h to establish an HR cell model. Subsequently, an adenovirus was used to overexpress Mydgf in CMECs. Flow cytometry and TUNEL staining were used to detect the extent of apoptosis, whereas qPCR was used to detect the relative expression of Mydgf mRNA. Western blotting was also performed to detect the expression of apoptosis-related proteins and endoplasmic reticulum stress (ERS)–related proteins, including C/EBP Homologous Protein (CHOP), glucose-regulated protein 78 (GRP 78), and cleaved Caspase-12. The endoplasmic reticulum stress agonist tunicamycin (TM) was used to stimulate CMECs for 24 h as a rescue experiment for Mydgf. Flow cytometry revealed that the HR model effectively induced endothelial cell apoptosis, whereas qPCR and western blotting showed that Mydgf mRNA and protein levels decreased significantly after HR treatment (P < 0.05). Overexpression of Mydgf in cells effectively reduced apoptosis after HR. Furthermore, western blotting showed that HR induced a significant upregulation of CHOP, GRP78, and cleaved-Caspase-12 expression in CMECs, whereas HR-treated cells downregulated the expression of CHOP, GRP78, and cleaved-Caspase-12 after Mydgf overexpression. Under HR conditions, TM significantly reversed the protective effect of Mydgf on CMECs. Mydgf may reduce CMEC apoptosis induced by HR by regulating oxidative stress in ERS. [ABSTRACT FROM AUTHOR]

Endothelial cell apoptosis and renin-angiotensin-aldosterone system (RAAS) activation are the major pathological mechanisms for cardiovascular disease and heart failure; however, the interaction and mechanism between them remain unclear. Investigating the role of PTP1B in angiotensin II (Ang II)–induced apoptosis of primary cardiac microvascular endothelial cells (CMECs) may provide direct evidence of the link between endothelial cell apoptosis and RAAS. Isolated rat CMECs were treated with different concentrations of Ang II to induce apoptosis, and an Ang II concentration of 4 nM was selected as the effective dose for the subsequent studies. The CMECs were cultured for 48 h with or without Ang II (4 nM) in the absence or presence of the PTP1B inhibitor TCS 401 (8 μM) and the PI3K inhibitor LY294002 (10 μM). The level of CMEC apoptosis was assessed by TUNEL staining and caspase-3 activity. The protein expressions of PTP1B, PI3K, Akt, p-Akt, Bcl-2, Bax, caspase-3, and cleaved caspase-3 were determined by Western blot (WB). The results showed that Ang II increased apoptosis of CMECs, upregulated PTP1B expression, and inhibited the PI3K/Akt pathway. Furthermore, cotreatment with PTP1B inhibitor significantly decreased the number of apoptotic CMECs induced by Ang II, along with increased PI3K expression, phosphorylation of Akt and the ratio of Bcl-2/Bax, decreased caspase-3 activity, and a cleaved caspase-3/caspase-3 ratio, while treatment with LY294002 partly inhibited the anti-apoptotic effect of the PTP1B inhibitor. Ang II induces apoptosis of primary rat CMECs via regulating the PTP1B/PI3K/Akt pathway. [ABSTRACT FROM AUTHOR]

This study is performed to figure out the role of long‐chain noncoding RNA growth‐arrest specific transcript 5 (GAS5) in homocysteine (HCY)‐induced cardiac microvascular endothelial cells (CMECs) injury. CMECs were cultured and the model of CMECs injury was established by coincubation with HCY. To construct stable overexpression of GAS5 cells, the expression of GAS5, microRNA‐33a‐5p (miR‐33a‐5p) and ATP‐binding cassette transporter A1 (ABCA1), and biological characteristics of cells were determined. The messenger RNA (mRNA) level and secretion of vascular endothelial growth factor (VEGF), activity of reactive oxygen species (ROS) and superoxide dismutase (SOD), and the content of malondialdehyde (MDA) were measured. The binding site between GAS5 and miR‐33a‐5p and between miR‐33a‐5p and ABCA1 was verified. CMECs were successfully cultured. Reduction of GAS5 expression and ABCA1 expression together with increased expression of miR‐33a‐5p was found in CMECs induced by HCY. After overexpression of GAS5, there showed increased proliferative activity, decreased cell apoptosis rate and apoptosis index, enhanced cell migration ability, increased number of lumen formation, increased mRNA expression of VEGF in cells and the secretion in the supernatant, decreased activity of ROS and SOD in cells, and decreased content of ROS in cells. miR‐33a‐5p could promote the enrichment of GAS5 and ABCA1 was the direct target gene of miR‐33a‐5p. Our study suggests that the low expression of GAS5 was observed in HCY‐induced CMECs injury, and the upregulation of GAS5 could attenuate HCY‐induced CMECs injury by mediating oxidative stress, and its mechanism is related to the upregulation of ABCA1 expression by competitively binding with miR‐33a‐5p. [ABSTRACT FROM AUTHOR]

Hypoxia/reoxygenation (H/R) often results in cellular oxidative stress and the subsequent apoptosis of cardiac microvascular endothelial cells (CMECs). More recently, studies have highlighted the therapeutic effects of matrine on various cardiovascular diseases. Thus, the aim of the present study was to investigate the underlying mechanism and effects of matrine on hypoxia/reoxygenation (H/R)-induced apoptosis of CMECs in rats. CMECs from Sprague Dawley (SD) rats were primarily treated with H/R, ld (low-dose, 0.5 mg/mL)-Ma + H/R, md (middle-dose, 1 mg/mL)-Ma + H/R, hd (high-dose, 2 mg/mL)-Ma + H/R, Ma + AG490 + H/R (2 mg/mL matrine and 50 μmol/L AG490, a JAK2/STAT3 signaling pathway inhibitor), and AG490 + H/R in an attempt to identify the underlying regulatory mechanisms of matrine. MTT assay was applied to determine cell viability. Hoechst staining was performed to detect the morphology of apoptotic CMECs, while cell cycle and the rate of apoptosis rate were determined by flow cytometry means. The mRNA and protein expression of the JAK2/STAT3 signaling pathway and apoptosis related genes were determined through the use of RT-qPCR and western blot assay methods respectively. An in vitro angiogenesis assay was employed to evaluate the value of matrine in tube formation. CMECs treated with ld-Ma+H/R, md-Ma+H/R, hd-Ma+H/R and Ma + AG490+H/R exhibited higher cell viability, greater cell ratio at the S phase, higher expression levels of p-JAK2 and p-STAT3, increased tube formation ability, and a lower apoptosis rate, with a lower ratio of cells at the G1 phase and Bax/Bcl-2 ratio. Meanwhile, the rats treated with AG490+H/R exhibited opposite results. Taken together, the key findings of the present study suggest that matrine inhibits the H/R-induced apoptosis of CMECs in rats via the JAK2/STAT3 signaling pathway, highlighting its therapeutic potential for H/R injury. [ABSTRACT FROM AUTHOR]

Background: Mitochondrial fission has been identified as the pathogenesis underlying the development of cardiac microvascular ischemia reperfusion (IR) injury, although the regulatory signaling upstream from fission is far from clear. Bax inhibitor is a novel anti-apoptotic factor, and, however, its role of cardiac microvascular IR injury and mitochondrial homeostasis remains unclear.Methods: The cardiac microvascular IR injury was performed in WT mice and BI1 transgenic (BITG) mice. The alterations of microvascular structure and function were detected via electron microscope, immunohistochemistry and immunofluorescence in vivo. Cardiac microvascular endothelial cells were isolated form WT and BITG mice and underwent hypoxia/reoxygenation injury in vitro. Cellular viability and apoptosis were analyzed via MTT assay and caspase-3 activity. Mitochondrial function, morphology and apoptosis were detected. Signaling pathways were analyzed via inhibitor, siRNA and mutant plasmid.Results: Herein, we demonstrated that Bax inhibitor 1 (BI1) was downregulated following cardiac microvascular IR injury, and its expression correlated negatively with microvascular collapse, endothelial cell apoptosis and mitochondrial damage. However, compared to wild-type mice, BI1 transgenic mice were actually protected from the acute microvascular injury and mitochondrial dysfunction. Functional studies illustrated that reintroduced BI1 directly interacted with and inhibited the Syk pathway, leading to the inactivation of Nox2. Subsequently, less Nox2 was associated with ROS downregulation, inhibiting Drp1 phosphorylated activation. Through repression of the Syk-Nox2-Drp1 signaling axis, BI1 strongly disrupted mitochondrial fission, abolishing mitochondrial apoptosis and thus sustaining endothelial cell viability.Conclusions: In summary, our report illustrates that BI1 functions as a novel microvascular guardian in cardiac IR injury that operates via inhibition of the Syk-Nox2-Drp1-mitochondrial fission signaling axis. Thus, novel therapeutic strategies to regulate the balance between BI1 and mitochondrial fission could provide a survival advantage to microvasculature following IR stress. [ABSTRACT FROM AUTHOR]

Hypoxic injury of cardiac microvascular endothelial cells (CMECs) is an important pathophysiological event in myocardial infarction, whereas, the underlying mechanism is still poorly understood. Autophagy, a highly conserved process of cellular degradation, is required for normal cardiac function and also has been implicated in various cardiovascular diseases. Here we investigated the potential role of autophagy in CMEC dysfunction under hypoxia. CMECs were isolated from SD rats. Hypoxia (6–24 h, 1% O 2 ) induced autophagy in CMECs as evidenced by formation of punctate LC3, increased conversion of LC3-I to LC3-II and increased p62 degradation. Importantly, hypoxia-induced apoptosis in CMECs was attenuated by 3-Methyladenine (5 mM), an autophagy inhibitor, and aggravated by rapamycin (1.0 μg/L), an autophagy inducer. Meanwhile, hypoxia increased the nuclear localization of FoxO3α, accompanying with the decreased phosphorylation of FoxO3α and Akt. FoxO3α silencing decreased hypoxia-induced autophagy and the resultant apoptosis. Furthermore, treatment with 3-Methyladenine (10 mg/kg/day) improved the endothelial-dependent diastolic function of coronary artery in rats with myocardial infarction. These results indicated that hypoxia-induced autophagy formation in CMECs is mediated by FoxO3α and contributes to hypoxic injury of hearts. [ABSTRACT FROM AUTHOR]

Objective: This study was designed to detect the role of Toll-like receptor 4 (TLR4) signaling in the dysfunction of cardiac microvascular endothelial cells (CMECs) after hypoxia/reoxygenation (H/R). Methods: The cell viability of CMECs was measured by MTT assay. The migration of CMECs was detected by cell scratch wound assay. The expressions of TLR4, nuclear factor-kappa B (NF-κB) and eNOS were analyzed by Western blot. Secretions of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were determined by NO detection kit and ELISA. Results: Lipopolysaccharide (LPS) incubation increased the expressions of TLR4, NF-κB, IL-6 and TNF-α in CMECs ( P < 0.05 vs. control). The CMECs after H/R injury had impaired cell viability ( P < 0.01 vs. control) and migration ability ( P < 0.05 vs. control). Moreover, the expressions of TLR4, NF-κB, IL-6 and TNF-α were elevated after H/R in CMECs ( P < 0.01 vs. control), while NO and the eNOS expression were significantly decreased. In contrast, administration of the TLR4-neutralizing antibody MTS510 prior to H/R injury down-regulated the expressions of IL-6 and TNF-α and attenuated the dysfunction of CMECs. Conclusion: TLR4 and its signaling components can be activated by LPS and H/R in CMECs. Blocking the TLR4 signal pathway before H/R injury attenuates CMEC dysfunction. [ABSTRACT FROM AUTHOR]

PKC-β inhibitor Ruboxistaurin (RBX or LY333531) can be used to reverse diabetic microvascular complication. However, it has not been previously established whether RBX can protect against ischemia/reperfusion (I/R) injury of cardiac microvessels in diabetic rats. STZ-induced diabetic rats were randomized into four groups and underwent I/R procedures. Cardiac barrier function and the region of cardiac microvascular lesion were examined. Cell monolayer barrier function was detected in cultured cardiac microvascular endothelial cells (CMECs) subjected to simulated I/R (SI/R). PKC-β siRNA was transfected into CMECs to silence PKC-β. Apoptosis Index of CMECs was detected by TUNEL assay and phosphor-LIMK2 protein expression was examined by Western blot analysis. RBX and insulin administration significantly reduced the cardiac microvascular lesion region and Apoptosis Index of endothelial cells (all P < 0.05 vs. no-treatment group). RBX decreased phosphor-LIMK2 expression ( P < 0.05 vs. no-treatment group). RBX pretreatment and transfection with PKC-β siRNA induced a rapid barrier enhancement in CMECs monolayer as detected by increased transendothelial electrical resistance (TER) and decreased FITC-dextran clearance (all P < 0.05 vs. no-treatment group). Meanwhile, RBX pretreatment and transfection with PKC-β siRNA significantly decreased TUNEL positive CMECs and phosphor-LIMK2 expression in cultured CMECs (all P < 0.05 vs. no-treatment group). RBX pretreatment reduced F-actin/G-actin in cultured CMECs, reproducing the same effect as PKC-β siRNA. These data indicate that PKC-β inhibitor (RBX) may be helpful in attenuating the risk of severe cardiac microvascular I/R injury in diabetic rats partly due to its maintenance of endothelial barrier function and anti-apoptotic effect. [ABSTRACT FROM AUTHOR]

Our previous work revealed the protective effect of Qiliqiangxin (QLQX) on cardiac microvascular endothelial cells (CMECs), but the underlying mechanisms remain unclear. We aimed to investigate whether QLQX exerts its protective effect against high-concentration angiotensin II (Ang II)-induced CMEC apoptosis through the autophagy machinery. CMECs were cultured in high-concentration Ang II (1 μM) medium in the presence or absence of QLQX for 48 h. We found that QLQX obviously inhibited Ang II-triggered autophagosome synthesis and apoptosis in cultured CMECs. QLQX-mediated protection against Ang II-induced CMEC apoptosis was reversed by the autophagy activator rapamycin. Specifically, deletion of ATG7 in cultured CMECs indicated a detrimental role of autophagy in Ang II-induced CMEC apoptosis. QLQX reversed Ang II-mediated ErbB2 phosphorylation impairment. Furthermore, inhibition of ErbB2 phosphorylation with lapatinib in CMECs revealed that QLQX-induced downregulation of Ang II-activated autophagy and apoptosis was ErbB2 phosphorylation-dependent via the AKT-FoxO3a axis. Activation of ErbB2 phosphorylation by Neuregulin-1β achieved a similar CMEC-protective effect as QLQX in high-concentration Ang II medium, and this effect was also abolished by autophagy activation. These results show that the CMEC-protective effect of QLQX under high-concentration Ang II conditions could be partly attributable to QLQX-mediated ErbB2 phosphorylation-dependent downregulation of autophagy via the AKT-FoxO3a axis. [Display omitted] • Ang II-triggered overactivated autophagy is inhibited by QLQX, resulting in the alleviation of CMECs apoptosis. • QLQX alleviates Ang II-induced CMECs apoptosis via the AKT-FoxO3a axis in an ErbB2 phosphorylation-dependent manner. • QLQX treatment restores the Ang II-impaired AKT-FoxO3a axis activity by enhancing ErbB2 phosphorylation activation. [ABSTRACT FROM AUTHOR]

[Display omitted] Cardiac microvascular endothelial cells (CMECs), derived from coronary circulation microvessel, are the main barrier for the exchange of energy and nutrients between myocardium and blood. However, microvascular I/R injury is a severely neglected topic, and few strategies can reverse this pathology. In this study, we investigated the mechanism of shear stress in microvascular I/R injury, and try to elucidate the downstream signaling pathways that inhibit CMECs apoptosis to reduce I/R injury. Our results demonstrated that shear stress inhibited the apoptosis protein, increased PECAM-1 expression and eNOS phosphorylation in hypoxia reoxygenated (H/R) CMECs. The mechanism of shear stress was related to up-regulated expression of YAP, the increased number of YAP entering the nucleus by dephosphorylation, the reduced number of TUNEL positive cells, increased miR-206 and inhibited protein level of PDCD4 in CMECs. However, siRNA-mediated knockdown of YAP abolished the protective effects of shear stress on CMECs apoptosis, similar results obtained from administration with AMO-miR-206, and also prevented PDCD4 (target gene of miR-206) increasing when treatment with both AMO-miR-206 and mimics-miR-206. In vivo, restoring the blood fluid with nitroglycerin (NTG) to mimic in vitro shear stress levels, which subsequently improved cardiac function, reduced infarcted area, lowered microvascular perfusion defects. Functional investigations clearly illustrated that increased the protein expression of PECAM-1 and eNOS phosphorylation, activated YAP, strengthened miR-206 expression, and suppressed PDCD4 expression. In summary, this study confirmed that shear stress reversed CMECs apoptosis, relieved microvascular I/R injury, the mechanism of which involving through YAP/miR-206/PDCD4 signaling pathway to finally suppress myocardial I/R injury. [ABSTRACT FROM AUTHOR]

Cardiac microvascular damage, which is often caused by anoxia and hypoglycemia, is associated with the development of cardiac injury. DJ-1 encodes a peptidase C56 protein family related protein, is has been linked to oxidative stress in various cells such as neurons, COPD epithelial cells, and macrophages. However, the effect of DJ-1 towards oxidative stress caused by anoxia and hypoglycemia of cardiac microvascular endothelial cells (CMEC) remains unclear. In this study, we investigated the role and underlying molecular mechanism of DJ-1 in CMEC with anoxia/hypoglycemic (A/H) injury. We found that the mRNA and the protein expression of DJ-1 in CMEC with A/H injury were significantly downregulated. DJ-1 overexpression by pcDNA.3.1-DJ-1 transfection elevated cell viability while it inhibited LDH leakage, cell apoptosis, caspase-3 activity, ROS level, and MDA contents, while knockdown of DJ-1 has the opposite results. In addition, tube formation was increased in DJ-1 overexpression, while it was decreased in DJ-1 knockdown CMEC with A/H injury. In addition, our results indicated that DJ-1 can regulate glutathione (GSH) levels by modulating AKT activity in CMEC with A/H injury. The downregulation of AKT and GSH may remove the protective role of DJ-1 against A/H injury in CMEC. Taken together, this study showed that DJ-1 upregulation protected CMEC against A/H injury via the AKT/GSH signaling pathway. • DJ-1 was decreased in CMEC with A/H injury. • DJ-1 overexpression neutralized A/H injury in CMEC. • DJ-1 overexpression depressed CMEC apoptosis caused by A/H injury. • DJ-1 induction constrained oxidative stress caused by A/H injury in CMEC. • DJ-1 could protect CMEC from A/H injury by regulating AKT/GSH. [ABSTRACT FROM AUTHOR]