Bone Morphogenetic Protein 9 Protects Against Myocardial Infarction by Improving Lymphatic Drainage Function and Triggering DECR1-Mediated Mitochondrial Bioenergetics.

BACKGROUND: BMP9 (bone morphogenetic protein 9) is a member of the TGF-ß (transforming growth factor ß) family of cytokines with pleiotropic effects on glucose metabolism, fibrosis, and lymphatic development. However, the role of BMP9 in myocardial infarction (MI) remains elusive. METHODS: The expressional profiles of BMP9 in cardiac tissues and plasma samples of subjects with MI were determined by immunoassay or immunoblot. The role of BMP9 in MI was determined by evaluating the impact of BMP9 deficiency and replenishment with adeno-associated virus-mediated BMP9 expression or recombinant human BMP9 protein in mice. RESULTS: We show that circulating BMP9 and its cardiac levels are markedly increased in humans and mice with MI and are negatively associated with cardiac function. It is important to note that BMP9 deficiency exacerbates left ventricular dysfunction, increases infarct size, and augments cardiac fibrosis in mice with MI. In contrast, replenishment of BMP9 significantly attenuates these adverse effects. We further demonstrate that BMP9 improves lymphatic drainage function, thereby leading to a decrease of cardiac edema. In addition, BMP9 increases the expression of mitochondrial DECR1 (2,4-dienoyl-CoA reductase 1), a rate-limiting enzyme involved in ß-oxidation, which, in turn, promotes cardiac mitochondrial bioenergetics and mitigates MI-induced cardiomyocyte injury. Moreover, DECR1 deficiency exacerbates MI-induced cardiac damage in mice, whereas this adverse effect is restored by the treatment of adeno-associated virus-mediated DECR1. Consistently, DECR1 deletion abrogates the beneficial effect of BMP9 against MI-induced cardiomyopathy and cardiac damage in mice. CONCLUSIONS: These results suggest that BMP9 protects against MI by fine-tuning the multiorgan cross-talk among the liver, lymph, and the heart.

Macrophage-Derived GSDMD Plays an Essential Role in Atherosclerosis and Cross Talk Between Macrophages via the Mitochondria-STING-IRF3/NF-κB Axis.

BACKGROUND: Macrophages play a crucial role in atherosclerotic plaque formation, and the death of macrophages is a vital factor in determining the fate of atherosclerosis. GSDMD (gasdermin D)-mediated pyroptosis is a programmed cell death, characterized by membrane pore formation and inflammatory factor release. METHODS: ApoE-/- and Gsdmd-/- ApoE-/- mice, bone marrow transplantation, and AAV (adeno-associated virus serotype 9)-F4/80-shGSDMD (shRNA-GSDMD) were used to examine the effect of macrophage-derived GSDMD on atherosclerosis. Single-cell RNA sequencing was used to investigate the changing profile of different cellular components and the cellular localization of GSDMD during atherosclerosis. RESULTS: First, we found that GSDMD is activated in human and mouse atherosclerotic plaques and Gsdmd-/- attenuates the atherosclerotic lesion area in high-fat diet-fed ApoE-/- mice. We performed single-cell RNA sequencing of ApoE-/- and Gsdmd-/- ApoE-/- mouse aortas and showed that GSDMD is principally expressed in atherosclerotic macrophages. Using bone marrow transplantation and AAV-F4/80-shGSDMD, we identified the potential role of macrophage-derived GSDMD in aortic pyroptosis and atherosclerotic injuries in vivo. Mechanistically, GSDMD contributes to mitochondrial perforation and mitochondrial DNA leakage and subsequently activates the STING (stimulator of interferon gene)-IRF3 (interferon regulatory factor 3)/NF-κB (nuclear factor kappa B) axis. Meanwhile, GSDMD regulates the STING pathway activation and macrophage migration via cytokine secretion. Inhibition of GSDMD with GSDMD-specific inhibitor GI-Y1 (GSDMD inhibitor Y1) can effectively alleviate the progression of atherosclerosis. CONCLUSIONS: Our study has provided a novel macrophage-derived GSDMD mechanism in the promotion of atherosclerosis and demonstrated that GSDMD can be a potential therapeutic target for atherosclerosis.

Emodin attenuates cardiomyocyte pyroptosis in doxorubicin-induced cardiotoxicity by directly binding to GSDMD.

BACKGROUND: Doxorubicin (Dox), which is an anticancer drug, has significant cardiac toxicity and side effects. Pyroptosis occurs during Dox-induced cardiotoxicity (DIC), and drug inhibition of this process is one therapeutic approach for treating DIC. Previous studies have indicated that emodin can reduce pyroptosis. However, the role of emodin in DIC and its molecular targets remain unknown. HYPOTHESIS/PURPOSE: We aimed to clarify the protective role of emodin in mitigating DIC, as well as the mechanisms underlying this effect. METHODS: The model of DIC was established via the intraperitoneal administration of Dox at a dosage of 5 mg/kg per week for a span of 4 weeks. Emodin at two different doses (10 and 20 mg/kg) or a vehicle was intragastrically administered to the mice once per day throughout the Dox treatment period. Cardiac function, myocardial injury markers, pathological morphology of the heart, level of pyroptosis and mitochondrial function were assessed. Protein microarray, biolayer interferometry and pull-down assays were used to confirm the target of emodin. Moreover, GSDMD-overexpressing plasmids were transfected into GSDMD-/- mice and HL-1 cells to further verify whether emodin suppressed GSDMD activation. RESULTS: Emodin therapy markedly enhanced cardiac function and reduced cardiomyocyte pyroptosis in mice induced by Dox. Mechanistically, emodin binds to GSDMD and inhibits the activation of GSDMD by targeting the Trp415 and Leu290 residues. Moreover, emodin was able to mitigate Dox-induced cardiac dysfunction and myocardial injury in GSDMD-/- mice overexpressing GSDMD, as shown by increased EF and FS, decreased serum levels of CK-MB, LDH and IL-1ß and mitigated cell death and cell morphological disorder. Additionally, emodin treatment significantly reduced GSDMD-N expression and plasma membrane disruption in HL-1 cells overexpressing GSDMD induced by Dox. In addition, emodin reduced mitochondrial damage by alleviating Dox-induced GSDMD perforation in the mitochondrial membrane. CONCLUSION: Emodin has the potential to attenuate DIC by directly binding to GSDMD to inhibit pyroptosis. Emodin may become a promising drug for prevention and treatment of DIC.

Gasdermin D affects aortic vascular smooth muscle cell pyroptosis and Ang II-induced vascular remodeling.

Vascular smooth muscle cells (VSMCs) are primarily responsible for vasoconstriction and the regulation of blood pressure1. Pyroptosis, a particular form of regulated cell death, is involved in multiple vascular injuries, including hypertensive vascular dysfunction. This pyroptotic cell death is mediated by the pore-forming protein of Gasdermin D (GSDMD). This study was designed to examine the direct effect of GSDMD on smooth muscle cell pyroptosis and vascular remodeling. Findings revealed that GSDMD was activated in Angiotensin (Ang) II- treated aortas. We then showed that genetic deletion of Gsdmd reduced vascular remodeling and aorta pyroptosis induced by Ang II in vivo. Aberrant expression of GSDMD by recombinant AAV9 virus carrying Gsdmd cDNA aggravated the level of pyroptosis in aortas of Ang II mice. Gain- and loss-of- function analysis further confirmed that GSDMD regulated the pyroptosis of murine aortic vascular smooth muscle cells (MOVAS) in an in vitro model of tumor necrosis factor (TNF)-α treatment, which was achieved by transfecting expressing plasmid or siRNA, respectively. Overall, this study provided evidence supporting the active involvement of GSDMD in smooth muscle cell pyroptosis and Ang II-induced mice vascular injury. This finding lends credence to GSDMD as a potential therapeutic target for hypertensive vascular remodeling via inhibiting pyroptosis.

Tussilagone attenuates atherosclerosis through inhibiting MAPKs-mediated inflammation in macrophages.

Atherosclerosis is a common chronic inflammatory disease. Recent studies have highlighted the key role of macrophages and inflammation in process of atherosclerotic lesion formation. A natural product, tussilagone (TUS), has previously exhibited anti-inflammatory activities in other diseases. In this study, we explored the potential effects and mechanisms of TUS on the inflammatory atherosclerosis. Atherosclerosis was induced in ApoE-/- mice by feeding them with a high-fat diet (HFD) for 8 weeks, followed by administration of TUS (10, 20 mg ·kg-1·d-1, i.g.) for 8 weeks. We demonstrated that TUS alleviated inflammatory response and reduced atherosclerotic plaque areas in HFD-fed ApoE-/- mice. Pro-inflammatory factor and adhesion factors were inhibited by TUS treatment. In vitro, TUS suppressed foam cell formation and oxLDL-induced inflammatory response in MPMs. RNA-sequencing analysis indicated that MAPK pathway was related to the anti-inflammation and anti-atherosclerosis effects of TUS. We further confirmed that TUS inhibited MAPKs phosphorylation in plaque lesion of aortas and cultured macrophages. MAPK inhibition blocked oxLDL-induced inflammatory response and prevented the innately pharmacological effects of TUS. Our findings present a mechanistic explanation for the pharmacological effect of TUS against atherosclerosis and indicate TUS as a potentially therapeutic candidate for atherosclerosis.

Cardiac sirtuin1 deficiency exacerbates ferroptosis in doxorubicin-induced cardiac injury through the Nrf2/Keap1 pathway.

Doxorubicin (DOX), a broad-spectrum chemotherapeutic agent for various cancers, has limited clinical application because of its serious cardiotoxicity, which is due to different mechanisms, including cardiac ferroptosis and oxidative stress. Some drugs, such as berberine or dioscin, show efficacy in impeding DOX-induced cardiotoxicity by activating Sirtuin 1 (Sirt1). However, there is no direct evidence to clarify the role of Sirt1 in DOX-induced cardiomyopathy and its underlying role in cardiac ferroptosis. In this study, C57BL/6 and cardiac-specific Sirt1-/- knockout mice were used as a DOX-induced cardiotoxicity model. We found that cardiac Sirt1 was downregulated, oxidative stress was increased and ferroptosis were obviously enhanced, as reflected by decreased Glutathione peroxidase 4 (GPX4) and increased Heme oxygenase 1 (Hmox-1), exposure to DOX treatment in mice and H9c2 cells compared with the control. And Sirt1 activation was resistant to cardiac injury induced by DOX, as observed the improvement of cardiac dysfunction, and the reduction of cardiac fibrosis. However, cardiac Sirt1 deficiency aggravated Dox-induced cardiac dysfunction and cardiac remodeling, further downregulated GPX4, upregulated Hmox-1 expression and increased ROS level. In addition, Sirt1-siRNA exacerbated DOX-induced cardiotoxicity in H9c2 cells, which is similar to the results obtained in vivo. Furthermore, DOX decrease Nrf2 translocation from the cytosol to the nucleus, and Sirt1 deficiency further restrain the process, as well as the downstream Keap1 pathways, in DOX-induced cardiotoxicity. This study provides direct evidence that Sirt1 plays a protective role in DOX-induced cardiotoxicity by mediating ferroptosis reduction via the Nrf2/Keap1 pathway.

Hsa_circ_0007478 aggravates NLRP3 inflammasome activation and lipid metabolism imbalance in ox-LDL-stimulated macrophage via miR-765/EFNA3 axis.

Coronary heart disease can be effectively prevented by alleviating atherosclerotic plaque progression. Ox-LDL-induced inflammatory response in macrophages is a critical factor in the pathophysiology of atherosclerosis. It is well known that circular RNAs (circRNAs) are associated with the progression of several human diseases, such as coronary artery diseases, by sponging microRNAs (miRNAs), but the function and hidden mechanisms of circRNAs in macrophage inflammation and lipid metabolism remain unclear. In our study, we established an ox-LDL-stimulated macrophage model and used microarray to detect circRNA expression in macrophages. The results revealed distinct profiles of circRNA expression across the ox-LDL-stimulated macrophage group and the control group. Among them, hsa_circ_0007478 was upregulated in ox-LDL-stimulated macrophages, accompanied by reduced miR-765 and increased EFNA3 expression. Activation of NLRP3 inflammasome and IL-1ß in macrophages was decreased following silencing of hsa_circ_0007478 or transfection of miR-765 mimics. In addition, we demonstrated that as a direct target gene of miR-765, the expression of EFNA3 regulated NLRP3 inflammasome and IL-1ß levels in macrophages. Besides, hsa_circ_0007478 promoted EFNA3 expression by acting as a miR-765 sponge. We further showed that hsa_circ_0007478/miR-765/EFNA3 axis could also be involved in the inhibition of the lipid metabolism and foam cells formation in ox-LDL-macrophages. Taken together, these findings suggest that Hsa_circ_0007478 may be a potential molecular target against the inflammatory response and foam cells during atherosclerosis.

Suppression of Netrin-1 attenuates angiotension II-induced cardiac remodeling through the PKC/MAPK signaling pathway.

BACKGROUND: Myocardial remodeling caused by angiotensin II (Ang II) is essential for the pathological process of heart failure. Netrin-1, which is an axonal guidance cue, has been shown to be involved in the inflammatory response, tumorigenesis, and angiogenesis in non-neuronal tissues. However, the role of Netrin-1 in cardiac remodeling has not been fully elucidated. METHODS: The rat cardiomyocyte cell line H9c2 and primary neonatal rat cardiomyocytes were treated with Ang II. Cells were transfected with siRNA to silence Netrin-1 expression. Real-time polymerase chain reaction and Western blot analysis were used to detect the markers for fibrosis, apoptosis, and hypertrophy in cardiomyocytes. An Annexin V-EGFP/PI cell apoptosis detection kit was used to measure the level of apoptosis caused by angiotensin II. RESULTS: We found that Netrin-1 expression was upregulated in the H9c2 cells and the neonatal rat cardiomyocytes stimulated by Ang II. The increased Netrin-1 expression was decreased by valsartan to block AT1R. Importantly, the application of Netrin-1 siRNA significantly alleviated the degrees of myocardial hypertrophy, fibrosis (reflected by Myhc, collagen I, and TGF-ß) and apoptosis (reflected by the level of Caspase 3, Bax, and Bcl-2) induced by Ang II. In addition, the silencing of Netrin-1 substantially decreased the phosphorylation of PKCα, JNK, and P38. We treated H9c2 cells with LY317615, SP600125, and SB203580, inhibitors of PKCα, JNK, and P38, respectively, thereby resulting in a substantial decrease in hypertrophy, fibrosis, and apoptosis. CONCLUSIONS: Ang II produces cardiac hypertrophy, fibrosis, and apoptosis through the upregulation of Netrin-1 and the activation of the AT1R/PKCα/MAPK (JNK, P38) pathway. Suppression of Netrin-1 can relieve Ang II-induced cardiac remodeling via inhibition of the PKCα/MAPK (JNK and P38) signaling pathway. Thus, Netrin-1 may be a novel therapeutic target for Ang II-mediated cardiac remodeling.

1,25(OH)2D3 Strengthens the Vasculogenesis of Multipotent Mesenchymal Stromal Cells from Rat Bone Marrow by Regulating the PI3K/AKT Pathway.

BACKGROUND: Multipotent mesenchymal stromal cells (MSCs) have recently been reported to promote vasculogenesis by differentiating into endothelial cells and releasing numerous cytokines and paracrine factors. However, due to low cell activity, their potential for clinical application is not very satisfactory. This study aimed to explore the effects and mechanisms of 1,25-dihydroxyvitamin D (1,25(OH)2D3) on the vasculogenesis of MSCs. METHODS: MSCs were isolated from the femurs and tibias of rats and characterized by flow cytometry. After treatment with different concentrations of 1,25(OH)2D3 (0 µM, 0.1 µM and 1 µM), the proliferation of MSCs was analyzed by Cell Counting Kit-8 (CCK-8), and the migratory capability was measured by Transwell assays and cell scratch tests. Capillary-like structure formation was observed by using Matrigel. Western blotting was used to detect the expression of FLK-1 and vWF to investigate the differentiation of MSCs into endothelial cells. Western blotting and gelatin zymography were used to detect the expression and activities of VEGF, MMP-2 and MMP-9 secreted by MSCs under the influence of 1,25(OH)2D3. Finally, the VDR antagonist pyridoxal-5-phosphate (P5P) and the PI3K/AKT pathway inhibitor LY294002 were utilized to test the phosphorylation levels of key kinases in the PI3K/AKT pathway by Western blotting and the formation of capillary-like structures in Matrigel. RESULTS: The proliferation and migratory capability of MSCs and the ability of MSCs to form a tube-like structure in Matrigel were enhanced after treatment with 1,25(OH)2D3. Moreover, MSCs treated with 1,25(OH)2D3 showed high expression of vWF and Flk-1. There was a significant increase in the expression of VEGF, MMP-2 and MMP-9 secreted by MSCs treated with 1,25(OH)2D3, as well as in the activity of MMP-2 and MMP-9. The phosphorylation level of AKT increased with time after 1,25(OH)2D3 treatment, while LY294002 weakened AKT phosphorylation. In addition, the ability to form capillary-like structures was reduced when the VDR and PI3K/AKT pathways were blocked. CONCLUSION: This study confirmed that 1,25(OH)2D3 treatment can strengthen the ability of MSCs to promote vasculogenesis in vitro, and the mechanism may be related to the activation of the PI3K/AKT pathway.

Relationship between plasma cancer antigen (CA)-125 level and one-year recurrence of atrial fibrillation after catheter ablation.

BACKGROUND: Previous studies have shown that plasma cancer antigen-125 (CA-125) is closely related to heart failure and new-onset atrial fibrillation (AF), but no study reported the relationship between CA-125 concentrations and advanced recurrence of AF ablation. This research is the first to describe CA-125 as a biomarker for the recurrence of AF after ablation. METHODS: A total of 422 AF patients undergoing catheter ablation were included in this study. RESULTS: During the 1-y follow-up, 326 patients (77.25%) maintained a sinus rhythm, whereas 83 patients (20.44%) presented AF recurrence. The patients with AF recurrence showed higher CA-125 concentrations at baseline than those with maintained sinus rhythm (P = 0.0001). Multivariate Cox proportional hazards regression analyses revealed that persistent AF (HR 2.212; 95% CI: 1.396-3.504, P = 0.001) and CA-125 concentration (HR, 1.003; 95% (CI): 1.000-1.005, P = 0.019) were independent predictors of AF recurrence. According to the receiver operating characteristic (ROC) analysis, CA-125 yielded an optimal cut-off value of 11.05 U/ml, and its sensitivity and specificity reached 65.6% and 85.0%, respectively. In addition, the area under the curve (AUC) value spanned 80.3% (95% CI: 0.750-0.857, P < 0.0001). Moreover, the results of the subgroup analysis indicated that patients with persistent atrial fibrillation have higher concentrations of CA-125 and have an increased risk of the recurrence of AF. CONCLUSIONS: High CA-125 concentration is an independent predictor of AF recurrence after 1 y of AF ablation, especially in patients with persistent AF.

P2X7 receptor regulates EMMPRIN and MMP­9 expression through AMPK/MAPK signaling in PMA­induced macrophages.

The rupture of atherosclerotic plaques may result in the formation of thrombi, which may induce subsequent cardiac events such as acute myocardial infarction. Overproduction of matrix metalloproteinases (MMPs) and extracellular matrix metalloproteinase inducers (EMMPRINs) by monocytes and macrophages may lead to rupture of atherosclerotic plaques as a result of the degradation of the extracellular matrix. The purinergic 2X7 receptor (P2X7R) is expressed in macrophages that are assembled in atherosclerotic lesions of human carotid arteries. P2X7R may serve a crucial role in the development of atherosclerosis; therefore, the present study aimed to determine whether P2X7R regulated the expression of EMMPRIN and MMP­9 in phorbol 12­myristate 13­acetate (PMA)­induced macrophages. In addition, the potential molecular mechanisms involved in this process were investigated. THP­1 human monocytic cells were pretreated with A­438079 (a specific inhibitor of P2X7R) for 1 h and subsequently incubated with or without PMA for 48 h. Exposure to A­438079 significantly decreased the expression of MMP­9 and EMMPRIN in the PMA­induced macrophages and attenuated the activation (phosphorylation) of mitogen­activated protein kinase (MAPK) signaling, including c­Jun N­terminal kinase, p38 and extracellular signal­regulated kinase. The present study also demonstrated that 5'­AMP­activated protein kinase (AMPK) was activated by PMA exposure during differentiation from monocytes to macrophages. This activation was reversed by A­438079 treatment through the inhibition of P2X7R expression. These results suggested that the inhibition of P2X7R may be able to suppress the AMPK/MAPK signaling pathway and consequently downregulate both EMMPRIN and MMP­9 expression in PMA­induced macrophages.

Inhibition of LncRNA-HRIM Increases Cell Viability by Regulating Autophagy Levels During Hypoxia/Reoxygenation in Myocytes.

Backgrund/Aims: Ischemia reperfusion (I/R) promotes the severity of cardiomyocyte injury. Long noncoding RNAs (LncRNAs) are key regulators in cardiovascular diseases. However, the association between LncRNAs and myocardial I/R injury has not been thoroughly characterized to date. We attempted to clarify the potential biological role of a LncRNA (E230034O05Rik), which we named hypoxia/reoxygenation (H/R) injury-related factor in myocytes (HRIM), by investigating the differential expression of LncRNAs between groups of myocytes exposed to either a normal level of oxygen or to H/R. METHODS: Microarray analysis was used to determine analyze the global differential expression of LncRNAs in H9c2 myocytes exposed either to a normal level of oxygen or to H/R. Target LncRNA levels were further verified in vitro and ex vivo by real-time polymerase chain reaction (qPCR). Cell viability was analyzed using the Cell Counting Kit-8 assay. Autophagy levels were confirmed by Western blotting, transmission electron microscopy, and autophagic double-labeled (mRFP-GFP-LC3) adenovirus analyses. RESULTS: Gene expression profiling revealed that 797 LncRNAs and 1898 mRNAs were differentially expressed in the H/R group compared with the normal oxygen group. Among these LncRNAs and mRNAs, 6 upregulated LncRNAs and 2 downregulated LncRNAs in the H/R group were selected and further validated by qPCR in vitro and ex vivo. Additionally, LncRNA-HRIM was inhibited by specific siRNAs in H9c2 myocytes exposed to H/R. The inhibition of LncRNA-HRIM by siRNA prevented cell death by suppressing excessive autophagic activity in myocytes, This finding suggests a detrimental role of LncRNA-HRIM in the regulation of I/R injury. CONCLUSIONS: LncRNAs are involved in H/R injury of H9c2 myocytes. Inhibition of LncRNA-HRIM increased cell viability by reducing autophagy in myocytes during H/R.

Inhibition of hsa-miR-6086 protects human umbilical vein endothelial cells against TNFα-induced proliferation inhibition and apoptosis via CDH5.

MiRNAs are considered as a novel class of biomarkers or treatment targets for cardiovascular diseases. Hsa-miR-6086, a novel mi-RNA, was reported to be downregulated during the differentiation of human embryonic stem cells into endothelial cells (ECs). Interestingly, CDH5 (cadherin 5), encoding a classical cadherin of the cadherin superfamily, is a cellular marker of ECs and has been reported to be a target of hsa-miR-6086. However, the role of hsa-miR-6086 in ECs is virtually unknown. Herein, we report that hsa-miR-6086 was markedly induced by TNFα stimulation in human umbilical vein endothelial cells (HUVECs), whereas CDH5 expression was greatly reduced. Importantly, TNFα-induced suppression of CDH5 expression was largely prevented by inhibiting hsa-miR-6086, and hsa-miR-6086 mimic greatly decrease CDH5 expression in HUVECs, suggesting that the induction of hsa-miR-6086 is responsible for CDH5 downregulation by TNFα. In addition, restoration of CDH5 expression level by either inhibiting hsa-miR-6086 or exogenously expressing CDH5 cDNA that is not affected by hsa-miR-6086 protected HUVECs against TNFα-induced apoptosis and cell growth inhibition. Taken together, our study reveals that hsa-miR-6086 is induced by TNFα and mediates TNFα-induced HUVEC growth inhibition through downregulating CDH5 expression. Hence, hsa-miR-6086 might be a new target for treating TNFα-induced endothelial dysfunction.

NACHT, LRR and PYD domains-containing protein 3 inflammasome is activated and inhibited by berberine via toll-like receptor 4/myeloid differentiation primary response gene 88/nuclear factor-κB pathway, in phorbol 12-myristate 13-acetate-induced macrophages.

The nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP-3) inflammasome has recently emerged as a pivotal regulator of chronic inflammation. The present study investigated the expression of NLRP3 inflammasome in phorbol 12-myristate 13-acetate (PMA)-induced macrophages, and aimed to identify the effects of berberine on the inflammasome. Human monocytic THP-1 cells were pretreated with berberine for 1 h and then induced with PMA for 48 h. Total RNA and protein were collected for reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. Supernatants were collected to determine IL-1ß levels by using ELISA. The present study demonstrated that NLRP3 inflammasome and IL-1ß were activated in PMA-induced macrophages in a time-dependent manner, whereas berberine significantly inhibited their expression in a dose-dependent manner in PMA-induced macrophages. Furthermore, berberine also suppressed the toll-like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (Myd88)/nuclear factor (NF)-κB signaling pathway which was activated during the conversion of THP-1 cells to macrophages by PMA. In conclusion, berberine reduced NLRP3 inflammasone expression by suppressing the activation of the TLR4/Myd88/NF-κB signaling pathway in PMA-induced macrophages. This inhibitory effect may imply an important role of berberine on chronic inflammation and atherogenic progression in coronary artery disease.

Inhibition of autophagy by berberine enhances the survival of H9C2 myocytes following hypoxia.

Hypoxia may induce apoptosis and autophagy to promote cardiomyocyte injury. The present study investigated the effect of berberine, a natural extract of Rhizoma Coptidis, on hypoxia­induced autophagy and apoptosis in the H9c2 rat myocardial cell line. Expression levels of apoptosis and autophagy markers were upregulated in H9c2 myocytes during hypoxia and cell viability was reduced. However, berberine significantly reduced hypoxia­induced autophagy in H9c2 myocytes, as demonstrated by the ratio of microtubule­associated proteins 1A/1B light chain 3 I/II and the expression levels of B­cell lymphoma 2 (Bcl­2)/adenovirus E1B 19 kDa protein­interacting protein 3, and promoted cell viability. In addition, expression levels of the Bcl­2 anti­apoptotic protein were significantly downregulated, and expression levels of pro­apoptotic proteins Bcl­2­associated X protein and cleaved caspase­3 were upregulated during hypoxia injury in cardiac myocytes. This was reversed by treatment with berberine or the autophagy inhibitor 3­methyladenine, whereas the autophagy agonist rapamycin had the opposite effects, suggesting that berberine reduces myocyte cell death via inhibition of autophagy and apoptosis during hypoxia. In addition, Compound C, a 5' adenosine monophosphate­activated protein kinase (AMPK) inhibitor, reduced apoptosis and autophagy in hypoxic myocytes, suggesting that the activation of the AMPK signaling pathway may be involved in this process. These findings suggested that berberine protects cells from hypoxia­induced apoptosis via inhibition of autophagy and suppression of AMPK activation. Therefore, berberine may be a potential therapeutic agent for the treatment of patients with cardiac myocyte injury and ischemia.

Inhibition of epidermal growth factor receptor attenuates atherosclerosis via decreasing inflammation and oxidative stress.

Atherosclerosis is a progressive disease leading to loss of vascular homeostasis and entails fibrosis, macrophage foam cell formation, and smooth muscle cell proliferation. Recent studies have reported that epidermal growth factor receptor (EGFR) is involved vascular pathophysiology and in the regulation of oxidative stress in macrophages. Although, oxidative stress and inflammation play a critical role in the development of atherosclerosis, the underlying mechanisms are complex and not completely understood. In the present study, we have elucidated the role of EGFR in high-fat diet-induced atherosclerosis in apolipoprotein E null mice. We show increased EGFR phosphorylation and activity in atherosclerotic lesion development. EGFR inhibition prevented oxidative stress, macrophage infiltration, induction of pro-inflammatory cytokines, and SMC proliferation within the lesions. We further show that EGFR is activated through toll-like receptor 4. Disruption of toll-like receptor 4 or the EGFR pathway led to reduced inflammatory activity and foam cell formation. These studies provide evidence that EGFR plays a key role on the pathogenesis of atherosclerosis, and suggests that EGFR may be a potential therapeutic target in the prevention of atherosclerosis development.

MicroRNA-21 protects against cardiac hypoxia/reoxygenation injury by inhibiting excessive autophagy in H9c2 cells via the Akt/mTOR pathway.

MicroRNAs and autophagy play critical roles in cardiac hypoxia/reoxygenation (H/R)-induced injury. Here, we investigated the function of miR-21 in regulating autophagy and identified the potential molecular mechanisms involved. To determine the role of miR-21 in regulating autophagy, H9c2 cells were divided into the following six groups: control group, H/R group, (miR-21+ H/R) group, (miR-21-negative control + H/R) group, (BEZ235+ H/R) group and (miR-21+ BEZ235+ H/R) group. The cells underwent hypoxia for 1 hr and reoxygenation for 3 hrs. Cell count kit-8 was used to evaluate cell function and apoptosis was analysed by Western blotting. Western blotting and transmission electron microscopy were used to investigate autophagy. We found that miR-21 expression was down-regulated, and autophagy was remarkably increased in H9c2 cells during H/R injury. Overexpression of miR-21 with a miR-21 precursor significantly inhibited autophagic activity and decreased apoptosis, accompanied by the activation of the AKT/mTOR pathway. In addition, treatment with BEZ235, a novel dual Akt/mTOR inhibitor, resulted in a significant increase in autophagy and apoptosis. However, we found that miR-21-mediated inhibition of apoptosis and autophagy was partly independent of Akt/mTOR activation, as demonstrated in cells treated with both miR-21 and BEZ235. We showed that miR-21 could inhibit H/R-induced autophagy and apoptosis, which may be at least partially mediated by the Akt/mTOR signalling pathway.

Combined Treatment with Amlodipine and Atorvastatin Calcium Reduces Circulating Levels of Intercellular Adhesion Molecule-1 and Tumor Necrosis Factor-α in Hypertensive Patients with Prediabetes.

OBJECTIVE: To assess the effect of amlodipine and atorvastatin on intercellular adhesion molecule (ICAM)-1 and tumor necrosis factor (TNF)-α expression, as endothelial function and inflammation indicators, respectively, in hypertensive patients with and without prediabetes. METHODS: Forty-five consecutive patients with hypertension, diagnosed according to JNC7, were divided into two groups based on the presence (HD group, n = 23) or absence (H group, n = 22) of prediabetes, diagnosed according to 2010 ADA criteria, including impaired glucose tolerance (IGT) and fasting glucose tests. All patients simultaneously underwent 12-week treatment with daily single-pill amlodipine besylate/atorvastatin calcium combination (5/10 mg; Hisun-Pfizer Pharmaceuticals Co. Ltd). Serum isolated before and after treatment from overnight fasting blood samples was analyzed by ELISA. RESULTS: In the HD and H groups after vs. before 12-week amlodipine/atorvastatin treatment, there were significantly (all P < 0.01) lower levels of ICAM-1 (3.06 ± 0.34 vs. 4.07 ± 0.70 pg/ml; 3.26 ± 0.32 vs. 3.81 ± 0.60 pg/ml, respectively) and TNF-α (78.71 ± 9.19 vs. 110.94 ± 10.71 pg/ml; 80.95 ± 9.33 vs. 101.79 ± 11.72 pg/ml, respectively), with more pronounced reductions in HD vs. H group (ICAM-1Δ: 1.01 ± 0.80 vs. 0.55 ± 0.64 pg/ml, respectively, P = 0.037; TNF-αΔ: 32.23 ± 14.33 vs. 20.84 ± 14.89 pg/ml, respectively, P = 0.011), independent of the blood pressure (BP) and cholesterol level reduction. CONCLUSIONS: Amlodipine/atorvastatin improved endothelial function and inflammation, as reflected by lower circulating levels of ICAM-1 and TNF-α, more prominently in hypertensives with than without prediabetes. Starting statin treatment before overt diabetes in hypertensives might thus improve cardiovascular outcomes.

Atorvastatin suppresses NLRP3 inflammasome activation via TLR4/MyD88/NF-κB signaling in PMA-stimulated THP-1 monocytes.

AIMS: Increasing evidence shows that NLRP3 inflammasome is closely associated with the progression of atherosclerosis. The purpose of the present study was to evaluate the effects of atorvastatin on NLRP3 inflammasome in PMA-stimulated THP-1 cells and explore its underlying mechanism. METHODS: Human monocytic THP-1 cells were pretreated with atorvastatin for 1h and then induced by PMA for 48h. Total protein was collected for real-time PCR and Western blot analysis. Cytokine IL-1ß release was detected by ELISA assay. And the NF-κB p65 translocation was detected by cellular NF-κB translocation kit. RESULTS: It was shown that atorvastatin significantly reduced the expression of NLRP3, the cleavage of caspase-1 and IL-1ß in PMA-induced THP-1 cells. Moreover, Bay (a NF-κB inhibitor) treatment greatly suppressed the expression of NLRP3, the cleavage of caspase-1 and IL-1ß in PMA-induced THP-1 cells, suggesting that the activation of NF-κB pathway takes part in regulating the expression of NLRP3 inflammasome. In addition, atorvastatin markedly inhibited the up-regulation of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and the activation of nuclear factor kappa b (NF-κB) in PMA-stimulated THP-1 cells. CONCLUSIONS: Atorvastatin exerts an anti-inflammatory effect by inhibiting NLRP3 inflammasome through suppressing TLR4/MyD88/NF-κB pathway in PMA-induced THP-1 monocytes.

Serum Markers of Endothelial Dysfunction and Inflammation Increase in Hypertension with Prediabetes Mellitus.

AIMS: The aim of this study was to examine endothelial dysfunction and inflammation in hypertension and prediabetes by studying adhesion molecules and inflammatory factors. METHODS AND RESULTS: This study included 133 outpatients. Participants were categorized into three groups based on the presence or absence of hypertension and prediabetes: control subjects without prediabetes and hypertension (N group, n = 39); patients with hypertension only (H group, n = 34); and patients with hypertension and prediabetes (HD group, n = 60). Hypertension was diagnosed according to JNC7 criteria. Prediabetes was defined according to 2010 American Diabetes Association criteria. Plasma was isolated from overnight fasting blood samples for enzyme-linked immunosorbent assay (ELISA) analysis of concentrations of intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α), P-selectin, and interleukin-6 (IL-6) as indicators of endothelial function and inflammation. We found that the H and HD groups showed significantly higher levels of all four biomarkers compared with the N group (all p < 0.01). The HD group also showed significantly higher levels of ICAM-1 (p = 0.042) and TNF-α (p < 0.01) compared with the H group; no significant differences in P-selectin (p = 0.59) and IL-6 (p = 0.70) levels were observed among these groups. CONCLUSIONS: Prediabetes and hypertension induce endothelial dysfunction and inflammation by elevating levels of soluble adhesion molecules and inflammatory cytokines. The comorbidity of these diseases may exacerbate inflammation and endothelial dysfunction by enhancing the expression of ICAM-1 and TNF-α.