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Few immortalized cardiac microvascular endothelial cell (CMEC) lines are available, particularly mouse lines. We purchased the CLU510 mCMEC line (Cedarlane), isolated by fluorescence-activated cell sorting for CD31 and VE-cadherin. The cell line has been used in previous studies, although none report CD31 or VE-cadherin expression. We analyzed endothelial profile of two vials of passage 38 cells. CD31 and VE-cadherin mRNA were hardly expressed in mCMECs compared to primary mouse lung ECs. CD31 and VE-cadherin protein levels were also negligible compared to multiple EC lines. Thus, CLU510 mCMECs beyond P38 do not harbor an endothelial phenotype. Caution should be warranted when using commercial cells and journals should carefully consider the validity of results when essential characterization of cell lines is omitted.

Human cardiac microvascular endothelial cells (HCMECs) are sensitive to ischemia and vulnerable to damage during reperfusion. The release of damage-associated molecular patterns (DAMPs) during reperfusion induces additional tissue damage. The current study aimed to identify early protein DAMPs in human cardiac microvascular endothelial cells subjected to ischemia-reperfusion injury (IRI) using a proteomic approach and their effect on endothelial cell injury. HCMECs were subjected to 60 min of simulated ischemia and 6 h of reperfusion, which can cause lethal damage. DAMPs in the culture media were subjected to liquid chromatography-tandem mass spectrometry proteomic analysis. The cells were treated with endothelial IRI-derived DAMP medium for 24 h. Endothelial injury was assessed by measuring lactate dehydrogenase activity, morphological features, and the expression of endothelial cadherin, nitric oxide synthase (eNOS), and caveolin-1. The top two upregulated proteins, DNAJ homolog subfamily B member 11 and pyrroline-5-carboxylate reductase 2, are promising and sensitive predictors of cardiac microvascular endothelial damage. HCMECs expose to endothelial IRI-derived DAMP, the lactate dehydrogenase activity was significantly increased compared with the control group (10.15 ± 1.03 vs 17.67 ± 1.19, respectively). Following treatment with endothelial IRI-derived DAMPs, actin-filament dysregulation, and downregulation of vascular endothelial cadherin, caveolin-1, and eNOS expressions were observed, along with cell death. In conclusion, the early protein DAMPs released during cardiac microvascular endothelial IRI could serve as novel candidate biomarkers for acute myocardial IRI. Distinct features of impaired plasma membrane integrity can help identify therapeutic targets to mitigate the detrimental consequences mediated of endothelial IRI-derived DAMPs.

Hyperhomocysteinemia (HHcy) is associated with the development and progression of chronic cardiovascular diseases through the deleterious effects of high levels of homocysteine (Hcy) on the cardiovascular system. However, the exact mechanism of action of Hcy on the acute injury of the cardiovascular system following ischemia/reperfusion (I/R) remains unclear. The present study demonstrated that copper mobilization occurs during cardiac I/R, and the interactive toxic effect of Hcy and mobile Cu 2+ during cardiac I/R induces necroptosis of cardiac microvascular endothelial cells (CMECs) and thus enhances cardiac dysfunction. In the present study, we utilized three cardiac I/R model: isolated rat heart, in vivo model as well as cell culture, and demonstrated that copper mobilization occurs during cardiac I/R, and the interactive toxic effect of Hcy and mobile Cu 2+ during cardiac I/R induces necroptosis of cardiac microvascular endothelial cells (CMECs) and thus enhances cardiac dysfunction. Furthermore, we proved that the Cu 2+ chelator TTM significantly mitigated the deleterious effects of Hcy and Cu 2+ on CMECs and cardiac function both in vitro and in vivo. Mechanismly, the combinative effect of Hcy and Cu 2+ are associated with the production of reactive oxygen species (ROS) and nitric oxide (NO) by NADPH oxidase (NOX) and endothelial nitric oxide synthase (eNOS), respectively. Subsequently, the overproduction of toxic peroxynitrite (ONOO - ) induces CMECs necroptosis. The application of ROS scavengers in CMECs resulted in a notable reduction in necroptosis mediated by Hcy and Cu 2+ under hypoxia/reperfusion (H/R) condition. These findings indicate that the mechanism by which Hcy and Cu 2+ enhances cardiac dysfunction under I/R condition may be attributed to the stimulation of both NOX and eNOS activity, resulting in the generation of excessive ONOO - and subsequent necroptosis of CMECs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Microvascular dysfunction caused by cardiac ischemia-reperfusion (I/R) leads to multiple severe cardiac adverse events, such as heart failure and ventricular modeling, which plays a critical role in outcomes. Though marrow mesenchymal stem cell (MSC) therapy has been proven effective for attenuating I/R injury, the limitations of clinical feasibility cannot be ignored. Since exosomes are recognized as the main vehicles for MSCs paracrine effects, we assumed that MSC-derived exosomes could prevent microvascular dysfunction and further protect cardiac function. By establishing a rat cardiac I/R model in vivo and a cardiac microvascular endothelial cells (CMECs) hypoxia-reperfusion (H/R) model in vitro, we demonstrated that MSC-derived exosomes enhanced microvascular regeneration under stress, inhibited fibrosis development, and eventually improved cardiac function through platelet-derived growth factor receptor-β (PDGFR-β) modulation. Furthermore, we found that MSC-derived exosomes possessed better therapeutic effects than MSCs themselves., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: The aim of this study is to investigate local cardiac and circulating AVP secretion during heart failure and to determine whether AVP mediates ventricular remodeling., Methods: We assessed cardiac function and AVP levels of post-myocardial infarction (MI) heart-failure rats 3 weeks (n = 10), 4 weeks (n = 10), 6 weeks (n = 10), 9 weeks (n = 15) after the proximal left anterior descending coronary artery (LAD) ligation. Ten sham-operated rats were used as the control group. In vitro, cardiac microvascular endothelial cells (CMECs) were initiated from isolated Wistar rat hearts and subjected to Ang II to induce AVP expression and secretion. Besides, the effects of AVP stimulation on CMECs and cardiac fibroblasts (CFs) were studied using methylthiazol tetrazolium assay, Western blotting and real-time PCR., Results: With cardiac dysfunction, plasma and local cardiac AVP, aldosterone levels increased over time, peaking at 9 weeks post-MI. AVP levels were negatively correlated with serum Na(+) and LVEF but positively correlated with LVEDD and myocardial hydroxyproline. In CMECs treated with Ang II, AVP mRNA and protein expression increased. In addition, AVP promoted CFs proliferation and up-regulated the expression of endothelin-1 and connective tissue growth factor., Conclusion: CMECs are the cellular sources of elevated local heart AVP stimulated with Ang II/AT1. An intrinsic cardiac AVP system exists. Local cardiac and circulating AVP secretion were enhanced by deteriorating cardiac function. AVP may promote ventricular remodeling. Thus, AVP could be an important mediator of myocardial fibrosis in late-stage heart failure., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

FoxO3a, a member of the forkhead transcription factors, has been demonstrated to be involved in myocardial ischemia/reperfusion (I/R) injury. Cardiac microvascular endothelial cells (CMECs) are some of the predominant cells damaged immediately after myocardial I/R injury. Despite the importance of injured CMECs in an ischemic heart, little is known about the involvement of FoxO3a in regulating CMECs injury. Thus, we used rat CMECs following simulated I/R to examine FoxO3a activation and signaling in relation to survival, the cell cycle and apoptosis in CMECs. We found that Akt negatively regulates activation of the FoxO3a pathway by phosphorylating FoxO3a in CMECs as demonstrated with an Akt inhibitor and activator. Upon I/R injury, the FoxO3a pathway was significantly activated in CMECs, which was accompanied by Akt deactivation. In parallel, the I/R of CMECs induced G1-phase arrest through p27(Kip1) up-regulation and significant activation of caspase-3. Accordingly, inhibition of the FoxO3a pathway by IGF-1, an Akt activator, could significantly block the I/R-enhanced activation of p27(Kip1) and caspase-3 in CMECs. Collectively, our results indicate that the FoxO3a pathway is involved in the I/R injury of CMECs at least in part through the regulation of cell cycle arrest and apoptosis, suggesting that the FoxO3a pathway may be a novel therapeutic target that protects against microvascular endothelial damage in ischemic hearts., (© 2013.)