Serum Of Coronary Artery Disease Patients Research Articles

Role of miR-128-3p and miR-195-5p as biomarkers of coronary artery disease in Indians: a pilot study

Coronary artery disease (CAD) imposes a significant economic burden in developing countries like India. Timely diagnosis and treatment should be prioritized to mitigate the disease. Current diagnostic tools being invasive and less specific raise the need to develop less invasive and more reliable molecular biomarkers. MicroRNAs (miRNAs) are an emerging class of molecules that can serve as a potential source of non-invasive biomarkers for CAD. The objective of this study was to determine the potential of circulatory miRNAs as diagnostic biomarkers in CAD. In this study, we have reported two microRNAs, miR-128-3p and miR-195-5p in the serum of CAD patients in Indian Population. A total of 124 subjects were recruited which included 89 angiographically proven CAD patients and 35 control subjects. Our results show a significant decrease in the levels of miR-128-3p in CAD patients while there were no significant changes in the levels of miR-195-5p. Further bioinformatics analysis revealed the potential role of miR-128-3p in cholesterol homeostasis. Altered homeostasis due to cholesterol accumulation in macrophages is the driving force behind formation of foam cells which in turn accelerates the progression of CAD. Here, we have shown that miR-128-3p increases cholesterol levels in macrophages by decreasing cholesterol efflux in-vitro.

Correlation of circulatory VEGF and miRNA 126 in angiographically proven coronary artery disease and its clinical importance

Background: Atherosclerotic ischemic coronary artery disease (CAD) is a major cause of morbidity and mortality in both developed and developing countries, indicating the need for the discovery of deeper molecular insights of CAD mechanisms, biomarkers, and innovative therapeutic targets. Vascular endothelial growth factor (VEGF) may have some role, in the progression of human coronary atherosclerosis. Circulating microRNAs (miR) have a potential as a diagnostic as well as a prognostic biomarker of cardiovascular disease and dysfunctions. Aims and Objectives: The aim of the study was to correlate the levels of miRNA-126 and VEGF in the serum of CAD patients. Materials and Methods: The study was conducted on 100 cases of freshly diagnosed CAD patients (angiographically proven) and 80 normal healthy patients. Various serum biomarkers were estimated. The level of serum VEGF and Mi-RNA was correlated for CAD patients. Results: Highly significantly difference was found in levels of hemoglobin A1C, low density lipoprotein, total cholesterol, and total homocysteine while high-density lipoproteins and triglycerides were less significantly different for controls and cases. VEGF levels of CAD cases were found to be very significantly higher as compared to controls. The miRNA-126 expression was found to be lower in the CAD subjects compared to non-CAD subjects. Conclusion: The study was suggested the role of VEGF in atherogenesis and plaque instability. No significant correlation was found among between miR-126 levels and VEGF levels among CAD cases. The study is limited by a smaller sample size and was single-centered; therefore, multicentric study with a larger patient cohort needs to be done and to validate our findings.

NR3C2 mediates oxidised low-density lipoprotein-induced human coronary endothelial cells dysfunction via modulation of NLRP3 inflammasome activation

Nuclear receptor subfamily 3 group C member 2 (NR3C2) has been revealed to affect the progression of multiple inflammatory diseases, while NR3C2’s efficacy in coronary artery disease (CAD) remains largely unsolved. The study intended to elucidate the possible mechanisms of NR3C2 in oxidised low density lipoprotein (ox-LDL)-induced inflammation in human coronary endothelial cells (HCAECs) via regulating NACHT, LRR, and PYD domains-containing protein 3 (NLRP3). Patients who underwent CT angiography or coronary angiography for suspected CAD in our hospital were collected. The patients were divided into the CAD and the non-CAD (NCAD) groups. The expression of NR3C2 and NLRP3 in the peripheral blood of patients in both groups was examined by RT-qPCR. HCAECs were treated with ox-LDL to establish the model. The expression of NR3C2 and NLRP3 in ox-LDL-induced HCAECs was tested by RT-qPCR. The proliferation of HCAECs was measured using CCK-8 assay, the apoptosis of HCAECs was assessed by flow cytometry, and the levels of inflammation-related factors IL-1β and IL-18 in the cell supernatant were evaluated by ELISA. The molecular mechanisms of these factors in the proliferation and apoptosis of HCAECs and in the inflammatory response were further determined by knockdown and overexpression systems. The relationship between NR3C2 and NLRP3 was determined by ChIP and luciferase activity assays and bioinformatics analysis. NR3C2 and NLRP3 levels were elevated in the serum of CAD patients. The ox-LDL treatment elevated NR3C2 levels, evoked apoptosis and inflammation, and impeded cell viability in HCAECs, whereas downregulation of NR3C2 increased cell viability and reduced apoptosis and inflammatory response in ox-LDL-induced inflammation in HCAECs. NR3C2 levels were positively correlated with NLRP3, and NR3C2 elevated NLRP3 expression through transcription. Overexpression of NLRP3 counteracted the impacts of silencing NR3C2 on cell viability, cell apoptosis, and inflammatory response in ox-LDL-induced HCAECs. Our research stresses that NR3C2 transcription promotes NLRP3 to induce inflammatory responses in ox-LDL-induced HCAECs.

Circ_HECW2 regulates ox-LDL-induced dysfunction of cardiovascular endothelial cells by miR-942-5p/TLR4 axis.

Coronary artery disease (CAD) is a common coronary artery disease. The functional mechanism of circular RNA (circRNA) HECT, C2 and WW domain containing E3 ubiquitin protein ligase 2 (circ_HECW2, hsa_circ_0057583) in ox-LDL-treated human cardiac microvascular endothelial cells (hCMECs) is still unclear. Expression levels of circ_HECW2, microRNA (miR)-942-5p, and toll-like receptor 4 (TLR4) were analyzed by quantitative real-time PCR (qRT-PCR) and western blot assays. Cell proliferation and apoptosis were analyzed by 5-ethynyl-2'-deoxyuridine (EdU) assay, cell counting kit-8 (CCK8) assay, and flow cytometry, respectively. Tube formation assay was performed to analyze the angiogenesis of cells. Luciferase reporter and RNA pull-down assays were performed to analyze the target relationship among circ_HECW2, miR-942-5p and TLR4. Circ_HECW2 and TLR4 expression levels were up-regulated and miR-942-5p expression was decreased in the serum of CAD patients and oxidized low-density lipoprotein (ox-LDL)-induced hCMECs. Knockdown of circ_HECW2 enhanced cell proliferation and inhibited cell apoptosis in ox-LDL-treated hCMECs. MiR-942-5p was the target of circ_HECW2 and directly targeted TLR4. Moreover, the effect of circ_HECW2 knockdown could be weakened by anti-miR-942-5p, and TLR4 could restore the function of miR-942-5p on cell damage of ox-LDL-induced hCMECs. Circ_HECW2 could regulate ox-LDL-induced cardiovascular endothelial cell dysfunction through targeting miR-942-5p/TLR4 axis.

Clinical significance of lncRNA-AWPPH in coronary artery diseases.

The aim of this study was to determine serum level of long non-coding RNA (lncRNA)-AWPPH in coronary artery disease (CAD) patients and its clinical significance as a serum marker. Serum levels of lncRNA-AWPPH in 132 CAD patients and 50 controls were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Based on medical history of statin therapy, differential expressions of lncRNA-AWPPH in CAD patients were examined. Then, the correlation between lncRNA-AWPPH level and clinical data of CAD patients was analyzed. Moreover, risk factors influencing prognosis of CAD were assessed by multivariate logistic regression analysis. It was found that lncRNA-AWPPH was highly expressed in serum of CAD patients, especially those receiving rosuvastatin therapy. LDL-C, hs-CRP, and serum lncRNA-AWPPH were independent risk factors for CAD, while HDL-C was favorable to CAD. LncRNA-AWPPH is highly expressed in serum of CAD patients, which can be reduced by statin therapy, and it may be a potential serum marker for predicting the prognosis of CAD.

Expression level of lncRNA PVT1 in serum of patients with coronary atherosclerosis disease and its clinical significance.

The purpose of this study was to detect the expression level of PVT1 in the serum of coronary artery disease (CAD) patients, and to explore the clinical significance of PVT1 in CAD. A total of 200 CAD patients and 200 healthy controls in the same period were included. The serum level of PVT1 in every subject was detected. Then, the correlation between PVT1 level and Gensini score in CAD patients was analyzed by Spearman correlation test. Finally, multivariable Logistic regression test was conducted to assess risk factors influencing coronary atherosclerosis disease. It was found that PVT1 was highly expressed in the serum of CAD patients and its level was correlated with Gensini score (r=0.761, p=0.023). Besides, multivariable Logistic regression test obtained that PVT1 was the risk factor influencing coronary atherosclerosis disease (crude OR = 2.074, 95% CI: 1.642-3.529; adjusted OR = 1.762, 95% CI: 1.382-2.096). Serum level of PVT1 helps to distinguish mild and severe CAD. Meanwhile, PVT1 is an independent risk factor influencing the development of coronary atherosclerosis disease.

MiR-126 on mice with coronary artery disease by targeting S1PR2.

To screen the differentially expressed micro ribonucleic acids (miRNAs) in the serum of coronary atherosclerosis patients, and to investigate their possible mechanisms of action. The differentially expressed serum miRNAs were screened from 3 coronary artery disease (CAD) patients and 3 healthy controls using miRNA expression profiles, which were verified using low-throughput quantitative Reverse Transcription-Polymerase Chain Reaction (RT-qPCR) assay. 60 apolipoprotein E (ApoE)-/- mice were divided into model group, agomir-126 group, agomir-control (con) group, and antagomir-126 group using a random number table. They were fed with high-fat diets (21% fat and 0.15% cholesterol) ad libitum for 15 weeks to establish the mouse model of CAD. Then, hematoxylin and eosin (HE) staining was applied to detect the impact of miR-126 expression level on the tissue morphology in the thoracic aortic region. The influences of miR-126 expression level on the secretion levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-10 were determined via enzyme-linked immunosorbent assay (ELISA). Western blotting assay was performed to examine the effects of miR-126 expression level on the expression levels of nuclear factor-kappa B (NF-κB) and vascular cell adhesion molecule-1 (VACM-1) in the tissues of the thoracic aortic region of the mice. The correlation between miR-126 expression level and sphingosine-1-phosphate receptor 2 (S1PR2) in the serum of CAD patients and animal models was analyzed by the Pearson correlation coefficient method. The targets of miR-126 were predicted using the bioinformatics method, and the direct targets were verified through investigations. Western blotting assay and ELISA were adopted to detect the impacts of miR-126 expression level on the expression and secretion levels of TNF-α, IL-1β, and IL-10 in S1P + oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs). Lentivirus-small hairpin RNA (shRNA) was utilized to knock down the expression level of S1RP2 to determine whether miR-126 affected the increase in the inflammation level in S1P + ox-LDL-induced HUVECs by targeting S1RP2. Compared with those in control group, 4 miRNAs (miR-126, miR-206, miR-4297, and miR-3646) in the serum of CAD patients exhibited the most significant expression differences, which increased by 6.72, 7.11, 13.57, and 21.22 times, respectively. The verification results of low-throughput RT-qPCR assay indicated that there were remarkable changes in the expression levels of the 4 selected miRNAs with differential expressions in comparison with those in control group, displaying statistically significant differences (p<0.01). The results of HE staining manifested that the coronary atherosclerotic plaques were reduced markedly in agomir-126 group, while notably more coronary atherosclerotic plaques were formed in the thoracic aortic region in antagomir-126 group. Meanwhile, the elevated expression level of miR-126 evidently lowered the expressions of serum TNF-α and IL-1β, but significantly increased the expression of IL-10 in the mouse model of CAD. According to the analysis results of the Pearson correlation coefficient method, the miR-126 expression level was negatively correlated with S1PR2 expression level in the serum of both CAD patients and animal models (r=-0.6123, r=-5.37). It was shown in bioinformatics prediction and luciferase reporter gene assay that miR-126 negatively regulated the S1PR2 expression by targeting the 3' untranslated region (UTR) of S1PR2 messenger RNA (mRNA). In the in vitro inflammation model, the increased expression level of miR-126 could relieve the inflammation in cells induced by S1P + ox-LDL. Based on the results of both Western blotting assay and ELISA, the differences in the expression and secretion levels of TNF-α, IL-1β, and IL-10, as well as the expression levels of signaling molecules of the NF-κB signaling pathway, in the cells were not statistically significant among miR-126 mimic treatment group, sh-S1PR2 group, and miR-126 mimic + sh-S1PR2 group, indicating that miR-126 affects the inflammation level in HUVECs by targeting S1PR2. MiR-126 represses the progression of coronary atherosclerosis in the mice by binding to S1PR2. The results of this research may propose a new mechanism of miR-126 in exerting its therapeutic effects and possess potential value for the treatment of CAD in the future.

Characterization of a novel endothelial biosensor assay reveals increased cumulative serum inflammatory potential in stabilized coronary artery disease patients.

BackgroundVascular disease is promoted by systemic inflammation that can arise from sites distal to the affected vessels. We sought to characterize the net inflammatory potential of serum from patients with coronary artery disease (CAD) using cultured endothelial cells as a cumulative biosensor.Methods and resultsSerum samples from CAD patients (N = 45) and healthy control subjects (N = 48) were incubated with primary human coronary artery endothelial cells at a 1:10 dilution for 4 h, followed by isolation of the cellular RNA. Alteration of inflammation-responsive elements (adhesion molecules and cytokines) was assessed by gene expression. Specific indicators included intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and interleukin-8 (IL-8). Additionally, the cytokine levels in serum samples from all subjects were quantified. Serum from CAD subjects induced greater endothelial ICAM-1, VCAM-1, and IL-8 expression compared to healthy control serum (p < 0.001 for each analysis). The three indicators of inflammatory potential (ICAM-1, VCAM-1, and IL-8 mRNA) trended independently of each other and also of serum inflammatory biomarkers. IL-8 expression correlated negatively with serum HDL levels but positively correlated with VLDL, plasminogen activator inhibitor-1 and C-reactive protein. Interestingly, serum levels of cytokines in CAD patients were not statistically different from healthy control subjects. A year of follow-up in a sub-group of CAD subjects revealed relatively stable measures.ConclusionsAs yet unidentified circulating factors in the serum of CAD patients appear to activate endothelial cells, leading to upregulation of adhesion molecules and chemokines. This cumulative assay performed well in terms of discriminating patients with CAD compared to healthy subjects, with greater range and specificity than specific inflammatory markers.

Abstract 20478: Circulating Inflammatory Potential Measured by an Endothelial Biosensor Assay in Coronary Artery Disease Patients

Introduction: Vascular disease is driven by systemic inflammation that can arise from sites distal to the affected coronary or cerebral vessels. The objective of this study is to characterize the inflammatory potential of serum from patients with coronary artery disease (CAD) using cultured endothelial cells as a biosensor of the circulating milieu. Methods: Serum samples from CAD patients (N=48) and healthy control subjects (N=50) were incubated with primary human coronary artery endothelial cells at a 1:10 dilution for 4h, following by isolation of the cellular RNA. Alteration of inflammation-responsive elements (adhesion molecules and cytokines) was assessed on a gene expression level. Specific indicators included intercellular adhesion molecule-1 (ICAM), vascular cell adhesion molecule-1 (VCAM), and interleukin-8 (IL-8). Additionally, the serum samples from the CAD patients and healthy individuals were quantitatively analyzed for IL-1β, IL-6, IL-8, and TNF-α using an electrochemical luminescence platform. Results: For the CAD subjects’ serum, the mean values of ICAM, VCAM, and IL-8 expression were all elevated compared to healthy control subjects (p&lt;0.001 for each by Students T-test). Correlational analysis revealed the three indicators (ICAM, VCAM, and IL-8) to be independent of each other and also other inflammatory markers such as C-reactive protein. IL-8 expression was negatively correlated with serum HDL levels but positively correlated with body fat composition. Interestingly, serum levels of IL-1β, IL-6, IL-8, and TNF-α in CAD patients were not statistically different from healthy control subjects. Conclusions: As yet uncharacterized circulating factors in the serum of CAD patients appear to activate endothelial cells. This assay paradigm performed well in terms of discriminating patients with CAD compared to healthy subjects, with greater range and specificity than specific inflammatory markers.

MicroRNA-31 controls phenotypic modulation of human vascular smooth muscle cells by regulating its target gene cellular repressor of E1A-stimulated genes

Phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of a variety of proliferative vascular diseases. The cellular repressor of E1A-stimulated genes (CREG) has been shown to play an important role in phenotypic modulation of VSMCs. However, the mechanism regulating CREG upstream signaling remains unclear. MicroRNAs (miRNAs) have recently been found to play a critical role in cell differentiation via target-gene regulation. This study aimed to identify a miRNA that binds directly to CREG, and may thus be involved in CREG-mediated VSMC phenotypic modulation. Computational analysis indicated that miR-31 bound to the CREG mRNA 3′ untranslated region (3′-UTR). miR-31 was upregulated in quiescent differentiated VSMCs and downregulated in proliferative cells stimulated by platelet-derived growth factor and serum starvation, demonstrating a negative relationship with the VSMC differentiation marker genes, smooth muscle α-actin, calponin and CREG. Using gain-of-function and loss-of-function approaches, CREG and VSMC differentiation marker gene expression levels were shown to be suppressed by a miR-31 mimic, but increased by a miR-31 inhibitor at both protein and mRNA levels. Notably, miR-31 overexpression or inhibition affected luciferase expression driven by the CREG 3′-UTR containing the miR-31 binding site. Furthermore, miR-31-mediated VSMC phenotypic modulation was inhibited in CREG-knockdown human VSMCs. We also determined miR-31 levels in the serum of patients with coronary artery disease (CAD), with or without in stent restenosis and in healthy controls. miR-31 levels were higher in the serum of CAD patients with restenosis compared to CAD patients without restenosis and in healthy controls. In summary, these data demonstrate that miR-31 not only directly binds to its target gene CREG and modulates the VSMC phenotype through this interaction, but also can be an important biomarker in diseases involving VSMC phenotypic modulation. These novel findings may have extensive implications for the diagnosis and therapy of a variety of proliferative vascular diseases.

Insight into the Mode of Action of ACE Inhibition in Coronary Artery Disease

ACE inhibition is now recognized as superior to placebo on outcomes in stable coronary artery disease (CAD), including total and cardiovascular mortality, fatal and nonfatal myocardial infarction, heart failure, revascularization and stroke. This review examines clinical evidence for the mode of action of ACE inhibitors in CAD, which is dominated by the results of a single trial, EUROPA, and its substudies. The generally accepted mode of action for ACE inhibitors in CAD is blood pressure reduction. However, the EUROPA data demonstrate that endothelial protection, with the effect of arresting or reducing the processes of atherosclerosis is also important. Chronic overexpression of tissue ACE in CAD disrupts the angiotensin II/bradykinin balance with a net result of endothelial dysfunction. ACE inhibitors reduce production of angiotensin II, which prevents vasoconstriction, reduces adhesion molecules and growth factors, decreases oxidative stress and prevents apoptosis. A concomitant decrease in the degradation of bradykinin as a result of ACE inhibition raises levels of this kinin, leading to vasodilation and an antiapoptotic action, as well as opposition of the negative actions of angiotensin II. We now have clinical trial evidence of these processes in CAD patients participating in the EUROPA study by measurement of markers of endothelial function, including nitric oxide synthase (eNOS), the rate of apoptosis and levels of von Willebrand factor (vWf). Serum from CAD patients was found to significantly downregulate eNOS protein expression and activity versus that of healthy controls (p < 0.01), most probably as a result of upregulation of tissue ACE. One year of treatment with perindopril upregulated eNOS protein expression and activity (19% and 27% vs placebo; p < 0.05). Similarly, vWf was elevated at baseline and significantly reduced after 1 year of treatment with perindopril (p < 0.001). Increased endothelial apoptosis by serum of CAD patients was accompanied by excess angiotensin II and tumour necrosis factor-alpha and a reduction in bradykinin; all of these parameters were reversed by treatment. We therefore have clinical results showing that perindopril normalizes the angiotensin II/bradykinin balance, reduces inflammation and prevents endothelial apoptosis. Accumulating preclinical evidence for the absence of a class effect for ACE inhibitors includes differences in terms of effect on eNOS and rate of endothelial apoptosis. These differences appear to be related to tissue affinity, penetration of atherosclerotic plaque and affinity for the target enzyme. Consideration of these features is important when administering ACE inhibition as secondary prevention in CAD patients. In this context, current European guidelines for stable angina pectoris recommend prescription of agents and doses with proven efficacy in secondary prevention.