Cardiac Microvascular Endothelial Cells Research Articles

Integrin subunit alpha 5 maintains mitochondrial function in ox-LDL-induced cardiac microvascular endothelial cells via activating the PI3K/AKT signaling pathway.

Cardiac microvascular endothelial cells (CMECs) assume a pivotal role in the regulation of blood flow, and their impairment precipitates a spectrum of pathological transformations. Our previous study unveiled a notable mitigation of CMECs dysfunction through the intervention of integrin subunit alpha 5 (ITGA5), a member of the integrin protein family. This study delves into the effect of ITGA5 on the mitochondrial function in CMECs and reveals the regulation pathway. CMECs were stimulated with oxidized low-density lipoprotein (ox-LDL) to mimic coronary artery disease (CAD). The effects of ITGA5 on diverse facets of CMEC behavior, encompassing viability, apoptosis, angiogenesis, oxidative stress, and mitochondrial function, was systematically ascertained. Employing the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway as a focal point of investigation, the mediation of this pathway was substantiated utilizing the PI3K inhibitor LY294002. ITGA5 overexpression exerted a mitigating influence upon the ox-LDL-induced detriment to CMECs, manifested as increased viability, angiogenesis, mitochondrial function, and diminished apoptosis and oxidative stress. The counteraction of these salubrious effects by the administration of the PI3K inhibitor attests to the engagement of the PI3K/AKT signaling pathway. Overall, this study has discerned that ITGA5 activates the PI3k/Akt signaling pathway to orchestrate mitochondrial function and diminish ox-LDL-induced CMEC dysfunction. Thus, the targeted amelioration of this cellular injury emerges as a strategically pivotal endeavor for the prevention and amelioration of this ailment.

MAP4K4 exacerbates cardiac microvascular injury in diabetes by facilitating S-nitrosylation modification of Drp1

Dynamin-related protein 1 (Drp1) is a crucial regulator of mitochondrial dynamics, the overactivation of which can lead to cardiovascular disease. Multiple distinct posttranscriptional modifications of Drp1 have been reported, among which S-nitrosylation was recently introduced. However, the detailed regulatory mechanism of S-nitrosylation of Drp1 (SNO-Drp1) in cardiac microvascular dysfunction in diabetes remains elusive. The present study revealed that mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) was consistently upregulated in diabetic cardiomyopathy (DCM) and promoted SNO-Drp1 in cardiac microvascular endothelial cells (CMECs), which in turn led to mitochondrial dysfunction and cardiac microvascular disorder. Further studies confirmed that MAP4K4 promoted SNO-Drp1 at human C644 (mouse C650) by inhibiting glutathione peroxidase 4 (GPX4) expression, through which MAP4K4 stimulated endothelial ferroptosis in diabetes. In contrast, inhibition of MAP4K4 via DMX-5804 significantly reduced endothelial ferroptosis, alleviated cardiac microvascular dysfunction and improved cardiac dysfunction in db/db mice by reducing SNO-Drp1. In parallel, the C650A mutation in mice abolished SNO-Drp1 and the role of Drp1 in promoting cardiac microvascular disorder and cardiac dysfunction. In conclusion, our findings demonstrate that MAP4K4 plays an important role in endothelial dysfunction in DCM and reveal that SNO-Drp1 and ferroptosis activation may act as downstream targets, representing potential therapeutic targets for DCM.

Poricoic acid A promotes angiogenesis and myocardial regeneration by inducing autophagy in myocardial infarction

Myocardial infarction (MI) is a kind of cardiovascular diseases with high morbidity and mortality. Poricoic acid A (PAA) is the main active substance in Poria cocos, which has been discovered to exhibit an ameliorative role in the progression of many diseases. However, no report has been focused on the regulatory effects of PAA on MI progression. In this study, at first, oxygen glucose deprivation (OGD) treatment was performed in human cardiac microvascular endothelial cells (HCMECs) to mimic MI cell model. Our findings demonstrated that cell proliferation was reduced post OGD treatment, but which was reversed by PAA treatment. Moreover, PAA suppressed cell apoptosis in OGD-triggered HCMEC cells. Next, it revealed that PAA induced autophagy in OGD-treated HCMEC cells through enhancing LC3-II/LC3-I level and reducing P62 level. In addition, PAA strengthened the angiogenesis ability and migration ability in OGD-induced HCMEC cells. Lastly, it was uncovered that PAA modulated the AMPK/mTOR signaling pathway through affecting the p-mTOR/mTOR and p-AMPK/AMPK levels. In conclusion, PAA can promote angiogenesis and myocardial regeneration after MI by inducing autophagy through modulating the AMPK/mTOR pathway. This work suggested that PAA may be a potential and useful drug for MI treatment.

Salvianic acid A sodium facilitates cardiac microvascular endothelial cell proliferation by enhancing the hypoxia-inducible factor-1 alpha/vascular endothelial growth factor signalling pathway post-myocardial infarction.

Cardiac microvascular endothelial cells (CMECs) are important cells surrounding the cardiomyocytes in the heart that maintain microenvironment homeostasis. Salvianic acid A sodium (SAAS) has been reported to prevent myocardial infarction (MI) injury. However, the role of SAAS on CMEC proliferation remains unclear. CEMCs exposed to oxygen glucose deprivation (OGD) were used to explore the angiogenic abilities of SAAS. In vivo, C57BL/6 mice were divided into three groups: sham, MI and SAAS + MI groups. Compared to OGD group, SAAS led to a reduction in the apoptotic rate and an increase of the proliferation in vitro. Additionally, SAAS increased the protein levels of Bcl2, HIF-1α and vascular endothelial growth factor (VEGF) with the reduction of Bax. In terms of the specific mechanisms, SAAS might inhibit HIF-1α ubiquitination and enhance the HIF-1α/VEGF signalling pathway to increase CMEC proliferation. Furthermore, SAAS increased the density of vessels, inhibited myocardial fibrosis and improved cardiac dysfunction in vivo. The present study has revealed that SAAS could potentially be used as an active substance to facilitate CMEC proliferation post-MI.

Endothelial identity not found – Beyond passage 38, commercial cardiac microvascular endothelial cells do not express CD31 and VE-cadherin

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.

Role and mechanism of miRNA in cardiac microvascular endothelial cells in cardiovascular diseases.

The occurrence and development of myocardial dysfunction are associated with damage in the cardiac microvascular endothelial cells (CMECs), which can regulate nutrient exchange and oxy-gen-carbon cycling to protect cardiomyocytes. Interventions targeting microRNAs (miRNAs) can effectively mitigate CMEC injury and thus improve cardiovascular diseases. MiRNAs are a class of noncoding single-strand RNA molecules typically 21-23 nucleotides in length that are encoded by endogenous genes. They are critical regulators of organism development, cell differentiation, metabolism, and apoptosis. Current clinical trials on miRNA drugs indicate that patient-specific miRNA levels are now being used as one of the criteria for predicting heart disease. However, the cellular process of various miRNAs in CMECs in cardiovascular diseases has not been fully elucidated. These mechanisms are a field that immediately requires further investigation. Accordingly, this review summarizes the roles and mechanisms of various miRNAs in CMECs in cardiovascular disease and includes the process of CMEC crosstalk between miRNAs and other cell types in the heart. Our study serves as a theoretical basis for the formal introduction of miRNA use into the treatment of cardiovascular diseases in the future.

TLN1 synergizes with ITGA5 to ameliorate cardiac microvascular endothelial cell dysfunction.

The complex process of atherosclerosis is thought to begin with endothelial cell dysfunction, and advanced atherosclerosis is the underlying cause of coronary artery disease (CAD). Uncovering the underlying mechanisms of CAD-related endothelial cell injury may contribute to the treatment. Cardiac microvascular endothelial cells (CMVECs) were treated with oxidised low-density lipoprotein (ox-LDL) to mimic an injury model. The involvement of Talin-1 (TLN1) and integrin alpha 5 (ITGA5) in the proliferation, apoptosis, angiogenesis, inflammatory response, and oxidative stress in CMVECs were assessed. TLN1 overexpression assisted CMVECs in resistance to ox-LDL stimulation, with alleviated cell proliferation and angiogenesis, reduced apoptosis, inflammatory response, and oxidative stress. TLN1 overexpression triggered increased ITGA5, and ITGA5 knockdown reversed the effects of TLN1 overexpression on the abovementioned aspects. Together, TLN1 synergized with ITGA5 to ameliorate the dysfunction in CMVECs. This finding suggests their probable involvement in CAD, and increasing their levels is beneficial to disease relief.

FOXO6 transcription inhibition of CTRP3 promotes OGD/R-triggered cardiac microvascular endothelial barrier disruption via SIRT1/Nrf2 signalling.

C1q/TNF-related protein 3 (CTRP3) has been clarified to display its protective roles in cardiac function. The current study is concentrated on exploring the impacts of CTRP3 on myocardial ischaemia. Oxygen and glucose hypoxia/reoxygenation (OGD/R) model was constructed in human cardiac microvascular endothelial cells (HCMECs). Reverse transcription-quantitative polymerase chain reaction and western blot analysis of CTRP3 expression were conducted. CCK-8 assay was to estimate cell activity and lactate dehydrogenase (LDH) assay kit was to test LDH release. TUNEL assay and western blot were to judge apoptosis. Endothelial barrier function was detected by in vitro vascular permeability assay kit. Zonula occludens-1 (ZO-1) expression was evaluated by immunofluorescence assay. The interaction between CTRP3 promoter and Forkhead Box O6 (FOXO6) was predicted by JASPAR database and verified by chromatin immunoprecipitation and luciferase reporter assays. After OGD/R-induced HCMECs were co-transfected with CTRP3 overexpression and FOXO6 overexpression plasmids, the above functional experiments above were conducted again. Lastly, the expression of sirtuin 1 (SIRT1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signalling-related proteins was examined by western blot. CTRP3 was down-regulated in OGD/R-induced HCMECs. CTRP3 enhanced the viability and barrier integrity while reduced the apoptosis and permeability of OGD/R-insulted HCMECs. This process may be regulated by FOXO6 transcription. Also, FOXO6 inhibition-mediated CTRP3 up-regulation activated the SIRT1/Nrf2 signalling. FOXO6 transcription inhibition of CTRP3 promotes OGD/R-triggered cardiac microvascular endothelial barrier disruption via SIRT1/Nrf2 signalling.

Superoxide dismutase mimetic nanozymes attenuate cardiac microvascular ischemia–reperfusion injury associated with hyperhomocysteinemia

The combination of homocysteine (Hcy) and copper ion (Cu2+) induces cardiac microvascular ischemia–reperfusion injury (IRI) by causing oxidative stress in the reperfusion treatment of ischemic cardiopathy. Therefore, antioxidant elimination of reactive oxygen species (ROS) shows great potential in the treatment of IRI. Herein, cerium vanadate nanorods (CVNRs) were designed and developed as an efficient ROS scavenger for the treatment of myocardial IRI in hyperhomocysteinemic rat (HHcyR) model. Thanks to its outstanding superoxide dismutase (SOD)-like activity, the CVNRs showed a significant antioxidant effect in human cardiac microvascular endothelial cells treated with Hcy and Cu2+ under hypoxia/reperfusion condition and greatly preserved the morphology and function of mitochondria. Further intravenous administration of CVNRs significantly maintained the integrity and perfusion of microvasculature, minimized the microcirculation malfunction, and finally reduced the myocardial infarction and maintained the heart function after IRI. Moreover, the administration of CVNRs in HHcyR exhibited negligible organ toxicity and inflammation response, indicating their excellent biocompatibility and biosafety. Collectively, the SOD-mimicking CVNRs can serve as a promising antioxidant nanozyme to treat cardiac microvascular IRI complicated with hyperhomocysteinemia, providing a new prospect for the development of antioxidant nanozyme in IRI treatment even with other risk factors.

Circulating exosomal mir-16-2-3p is associated with coronary microvascular dysfunction in diabetes through regulating the fatty acid degradation of endothelial cells

BackgroundCoronary microvascular dysfunction (CMD) is a frequent complication of diabetes mellitus (DM) characterized by challenges in both diagnosis and intervention. Circulating levels of microRNAs are increasingly recognized as potential biomarkers for cardiovascular diseases.MethodsSerum exosomes from patients with DM, DM with coronary microvascular dysfunction (DM-CMD) or DM with coronary artery disease (DM-CAD) were extracted for miRNA sequencing. The expression of miR-16-2-3p was assessed in high glucose-treated human aortic endothelial cells and human cardiac microvascular endothelial cells. Fluorescence in situ hybridization (FISH) was used to detect miR-16-2-3p within the myocardium of db/db mice. Intramyocardial injection of lentivirus overexpressing miR-16-2-3p was used to explore the function of the resulting gene in vivo. Bioinformatic analysis and in vitro assays were carried out to explore the downstream function and mechanism of miR-16-2-3p. Wound healing and tube formation assays were used to explore the effect of miR-16-2-3p on endothelial cell function.ResultsmiR-16-2-3p was upregulated in circulating exosomes from DM-CMD, high glucose-treated human cardiac microvascular endothelial cells and the hearts of db/db mice. Cardiac miR-16-2-3p overexpression improved cardiac systolic and diastolic function and coronary microvascular reperfusion. In vitro experiments revealed that miR-16-2-3p could regulate fatty acid degradation in endothelial cells, and ACADM was identified as a potential downstream target. MiR-16-2-3p increased cell migration and tube formation in microvascular endothelial cells.ConclusionsOur findings suggest that circulating miR-16-2-3p may serve as a biomarker for individuals with DM-CMD. Additionally, miR-16-2-3p appears to alleviate coronary microvascular dysfunction in diabetes by modulating ACADM-mediated fatty acid degradation in endothelial cells.

Macrophages promote the transition from myocardial ischemia reperfusion injury to cardiac fibrosis in mice through GMCSF/CCL2/CCR2 and phenotype switching.

Following acute myocardial ischemia reperfusion (MIR), macrophages infiltrate damaged cardiac tissue and alter their polarization phenotype to respond to acute inflammation and chronic fibrotic remodeling. In this study we investigated the role of macrophages in post-ischemic myocardial fibrosis and explored therapeutic targets for myocardial fibrosis. Male mice were subjected to ligation of the left coronary artery for 30 min. We first detected the levels of chemokines in heart tissue that recruited immune cells infiltrating into the heart, and found that granulocyte-macrophage colony-stimulating factor (GMCSF) released by mouse cardiac microvascular endothelial cells (MCMECs) peaked at 6 h after reperfusion, and c-c motif chemokine ligand 2 (CCL2) released by GMCSF-induced macrophages peaked at 24 h after reperfusion. In co-culture of BMDMs with MCMECs, we demonstrated that GMCSF derived from MCMECs stimulated the release of CCL2 by BMDMs and effectively promoted the migration of BMDMs. We also confirmed that GMCSF promoted M1 polarization of macrophages in vitro, while GMCSF neutralizing antibodies (NTABs) blocked CCL2/CCR2 signaling. In MIR mouse heart, we showed that GMCSF activated CCL2/CCR2 signaling to promote NLRP3/caspase-1/IL-1β-mediated and amplified inflammatory damage. Knockdown of CC chemokine receptor 2 gene (CCR2-/-), or administration of specific CCR2 inhibitor RS102895 (5 mg/kg per 12 h, i.p., one day before MIR and continuously until the end of the experiment) effectively reduced the area of myocardial infarction, and down-regulated inflammatory mediators and NLRP3/Caspase-1/IL-1β signaling. Mass cytometry confirmed that M2 macrophages played an important role during fibrosis, while macrophage-depleted mice exhibited significantly reduced transforming growth factor-β (Tgf-β) levels in heart tissue after MIR. In co-culture of macrophages with fibroblasts, treatment with recombinant mouse CCL2 stimulated macrophages to release a large amount of Tgf-β, and promoted the release of Col1α1 by fibroblasts. This effect was diminished in BMDMs from CCR2-/- mice. After knocking out or inhibiting CCR2-gene, the levels of Tgf-β were significantly reduced, as was the level of myocardial fibrosis, and cardiac function was protected. This study confirms that the acute injury to chronic fibrosis transition after MIR in mice is mediated by GMCSF/CCL2/CCR2 signaling in macrophages through NLRP3 inflammatory cascade and the phenotype switching.

Nicorandil alleviates cardiac microvascular ferroptosis in diabetic cardiomyopathy: Role of the mitochondria-localized AMPK-Parkin-ACSL4 signaling pathway

Mitochondria-associated ferroptosis exacerbates cardiac microvascular dysfunction in diabetic cardiomyopathy (DCM). Nicorandil, an ATP-sensitive K+ channel opener, protects against endothelial dysfunction, mitochondrial dysfunction, and DCM; however, its effects on ferroptosis and mitophagy remain unexplored. The present study aimed to assess the beneficial effects of nicorandil against endothelial ferroptosis in DCM and the underlying mechanisms. Cardiac microvascular perfusion was assessed using a lectin perfusion assay, while mitophagy was assessed via mt-Keima transfection and transmission electron microscopy. Ferroptosis was examined using mRNA sequencing, fluorescence staining, and western blotting. The mitochondrial localization of Parkin, ACSL4, and AMPK was determined via immunofluorescence staining. Following long-term diabetes, nicorandil treatment improved cardiac function and remodeling by alleviating cardiac microvascular injuries, as evidenced by the improved microvascular perfusion and structural integrity. mRNA-sequencing and biochemical analyses showed that ferroptosis occurred and Pink1/Parkin-dependent mitophagy was suppressed in cardiac microvascular endothelial cells after diabetes. Nicorandil treatment suppressed mitochondria-associated ferroptosis by promoting the Pink1/Parkin-dependent mitophagy. Moreover, nicorandil treatment increased the phosphorylation level of AMPKα1 and promoted its mitochondrial translocation, which further inhibited the mitochondrial translocation of ACSL4 via mitophagy and ultimately suppressed mitochondria-associated ferroptosis. Importantly, overexpression of mitochondria-localized AMPKα1 (mitoAα1) shared similar benefits with nicorandil on mitophagy, ferroptosis and cardiovascular protection against diabetic injury. In conclusion, the present study demonstrated the therapeutic effects of nicorandil against cardiac microvascular ferroptosis in DCM and revealed that the mitochondria-localized AMPK-Parkin-ACSL4 signaling pathway mediates mitochondria-associated ferroptosis and the development of cardiac microvascular dysfunction.

Irisin links Claudin-5 preservation and Mfn2-mediated mitochondrial dynamics to resist doxorubicin-induced cardiac endothelial damage

Irisin is protective in the cardiac microenvironment and can resist doxorubicin-induced cardiotoxicity. The purpose of this study was to investigate the connection between Irisin, endothelial cell integrity, and mitochondrial dynamics. Primary cardiac microvascular endothelial cells (CMECs) were used to explore the regulatory effects of Irisin on tight junction proteins, mitochondrial dynamics, β-catenin expression, and transcriptional activity. Results showed that Irisin can suppress doxorubicin-induced upregulation of MMP2 and MMP9, thereby reducing the degradation of tight junction proteins (ZO-1 and Claudin-5) and VE-cadherin. The preservation of Claudin-5 contributes to maintaining Mfn2 expression, which in turn supports mitochondrial fusion. Although Irisin restores doxorubicin-induced downregulation of β-catenin, it concurrently limits β-catenin transcriptional activity via Mfn2-mediated sulfenylation. Therefore, this study revealed a novel mechanism linking the protective effects of Irisin on the tight junction proteins and mitochondrial dynamics upon doxorubicin exposure.

MiRNA-146a-5p Inhibits Hypoxia-Induced Myocardial Fibrosis Through EndMT.

Cardiac Vascular disease particularly myocardial infarction (MI) is a threat to health worldwide. microRNAs (miRNAs) have been shown to regulate myocardial fibrosis. Therefore, it is potential to investigate the mechanism of miRNA and fibrosis following myocardial infarction. Hypoxia human cardiac microvascular endothelial cells (HCMECs) were selected for the vitro experimental model. The miR-146a-5p expression was tested via RT-qPCR. The level of endothelial-to-mesenchymal transition (EndMT) and fibrosis markers were detected by Western blotting and immunofluorescence. Then, the inflammation, cell viability and apoptosis were investigated. The target was predicted by an online database and verified by a dual-luciferase activity assay. An MI mouse model was created to validate that miR-146a-5p regulates cardiac fibrosis in vivo. MI mouse was transfected with miR-146a-5p lentivirus. Subsequently, its effect on cardiac fibrosis of infarcted hearts was assessed by In situ hybridization (ISH), Immunohistochemistry (IHC), Triphenylterazolium chloride (TTC) staining and Masson staining. Herein, we confirmed that miR-146a-5p was down-regulated in hypoxia HCMECs. Overexpression of miR-146a-5p inhibited hypoxia-induced cardiac fibrosis following myocardial infarction by inhibiting EndMT in HCMECs. Thioredoxin-interacting protein (TXNIP) was a target that was negatively regulated by miR-146a-5p. Up-regulation of miR-146a-5p inhibited cardiac fibrosis via regulating EndMT by targeting TXNIP, and it also regulated EndMT to inhibit cardiac fibrosis in vivo.

Pinacidil ameliorates cardiac microvascular ischemia-reperfusion injury by inhibiting chaperone-mediated autophagy of calreticulin.

Calcium overload is the key trigger in cardiac microvascular ischemia-reperfusion (I/R) injury, and calreticulin (CRT) is a calcium buffering protein located in the endoplasmic reticulum (ER). Additionally, the role of pinacidil, an antihypertensive drug, in protecting cardiac microcirculation against I/R injury has not been investigated. Hence, this study aimed to explore the benefits of pinacidil on cardiac microvascular I/R injury with a focus on endothelial calcium homeostasis and CRT signaling. Cardiac vascular perfusion and no-reflow area were assessed using FITC-lectin perfusion assay and Thioflavin-S staining. Endothelial calcium homeostasis, CRT-IP3Rs-MCU signaling expression, and apoptosis were assessed by real-time calcium signal reporter GCaMP8, western blotting, and fluorescence staining. Drug affinity-responsive target stability (DARTS) assay was adopted to detect proteins that directly bind to pinacidil. The present study found pinacidil treatment improved capillary density and perfusion, reduced no-reflow and infraction areas, and improved cardiac function and hemodynamics after I/R injury. These benefits were attributed to the ability of pinacidil to alleviate calcium overload and mitochondria-dependent apoptosis in cardiac microvascular endothelial cells (CMECs). Moreover, the DARTS assay showed that pinacidil directly binds to HSP90, through which it inhibits chaperone-mediated autophagy (CMA) degradation of CRT. CRT overexpression inhibited IP3Rs and MCU expression, reduced mitochondrial calcium inflow and mitochondrial injury, and suppressed endothelial apoptosis. Importantly, endothelial-specific overexpression of CRT shared similar benefits with pinacidil on cardiovascular protection against I/R injury. In conclusion, our data indicate that pinacidil attenuated microvascular I/R injury potentially through improving CRT degradation and endothelial calcium overload.

Cardiac endothelial ischemia/reperfusion injury-derived protein damage-associated molecular patterns disrupt the integrity of the endothelial barrier

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.

DNA-PKcs Phosphorylates Cofilin2 to Induce Endothelial Dysfunction and Microcirculatory Disorder in Endotoxemic Cardiomyopathy

The presence of endotoxemia is strongly linked to the development of endothelial dysfunction and disruption of myocardial microvascular reactivity. These factors play a crucial role in the progression of endotoxemic cardiomyopathy. Sepsis-related multiorgan damage involves the participation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). However, whether DNA-PKcs contributes to endothelial dysfunction and myocardial microvascular dysfunction during endotoxemia remains unclear. Hence, we conducted experiments in mice subjected to lipopolysaccharide (LPS)-induced endotoxemic cardiomyopathy, as well as assays in primary mouse cardiac microvascular endothelial cells. Results showed that endothelial-cell-specific DNA-PKcs ablation markedly attenuated DNA damage, sustained microvessel perfusion, improved endothelial barrier function, inhibited capillary inflammation, restored endothelium-dependent vasodilation, and improved heart function under endotoxemic conditions. Furthermore, we show that upon LPS stress, DNA-PKcs recognizes a TQ motif in cofilin2 and consequently induces its phosphorylation at Thr 25 . Phosphorylated cofilin2 shows increased affinity for F-actin and promotes F-actin depolymerization, resulting into disruption of the endothelial barrier integrity, microvascular inflammation, and defective eNOS-dependent vasodilation. Accordingly, cofilin2-knockin mice expressing a phospho-defective (T25A) cofilin2 mutant protein showed improved endothelial integrity and myocardial microvascular function upon induction of endotoxemic cardiomyopathy. These findings highlight a novel mechanism whereby DNA-PKcs mediates cofilin2 Thr25 phosphorylation and subsequent F-actin depolymerization to contribute to endotoxemia-related cardiac microvascular dysfunction.

Retracted: Exosomal miR-27b-3p Derived from Hypoxic Cardiac Microvascular Endothelial Cells Alleviates Rat Myocardial Ischemia/Reperfusion Injury through Inhibiting Oxidative Stress-Induced Pyroptosis via Foxo1/GSDMD Signaling.

[This retracts the article DOI: 10.1155/2022/8215842.].

Identifying pro-angiogenic lncRNAs to regenerate the damaged myocardium following myocardial infarction

Abstract Cardiovascular diseases are the most common cause of death worldwide and Myocardial Infarction (MI) is the primary symptom of cardiovascular disease. In MI, Damage-Associated Molecular Patterns (DAMPs) released by dying cells play an important role in driving the inflammatory response following myocardial damage with potentially adverse or pro-regenerative outcomes. Since angiogenesis is a key process to ameliorate the sequelae of MI and the effect of DAMPs on cardiac cells are still widely unknown, hereby we present a novel model to study how DAMPs affect the gene expression of human Cardiac Microvascular Endothelial Cells (CMECs), one of the most abundant cell types in the heart, aiming to identify lncRNA targets to induce regenerative angiogenesis in patients with MI. To generate a cocktail of DAMPs, cells were isolated from the heart tissue of patients undergoing septal myectomy via collagenase treatment and exposed to repeated cycles of freeze-thawing to induce cell rupture. The release of endogenous molecules (DAMPs), such as DNA, was confirmed by nano drop. CMECs were then stimulated with DAMPs for 24h and RNA sequencing was performed. Finally, to predict potential co-regulation between lncRNAs and angiogenic proteins, a combination of PLAIDOH and NoRCE were used. The RNA sequencing revealed a total of 1294 Differently Expressed Genes (DEGs) in CMECs stimulated with DAMPs, of which 218 were lncRNAs annotated in GENCODE v42. Among the top 50 DEGs were found proteins involved in cell growth, proliferation, and immune response such as FGF5, CEP128, and IL32 and the GSE analysis revealed that proliferation (mitosis), migration, and angiogenesis were among the most enriched terms. The analysis with PLAIDOH and NoRCe revealed ∼25 differentially expressed lncRNAs which correlate with proteins involved in cell cycle and mitotic entry (Pearson 0.6-0.99 and p-value < 0.05). Among these were found HIF1A-AS3, TGFB-OT1, TGFB-AS1. Following the in-silico analysis, results were confirmed by qPCR. qPCR for HIFA1A-AS3 showed a significant reduction of 40% when CMECs were stimulated with DAMPs (p-value: 0.04) confirming the sequencing results. Whether HIFA1A-AS3 is involved in cell proliferation will now be validated with KO experiments. In conclusion, the administration of DAMPs to CMECs for 24h induced genes involved in proliferation, migration, and angiogenesis, processes that are also highly regulated in MI. By combining PLAIDOH and NoRCE we were able to identify lncRNAs which correlate in expression with protein-coding genes involved in these processes indicating co-regulation. These experiments recapitulate the main endothelial cell processes found in MI via administration of DAMPs and enable us to identify lncRNA targets to promote regenerative angiogenesis. Hence, this brings us one step closer to identifying gene therapy targets capable of inducing regenerative angiogenesis in patients with MI.

Novel endothelial-enriched long non-coding RNA AL590004.3 (IRENE) is dysregulated in HFpEF and regulates endothelial enhancement of cardiomyocyte function

Abstract Background Heart failure with preserved ejection fraction (HFpEF) is the most prevalent form of HF to date. However, there is a lack of medication which stages HFpEF as an unmet clinical need. HFpEF is characterized by cardiac microvascular dysfunction, promoted by co-morbidities-associated low-grade pro-inflammatory state. We have shown that cardiac endothelial cells regulate cardiomyocyte relaxation, which is impaired when endothelial cells are inflamed. Long non-coding RNAs (lncRNAs) have been implicated in various cardiovascular diseases. However, their role in HFpEF is unknown. Here, we discovered a novel lncRNA AL590004.3 which is significantly reduced in the heart of patients with HFpEF. We hypothesized that AL590004.3 is important for cardiac endothelial function, and dysregulation of endothelial AL590004.3 impairs cardiomyocyte relaxation. Methods and results We demonstrated that AL590004.3 is significantly downregulated (∼7 fold, p<0.001) in HFpEF as assessed by in silico analysis of RNA sequencing of HFpEF patient cardiac biopsies. To assess the tissue distribution, we performed RNA sequencing comparing cells from various tissues and found the highest expression of AL590004.3 in the cardiac microvascular endothelial cells (CMECs), indicating its importance in cardiac endothelial cell biology. The expression of AL590004.3 was reduced when CMECs were exposed to pro-inflammatory cytokine TNFα and IL1β, indicating the detrimental effect of inflammation, as displayed by patients with HFpEF. Further, replicative senescent of CMECs led to reduced AL590004.3 expression (2 fold, p<0.005), showing the deleterious contribution of ageing, similar to what occurs in HFpEF patients. We showed that AL590004.3 is mostly expressed in the chromatin fraction (∼90%) of CMECs, suggesting its function in chromatin modification. To assess the function of AL590004.3, we performed loss-of-function. Using endothelial-cardiomyocyte co-culture set-up, we showed silencing of AL590004.3 in CMECs impaired endothelial function to enhance cardiomyocyte relaxation (∼1.5 fold, p<0.01). To dissect the molecular mechanism, we performed RNA sequencing of CMECs devoid of AL590004.3 and revealed four protein-coding genes (PXDC1, FAM50, ECI2, PRPF4B) that are differentially co-regulated, indicating their potential as protein-binding partners, which warrant further investigation. Conclusion We showed that AL590004.3 is a cardiac endothelial cell-specific lncRNA that is significantly downregulated in cardiac biopsy from HFpEF patients. AL590004.3 is important for cardiac endothelial cell function as loss-of-function in CMECs impaired endothelial enhancement of cardiomyocyte relaxation. Herewith, we name this lncRNA Important for the Regulation of ENdothelial function in HFpEF (IRENE).