Coronary Endothelial Injury Research Articles

A novel format of TNF-α binding affibody molecule ameliorate coronary artery endothelial injury in a mouse model of Kawasaki disease

Kawasaki disease (KD) is a disease characterized by systemic immune vasculitis that often involves coronary arteries and can result in long-term cardiovascular sequelae. Different strategies for treatment of KD-and KD-induced coronary artery lesions are currently under investigation, including passive immunization with anti-TNFα monoclonal antibodies (mAbs). Herein, we examine the potential therapeutic capabilities of a novel type of TNFα-targeting agent based on an affibody molecule possessing fundamentally different properties than mAbs. Using phage display technology, we successfully screened and obtained three TNF-α binding affibody molecules and confirmed their high binding affinity and specificity for recombinant and native TNF-α by surface plasmon resonance (SPR), confocal double immunofluorescence and coimmunoprecipitation assays. Moreover, by binding to TNF-α, the affibody molecules could effectively neutralize TNFα-induced L929 cytotoxicity. To increase the targeting properties and serum half-life, one preferred affibody molecule ZTNF-α263 was redesigned to assemble drugs with bivalent TNFα binding with added specificity for serum albumin (ZTNF-α263-ABD035-ZTNF-α263, hereinafter denoted ZTAT). We further determined its binding ability, TNF-α signal blocking and neutralizing capacity, serum half-life and immunogenicity. Most importantly, our study provides strong evidence that the engineered ZTAT protein was therapeutically effective against KD induced-endothelial injury, as judged by both in vitro and in vivo assessments. These data suggested that because of the flexibility inherent, low-molecular weight anti-TNFα affibody construct ZTAT, can be developed into a potent therapeutic agent that can be produced and purified cost-effectively.

Protective Effect and Mechanism of miR-328-3p on Coronary Artery Endothelial Cell Injury Induced by Oxidized Low-density Lipoprotein

To investigate the protective effect of miR-328-3p on oxidized low-density lipoprotein (ox-LDL)-induced coronary artery endothelial cell injury and the potentially relevant mechanisms. Human coronary artery endothelial cells (HCAECs) were induced with ox-LDL, and the cells were divided into a control group consisting of normal cells, an ox-LDL group receiving ox-LDL treatment, an ox-LDL+miR-NC group transfected with miR-NC and treated with ox-LDL, an ox-LDL+miR-328-3p group transfected with miR-328-3p and treated with ox-LDL, and ox-LDL+miR-328-3p+pcDNA group co-transfected miR-328-3p and pcDNA and treated with ox-LDL, and an ox-LDL+miR-328-3p+insulin-like growth factor 2 (IGF2) group co-transfected miR-328-3p and IGF2 and treated with ox-LDL. The expression level of miR-328-3p was determined with RT-qPCR. Cell proliferation was determined by MTT. Cell apoptosis was measured by flow cytometry. Western blot was conducted to examine the protein expression levels of cleaved cas-3 and IGF2. ELISA was performed to determine the levels of tumor necrosis factor α (TNF-α), interleukin (IL)-6, and IL-1β. Dual luciferase reporter experiment was performed to verify the targeting relationship between miR-328-3p and IGF2. Compared with those of the control group, miR-328-3p expression level and cell activity were significantly reduced in the ox-LDL group (P<0.05), while the apoptotic rate, the protein expression levels of cleaved cas-3, IGF2, Bax, and Bcl-2, and the levels of TNF-α, IL-6, and IL-1β were significantly increased (P<0.05). Compared with those of the ox-LDL+miR-NC group, miR-328-3p expression level and cell activity significantly increased in the ox-LDL+miR-328-3p group (P<0.05), while the apoptosis rate, the protein expression levels of cleaved cas-3 and IGF2, and the levels of TNF-α, IL-6, and IL-1β were significantly reduced. IGF2 was a functional target of miR-328-3p. Compared with those of the ox-LDL+miR-328-3p+pcDNA co-transfection group, the IGF2 protein level was significantly increased (P<0.05) and cell activity was significantly decreased (P<0.05) in the ox-LDL+miR-328-3p+IGF2 co-transfection group, while the apoptosis rate, cleaved cas-3 protein level, and the levels of TNF-α, IL-6, and IL-1β were significantly elevated (P<0.05). miR-328-3p inhibits ox-LDL-induced apoptosis and inflammatory in coronary artery endothelial cell injury through targeted negative regulation of IGF2.

Platelet-derived exosomes regulate endothelial cell inflammation and M1 macrophage polarization in coronary artery thrombosis via modulating miR-34a-5p expression

As the important factors in coronary artery thrombosis, endothelial injury and M1 macrophage polarization are closely related to the expression of miR-34a-5p. Exosomes in plasma are mainly derived from platelets and play an important role in thrombosis. Based on these facts, this study was conducted to investigate the acting mechanism of platelet-derived exosomes (PLT-exo) in the effects of endothelial injury and M1 macrophage polarization on coronary artery thrombosis. Firstly, rats were divided into the sham-operated group and the coronary microembolization (CME) group, and their plasma-derived exosomes were extracted to detect the expression of miR-34a-5p. Next, the PLT-exo were extracted from healthy volunteers and then co-cultured with ox-LDL-induced endothelial cells and LPS-induced macrophages, respectively. Subsequently, the expression of IL-1β, IL-6, TNF-α, and ICAM-1 in endothelial cells was measured, and the level of markers related to M1 macrophage polarization and Sirt1/NF-κB pathway was detected. Finally, the above indicators were examined again after PLT-exo combined with miR-34a-5p mimic were co-cultured with endothelial cells and macrophages, respectively. The results demonstrated that the expression of miR-34a-5p in the CME group was up-regulated compared with the sham-operated group. In cell experiments, PLT-exo modulated the Sirt1/NF-κB pathway by inhibiting the expression of intracellular miR-34a-5p and down-regulated the expression of IL-1β, IL-6, TNF-α, and ICAM-1 in endothelial cells and M1 macrophage polarization. After the transfection with miR-34a-5p mimic, endothelial cell inflammatory injury and M1 macrophage polarization increased to varying degrees. In conclusion, PLT-exo can alleviate coronary artery thrombosis by reducing endothelial cell inflammation and M1 macrophage polarization via inhibiting miR-34a-5p expression. In contrast, miR-34a-5p overexpression in PLT-exo may exacerbate these pathological injuries in coronary artery thrombosis.

Methylglyoxal induces endothelial cell apoptosis and coronary microvascular dysfunction through regulating AR-cPLA2 signaling

ObjectiveSince diabetic patients with coronary microvascular dysfunction (CMD) exhibit high cardiac mortality and women have higher prevalence of non-obstructive coronary artery disease than men, we tried to expand the limited understanding about the etiology and the sex difference of diabetic CMD. Approach and resultsAccumulated methylglyoxal (MGO) due to diabetes promotes vascular damage and it was used for mimicking diabetic status. Flow cytometry analysis and isometric tension measurement were performed to evaluate coronary artery endothelial injury. MGO induced apoptosis of coronary endothelial cells, accompanied by downregulation of androgen receptor (AR). Lentivirus-mediated stable expression of AR in coronary endothelial cells increased anti-apoptotic Bcl-2 expression and attenuated MGO-induced cell apoptosis. cPLA2 activation was the downstream of AR downregulation by MGO treatment. Moreover, MGO also activated cPLA2 rapidly to impair endothelium-dependent vasodilation of coronary arteries from mice. Reactive oxygen species (ROS) overproduction was demonstrated to account for MGO-mediated cPLA2 activation and endothelial dysfunction. Importantly, AR blockade increased endothelial ROS production whereas AR activation protected coronary artery endothelial vasodilatory function from the MGO-induced injury. Although galectin-3 upregulation was confirmed by siRNA knockdown in endothelial cells not to participate in MGO-induced endothelial apoptosis, pharmacological inhibitor of galectin-3 further enhanced MGO-triggered ROS generation and coronary artery endothelial impairment. ConclusionsOur data proposed the AR downregulation-ROS overproduction-cPLA2 activation pathway as one of the mechanisms underlying diabetic CMD and postulated a possible reason for the sex difference of CMD-related angina. Meanwhile, MGO-induced galectin-3 activation played a compensatory role against coronary endothelial dysfunction.

Preventing 5-fluorouracil-induced ischemic events in very high-risk cardiac patients: A retrospective cohort analysis (SQCCCRC protocol).

e24112 Background: 5-Fluorouracil (5-FU), a potent chemotherapy agent for various cancers, is linked to cardiotoxicity, particularly in patients with pre-existing ischemic heart disease. The incidence varies, necessitating effective preventive strategies. Among the several mechanisms responsible for 5-FU-induced myocardial ischemia are coronary vasospasm and endothelial injury. Therefore, prevention of ischemic events in very high-risk patients by controlling modifiable cardiac risk factors and good medical treatment including vasodilators are a potential strategy in preventing 5-FU ischemic events. Methods: To assess the effectiveness and safety of a protocol involving Amlodipine and Isosorbide dinitrate in preventing 5-FU-induced ischemic events in very high-risk cardiac patients with documented ischemic heart disease. Nineteen patients underwent 252 cycles of 5-FU chemotherapy, with 12 patients (181 cycles) following the pre-defined protocol (5-FU Protocol Group) and 7 patients (71 cycles) not adhering (Missed 5-FU Protocol Group). The primary outcome measure was the prevention of 5-FU-induced ischemic events, evidenced by the absence of chest pain, elevated Troponin levels, or ECG changes during 5-FU infusion. Results: The 5-FU Protocol Group demonstrated significant reductions in ischemic events, chest pain with p-values of 0.009 for both outcomes. Additionally, the occurrence of ECG changes post-5FU and an increase in Troponin levels was significantly lower in the 5-FU Protocol Group, with p-values of 0.036 for both parameters. Although not statistically significant, there was a lower 5FU-related mortality rate in the 5-FU Protocol Group compared to the missed 5-FU Protocol Group (0% vs. 14.3%, p = 0.37). The incidence of cancer-related mortality did not exhibit a statistically significant difference between the two groups. To evaluate this, we specifically examined and compared the occurrence of ischemic events in both the 5-FU Protocol Group and the Missed 5-FU Protocol Group. The comparison was conducted using the Fisher exact test. Conclusions: The use of vasodilators may be effective in preventing 5-Fluorouracil-Induced ischemic events in very high-risk cardiac patients with documented coronary artery disease. This approach may help reduce the risk of cardiac events and improve the safety of chemotherapy in this vulnerable patient population. Monitoring cardiotoxicity with maximum tolerated medical treatment and dedicated hospital protocols may be a good prophylaxis approach. Further studies are needed to confirm the safety and efficacy of this approach in a larger patient population, and to assess its impact on long-term cardiac outcomes.

BuyangHuanwu Decoction alleviates Endothelial Cell Apoptosis and Coronary Microvascular Dysfunction via Regulation of the MAPKK4/p38 Signaling Axis.

MAPKK4 has been implicated in the pathological mechanisms underlying myocardial and vascular injury, specifically influencing endothelial cell damage and programmed cell death via subcellular pathways. Nevertheless, the regulatory role of MAPKK4 in coronary microvascular injury following myocardial infarction remains unconfirmed, and the exploration of targeted mitochondrial protective therapeutic agents remains unaddressed. In light of this gap, we established a MAPKK4 gene-modified mouse model of ischemia-reperfusion injury and employed Buyang Huanwu decoction (BYHW), a traditional cardiovascular therapeutic formula, to assess its efficacy in treating coronary microvascular injury post-ischemia-reperfusion. The study aimed to elucidate the mechanism by which BYHW mitigates coronary microvascular injury induced by ischemia-reperfusion through the attenuation of endothelial cell apoptosis. Experimental outcomes revealed that high-dose BYHW significantly ameliorated coronary microvascular injury post-ischemia-reperfusion, restoring the structural integrity of the coronary microvasculature and reducing inflammation and oxidative stress. Contrarily, in transgenic mice overexpressing MAPKK4, BYHW intervention failed to attenuate microvascular inflammation and oxidative stress. To further investigate, we simulated hypoxia/reoxygenation injury in vascular endothelial cells using a MAPKK4-related cellular gene modification model. The results indicated that BYHW attenuates inflammatory damage and enhances the viability of vascular endothelial cells following hypoxic stress, inhibiting apoptosis via the mitochondrial pathway. However, overexpression of MAPKK4/p38 negated the therapeutic effects of BYHW, showing no impact on endothelial cell apoptosis and oxidative stress under hypoxic conditions. Molecular interaction studies confirmed that the active components of BYHW, Astragaloside IV and Ligustrazine, interact with the MAPKK4/P38 axis. In vitro experiments further suggested that the interaction between MAPKK4 and P38 play a crucial role in the ability of BYHW to inhibit apoptosis in coronary microvascular endothelial cells. Therapeutically, MAPKK4 may potentiate the apoptotic pathway in microvascular endothelial cells by modulating downstream P38 expression and phosphorylation, thereby exacerbating ischemia-reperfusion-induced coronary microvascular endothelial injury. From an in vivo perspective, the transgenic overexpression of MAPKK4 and P38 inhibited the microvascular protective effects of BYHW. These findings collectively underscore the significance of the MAPKK4-P38 axis in the protection of coronary microvascular endothelial cells.

Methylation and transcriptomic expression profiles of HUVEC in the oxygen and glucose deprivation model and its clinical implications in AMI patients.

The obstructed coronary artery undergoes a series of pathological changes due to ischemic-hypoxic shocks during acute myocardial infarction (AMI). However, the altered DNA methylation levels in endothelial cells under these conditions and their implication for the etiopathology of AMI have not been investigated in detail. This study aimed to explore the relationship between DNA methylation and pathologically altered gene expression profile in human umbilical vein endothelial cells (HUVECs) subjected to oxygen-glucose deprivation (OGD), and its clinical implications in AMI patients. The Illumina Infinium MethylationEPIC BeadChip assay was used to explore the genome-wide DNA methylation profile using the Novaseq6000 platform for mRNA sequencing in 3 pairs of HUVEC-OGD and control samples. GO and KEGG pathway enrichment analyses, as well as correlation, causal inference test (CIT), and protein-protein interaction (PPI) analyses identified 22 hub genes that were validated by MethylTarget sequencing as well as qRT-PCR. ELISA was used to detect four target molecules associated with the progression of AMI. A total of 2,524 differentially expressed genes (DEGs) and 22,148 differentially methylated positions (DMPs) corresponding to 6,642 differentially methylated genes (DMGs) were screened (|Δβ|>0.1 and detection p < 0.05). After GO, KEGG, correlation, CIT, and PPI analyses, 441 genes were filtered. qRT-PCR confirmed the overexpression of VEGFA, CCL2, TSP-1, SQSTM1, BCL2L11, and TIMP3 genes, and downregulation of MYC, CD44, BDNF, GNAQ, RUNX1, ETS1, NGFR, MME, SEMA6A, GNAI1, IFIT1, and MEIS1. DNA fragments BDNF_1_ (r = 0.931, p < 0.0001) and SQSTM1_2_NEW (r = 0.758, p = 0.0043) were positively correlated with the expressions of corresponding genes, and MYC_1_ (r = -0.8245, p = 0.001) was negatively correlated. Furthermore, ELISA confirmed TNFSF10 and BDNF were elevated in the peripheral blood of AMI patients (p = 0.0284 and p = 0.0142, respectively). Combined sequencing from in vitro cellular assays with clinical samples, aiming to establish the potential causal chain of the causal factor (DNA methylation) - mediator (mRNA)-cell outcome (endothelial cell ischemic-hypoxic injury)-clinical outcome (AMI), our study identified promising OGD-specific genes, which provided a solid basis for screening fundamental diagnostic and prognostic biomarkers of coronary endothelial cell injury of AMI. Moreover, it furnished the first evidence that during ischemia and hypoxia, the expression of BNDF was regulated by DNA methylation in endothelial cells and elevated in peripheral blood.

NFIL3 aggravates human coronary artery endothelial cell injury by promoting ITGAM transcription in Kawasaki disease

ABSTRACTObjectiveHigh expression of nuclear factor interleukin-3 (NFIL3) and integrin Alpha M (ITGAM) was found in serum samples from Kawasaki disease (KD) patients through bioinformatics analysis. Hence, this study aimed to explore the biological functions of NFIL3 and ITGAM in KD serum-stimulated human coronary artery endothelial cells (HCAECs).MethodsThe differentially-expressed genes in KD were analyzed through bioinformatics analysis. Serum samples were obtained from 18 KD patients and 18 healthy volunteers, followed by detection of NFIL3 and ITGAM levels in KD serum. After HCAECs were transfected with sh-NFIL3, sh-ITGAM, or sh-NFIL3 + oe-ITGAM and underwent 24-h KD serum stimulation, cell viability and apoptosis and the levels of inflammation-related factors were measured. The binding between NFIL3 and ITGAM was validated by dual-luciferase and chromatin immunoprecipitation (ChIP) assays.ResultsNFIL3 and ITGAM were up-regulated in serum from KD patients and KD serum-stimulated HCAECs. Down-regulation of NFIL3 or ITGAM inhibited KD serum-induced cell apoptosis and inflammatory response of HCAECs and promoted cell viability. Mechanistically, NFIL3 promoted ITGAM transcription level. Up-regulation of ITGAM reversed the improvement of NFIL3 down-regulation on KD serum-induced HCAEC injury.ConclusionNFIL3 aggravated KD serum-induced HCAEC injury by promoting ITGAM transcription, which provided new insights into the treatment of KD.

The Therapeutic Effects of EFNB2-Fc in a Cell Model of Kawasaki Disease.

The EphrinB2/EphB4 signaling pathway involves the regulation of vascular morphogenesis and angiogenesis. However, little is known about EphrinB2/EphB4 in the pathogenesis of Kawasaki disease (KD) and coronary artery aneurysm formation. Hence, this study aimed to explore the role of EphrinB2/EphB4 and the potential therapeutic effect of EphrinB2-Fc in the coronary arterial endothelial injury of KD. The levels of EphB4 were compared between KD patients and healthy children. Human coronary artery endothelial cells (HCAECs) were stimulated with sera from acute KD patients to establish the KD cell model. The overexpression of EphB4 or treatment with EphrinB2-Fc was found to intervene in the cell model. The cell migration, angiogenesis, and proliferation ability were assessed, and the expression of inflammation-related factors was measured. Our study showed that EphB4 showed low expression in both KD patients and the cell model of KD. The EphB4 protein levels in the CECs of CAA+ KD patients were much lower than those in healthy children. EphrinB2-Fc treatment of KD sera-activated HCAECs suppressed cell proliferation, reduced the expression of inflammation-related factors (such as IL-6 and P-selectin), and elevated cell angiogenesis ability. The results reveal that EphrinB2-Fc has a protective function in endothelial cells and has promising clinical applications for protecting vascular endothelium in patients with KD.

A Vegfc-Emilin2a-Cxcl8a Signaling Axis Required for Zebrafish Cardiac Regeneration.

Ischemic heart disease following the obstruction of coronary vessels leads to the death of cardiac tissue and the formation of a fibrotic scar. In contrast to adult mammals, zebrafish can regenerate their heart after injury, enabling the study of the underlying mechanisms. One of the earliest responses following cardiac injury in adult zebrafish is coronary revascularization. Defects in this process lead to impaired cardiomyocyte repopulation and scarring. Hence, identifying and investigating factors that promote coronary revascularization holds great therapeutic potential. We used wholemount imaging, immunohistochemistry and histology to assess various aspects of zebrafish cardiac regeneration. Deep transcriptomic analysis allowed us to identify targets and potential effectors of Vegfc (vascular endothelial growth factor C) signaling. We used newly generated loss- and gain-of-function genetic tools to investigate the role of Emilin2a (elastin microfibril interfacer 2a) and Cxcl8a (chemokine (C-X-C) motif ligand 8a)-Cxcr1 (chemokine (C-X-C) motif receptor 1) signaling in cardiac regeneration. We first show that regenerating coronary endothelial cells upregulate vegfc upon cardiac injury in adult zebrafish and that Vegfc signaling is required for their proliferation during regeneration. Notably, blocking Vegfc signaling also significantly reduces cardiomyocyte dedifferentiation and proliferation. Using transcriptomic analyses, we identified emilin2a as a target of Vegfc signaling and found that manipulation of emilin2a expression can modulate coronary revascularization as well as cardiomyocyte proliferation. Mechanistically, Emilin2a induces the expression of the chemokine gene cxcl8a in epicardium-derived cells, while cxcr1, the Cxcl8a receptor gene, is expressed in coronary endothelial cells. We further show that Cxcl8a-Cxcr1 signaling is also required for coronary endothelial cell proliferation during cardiac regeneration. These data show that after cardiac injury, coronary endothelial cells upregulate vegfc to promote coronary network reestablishment and cardiac regeneration. Mechanistically, Vegfc signaling upregulates epicardial emilin2a and cxcl8a expression to promote cardiac regeneration. These studies aid in understanding the mechanisms underlying coronary revascularization in zebrafish, with potential therapeutic implications to enhance revascularization and regeneration in injured human hearts.

Abstract 11758: Prevention of Radiation-Induced Coronary Microvascular Leakage by the Restoration of an Endothelial Tight-Junction Protein

Introduction: In cancer survivors exposed to ionizing radiation (IR) exposure to the chest and heart, coronary endothelial injury and disruption of vascular barrier integrity is noted. The mechanisms driving the IR-induced leakiness of the coronary microvasculature are not known. There is no therapy to prevent such injury. Hypothesis: IR exposure downregulates a tight junction protein claudin-1 (cldn1), which can lead to altered endothelial barrier integrity. Ac-SDKP, an endogenous peptide, prevents cldn1 loss and preserves coronary vascular barrier function. Methods: We tested the constitutive and physiological effects of cldn1 both in vitro and in vivo . Human coronary vascular endothelial cells (HMVECs) lacking cldn1 by siRNA silencing were used for loss-of-function studies. Newly developed vascular-specific (VE-cadherin promotor-linked) cldn1 overexpressing transgenic (TG) mice were used for cldn1 gain-of-function studies. Mice were exposed to 45Gy chest radiation in fractionated doses (3Gy/day). A subgroup of irradiated mice was treated with Ac-SDKP (3.2mg/kg/day) for 3-weeks. Evans blue dye was injected via tail-vein 30 mins before euthanasia and dye extravasation through coronary microvasculature was quantified. Cldn1 expression was examined by Western blot and in vitro cell permeability was examined by FITC-dextran permeability assays. Results: Cldn1 silencing with siRNA showed increased permeability in HMVECs (control, 1.0±0.01, siRNA, 1.3±0.01; p=0.0002). Cardiac irradiation significantly decreased myocardial cldn1 (control 1.0±0.27, IR 0.45±0.31; p=0.001). Consequently, there was a significant increase in myocardial Evans blue dye extravasation compared to controls (control 1.0±0.22, IR 1.3±0.23, p=0.039). Cldn1 overexpression mice had decreased dye extravasation compared to WT controls (WT 1.0±0.23, TG 0.38±0.27 p=0.002). Ac-SDKP restored cldn1 protein expression, and inhibited dye leakage in the myocardium after IR exposure (0.57±0.21, p=0.048 vs IR). Conclusions: Our study shows that IR exposure induces endothelial barrier disruption through a reduced expression of endothelial tight-junction protein, cldn1. Ac-SDKP prevents these effects, at least in part, by preventing loss of claudin-1.

KCa3.1 Inhibition of Macrophages Suppresses Inflammatory Response Leading to Endothelial Damage in a Cell Model of Kawasaki Disease.

PurposeMacrophages-mediated inflammation is linked with endothelial damage of Kawasaki disease (KD). KCa3.1, a calcium-activated potassium channel, modulates inflammation of macrophages. However, little is known about the role of KCa3.1 in inflammation by macrophages involved in KD. Hence, this study is aimed to explore the potential role of KCa3.1 in regulating inflammatory response by macrophages and subsequent vascular injury in an in vitro model of KD.MethodsRAW264.7 cells were stimulated with Lactobacillus casei cell wall extract (LCWE) with or without TRAM-34 or PDTC or AG490. Subsequently, mouse coronary artery endothelial cells (MCAECs) were incubated with RAW264.7 cells-conditioned medium to mimic local inflammatory lesions in KD. CCKi8 assay was used to evaluate cell viability. The mRNA levels of inflammatory mediators were detected by qRT-PCR. Expressions of KCa3.1, MCAECs injury-associated molecules, proteins involved in signal pathways of nuclear factor-κB (NF-κB), signal transducers and activators of transcription (STAT) 3 and p38 were evaluated by Western blot.ResultsOur study showed that LCWE increased KCa3.1 protein level in RAW264.7 macrophages and KCa3.1 inhibition by TRAM-34 notably suppressed the expression of pro-inflammatory molecules in LCWE-treated macrophages via blocking the activation of NF-κB and STAT3 pathways. Besides, the inflammation and damage of MCAECs were attenuated in the TRAM-34-treated group compared with the KD model group. This vascular protective role was dependent on the down-regulation of NF-κB and STAT3 signal pathways, which was confirmed by using inhibitors of NF-κB and STAT3.ConclusionThis study demonstrates that KCa3.1 blockade of macrophages suppresses inflammatory reaction leading to mouse coronary artery endothelial cell injury in a cell model of KD by hampering the activation of NF-κB and STAT3 signaling pathway. These findings imply that KCa3.1 may be a potential therapeutic target for KD.

Coronary Endothelium No-Reflow Injury Is Associated with ROS-Modified Mitochondrial Fission through the JNK-Drp1 Signaling Pathway.

Coronary artery no-reflow is a complex problem in the area of reperfusion therapy, and the molecular mechanisms underlying coronary artery no-reflow injury have not been fully elucidated. In the present study, we explored whether oxidative stress caused damage to coronary endothelial cells by inducing mitochondrial fission and activating the JNK pathway. The hypoxia/reoxygenation (H/R) model was induced in vitro to mimic coronary endothelial no-reflow injury, and mitochondrial fission, mitochondrial function, and endothelial cell viability were analyzed using western blotting, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence. Our data indicated that reactive oxygen species (ROS) were significantly induced upon H/R injury, and this was followed by decreased endothelial cell viability. Mitochondrial fission was induced and mitochondrial bioenergetics were impaired in cardiac endothelial cells after H/R injury. Neutralization of ROS reduced mitochondrial fission and protected mitochondrial function against H/R injury. Our results also demonstrated that ROS stimulated mitochondrial fission via JNK-mediated Drp1 phosphorylation. These findings indicate that the ROS-JNK-Drp1 signaling pathway may be one of the molecular mechanisms underlying endothelial cell damage during H/R injury. Novel treatments for coronary no-reflow injury may involve targeting mitochondrial fission and the JNK-Drp1 signaling pathway.

Quantitative proteomics and bioinformatics analyses of human coronary artery endothelial cell injury induced by Kawasaki disease

To study the biomarkers for human coronary artery endothelial cell (HCAEC) injury induced by Kawasaki disease (KD) using isobaric tags for relative and absolute quantitation (iTRAQ) proteomics. HCAECs cultured with the serum of children with KD were used as the KD group, and those cultured with the serum of healthy children was used as the healthy control group. The iTRAQ technique was used to measure the expression of proteins in two groups. The data on proteins were analyzed by bioinformatics. Western blot was used for the validation of protein markers. A total of 518 significantly differentially expressed proteins were identified (with an absolute value of difference fold of >1.2, P<0.05). The gene ontology analysis showed that the differentially expressed proteins were significantly enriched in biological processes (including cellular processes, metabolic processes, and biological regulation), cellular components (including cell parts, cells, and organelles), and molecular functions (including binding, catalytic activity, and molecular function regulators). The KEGG analysis showed that the proteins were significantly enriched in the signaling pathways of ribosomes, PI3K-Akt signaling pathway, and transcriptional dysregulation in cancer. The PPI network showed that the top 9 protein markers in relation density were PWP2, MCM4, MCM7, MCM5, MCM3, MCM2, SLD5, HDAC2, and MCM6, which were selected as the protein markers for coronary endothelial injury in KD. Western blot showed that the KD group had significantly lower expression levels of the protein markers HDAC2, PWP2, and MCM2 than the healthy control group (P<0.05). The serum of children with KD significantly changes the protein expression pattern of HCAECs and affects the signaling pathways associated with the cardiovascular system, which provides a new basis for the pathophysiological mechanism and therapeutic targets of KD.

Endovascular procedures cause transient endothelial injury but do not disrupt mature neointima in Drug Eluting Stents

Extensive application of coronary intravascular procedures has led to the increased need of understanding the injury inflicted to the coronary arterial wall. We aimed to investigate acute and prolonged coronary endothelial injury as a result of guidewire use, repeated intravascular imaging and stenting. These interventions were performed in swine (N = 37) and injury was assessed per coronary segment (n = 81) using an Evans Blue dye-exclusion-test. Scanning electron microscopy and light microscopy were then used to visualize the extent and nature of acute (<4 hours) and prolonged (5 days) endothelial injury. Guidewire and imaging injury was mainly associated with denudation and returned to control levels at 5 days. IVUS and OCT combined (Evans Blue staining 28 ± 16%) did not lead to more acute injury than IVUS alone (33 ± 15%). Stent placement caused most injury (85 ± 4%) and despite early stent re-endothelialization at 5 days, the endothelium proved highly permeable (97 ± 4% at 5 days; p < 0.001 vs acute). Imaging of in-stent neointima at 28 days after stent placement did not lead to neointimal rupture. Guidewire, IVUS and OCT induce acute endothelial cell damage, which does not increase during repeated imaging, and heals within 5 days. Interestingly, endothelial permeability increases 5 days post stenting despite near complete re-endothelialization.

Overexpression Of miR138 Ameliorates Spared Sciatic Nerve Injury-Induced Neuropathic Pain Through The Anti-Inflammatory Response In Mice.

BackgroundThe emerging role of inflammation in the initiation and maintenance of neuropathic pain has been confirmed. Previous studies have reported that miR138 has neuroprotective and anti-inflammatory effects in animal models of spinal cord injury and in human coronary artery endothelial cell injury, while its effect on neuropathic pain is still not known. As the mechanism of neuropathic pain remains unclear, we investigated whether miR138 is involved in the development of neuropathic pain and the role of miR138 in the modulation of inflammation in the spinal cord in a mouse model of neuropathic pain induced by spared sciatic nerve injury (SNI).Materials and methodsFirstly, the expression of miR138 in spinal cord was evaluated on days 1, 3, 5, 7, 9 and 14 after SNI. And then, LV-miR-control and LV-miR138 were intrathecally injected 1 week before the surgery followed by investigation of the expression of miR138, mechanical allodynia and thermal hyperalgesia on day 1, 3, 5, 7, 9, 14 after SNI. Ipsilateral L4-L6 spinal cord tissue was harvested on day 14 post-SNI and detected by Western blotting, enzyme-linked immunosorbent assay or immunohischemistry.ResultsWe observed decreased expression of miR138 and increased expression of proinflammatory cytokines, along with activated microglia, astrocytes and nuclear factor-κВ (NF-κВ), in the spinal cord dorsal horn after SNI. Moreover, the intrathecal upregulation of miR138 significantly alleviated SNI-induced mechanical allodynia and thermal hyperalgesia, downregulated the production of proinflammatory cytokines, and deactivated microglia, astrocytes and NF-κВ.ConclusionThe results indicate that miR138 contributes to the development of neuropathic pain and that the overexpression of miR138 alleviates pain hypersensitivity by inhibiting proinflammatory cytokine production and glial activation, which suggests a novel target for reducing neuropathic pain.

Cardiac allograft vasculopathy after heart transplantation: current prevention and treatment strategies.

Cardiac allograft vasculopathy (CAV) is a leading cause of mortality in heart transplantation patients. Despite optimal immunosuppression therapy, the rate of CAV post-transplantation remains high. In this review, we gathered all recent studies as well as experimental evidence focusing on the prevention and treatment strategies regarding CAV after heart transplantation. A complete literature survey was performed using the PubMed database search to gather available information regarding prevention and treatment strategies of CAV after heart transplantation. Several non-immune and immune factors have been linked to CAV such as ischemic reperfusion injury, metabolic disorders, cytomegalovirus infection, coronary endothelial dysfunction, injury and inflammation respectively. Serial coronary angiography combined with intravascular ultrasound is currently the method of choice for detecting early disease. Biomarkers and noninvasive imaging can also assist in the early identification of CAV. Treatment strategies such as mammalian target of rapamycin inhibitors proceed to grow, but prevention remains the objective. Early detection is the key to therapy management. It enables early identification and diagnosis of patients with CAV, who would gain the most from prompt treatment. Further investigation is needed to elucidate the multifactorial pathophysiological process of CAV, develop detection methods and find treatments that prevent or slow disease progression.

Qiliqiangxin improves oxidized low-density lipoprotein-induced human coronary artery endothelial cells injury

Qiliqiangxin (QL) has protective effect for the cardiovascular diseases treatment. This study aim to evaluate the effect of QL on the cell viability, cell apoptosis and caspase-3 activity in oxidized LDL (oxLDL)-induced Human Coronary Artery Endothelial Cells (HCAECs). HCAECs were treated with QL (0-1μg/ml) and oxLDL (150 μg/ml). The HCAECs viability was detected by cell proliferation assay and Lactate Dehydrogenase (LDH) release assay. The HCAECs apoptosis was evaluated by the annexin V-FITC assay. The activity of caspase-3 of HCAECs was measured by colorimetric caspase-3 assay. Cell migration assay and capillary-like tube formation assay on Matrigel were performed. Treatment of HCAECs to ox-LDL (150 μg/ml) decreased cell viability, stimulated apoptosis and enhanced caspase-3 activity. In addition, treatment with QL (0.5 μg/ml) alone did not affect cell viability and LDH release. Furthermore, the treatment with QL (0.5 μg/ml) significantly increased ox-LDL (150 μg/ml) decreased cell viability. Treatment with QL (0.5 μg/ml) reduced ox-LDL-stimulated apoptosis and caspase-3 activity in HCAECs in a dose-dependent manner. QL (0.5 μg/ml) attenuated ox-LDL (150 μg/ml) abolished cell migration and tube formation. Our study demonstrated that QL prevents ox-LDLinduced HCAECs injury by decreasing the apoptosis via caspase-3 activity.

Correlation between NF-κB signal pathway-mediated caspase-4 activation and Kawasaki disease.

The aim of the study was to investigate the role and mechanisms of action of nuclear factor-κB (NF-κB)-mediated caspase-4 activation in the induction of inflammatory cytokines during Kawasaki disease (KD) and coronary artery endothelial cell injury. Peripheral blood mononuclear cells (PBMCs) were isolated from KD patients and healthy controls and cultured. Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was applied to detect tumor necrosis factor (TNF)-α levels in activated PBMC-conditioned culture media. To establish a culture model for human coronary artery endothelial cells (HCAECs), we employed KD patient-origin PBMC culture-conditioned media to induce HCAEC transformation and detected the nuclear activation of NF-κB p65 and intracellular caspase-4 protein concentrations using western blot analysis. We also investigated the nuclear transfer of NF-κB p65 using immunofluorescence, as well as HCAEC interleukin (IL)-6 and IL-1β secretion using ELISA. Finally, we investigated HCAEC apoptosis using using Annexin V/PI double staining. After PBMCs were stimulated in vitro, TNF-α secretion was significantly higher in the KD group versus controls (P<0.01). HCAEC cells treated with supernatant conditioned by cells from KD patients showed a significant elevation of NF-κB p65 and caspase-4 protein expression versus HCAEC cells treated with supernatant conditioned by control cells (P<0.01). Similarly, IL-6 and IL-1β secretion, as well as apoptotic rate, were significantly elevated (P<0.01). SN50, an NF-κB inhibitor, significantly attenuated caspase-4 expression, secretion of IL-6, IL-1β, and TNF-α, as well as HCAEC apoptosis in cells treated with KD patient PBMC-conditioned media. NF-κB can induce the generation of various inflammatory factors including IL-6 and IL-1β, mediate the expression of caspase-4 in HCAEC cells, and affect apoptosis and injury of HCAEC cells. Therefore, the expression of caspase-4, mediated by NF-κB signal pathway, plays a critical role in KD.

Overexpression of microRNA-138 alleviates human coronary artery endothelial cell injury and inflammatory response by inhibiting the PI3K/Akt/eNOS pathway.

This study aimed to investigate the role of miR‐138 in human coronary artery endothelial cell (HCAEC) injury and inflammatory response and the involvement of the PI3K/Akt/eNOS signalling pathway. Oxidized low‐density lipoprotein (OX‐LDL)‐induced HCAEC injury models were established and assigned to blank, miR‐138 mimic, miR‐138 inhibitor, LY294002 (an inhibitor of the PI3K/Akt/eNOS pathway), miR‐138 inhibitor + LY294002 and negative control (NC) groups. qRT‐PCR and Western blotting were performed to detect the miR‐138, PI3K, Akt and eNOS levels and the protein expressions of PI3K, Akt, eNOS, p‐Akt, p‐eNOS, Bcl‐2, Bax and caspase‐3. ELISAs were employed to measure the expressions of TNF‐α, IL‐4, IL‐6, IL‐8, IL‐10 and nitric oxide (NO) and the activities of lactate dehydrogenase (LDH) and eNOS. MTT and flow cytometry were performed to assess the proliferation and apoptosis of HCAECs. Compared to the blank group, PI3K, Akt and eNOS were down‐regulated in the miR‐138 mimic and LY294002 groups but were up‐regulated in the miR‐138 inhibitor group. The miR‐138 mimic and LY294002 groups showed decreased concentrations of TNF‐α, IL‐6, IL‐8 and NO and reduced activities of LDH and eNOS, while opposite trends were observed in the miR‐138 inhibitor group. The concentrations of IL‐4 and IL‐10 increased in the miR‐138 mimic and LY294002 groups but decreased in the miR‐138 inhibitor group. The miR‐138 mimic and LY294002 groups had significantly decreased cell proliferation and increased cell apoptosis compared to the blank group. These findings indicate that up‐regulation of miR‐138 alleviates HCAEC injury and inflammatory response by inhibiting the PI3K/Akt/eNOS signalling pathway.