Identification and Characterization of Necroptosis-Related Differentially Expressed Genes in Acute Myocardial Infarction: Insights into Immune-Related Pathways and Protein-Protein Interactions.

Acute myocardial infarction (AMI) is a serious cardiovascular medical emergency that can lead to death. Necroptosis, a programmed cell death pathway, has been implicated in the development and progression of AMI. The aim of our study was to identify necroptosis-related differentially expressed genes (NRDEGs) in AMI and investigate their interactions and functions. The GSE66360 dataset was screened for NRDEGs using the 'limma' R package, with a threshold of p 0.05. A set of 159 necroptosis-related genes (NRGs) was retrieved from the KEGG database. The protein-protein interactions (PPI) network was constructed using the STRING data resource. Molecular Complex Detection (MCODE) and cytohHubba plugin was applied to find the major modules and genes. Gene ontology (GO) and KEGG pathway analyses were performed using the R 'clusterProfiler' package. The enrichment scores for immune cell types and associated biological pathways or functions were gained using the ssGSEA method. Our study identified 5 down-regulated and 16 up-regulated NRDEGs in AMI. The PPI network analysis revealed several important modules and hub genes, including TNF, IL1B, TLR4, STAT3, NLRP3, TNFAIP3, CYBB, IFNGR1, FADD, and IL33. GO analysis revealed that NRDEGs were enriched in multiple biological processes, cellular components, and molecular functions, including those related to cytokine production, response to cytokine stimulus, and necroptotic process. NRDEGs were found to be particularly abundant in a number of non-disease pathways, such as necroptosis and immune-related pathways like cytokine-cytokine receptor interaction and TNF signaling pathway, according to KEGG pathway analysis. The ssGSEA analysis revealed a correlation between immune cells and NRDEGs in AMI. The study identified NRDEGs and their interactions in AMI, providing insights into the potential function of necroptosis in the pathological process of AMI. The results imply that immune-related pathways and cytokines may be crucial in the initiation and development of AMI. The study provides a foundation for further research on the underlying mechanisms of necroptosis in AMI and the potential for developing novel therapies.

Risk factors for postoperative pulmonary complications in elderly patients receiving elective colorectal surgery: A retrospective study.

Study objective: Postoperative pulmonary complications (PPCs) are common and associated with adverse outcomes impairing long-term survival and quality of recovery. This single-centered retrospective study aimed to examine factors associated with PPCs in patients receiving elective colorectal surgery aged ≥60 years. Methods: Between January 2019 and December 2019, 638 patients at the Shanghai Changhai Hospital who had received elective surgery for colorectal cancer were enrolled in this study. Patients were divided into the PPC group (n=38) and non-PPC group (n=600). Neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), red blood cell distribution width (RDW), and systemic inflammatory index (SII) were selected and caculated to indicate preoperative and postoperative inflammatory status. Receiver operating characteristic curve and bivariate correlation analyses were performed to evaluate the identified risk factors. Main results: The overall incidence of PPCs was approximately 5.96%. Multivariate regression analysis identified age (OR = 1.094, 95%CI 1.038-1.153, P = 0.001), preoperative RDW (OR = 1.159, 95%CI 1.025-1.309, P = 0.018), and preoperative SII (OR = 1.001, 95%CI 1.000-1.003, P = 0.035) as independent risk factors for PPCs. The cut-off values of age, preoperative RDW, and preoperative SII for predicting PPCs were 69.5 (sensitivity 0.658, specificity 0.653), 13.2 (sensitivity 0.789, specificity 0.552) and 556.1 (sensitivity 0.579, specificity 0.672), respectively. Conclusions: Age, preoperative RDW, and preoperative SII were identified as independent risk factors for PPC occurrence in elderly patients receiving elective colorectal surgery. Further studies are warranted to evaluate whether normalization of preoperative RDW and SII, as modifiable risk factors, are associated with improved surgical outcomes.

Blood metabolomic profiling predicts postoperative gastrointestinal function of colorectal surgical patients under the guidance of goal-directed fluid therapy.

Postoperative gastrointestinal function influences postoperative recovery and length of hospital stay for patients undergoing colorectal surgery. Goal-directed fluid therapy (GDFT) restricts fluid administration to an amount required to prevent dehydration. Although the fluid management of GDFT could decrease the incidence of postoperative complications in patients who undergo high-risk surgery, certain patients may not respond to GDFT. Thus, to achieve optimal treatment, identification of patients suitable for GDFT is necessary. Metabolomic profiling of 48 patients undergoing surgery for colorectal cancer was performed. Patients were divided into delayed- and enhanced-recovered groups based on gastrointestinal function within 72 hours, and the results of omics analysis showed differential serum metabolites between the two groups of patients in the post anesthesia care unit 24 hours after surgery. A support vector machine model was applied to evaluate the curative effects of GDFT in different patients. Four metabolites, oleamide, ubiquinone-1, acetylcholine, and oleic acid, were found to be highly associated with postoperative gastrointestinal function and could be used as potential biomarkers. Moreover, four pathways were found to be highly related to postoperative gastrointestinal recovery. Among them, the vitamin B6 metabolism pathway may be a common pathway for improving postoperative recovery in various diseases. Our findings proposed a novel method to predict postoperative recovery of gastrointestinal function based on metabolomic profiling and suggested the potential mechanisms contributing to gastrointestinal function after surgical resection of colorectal cancer under the fluid management of GDFT.

Acetate attenuates perioperative neurocognitive disorders in aged mice.

Perioperative neurocognitive disorders are common in elderly patients who have undergone surgical procedures. Neuroinflammation induced by microglial activation is a hallmark of these neurological disorders. Acetate can suppress inflammation in the context of inflammatory diseases. We employed an exploratory laparotomy model with isoflurane anesthesia to study the effects of acetate on perioperative neurocognitive disorders in aged mice. Neurocognitive function was assessed with open-field tests and Morris water maze tests 3 or 7 days post-surgery. Acetate ameliorated the surgery-induced cognitive deficits of aged mice and inhibited the activation of IBA-1, a marker of microglial activity. Acetate also reduced expression of inflammatory proteins (tumor necrosis factor-α, interleukin-1ß and interleukin-6), oxidative stress factors (NADPH oxidase 2, inducible nitric oxide synthase and reactive oxygen species), and signaling molecules (nuclear factor kappa B and mitogen-activated protein kinase) in the hippocampus. BV2 microglial cells were used to verify the anti-inflammatory effects of acetate in vitro. Acetate suppressed inflammation in lipopolysaccharide-treated BV2 microglial cells, but not when GPR43 was silenced. These results suggest that acetate may bind to GPR43, thereby inhibiting microglial activity, suppressing neuroinflammation, and preventing memory deficits. This makes acetate is a promising therapeutic for surgery-induced neurocognitive disorders and neuroinflammation.

Acetate attenuates inflammasome activation through GPR43-mediated Ca2+-dependent NLRP3 ubiquitination.

Acetate has been indicated to be elevated and to regulate inflammation in inflammatory and metabolic diseases. The inflammasome serves as a key component of immune homeostasis, and its dysregulation can lead to various inflammatory disorders. However, little is known about the effects of acetate on inflammasome activation and the underlying mechanism. Here, we demonstrate that acetate attenuates inflammasome activation via GPR43 in a Ca2+-dependent manner. Through binding to GPR43, acetate activates the Gq/11 subunit and subsequent phospholipase C-IP3 signaling to decrease Ca2+ mobilization. In addition, acetate activates soluble adenylyl cyclase (sAC), promotes NLRP3 inflammasome ubiquitination by PKA, and ultimately induces NLRP3 degradation through autophagy. In vivo, acetate protects mice from NLRP3 inflammasome-dependent peritonitis and LPS-induced endotoxemia. Collectively, our research demonstrates that acetate regulates the NLRP3 inflammasome via GPR43 and Ca2+-dependent mechanisms, which reveals the mechanism of metabolite-mediated NLRP3 inflammasome attenuation and highlights acetate as a possible therapeutic strategy for NLRP3 inflammasome-related diseases.

Publisher Correction: Acetate attenuates inflammasome activation through GPR43-mediated Ca2+-dependent NLRP3 ubiquitination.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Exercise training lowers the enhanced tonically active glutamatergic input to the rostral ventrolateral medulla in hypertensive rats.

AIMS: It is well known that low-intensity exercise training (ExT) is beneficial to cardiovascular dysfunction in hypertension. The tonically active glutamatergic input to the rostral ventrolateral medulla (RVLM), a key region for control of blood pressure and sympathetic tone, has been demonstrated to be increased in hypertensive rats. The aim of this study was to determine the effect of ExT on the increased glutamatergic input to the RVLM in spontaneously hypertensive rat (SHR). METHODS: Normotensive rats Wistar-Kyoto (WKY) and SHR were treadmill trained or remained sedentary (Sed) for 12 weeks and classed into four groups (WKY-Sed, WKY-ExT, SHR-Sed, and SHR-ExT). The release of glutamate in the RVLM and its contribution to cardiovascular activity were determined in WKY and SHR after treatment of ExT. RESULTS: Blood pressure and sympathetic tone were significantly reduced in SHR after treatment with ExT. Bilateral microinjection of the glutamate receptor antagonist kynurenic acid (2.7 nmol in 100 nL) into the RVLM significantly decreased resting blood pressure, heart rate, and renal sympathetic nerve activity in SHR-Sed but not in WKY groups (WKY-Sed and WKY-ExT). However, the degree of reduction in these cardiovascular parameters evoked by KYN was significantly blunted in SHR-ExT compared with SHR-Sed group. The concentration of glutamate and the protein expression of vesicular glutamate transporter 2 in the RVLM were significantly increased in SHR-Sed compared with WKY-Sed, whereas they were reduced after treatment with ExT. CONCLUSION: Our findings suggest that ExT attenuates the enhancement in the tonically acting glutamatergic input to the RVLM of hypertensive rats, thereby reducing the sympathetic hyperactivity and blood pressure.

Association of serum a disintegrin and metalloproteinase with thrombospodin motif 4 levels with the presence and severity of coronary artery disease.

OBJECTIVE: A disintegrin and metalloproteinase with thrombospodin motif 4 (ADAMTS4) has been shown to be an important player in atherosclerosis. However, the clinical significance of measuring serum ADAMTS4 levels has not been fully elucidated. We therefore investigate whether serum ADAMTS4 levels are associated with the presence and severity of coronary artery disease (CAD). METHODS: Serum levels of ADAMTS4 were measured in 192 patients undergoing elective coronary angiography for suspected CAD, the severity of CAD was determined by the number of diseased vessels and the severity score of coronary stenosis. RESULTS: Patients with CAD showed significantly higher levels of ADAMTS4 than did patients with normal coronary arteries [57.82 (48.96-70.32) vs. 46.55 (41.16-51.72) ng/ml, P<0.001]. ADAMTS4 levels increased with the number of diseased vessels (P<0.05) and significantly associated with severity score of stenosis (rs=0.601, P<0.001). In the multivariate analysis, ADAMTS4 levels were found to be independently correlated with the presence and severity of CAD. Receiver-operating characteristic analysis revealed that a cut-off of serum ADAMTS4 levels of 51.63 ng/ml could predict CAD with a 76% sensitivity and a 69% specificity. ADAMTS4 levels were significantly lower in patients with statin treatment than in those without it [47.49 (42.30, 57.09) vs. 56.39 (47.05, 68.94) ng/ml, P<0.05]. CONCLUSION: In conclusion, serum ADAMTS4 levels are associated with the presence and the severity of CAD, ADAMTS4 might serve as an independent factor for predicting CAD. Statin therapy reduces the serum levels of ADAMTS4.

The quantification of ADAMTS4 and 8 expression and selection of reference genes for quantitative real-time PCR analysis in myocardial infarction.

INTRODUCTION: ADAMTS4 and ADAMTS8 are proteases involved in ECM proteolysis and antiangiogenesis, but little is known about their expression and function in myocardial infarction (MI). We examined ADAMTS4 and ADAMTS8 expression in a rat MI model by quantitative real-time polymerase chain reaction (qPCR) and enzyme linked immunosorbent assay (ELISA). The expressions of glyseraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB), acidic ribosomal phosphoprotein P0 (ARBP), and ribosomal protein L13A (RPL13A) were examined in order to validate the appropriate housekeeping genes after MI. METHODS: Male Wistar rats were subjected to MI, and infarcted myocardial tissue was collected at 3, 6, 12, 24h, 3, 7, 14 and 21days after MI. ADAMTS4, ADAMTS8, and the four housekeeping genes were quantified using qPCR and the expression stability of the four housekeeping genes was investigated using GeNorm software. The protein levels of ADAMTS4 were detected using ELISA kits. RESULTS: The M values of GAPDH, ACTB, ARBP and RPL13A were 0.721, 1.2, 0.812 and 0.812 respectively. GAPDH and ARBP were ranked the most stable genes. ADAMTS4 mRNA increased at 3h after MI, peaked at 6h, then decreased rapidly. ADAMTS8 mRNA increased at 6h, peaked at 24h, remained high at 3d, then decreased gradually. The protein levels of ADAMTS4 were significantly increased at 6h, 12h, 24h and 3d after MI. CONCLUSION: The results suggest that GAPDH and ARBP are two appropriate housekeeping genes for the rat MI model. Both ADAMTS4 and ADAMTS8 mRNA levels and ADAMTS4 protein level increased, but they exhibited different expression profiles.

ADAMTS4 level in patients with stable coronary artery disease and acute coronary syndromes.

OBJECTIVE: A recent study indicates that ADAMTS4 (a disintegrin and metalloprotease with thrombospondin motifs 4) was expressed in macrophage rich areas of human atherosclerotic carotid plaques and coronary unstable plaques suggesting a pathogenic role in the development of acute coronary syndromes (ACS). The aim of the study was to compare ADAMTS4 across the entire spectrum of coronary artery disease (CAD) and to investigate the temporal profiles of ADAMTS4. METHODS: Plasma levels of ADAMTS4 were measured in patients with stable effort angina pectoris (SAP), ACS and in controls. Venous blood was sampled upon admission before angiography and drug administration. In patients with ACS who underwent medical treatment, serial blood samples were also collected on days 1, 2, 3, 5 and 7 after admission. ADAMTS4 was measured using an enzyme immunoassay. RESULTS: Plasma ADAMTS4 level in cases was significantly greater than in controls (P<0.001). Higher levels of ADAMTS4 were found with progression of CAD from SAP to unstable angina pectoris (UAP) to non-ST-segment elevation acute myocardial infarction (NSTEMI) and to ST-segment elevation acute myocardial infarction (STEMI) (P<0.001). Elevated ADAMTS4 level was associated with ACS with an area under receiver operating characteristic (ROC) curve of 0.753 (95% CI 0.654-0.851; P<0.001). The pattern of ADAMTS4 release observed was clearly different in various forms of ACS. ADAMTS4 showed a weak correlation with high-sensitivity C-reactive protein (hs-CRP); however, no significant correlation was found between ADAMTS4 and troponin T (TnT) in ACS patients. CONCLUSIONS: Serial changes in plasma ADAMTS4 were documented in patients with ACS and may serve as a marker of plaque destabilization.

Elevated level of ADAMTS4 in plasma and peripheral monocytes from patients with acute coronary syndrome.

OBJECTIVES: A recent study shows that ADAMTS4 is expressed in macrophage-rich areas of human atherosclerotic carotid plaques and coronary unstable plaques, suggesting a pathogenic role of ADAMTS4 in the development of acute coronary syndrome (ACS). We investigated (a) whether the expression level of ADAMTS4 in plasma and peripheral blood mononuclear cells was affected; and (b) whether there was a relationship with hs-CRP level and the stability of coronary atherosclerotic plaque in patients with ACS. METHODS: Our study included 30 normal controls and 120 patients including 40 with stable angina (SA), 50 with unstable angina (UA), and 30 with acute myocardial infarction (AMI). The expression of ADAMTS4 in monocytes was analyzed by RT-PCR and plasma ADAMTS4 level was determined by ELISA. All coronary stenosis with >30% diameter reduction was assessed by angiographic coronary stenosis morphology. RESULTS: Patients with ACS showed a significant increase of ADAMTS4 (2.7 ± 0.4) expression in monocytes compared with controls (1.1 ± 0.2) and the SA group (1.3 ± 0.2) (P < 0.001). Plasma ADAMTS4 also showed a higher level in ACS patients (100.2 ± 31.6 ng/ml) than in control (47.5 ± 9.0 ng/ml, P < 0.001) and the SA group (54.3 ± 13.2 ng/ml, P < 0.001). Moreover, we found a positive correlation between hs-CRP and ADAMTS4 expression in monocytes as well as in plasma. There was also a positive correlation of ADAMTS4 expression in monocytes and plasma with complex coronary stenosis (r (1) = 0.61, r (2) = 0.57, P < 0.001). CONCLUSIONS: Patients with ACS showed increased ADAMTS4 expression, which may aggravate the development of atherosclerosis and instability of atherosclerotic plaques. Therefore, the ADAMTS4 expression may be a valuable marker for predicting the severity of ACS.

[Expression and purification of recombinant glycoprotein (GP) IIb/IIIa receptor antagonists].

To investigate the effect of GST-KGDX (glutathione S-transferase-Lys-Gly-Asp-X) fusion protein, GP IIb/IIIa receptor antagonist, on platelet function in vitro. The KGDX (Lys-Gly-Asp-X) gene was assembled from 2 synthetic oligonucleotides, 36 bp in length, using BamH I and Xho I restriction enzyme sites at the end of the gene for cloning into the expression vector pGEX4T-1. Expression of fusion protein was directed by the tac promoter. The Escherichia coli DH5a contained the plasmid pGEX-4T-1-KGDX was expressed by 37 degrees C heat induction. The fusion protein of KGDX with glutathione S-transferase (GST-KGDX) was purified in one step from the bacterial lysate by glutathione-agarose beads for affinity chromatography. GST-KGDX was found to be soluble and abundant, the yield of 35 mg/L of cultures was obtained. The GST-KGDX was expressed in E. coli to a level of 48.02% of total cellular protein. GST-KGDX inhibited ADP-induced human platelet aggregation stronger than GST (P < 0.05 or < 0.01). In flow cytometry assay for fibrinogen binding, both GST and GST-KGDX inhibited platelet aggregation by binding with high affinity to GPIIb/IIIa. Mean fluorescence intensity of GST-KGDX fusion protein was significantly higher than that of GST. It is concluded that the GST-KGDX fusion protein can be produced by E. coli and used as an antiplatelet agent.