[Mechanism of tetramethylpyrazine attenuates inflammatory injury in endothelial cells by activating the SIRT1 signaling pathway]. / 川芎嗪通过激活SIRT1ä¿¡å·é€šè·¯å‡è½»å† çš®ç»†èƒžç‚Žç—‡æŸä¼¤çš„æœºåˆ¶ç ”ç©¶.

OBJECTIVES: To study the effects and mechanisms of tetramethylpyrazine (TMP) on tumor necrosis factor-α (TNF-α)-induced inflammatory injury in human coronary artery endothelial cells (HCAEC). METHODS: HCAEC were randomly divided into four groups: the control group (no treatment), the model group (treated with TNF-α, 50 ng/mL for 24 hours), the TMP group (pre-treated with TMP, 80 µg/mL for 12 hours followed by TNF-α treatment for 24 hours), and the SIRT1 inhibitor group (pre-treated with TMP and the specific SIRT1 inhibitor EX527 for 12 hours followed by TNF-α treatment for 24 hours). Cell viability was assessed using the CCK-8 method, lactate dehydrogenase (LDH) activity was measured using an LDH assay kit, reactive oxygen species (ROS) levels were observed using DCFH-DA staining, expression of pyroptosis-related proteins was detected by Western blot, and SIRT1 expression was analyzed using immunofluorescence staining. RESULTS: Compared to the control group, the model group showed decreased cell viability, increased LDH activity, ROS level and expression of pyroptosis-related proteins, and decreased SIRT1 expression (P<0.05). Compared to the model group, the TMP group exhibited increased cell viability, decreased LDH activity, ROS level and expression of pyroptosis-related proteins, and increased SIRT1 expression (P<0.05). In comparison to the TMP group, the SIRT1 inhibitor group showed decreased cell viability, increased LDH activity, ROS level and expression of pyroptosis-related proteins, and decreased SIRT1 expression (P<0.05). CONCLUSIONS: TMP may attenuate TNF-α-induced inflammatory injury in HCAEC, which is associated with the inhibition of pyroptosis and activation of the SIRT1 signaling pathway.

PCSK9 Inhibitors Reduce PCSK9 and Early Atherogenic Biomarkers in Stimulated Human Coronary Artery Endothelial Cells.

Despite reports on the efficacy of proprotein convertase subtilisin-Kexin type 9 (PCSK9) inhibitors as a potent lipid-lowering agent in various large-scale clinical trials, the anti-atherogenic properties of PCSK9 inhibitors in reducing PCSK9 and atherogenesis biomarkers via the NF-ĸB and eNOS pathway has yet to be established. This study aimed to investigate the effects of PCSK9 inhibitors on PCSK9, targeted early atherogenesis biomarkers, and monocyte binding in stimulated human coronary artery endothelial cells (HCAEC). HCAEC were stimulated with lipopolysaccharides (LPS) and incubated with evolocumab and alirocumab. The protein and gene expression of PCSK9, interleukin-6 (IL-6), E-selectin, intercellular adhesion molecule 1 (ICAM-1), nuclear factor kappa B (NF-ĸB) p65, and endothelial nitric oxide synthase (eNOS) were measured using ELISA and QuantiGene plex, respectively. The binding of U937 monocytes to endothelial cell capacity was measured by the Rose Bengal method. The anti-atherogenic effects of evolocumab and alirocumab were contributed to by the downregulation of PCSK9, early atherogenesis biomarkers, and the significant inhibition of monocyte adhesion to the endothelial cells via the NF-ĸB and eNOS pathways. These suggest the beyond cholesterol-lowering beneficial effects of PCSK9 inhibitors in impeding atherogenesis during the initial phase of atherosclerotic plaque development, hence their potential role in preventing atherosclerosis-related complications.

CRISPR-Cas9 mediated gene knockout in human coronary artery endothelial cells reveals a pro-inflammatory role of TLR2.

Endothelial inflammatory responses promote the development and progression of atherosclerosis. It was reported that Toll-like receptors 2 (TLR2) is associated with endothelial inflammation. However, the effect of TLR2 on inflammatory responses in human coronary artery endothelial cells (HCAECs) remains largely unknown. Here, we tested the hypothesis that TLR2 can enhance inflammatory reactions in HCAECs after stimulated by TLR2 agonist. First, we used CRISPR-Cas9 technology to knockout TLR2 gene in HCAECs. Then, TLR2-KO and wild type HCAECs were treated with TLR2 agonist peptidoglycan (PGN). The expression levels of intercellular cell adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), and interleukin-8 (IL-8) were analyzed by real-time PCR, Western blot, and ELISA. The expression status of myeloid differentiation primary response gene 88 (MyD88), phosphorylated IRAK-1 (pIRAK-1) and phosphorylated NF-κB (pNF-κB) were detected by Western blot. Our results show that after treated with TLR2 agonist, the expression levels of ICAM-1, IL-6, and IL-8 were downregulated in TLR2-KO cells compared to those of wild type cells. Further, Western blots of MyD88, pIRAK-1, and pNF-κB show that the expression levels of these pro-inflammatory molecules were much lower in TLR2-KO cells compared to that of wild type cells by stimulating with TLR2 agonist. We suggest that TLR2 may affect inflammatory reaction in HCAECs by introducing pro-inflammatory molecules like MyD88, pIRAK-1, and pNF-κB.

Endothelial microparticle-promoted inhibition of vascular remodeling is abrogated under hyperglycaemic conditions.

BACKGROUND: Endothelial microparticles (EMPs) inhibit vascular remodeling by transferring functional microRNA (miRNA) into target vascular smooth muscle cells (VSMCs). Because EMPs are increased in diabetic patients and potentially linked to vascular complications in diabetes mellitus, we sought to determine whether effects of EMPs generated under high glucose concentration on vascular remodeling might differ from EMPs derived from untreated cells. METHODS AND RESULTS: EMPs were generated from human coronary endothelial cells (HCAEC) exposed to high glucose concentrations in order to mimic diabetic conditions. These EMPs were defined as 'hyperglycaemic' EMPs (hgEMPs) and their miRNA transfer capacity and functional effects were compared with EMPs generated from 'healthy' untreated HCAECs. In vitro, the intercellular transfer of antiproliferative miRNA-126-3p from ECs to VSMCs via EMPs was significantly reduced under hyperglycaemic conditions. Additionally, EMP-mediated inhibition of the miRNA-126-3p target LRP6 and of VSMC migration and proliferation was abrogated, when hgEMPs were used. In vivo, the inhibitory effect of EMPs on neointima formation, VSMC proliferation and macrophage infiltration was abolished in mice treated with hgEMPs. CONCLUSION: Pathological hyperglycaemic conditions weaken potentially protective intercellular communication mechanisms by affecting EMP content and function.

Differential effects of cyclooxygenase-2 (COX-2) inhibitors on endoplasmic reticulum (ER) stress in human coronary artery endothelial cells.

Selective cyclooxygenase-2 (COX-2) inhibitor rofecoxib was pulled off the market because of its association with increased risk of adverse cardiovascular effects. The precise underlying mechanism for the differential effects of COX-2 inhibitors on cardiovascular risk is not known. Since endoplasmic reticulum (ER) stress is implicated in atherogenesis, we examined the effects of COX-2 inhibitors on ER stress in primary human coronary artery endothelial cells (HCAEC), human umbilical vein endothelial cells (HUVEC), and human pulmonary artery endothelial cells (HPAEC). ER stress was measured in HCAEC treated with either tunicamycin (TM) or high-concentrations (27.5 mM) of dextrose (HD) using the secreted alkaline phosphatase (ES-TRAP) assay. Markers of the unfolded protein response (UPR) such as activating transcription factor 6 (ATF6), glucose-regulated protein 78 (GRP78), inositol-requiring enzyme 1α (IRE1α), phospho-IRE1α, protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), and phospho-PERK were measured by Western blot. Treatment of HCAEC with TM and HD decreased secreted alkaline phosphatase activity indicating increased ER stress. Treatment of cells exposed to TM or HD with celecoxib, meloxicam, ibuprofen, and acetylsalicylic acid, but not rofecoxib, resulted in a dose-dependent decrease in ER stress. High-dextrose and TM increased IRE1α and PERK phosphorylation and ATF6 and GRP78 expression. Treatment with celecoxib, but not rofecoxib, inhibited these markers of the UPR. Treatment with selective COX-2 inhibitors, with the exception of rofecoxib, suppressed ER stress as measured with both alkaline phosphatase activity assays and markers for the UPR. The inability of rofecoxib to inhibit ER stress, unlike the other cyclooxygenase inhibitors tested, may have contributed to its unfavorable effects on cardiovascular outcomes.

Tumor Necrosis Factor Receptor Superfamily Member 21 Induces Endothelial-Mesenchymal Transition in Coronary Artery Endothelium of Type 2 Diabetes Mellitus.

Diabetes mellitus (DM) is an increasing threat to human health and regarded as an important public issue. Coronary artery disease is one of the main causes of death in type 2 DM patients. However, the effect of hyperglycemia on coronary artery endothelial cells (CAECs) and the pathophysiologic mechanisms are still not well-explored. This study aims to explore the signal pathway and novel biomarkers of injury of CAECs in DM in understanding the microenvironment changes and mechanisms of diabetic heart disease. Next-generation sequence (NGS) and bioinformatics analysis to analyze the CAECs of one type 2 DM patient and one normal individual was performed, and it was found that tumor necrosis factor receptor superfamily member 21 (TNFRSF21) was a soluble factor in circulating system. Further experiments confirmed that advanced glycation end products (AGEs), the metabolite derived by hyperglycemia, increased the expression of TNFRSF21 in CAECs. TNFRSF21 induced endothelial-mesenchymal transition (EndoMT) in CAECs, resulting in increased permeability of CAECs. In addition, levels of serum TNFRSF21 were higher in type 2 DM patients with left ventricular hypertrophy (LVH) than those without LVH. Serum TNFRSF21 levels were also positively correlated with the LV mass index and negatively with LV systolic function. Serum TNFRSF21 levels were associated with changes in cardiac structure and function in patients with type 2 DM. In conclusion, TNFRSF21 plays a pathogenic role in heart disease of type 2 DM, and can be used as a biomarker of the impairment of cardiac structure and function in type 2 DM patients.

Reduced cellular glucose transport confers natural protection against dextrose-induced superoxide generation and endoplasmic reticulum stress in domestic hen.

Normal blood glucose levels in avian species are two to fourfold higher than that in humans and the higher blood glucose levels in birds do not cause adverse effects. Endothelial cells isolated from the aorta of the domestic hen (Gallus gallus domesticus) and chicken aortic smooth muscle cells (CAOSMC) were compared to human coronary artery endothelial cells (HCAEC) and human primary aortic smooth muscle cells (HASMC). Superoxide (SO) generation was measured using a superoxide-reactive probe. ER stress was measured using the placental alkaline phosphatase assay (ES-TRAP). Glucose transport kinetics were determined using the 3 H-2-deoxyglucose tracer. Dextrose-induced SO generation and ER stress were significantly blunted in avian endothelial cells compared to human cells. The Vmax of glucose uptake (in nmoles/mg protein/min) in avian endothelial cells (0.0018 ± 0.0001) and smooth muscle cells (0.0015 ± 0.0007) was approximately 18-25 fold lower compared to the Vmax in HCAEC (0.033 ± 0.0025) and HASMC (0.038 ± 0.004) (all p < 0.0001). The Michaelis-Menten constant (Km) of transport was also significantly different (p < 0.0001) in avian species. The relative resistance of avian cells to dextrose-induced oxidative stress and ER stress is mostly the result of reduced cellular dextrose transport.

Co-culture of peripheral blood mononuclear cell (PBMC) and human coronary artery endothelial cell (HCAEC) reveals the important role of autophagy implicated in Kawasaki disease.

BACKGROUND: Kawasaki disease (KD) is a systemic vasculitis syndrome that commonly occurs in children. Autophagy has been increasingly shown to be involved in various cardiovascular diseases, including endothelial dysfunction and vascular endothelial injury. However, whether autophagy is implicated in the pathogenesis of KD remains poorly understood, and particularly, how the dysfunction of human coronary artery endothelial cells (HCAECs) is associated with autophagy in peripheral blood mononuclear cells (PBMCs) from KD patients awaits further investigation. METHODS: Peripheral blood samples were collected from KD patients, common fever patients, and healthy controls. The PBMC samples were isolated from KD blood samples collected at three different phases: the acute phase before therapy (acute-KD), 1 week (subacute-KD), and 4 weeks (convalescent-KD) after drug administration. RESULTS: The autophagy flux was significantly increased in the PBMCs of KD patients at acute phase. The PBMCs of acute KD patients could induce autophagy in HCAECs and promote the secretion of chemokines and pro-inflammatory factors after cocultured with HCAECs whereas 3-methyladenine (3-MA) drug could partly reverse this process. CONCLUSIONS: Autophagy is involved in the inflammatory injury of vascular endothelial cells associated with PBMCs in KD patients, and may play a crucial role in regulating inflammation. Hence, we identify a novel regulatory mechanism of vascular injury in this disease.

Selective Interleukin-6 Trans-Signaling Blockade Is More Effective Than Panantagonism in Reperfused Myocardial Infarction.

Interleukin (IL)-6 is an emerging therapeutic target in myocardial infarction (MI). IL-6 has 2 distinct signaling pathways: trans-signaling, which mediates inflammation, and classic signaling, which also has anti-inflammatory effects. The novel recombinant fusion protein sgp130Fc achieves exclusive trans-signaling blockade, whereas anti-IL-6 antibodies (Abs) result in panantagonism. In a rat model of reperfused MI, sgp130Fc, but not anti-IL-6-Ab, attenuated neutrophil and macrophage infiltration into the myocardium, reduced infarct size, and preserved cardiac function 28 days after MI. These data demonstrate the efficacy of exclusive IL-6 trans-signaling blockade and support further investigation of sgp130Fc as a potential novel therapy in MI.

Berberine protects Kawasaki disease-induced human coronary artery endothelial cells dysfunction by inhibiting of oxidative and endoplasmic reticulum stress.

Kawasaki disease (KD) is an acute febrile illness characterized by systemic vasculitis especially in coronary arteries. Berberine (BBR) shows several beneficial effects on cardiovascular system. The present study is to investigate whether BBR exerts protective effect against KD-induced damage of human coronary artery endothelial cell (HCAECs) and the underlying mechanisms. HCAECs exposed to medium with 15% serum from KD patients or healthy volunteers for 24 h. Stimulated HCAECs were treated with vehicle (without BBR) and BBR (20 µM) for 24 h, the cell apoptosis, cell cycle, induction of intracellular reactive oxygen species (ROS) and protein expression were examined by flow cytometry and western blot. The KD-induced differentially expressed proteins in HCAECs were determined by quantitative proteomics. BBR inhibited HCAECs from apoptosis and arrested cell cycle at G0/G1 stage. BBR protected HCAECs from injury by inhibiting expression of THBD, vWF and EDN1. Bioinformatics analysis suggested that the oxidative and ER stress were involved in KD-induced damage in HCAECs. ROS production and the protein expression of ATF4, p-EIF2α, p-PERK, XBP1, p-IRE1, HSP90B1, HSPG2, DNAJC3, P4HB and VCP were increased by serum from KD patients and decreased by BBR treatment. BBR exerts its protective effects on KD-induced damage of HCAECs through its inhibitory effects on oxidative and ER stress indicating BBR as a therapeutic candidate for KD.

Strikingly Different Atheroprotective Effects of Apolipoprotein A-I in Early- Versus Late-Stage Atherosclerosis.

Preclinical studies have shown benefit of apolipoprotein A-I (apoA-I)/high-density lipoprotein (HDL) raising in atherosclerosis; however, this has not yet translated into a successful clinical therapy. Our studies demonstrate that apoA-I raising is more effective at reducing early-stage atherosclerosis than late-stage disease, indicating that the timing of HDL raising is a critical factor in its atheroprotective effects. To date, HDL-raising clinical trials have only been performed in aged patients with advanced atherosclerotic disease. Our findings therefore provide insight, related to important temporal aspects of HDL raising, as to why the clinical trials have thus far been largely neutral.

Octanoylated ghrelin attenuates angiogenesis induced by oxLDL in human coronary artery endothelial cells via the GHSR1a-mediated NF-κB pathway.

OBJECTIVE: Low concentrations of oxidized low-density lipoprotein (oxLDL) promote the in vitro angiogenesis of endothelial cells and play an important role in plaque angiogenesis, which may cause plaque vulnerability and enhance the risk of intravascular thrombosis. The aim of this research was to investigate the effects of octanoylated ghrelin on oxLDL-induced angiogenesis and the underlying molecular mechanisms involved in this process. MATERIALS/METHODS: Human coronary artery endothelial cells (HCAECs) were incubated with 5 µg/ml oxLDL and treated with various concentrations of octanoylated ghrelin (10(-9)-10(-6)M) with or without inhibitors for 24h. Cell proliferation, migration, and in vitro angiogenesis were analyzed by bromodeoxyuridine (BrdU) staining and BrdU enzyme-linked immunosorbent assay (ELISA), transwell assay, and tube formation on Matrigel, respectively. NF-κB (nuclear factor κB) expression was determined by Western-blot analysis. RESULTS: Treatment with oxLDL at 5 µg/ml enhanced the proliferation, migration and tube formation of HCAECs. In contrast, pretreatment with octanoylated ghrelin significantly attenuated in vitro angiogenesis in oxLDL-induced HCAECs. In addition, Western blot analysis indicated that NF-κB expression was increased after oxLDL treatment, and that this effect was significantly reversed by pretreatment with octanoylated ghrelin. However, the NF-κB inhibitor PDTC or the GHSR1a inhibitor [D-Lys3]-GHRP-6 abolished the effects of octanoylated ghrelin on the inhibition of angiogenesis and NF-κB p65 expression induced by oxLDL. CONCLUSIONS: These findings suggest that octanoylated ghrelin attenuates angiogenesis induced by oxLDL in HCAECs via the inhibition of GHSR1a-mediated NF-κB pathway. Furthermore, octanoylated ghrelin may promote the stability of vulnerable plaques by inhibiting plaque angiogenesis.