Splenic monocytes mediate inflammatory response and exacerbate myocardial ischemia/reperfusion injury in a mitochondrial cell-free DNA-TLR9-NLRP3-dependent fashion.

The spleen contributes importantly to myocardial ischemia/reperfusion (MI/R) injury. Nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) recruits inflammasomes, initiating inflammatory responses and mediating tissue injury. We hypothesize that myocardial cell-free DNA (cfDNA) activates the splenic NLRP3 inflammasome during early reperfusion, increases systemic inflammatory response, and exacerbates myocardial infarct. Mice were subjected to 40 min of ischemia followed by 0, 1, 5, or 15 min, or 24 h of reperfusion. Splenic leukocyte adoptive transfer was performed by injecting isolated splenocytes to mice with splenectomy performed prior to left coronary artery occlusion. CY-09 (4 mg/kg) was administered 5 min before reperfusion. During post-ischemic reperfusion, splenic protein levels of NLRP3, cleaved caspase-1, and interleukin-1ß (IL-1ß) were significantly elevated and peaked (2.1 ± 0.2-, 3.4 ± 0.4-, and 3.2 ± 0.2-fold increase respectively, p < 0.05) within 5 min of reperfusion. In myocardial tissue, NLRP3 was not upregulated until 24 h after reperfusion. Suppression by CY09, a specific NLRP3 inflammasome inhibitor, or deficiency of NLRP3 significantly reduced myocardial infarct size (17.3% ± 4.2% and 33.2% ± 1.8% decrease respectively, p < 0.01). Adoptive transfer of NLRP3-/- splenocytes to WT mice significantly decreased infarct size compared to transfer of WT splenocytes (19.1% ± 2.8% decrease, p < 0.0001). NLRP3 was mainly activated at 5 min after reperfusion in CD11b+ and LY6G- splenocytes, which significantly increased during reperfusion (24.8% ± 0.7% vs.14.3% ± 0.6%, p < 0.0001). The circulating cfDNA level significantly increased in patients undergoing cardiopulmonary bypass (CPB) (43.3 ± 5.3 ng/mL, compared to pre-CPB 23.8 ± 3.5 ng/mL, p < 0.01). Mitochondrial cfDNA (mt-cfDNA) contributed to NLRP3 activation in macrophages (2.1 ± 0.2-fold increase, p < 0.01), which was inhibited by a Toll-like receptor 9(TLR9) inhibitor. The NLRP3 inflammasome in splenic monocytes is activated and mediates the inflammatory response shortly after reperfusion onset, exacerbating MI/R injury in mt-cfDNA/TLR9-dependent fashion. The schema reveals splenic NLRP3 mediates the inflammatory response in macrophages and exacerbates MI/R in a mitochondrial cfDNA/ TLR9-dependent fashion.

Corrigendum: The Emerging Role of Rho Guanine Nucleotide Exchange Factors in Cardiovascular Disorders: Insights Into Atherosclerosis: A Mini Review.

[This corrects the article DOI: 10.3389/fcvm.2021.782098.].

RNA N6-methyladenosine modulates endothelial atherogenic responses to disturbed flow in mice.

Atherosclerosis preferentially occurs in atheroprone vasculature where human umbilical vein endothelial cells are exposed to disturbed flow. Disturbed flow is associated with vascular inflammation and focal distribution. Recent studies have revealed the involvement of epigenetic regulation in atherosclerosis progression. N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic mRNA, but its function in endothelial atherogenic progression remains unclear. Here, we show that m6A mediates the epidermal growth factor receptor (EGFR) signaling pathway during EC activation to regulate the atherosclerotic process. Oscillatory stress (OS) reduced the expression of methyltransferase like 3 (METTL3), the primary m6A methyltransferase. Through m6A sequencing and functional studies, we determined that m6A mediates the mRNA decay of the vascular pathophysiology gene EGFR which leads to EC dysfunction. m6A modification of the EGFR 3' untranslated regions (3'UTR) accelerated its mRNA degradation. Double mutation of the EGFR 3'UTR abolished METTL3-induced luciferase activity. Adenovirus-mediated METTL3 overexpression significantly reduced EGFR activation and endothelial dysfunction in the presence of OS. Furthermore, thrombospondin-1 (TSP-1), an EGFR ligand, was specifically expressed in atheroprone regions without being affected by METTL3. Inhibition of the TSP-1/EGFR axis by using shRNA and AG1478 significantly ameliorated atherogenesis. Overall, our study revealed that METTL3 alleviates endothelial atherogenic progression through m6A-dependent stabilization of EGFR mRNA, highlighting the important role of RNA transcriptomics in atherosclerosis regulation.

The Emerging Role of Rho Guanine Nucleotide Exchange Factors in Cardiovascular Disorders: Insights Into Atherosclerosis: A Mini Review.

Atherosclerosis is a leading cause of cardiovascular disease, and atherosclerotic cardiovascular disease accounts for one-third of global deaths. However, the mechanism of atherosclerosis is not fully understood. It is well-known that the Rho GTPase family, especially Rho A, plays a vital role in the development and progression of arteriosclerosis. Rho guanine nucleotide exchange factors (Rho GEFs), which act upstream of Rho GTPases, are also involved in the atheromatous pathological process. Despite some research on the role of Rho GEFS in the regulation of atherosclerosis, the number of studies is small relative to studies on the essential function of Rho GEFs. Some studies have preliminarily revealed Rho GEF regulation of atherosclerosis by experiments in vivo and in vitro. Herein, we review the advances in research on the relationship and interaction between Rho GEFs and atheroma to provide a potential reference for further study of atherosclerosis.

MicroRNA-34c Inhibits Osteogenic Differentiation and Valvular Interstitial Cell Calcification via STC1-Mediated JNK Pathway in Calcific Aortic Valve Disease.

Calcific aortic valve disease (CAVD), a common heart valve disease, is increasingly prevalent worldwide and causes high morbidity and mortality. Here, we aimed to investigate a possible role for miR-34c in the development of osteogenic differentiation during CAVD and to find out the underlying mechanisms. Valvular interstitial cells (VICs) were isolated from the clinical aortic valve tissue samples of CAVD patients and patients with acute aortic dissection and collected. Then, RT-qPCR was performed to determine miR-34c expression and western blot analysis was applied to confirm the relevant protein expression in these VICs. Dual luciferase reporter gene assay was applied to confirm the relation between miR-34c and STC1. Alkaline phosphatase (ALP) staining and alizarin red staining was performed to further confirm the degree of calcification in these samples. MiR-34c was lowly expressed and STC1 was highly expressed in the CAVD tissues. Furthermore, STC1 was the target of miR-34c and was negatively regulated by miR-34c. Overexpression of miR-34c in VICs was concomitant with suppression of both STC1 expression and phosphorylation level of c-Jun N-terminal kinase (JNK). In addition, significant decrease of bone morphogenetic protein-2 (BMP2) and osteocalcin, as well as the decrease of calcification degree were also observed in VICs with miR-34c overexpressed. Taken together, miR-34c could inhibit osteogenic differentiation and calcification of VICs by suppressing the STC1/JNK signaling pathway in CAVD, making miR-34c a novel therapeutic target for the treatment of CAVD.

Upregulation of microRNA-195 ameliorates calcific aortic valve disease by inhibiting VWF via suppression of the p38-MAPK signaling pathway.

BACKGROUND: Growing evidence has indicated that microRNAs (miRNAs) are involved in the progression of calcific aortic valve disease (CAVD), a progressive pathological condition with no effective pharmacological therapy. This study was set out with the aim to investigate possible roles of miR-195 in CAVD. METHODS AND RESULTS: Initially, the differential expressed genes (DEGs) associated with CAVD were screened out and miRNAs potentially regulating VWF were predicted from microarray analysis. Next, we quantified VWF and miR-195 expression in isolated aortic valve interstitial cells (AVICs) and aortic valve tissues, followed by confirmation of the target relationship between miR-195 and VWF using the dual luciferase reporter assay. Furthermore, we evaluated the biological functions of miR-195 and VWF on ALP activity, cell differentiation, and the levels of miR-195, VWF, Runx2, OCN, ALP, p38 and phosphorylated p38 in AVICs. VWF was highly expressed, while miR-195 was poorly expressed in CAVD. Furthermore, miR-195 targeted VWF and negatively regulated its expression. Upregulation of miR-195 or silencing VWF could reduce ALP activity, calcified deposition, and the mRNA and protein levels of Runx2, OCN, and ALP by inhibiting the p38-MAPK signaling pathway, thereby ameliorating aortic valve calcification in vitro. CONCLUSIONS: On all accounts, miR-195 can potentially inhibit aortic valve calcification by repressing VWF and p38-MAPK signaling pathway, highlighting a theoretical basis for pharmacological treatment of CAVD.

The Effects of Inhibition of MicroRNA-375 in a Mouse Model of Doxorubicin-Induced Cardiac Toxicity.

BACKGROUND Doxorubicin-induced myocardial toxicity is associated with oxidative stress, cardiomyocyte, apoptosis, and loss of contractile function. Previous studies showed that microRNA-375 (miR-375) expression was increased in mouse models of heart failure and clinically, and that inhibition of miR-375 reduced inflammation and increased survival of cardiomyocytes. This study aimed to investigate the effects and mechanisms of inhibition of miR-375 in a mouse model of doxorubicin-induced cardiac toxicity in vivo and in doxorubicin-treated rat and mouse cardiomyocytes in vitro. MATERIAL AND METHODS The mouse model of doxorubicin-induced cardiac toxicity was developed using an intraperitoneal injection of doxorubicin (15 mg/kg diluted in 0.9% saline) for eight days. Treatment was followed by a single subcutaneous injection of miR-375 inhibitor. H9c2 rat cardiac myocytes and adult murine cardiomyocytes (AMCs) were cultured in vitro and treated with doxorubicin, with and without pretreatment with miR-375 inhibitor. RESULTS Doxorubicin significantly upregulated miR-375 expression in vitro and in vivo, and inhibition of miR-375 re-established myocardial redox homeostasis, prevented doxorubicin-induced oxidative stress and cardiomyocyte apoptosis, and activated the PDK1/AKT axis by reducing the direct binding of miR-375 to 3' UTR of the PDK1 gene. Inhibition of PDK1 and AKT abolished the protective role of miR-375 inhibition on doxorubicin-induced oxidative damage. CONCLUSIONS Inhibition of miR-375 prevented oxidative damage in a mouse model of doxorubicin-induced cardiac toxicity in vivo and in doxorubicin-treated rat and mouse cardiomyocytes in vitro through the PDK1/AKT signaling pathway.

Genetic polymorphisms and transcription profiles associated with intracranial aneurysm: a key role for NOTCH3.

Intracranial aneurysm (IA) incidence is about 1~2%. However, the specific mechanisms of IA onset and development need further study. Our objective was to discover novel IA-related genes to determine possible etiologies further. We performed next-generation sequencing on nineteen Chinese patients with familial IA and one patient with sporadic IA. We obtained mRNA expression data of 129 samples from Gene Expression Omnibus (GEO) and made statistical computing to discover differentially expressed genes (DEGs). The screened IA-related gene NOTCH3 was determined by bioinformatic data mining. We verified the IA-related indicators of NOTCH3. Association was found between IA and the NOTCH3 SNPs rs779314594, rs200504060 and rs2285981. Levels of NOTCH3 mRNA were lower in IA tissue than in control tissue, but higher in peripheral blood neutrophils from IA patients than in neutrophils from controls. Levels of NOTCH3 protein were lower in IA tissue than in cerebral artery tissue. NOTCH3 also decreased the expression of angiogenesis factors in human umbilical vein endothelial cells. Variation in NOTCH3 and alteration of its expression in cerebral artery or neutrophils may contribute to IA. Our findings also describe a bioinformatic-experimental approach that may prove useful for probing the pathophysiology of other complex diseases.

Genetic polymorphisms in Sox17 associated with intracranial aneurysm in Chinese Han people: a genotype-phenotype study.

PURPOSE: Genetic factors play a vital role in intracranial aneurysm (IA) onset and development. Studying the relationship between IA and the Sox17 polymorphisms in diverse populations is essential for establishing credibility. PATIENTS AND METHODS: We collected blood samples derived from a total of 596 sporadic IA patients and 600 individual controls in several medical institutes in China. We used the Sequenom MassArray system for single nucleotide polymorphisms (SNPs) genotyping after DNA extraction. The SNPs data was tested and analyzed in PLINK (version 1.9). Multiple-testing was performed in PLINK to make the statistics more rigorous and accurate. RESULTS: We found that the allelic G of rs1072737 (OR=1.303, genomic-control corrected P-value =0.001032) is a risk allele, while the allelic G of rs9298506 (OR=0.7253, genomic-control corrected P-value =0.01559) is a protective allele in Chinese Han people. CONCLUSION: The allelic G of rs1072737 is a risk factor for IA, while the allelic G of rs9298506 serves as a protective factor for IA in Chinese Han people.

Infarct-Sparing Effect of Adenosine A2B Receptor Agonist Is Primarily Due to Its Action on Splenic Leukocytes Via a PI3K/Akt/IL-10 Pathway.

BACKGROUND: Adenosine A2B receptor (A2BAR) agonist reduces myocardial reperfusion injury by acting on inflammatory cells. Recently, a cardiosplenic axis was shown to mediate the myocardial postischemic reperfusion injury. This study aimed to explore whether the infarct-squaring effect of A2BAR agonist was primarily due to its action on splenic leukocytes. METHODS: C57BL6 (wild type [WT]) mice underwent 40 min of left coronary artery occlusion followed by 60 min of reperfusion. A2BAR knockout (KO) and interleukin (IL)-10KO mice served as donors for splenic leukocytes. Acute splenectomy was performed 30 min before ischemia. The acute splenic leukocyte adoptive transfer was performed by injecting 5 × 106 live splenic leukocytes into splenectomized mice. BAY 60-6583, an A2BAR agonist, was injected by i.v. 15 min before ischemia. The infarct size (IS) was determined using 2,3,5-triphenyltetrazolium chloride and Phthalo blue staining. The expression of p-Akt and IL-10 was estimated by Western blotting. Immunofluorescence staining assessed the localization of IL-10 expression. RESULTS: BAY 60-6583 reduced the myocardial IS in intact mice but failed to reduce the same in splenectomized mice, which had a smaller IS than intact mice. BAY 60-6583 reduced the IS in splenectomized mice with the acute transfer of WT splenic leukocytes; however, it did not protect the heart of splenectomized mice with the acute transfer of A2BRKO splenic leukocytes. Furthermore, BAY 60-6583 increased the levels of p-Akt and IL-10 in the WT spleen. Moreover, it did not exert any protective effect in IL-10KO mice. CONCLUSIONS: A2BAR activation before ischemia stimulated the IL-10 production in splenic leukocytes via a PI3K/Akt pathway, thereby exerting anti-inflammatory effects that limited the myocardial reperfusion injury.

Early and long-term results of combined cardiac surgery and neoplastic resection in patients with concomitant severe heart disease and neoplasms.

BACKGROUND: It is a surgical dilemma when patients present with both severe heart disease and neoplasms. The best surgical treatment remains controversial. This study aimed to analyze the early and long-term results of simultaneous surgical treatment of severe heart disease and neoplasms. METHODS: We reviewed the clinical records of 15 patients who underwent simultaneous neoplastic resection and cardiac surgery between September 2006 and January 2011. There were 5 male and 10 female patients. The mean age was (59.2 ± 12.5) years and the mean left ventricular ejection fraction was (57.4 ± 11.0)%. All patients were followed up completely for a period of 12 to 51 months (mean, (33.1 ± 11.2) months). RESULTS: Fifteen patients underwent simultaneous cardiac surgery and neoplastic resection. Cardiac procedures consisted of off pump coronary artery bypass grafting (n = 7), aortic valve replacement (n = 3), mitral valve replacement (n = 3), mitral valve replacement with coronary artery bypass grafting (n = 1) and left atrial myxoma resection (n = 1). Neoplastic resection consisted of lung cancer resection (n = 5), colonic cancer resection (n = 3), gallbladder resection (n = 1), colonic cancer resection with gallbladder resection (n = 1), hysterectomy (n = 2), hysterectomy with bilateral salpingo-oophorectomy (n = 2) and left ovariectomy (n = 1). Pathological examination confirmed malignant disease in 10 patients and benign disease in 5 patients. There were no perioperative myocardial infarctions, stroke, pericardial tamponade, renal failure or hospital deaths. The most frequent complications were atrial fibrillation (33.3%), pneumonia (26.7%), low cardiac output syndrome (6.7%) and delayed healing of surgical wounds (6.7%). There was 1 late death 42 months after surgery for recurrent malignant disease. At 1 and 3 years, survival rates were 100% (Kaplan-Meier method). CONCLUSIONS: Simultaneous cardiac surgery and neoplastic resection was not associated with increased early or late morbidity or mortality. Cardiopulmonary bypass does not appear to adversely affect survival in patients with malignant disease. The long-term survival was determined by tumor stage.

Postconditioning inhibits myocardial apoptosis during prolonged reperfusion via a JAK2-STAT3-Bcl-2 pathway.

BACKGROUND: Postconditioning (PostC) inhibits myocardial apoptosis after ischemia-reperfusion (I/R) injury. The JAK2-STAT3 pathway has anti-apoptotic effects and plays an essential role in the late protection of preconditioning. Our aim was to investigate the anti-apoptotic effect of PostC after prolonged reperfusion and the role of the JAK2-STAT3 pathway in the anti-apoptotic effect of PostC. METHODS: Wistar rats were subjected to 30 minutes ischemia and 2 or 24 hours (h) reperfusion, with or without PostC (three cycles of 10 seconds reperfusion and 10 seconds reocclusion at the onset of reperfusion). Separate groups of rats were treated with a JAK2 inhibitor (AG490) or a PI3K inhibitor (wortmannin) 5 minutes before PostC. Immunohistochemistry was used to analyze Bcl-2 protein levels after reperfusion. mRNA levels of Bcl-2 were detected by qRT-PCR. TTC staining was used to detect myocardial infarction size. Myocardial apoptosis was evaluated by TUNEL staining. Western-blot was used to detect p-STAT3 and p-Akt levels after reperfusion. RESULTS: There was more myocardial apoptosis at 24 h vs 2 h after reperfusion in all groups. PostC significantly reduced myocardial apoptosis and elevated Bcl-2 levels at both 2 and 24 hours after reperfusion. PostC increased p-STAT3 and p-Akt levels after reperfusion. Administration of AG490 reduced p-STAT3 and p-Akt levels and attenuated the anti-apoptotic effect of PostC. Wortmannin also reduced p-Akt levels and attenuated the anti-apoptotic effect of PostC but had no effect on p-STAT3 levels. AG490 abrogated the up-regulation of Bcl-2 by PostC. CONCLUSION: PostC may reduce myocardial apoptosis during prolonged reperfusion via a JAK2-STAT3-Bcl-2 pathway. As a downstream target of JAK2 signaling, activation of PI3K/Akt pathway may be necessary in the protection of PostC.

Perioperative brain natriuretic peptide in off-pump coronary artery bypass.

BACKGROUND: During the last decade brain natriuretic peptide (BNP) has been recognized as a useful marker for acute and chronic left ventricular dysfunction. The present study was designed to evaluate the clinical relevance of BNP before and after off-pump coronary artery bypass (OPCAB). METHODS: One hundred and twelve patients undergoing primary OPCAB were divided into two groups by preoperative BNP levels (group A, BNP < or = 100 pg/ml and group B, BNP > 100 pg/ml). Levels of BNP and MB isoenzyme of creatine kinase (CK-MB) were measured preoperatively, 6 hours and 1 day post-operatively. Echocardiographic and clinical data were collected. RESULTS: Patients in group A had smaller perioperative left ventricular end-diastolic dimensions (LVEDD) and greater left ventricular ejection fractions (LVEF) compared to group B (P < 0.05). Levels of BNP and CKMB increased postoperatively in both groups (P < 0.01). However, there was no relationship between postoperative BNP and CKMB at any time point. Logistic regression analyses showed that a preoperative BNP level > 100 pg/ml was an independent risk factor for ventilation > 24 hours (odds ratio, OR = 13.33; 95% CI: 1.42-125.03) and ICU stay > 72 hours (OR = 3.01; 95% CI: 1.09-8.33). CONCLUSION: The baseline BNP level correlated with preoperative ventricular function and longer durations of ventilation and hospital stay after OPCAB. BNP increased early after operation. However, postoperative BNP did not correlate with myocardial injury or clinical results after OPCAB.