Assessment of the Level of Matrix Metalloproteinases, VEGF and MicroRNA-34a in Patients With Non-obstructive and Obstructive Lesions of the Coronary Arteries.

AIM: To assess the levels of matrix metalloproteinases (MMP), vascular endothelial growth factor (VEGF), and miRNA-34a expression in patients with ischemic heart disease (IHD) and obstructive and nonobstructive coronary artery (CA) disease. MATERIAL AND METHODS: This cross-sectional observational study included 64 patients with IHD (diagnosis verified by coronary angiography or multislice computed tomography coronary angiography), of which 33 (51.6%) were men aged 64.9±8.1 years. 20 patients had nonobstructive CA disease (stenosis <50%), and 44 had hemodynamically significant stenoses. The control group consisted of 30 healthy volunteers. MMP-1, -9, -13, and -14, miRNA-34a, and VEGF were measured in all patients. RESULTS: The concentration of MMP-1 was significantly higher in patients with ischemia and nonobstructive CA disease (INOCAD) (p=0.016), and the concentration of MMP-9 was the highest in the group with obstructive CA disease (p<0.001). The concentrations of MMP-13 and MMP-14 did not differ significantly between the groups. The highest VEGF concentrations were observed in the INOCAD group (p<0.001). The expression of miRNA-34a significantly differed between the IHD groups with different types of CA disease and controls (p <0.001). Patients with hemodynamically significant stenosis showed moderate relationships between the concentrations of MMP-14 and VEGF (ρ=0.418; p=0.024), as well as between VEGF and miRNA-34a (ρ=0.425; p=0.022). Patients with INOCAD had a significant negative correlation between the concentrations of MMP-13 and VEGF (ρ= -0.659; p=0.003). Correlation analysis showed in all IHD patients a moderate relationship of the concentrations of MMP-1 and MMP-14 with VEGF (ρ=0.449; p=0.002 and p=0.341; p=0.019, respectively). According to ROC analysis, a MMP-9 concentration above 4.83 ng/ml can be a predictor for the presence of hemodynamically significant CA obstruction in IHD patients; a VEGF concentration higher than 27.23 pg/ml suggests the absence of hemodynamically significant CA stenosis. CONCLUSION: IHD patients with INOCAD had the greatest increase in MMP-1, whereas patients with obstructive CA disease had the highest level of MMP-9. According to our data, concentrations of MMP-9 and VEGF can be used to predict the degree of CA obstruction. The expression of miRNA-34a was significantly higher in IHD patients with INOCAD and CA obstruction than in the control group, which suggested a miRNA-34a contribution to the development and progression of coronary atherosclerosis. In the future, it may be possible to use this miRNA as a diagnostic marker for IHD.

Serum­derived exosomal hsa­let­7b­5p as a biomarker for predicting the severity of coronary stenosis in patients with coronary heart disease and hyperglycemia.

Exosomal microRNAs (miRNAs/miRs) are potential biomarkers for the diagnosis and treatment of cardiovascular disease, and hyperglycemia serves an important role in the development of atherosclerosis. The present study aimed to investigate the expression profile of serum­derived exosomal miRNAs in coronary heart disease (CHD) with hyperglycemia, and to identify effective biomarkers for predicting coronary artery lesions. Serum samples were collected from eight patients with CHD and hyperglycemia and eight patients with CHD and normoglycemia, exosomes were isolated and differentially expressed miRNAs (DEMIs) were filtered using a human miRNA microarray. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using standard enrichment computational methods for the target genes of DEMIs. Receiver operating characteristic (ROC) curve analysis was applied to evaluate the values of the selected DEMIs in predicting the severity of coronary stenosis. A total of 10 DEMIs, including four upregulated miRNAs (hsa­let­7b­5p, hsa­miR­4313, hsa­miR­4665­3p and hsa­miR­940) and six downregulated miRNAs (hsa­miR­4459, hsa­miR­4687­3p, hsa­miR­6087, hsa­miR­6089, hsa­miR­6740­5p and hsa­miR­6800­5p), were screened in patients with CHD and hyperglycemia. GO analysis showed that the 'cellular process', 'single­organism process' and 'biological regulation' were significantly enriched. KEGG pathway analysis revealed that the 'mTOR signaling pathway', 'FoxO signaling pathway' and 'neurotrophin signaling pathway' were significantly enriched. Among these DEMIs, only hsa­let­7b­5p expression was positively correlated with both hemoglobin A1C levels and Synergy between Percutaneous Coronary Intervention with Taxus and Cardiac Surgery score. ROC curves showed that hsa­let­7b­5p could serve as an effective biomarker for differentiating the severity of coronary stenosis. In conclusion, the present study demonstrated that serum­derived exosomal hsa­let­7b­5p is upregulated in patients with CHD and hyperglycemia, and may serve as a noninvasive biomarker for the severity of coronary stenosis.

Circulating long non-coding RNA Coromarker expression correlated with inflammation, coronary artery stenosis, and plaque vulnerability in patients with coronary artery disease.

BACKGROUND: The aim of the study was to assess the correlation between circulating long non-coding RNA (lncRNA) OTTHUMT00000387022 (named Coromarker) expression and disease severity, inflammatory cytokine levels, and plaque vulnerability in patients with coronary artery disease (CAD). METHODS: A total of 134 participants who received coronary angiography were enrolled and classified them as CAD patients (N = 89) and controls (N = 45). Blood samples were obtained from all subjects. Quantitative polymerase chain reaction was used to evaluate Coromarker expression. The enzyme-linked immunosorbent test was used to measure inflammatory cytokines including high sensitivity C reactive protein (hsCRP), interleukin (IL)-1ß (IL-1ß), IL-6, NOD-like receptor protein 3 (NLRP3), and markers of coronary plaque stability including matrix metallopeptidase 9 (MMP-9) and soluble CD40 ligand (sCD40L). The severity of coronary stenosis was determined from the Gensini Score. RESULTS: LncRNA Coromarker expression was elevated to a greater extent in CAD patients than in control subjects before and after adjustments for age/gender (both p < 0.001); it was an independent predictor of CAD risk (area under curve: 0.824, 95% CI: 0.732-0.915). Additionally, Coromarker expression was significantly associated with Gensini Score (r = 0.574, p < 0.001), hsCRP (r = 0.221, p = 0.015), IL-1ß (r = 0.351, p < 0.001), IL-6 (r = 0.286, p < 0.01), and NLRP3 levels (r = 0.312, p < 0.001). Coromarker expression was found to be linked with MMP-9 (r = 0.260, p < 0.01) and sCD40L (r = 0.441, p < 0.001). CONCLUSION: Circulating lncRNA Coromarker expression correlates with increased disease severity and inflammation as well as plaque vulnerability in patients with CAD.

Homozygous familial lipoprotein lipase deficiency without obvious coronary artery stenosis.

The prevalence of familial lipoprotein lipase deficiency (LPLD) is approximately one in 1,000,000 in the general population. There are conflicting reports on whether or not LPLD is atherogenic. We conducted coronary computed tomographic (CT) angiography on two patients in their 70 s who had genetically confirmed LPLD. Patient 1 was a 73 year old woman with a body mass index (BMI) of 27.5 kg/m2, no history of diabetes mellitus and no history of drinking alcohol or smoking. At the time of her first visit, her serum total cholesterol, triglycerides and high-density lipoprotein cholesterol levels were 4.8 mmol/L, 17.3 mmol/L, and 0.5 mmol/L, respectively. She was treated with a lipid-restricted diet and fibrate but her serum TG levels remained extremely high. Next-generation sequencing analysis revealed a missense mutation (homo) in the LPL gene, c.662T>C (p. Ile221Thr), leading to the diagnosis of homozygous familial LPL deficiency (LPLD). Patient 2 was another 73- year- old woman. She also had marked hypertriglyceridemia with no history of diabetes mellitus, drinking alcohol, or smoking. Previous genetic studies showed she had a nonsense mutation (homozygous) in the LPL gene, c.1277G>A (p.Trp409Ter). To clarify the degree of coronary artery stenosis in these two cases, we conducted coronary CT angiography and found that no coronary artery stenosis in either the right or left coronary arteries. Based on the findings in these two elderly women along with previous reports on patients in their 60 s with LPLD and hypertriglyceridemia, we suggest that LPLD may not be associated with the development or progression of coronary artery disease.

Relation of circulating lncRNA GAS5 and miR-21 with biochemical indexes, stenosis severity, and inflammatory cytokines in coronary heart disease patients.

BACKGROUND: Long noncoding RNA GAS5 (lnc-GAS5) and its target microRNA-21 (miR-21) regulate blood lipid, macrophages, Th cells, vascular smooth muscle cells to participate in atherosclerosis, and related coronary heart disease (CHD). The study aimed to further explore the linkage of their circulating expressions with common biochemical indexes, stenosis severity and inflammatory cytokines in CHD patients. METHODS: Ninety-eight CHD patients and 100 controls confirmed by coronary angiography were enrolled. Plasma samples were collected for lnc-GAS5 and miR-21 detection by reverse transcription-quantitative polymerase chain reaction and inflammatory cytokines determination by enzyme-linked immunosorbent assay. RESULTS: Lnc-GAS5 was increased in CHD patients compared with controls (2.270 (interquartile range [IQR]: 1.676-3.389) vs. 0.999 ([IQR: 0.602-1.409], p < 0.001), whereas miR-21 showed opposite tread (0.442 [IQR: 0.318-0.698] vs. 0.997 [IQR: 0.774-1.368], p < 0.001). In aspect of their intercorrelation, lnc-GAS5 negatively linked with miR-21 in CHD patients (p < 0.001) instead of controls (p = 0.211). Interestingly, among the common biochemical indexes, lnc-GAS5 related to decreased high-density lipoprotein cholesterol (p = 0.008) and increased C-reactive protein (CRP) (p < 0.001), while miR-21 correlated with lower total cholesterol (p = 0.024) and CRP (p < 0.001) in CHD patients. As stenosis degree, lnc-GAS5 positively correlated with Gensini score (p < 0.001), but miR-21 exhibited negative association (p = 0.003) in CHD patients. In terms of inflammatory cytokines, lnc-GAS5 positively related to tumor necrosis factor α (TNF-α) and interleukin (IL)-17A, while miR-21 negatively linked with TNF-α, IL-1ß, IL-6, and IL-17 in CHD patients (all p < 0.05). CONCLUSION: Circulating lnc-GAS5 and its target miR-21 exhibit potency to serve as biomarkers for CHD management.

Relations of physical signs to genotype, lipid and inflammatory markers, coronary stenosis or calcification, and outcomes in patients with heterozygous familial hypercholesterolemia.

BACKGROUND: Although the presence of physical signs [tendon xanthomas and/or corneal arcus (TX/CA)], are associated with the risk of coronary artery disease in patients with heterozygous familial hypercholesterolemia (HeFH), their relationship with genotypes and clinical characteristics has not been fully determined. This study aimed to examine the association of TX/CA with genetic mutation, lipid- and inflammation-related markers, the severity of coronary stenosis or calcification, and cardiovascular events (CVEs) in patients with HeFH. METHODS: LDLR, APOB, and PCSK9 genes were screened in 523 HeFH patients, and patients with TX/CA (n = 50) were 1:4 propensity score-matched to patients without TX/CA (n = 200) to adjust for age and sex. Laboratory markers (proprotein convertase subtilisin/kexin type 9 [PCSK9], lipoprotein(a) and high-sensitivity C-reactive protein [hsCRP]), computed tomography angiography, coronary angiography, and follow-up for CVEs were performed. RESULTS: Patients with physical signs had significantly higher low-density lipoprotein cholesterol levels; higher PCSK9 or hsCRP concentrations; more LDLR positive mutations; and higher prevalence of high tertiles of Gensini, SYNTAX and Jeopardy scores as well as coronary artery calcium scores than did those without. Over an average follow-up of 3.7 years, the incidence of CVEs was significantly higher in patients with TX/CA (log-rank p < 0.001). Patients with physical signs and mutation positivity had threefold higher risks of CVEs (adjusted hazard ratio 3.34, 95% confidence interval 1.04-10.72, p = 0.024). CONCLUSIONS: Physical signs were associated with genotypes and phenotypes, and worse outcomes in patients with HeFH, suggesting that these signs may help in risk stratification in these patients.

Upregulation of long non-coding RNA myocardial infarction-associated transcription is correlated with coronary artery stenosis and elevated inflammation in patients with coronary atherosclerotic heart disease.

Coronary atherosclerotic heart disease (CAD) is a chronic disease caused by multiple risk factors. Aberrant expression of long non-coding RNAs (lncRNAs) has been regarded as an independent risk factor of CAD. This study evaluated lncRNA myocardial infarction-associated transcription (MIAT) expression in CAD patients and its clinical significance. Totally, 155 CAD patients and 76 non-CAD controls were enrolled. MIAT expression was detected using reverse transcription quantitative polymerase chain reaction. The clinical diagnostic significance of MIAT was evaluated by plotting the receiver operating characteristic (ROC) curve. The levels of inflammatory cytokines were detected using enzyme-linked immunosorbent assay. microRNA (miR)-29b-3p expression and pregnancy-associated plasma protein A (PAPPA) level were detected. MIAT expression in CAD patients (4.23 [1.22-6.50]) was higher than that in non-CAD controls (1.64 [0.05-2.93]) (p < 0.01) and had an independent correlation with CAD. The area under ROC curve of predicting CAD was calculated as 0.790, the specificity as 71.40%, and the sensitivity as 70.00%. MIAT expression was positively correlated with the C-reactive protein level (r = 0.769, p < 0.0001) and pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and IL-8 levels, while negatively correlated with the anti-inflammatory cytokine IL-10. MIAT was positively correlated with Gensini score and had an independent correlation with it. LncRNA MIAT sponged miR-29b-3p and miR-29b-3p targeted PAPPA. In conclusion, lncRNA MIAT was upregulated in the peripheral blood of CAD patients and elicited clinical diagnostic significance. MIAT participated in the development of CAD via miR-29b-3p/PAPPA axis. This study provides insights into a potential target for the diagnosis and treatment of CAD.

Polygenic Risk Score-Enhanced Risk Stratification of Coronary Artery Disease in Patients With Stable Chest Pain.

BACKGROUND: Polygenic risk scores (PRSs) are associated with coronary artery disease (CAD), but the clinical potential of using PRSs at the single-patient level for risk stratification has yet to be established. We investigated whether adding a PRS to clinical risk factors (CRFs) improves risk stratification in patients referred to coronary computed tomography angiography on a suspicion of obstructive CAD. METHODS: In this prespecified diagnostic substudy of the Dan-NICAD trial (Danish study of Non-Invasive testing in Coronary Artery Disease), we included 1617 consecutive patients with stable chest symptoms and no history of CAD referred for coronary computed tomography angiography. CRFs used for risk stratification were age, sex, symptoms, prior or active smoking, antihypertensive treatment, lipid-lowering treatment, and diabetes. In addition, patients were genotyped, and their PRSs were calculated. All patients underwent coronary computed tomography angiography. Patients with a suspected ≥50% stenosis also underwent invasive coronary angiography with fractional flow reserve. A combined end point of obstructive CAD was defined as a visual invasive coronary angiography stenosis >90%, fractional flow reserve <0.80, or a quantitative coronary analysis stenosis >50% if fractional flow reserve measurements were not feasible. RESULTS: The PRS was associated with obstructive CAD independent of CRFs (adjusted odds ratio, 1.8 [95% CI, 1.5-2.2] per SD). The PRS had an area under the curve of 0.63 (0.59-0.68), which was similar to that for age and sex. Combining the PRS with CRFs led to a CRF+PRS model with area under the curve of 0.75 (0.71-0.79), which was 0.04 more than the CRF model (P=0.0029). By using pretest probability (pretest probability) cutoffs at 5% and 15%, a net reclassification improvement of 15.8% (P=3.1×10-4) was obtained, with a down-classification of risk in 24% of patients (211 of 862) in whom the pretest probability was 5% to 15% based on CRFs alone. CONCLUSIONS: Adding a PRS improved risk stratification of obstructive CAD beyond CRFs, suggesting a modest clinical potential of using PRSs to guide diagnostic testing in the contemporary clinical setting. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02264717.

Association of dipeptidyl peptidase IV polymorphism, serum lipid profile, and coronary artery stenosis in patients with coronary artery disease and type 2 diabetes.

ABSTRACT: Cardiovascular disease (CAD) is a devastating illness, but to date there are limited means of predicting a person's coronary stenosis severity and their prognosis. The study was performed to investigate the relationship between dipeptidyl peptidase 4(DPP4) gene polymorphisms and serum lipid profiles, as well as the severity of coronary artery stenosis in patients with CAD and type 2 diabetes (T2DM) for the first time.Herein, 201 patients with CAD and T2DM were enrolled in the Department of Cardiology, Shandong Provincial Qianfoshan Hospital. DPP4 rs3788979 and rs7608798 single nucleotide polymorphisms (SNPs) were genotyped. The general information of all patients was collected, and the associations between DPP4 SNPs and lipid profiles were detected. At the same time, association between SNP polymorphisms and the degree of coronary artery stenosis were analyzed.There was a significant difference in apolipoprotein B (ApoB) levels (P = .011) for the rs3788979 polymorphism, while no difference was identified in other blood lipids or with other mutations. SNP mutation of A to G in rs3788979 was associated with a reduced percentage of severe coronary artery stenosis in female patients (P = .023) as well as those with nosmoking (P = .030), nodrinking (P = 0.007), and nocardiovascular family history (P = 0.015).G allele of rs3788979 is associated with a reduced ApoB level. Besides, we suggest that G allele in rs3788979 may have a cardioprotective effect and prove to be a useful and specific measure when predicting a patient's coronary stenosis severity if diagnosed with CAD and T2DM.

Partial recovery of senescence in circulating follicular helper T cells after Dasatinib treatment.

Cellular senescence is an irreversible arrest of cell proliferation triggered by different stimuli, including DNA damage, telomere shortening and oncogenic stress. Senescent cells, by releasing the senescence-associated-secretory-phenotype (SASP), contribute to various diseases pathogenesis. Human atherosclerotic plaque contains cells with multiple markers of senescence that associate with disease severity. We characterized the frequency of senescent cTfh cells and genes expressions before and after treatment with Dasatinib in patients with different degrees of stenosis. Twelve high (≥50%), and twelve low (<50%) stenosis patients and six healthy controls were enrolled. The percentage of senescent CD3+CD4+CXCR5+CD153+CD57+ cells was significantly decreased in Dasatinib treated cells from individuals with low and high stenosis (P = 0.0007 and P = 0.0002, respectively). However, the frequency of total lymphocytes, CD3+ and CD4+ T cells were not significantly different between the groups before and after treatment. The expression levels of P53 (P = 0.0003 and P = 0.0001), P16 (P = 0.0005 and P = 0.0002), p21 (P = 0.0002 and P < 0.0001), SENEX (P = 0.0005 and P < 0.0001) and BCL-2 (P = 0.0005 and P = 0.0002) were decreased in PBMCs of low and high stenosis groups after treatment with Dasatinib, respectively. The percentage of senescent cTfh cells positively correlated with cholesterol (P = 0.034; r = 0.671), C-reactive protein (CRP) (P = 0.029; r = 0.707), Erythrocyte sedimentation rate (ESR) levels (P = 0.030; r = 0.598) and neutrophil counts (P = 0.021; r = 0.799) in patients with high stenosis. The decreased frequency of senescent cTfh cells and the expression levels of senescence genes after Dasatinib treatment in patients with atherosclerosis suggest a role for Dasatinib in partial clearance or rejuvenation of senescent cTfh cells, which may decrease inflammatory mediators and attenuate disease progression.

NFKB1 gene rs28362491 ins/del variation is associated with higher susceptibility to myocardial infarction in a Chinese Han population.

Myocardial infarction (MI), the leading cause of mortality and disability worldwide, is a disease in which multiple environmental and genetic factors are involved. Recently, researches suggested that insertion/deletion (ins/del) variation of NFKB1 gene rs28362491 is a functional polymorphism. In the present study, we aimed to explore the relation between variation of NFKB1 gene rs28362491 and MI by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 359 MI patients and 1085 control participants. Gensini score was used to evaluate the degree of coronary artery stenosis in MI patients. The plasma levels of interleukin-6 (IL-6), IL-8, malonaldehyde (MDA) and superoxide dismutase (SOD) were randomly measured by ELISA both in MI patients and control participants. We found that the detected frequencies of D allele (41.2% vs. 36.4%, P = 0.021) and DD genotype (17.5% vs. 12.0%, P = 0.022) were significantly higher in MI patients than in control participants. Compared with II or ID genotype carriers, the Gensini score in MI patients with DD genotype was 32-43% higher (both P < 0.001). Moreover, DD genotype carries had more diseased coronary arteries (P = 0.001 vs. II or ID genotype). Of note, IL-6 levels in MI patients carrying DD genotype were significantly higher than that in control participants and other genotype carriers in MI patients (both P < 0.05). In conclusion, NFKB1 gene rs28362491 DD genotype was associated with a higher risk of MI and more severe coronary artery lesion, which also had a potential influence on the level of inflammatory cytokine IL-6.

The Regulation of Exosome-Derived miRNA on Heterogeneity of Macrophages in Atherosclerotic Plaques.

Exosomes are nanosized vesicles secreted by most cells, which can deliver a variety of functional lipids, proteins, and RNAs into the target cells to participate in complex intercellular communications. Cells respond to certain physical, chemical, and biological stimuli by releasing exosomes. Exosomes are rich in small molecules of RNA, including miRNAs and mRNAs, which have been demonstrated to have certain functions in recipient cells. Recent studies on single-cell RNA sequences have revealed the transcription and the heterogeneity of macrophages in Ldlr-/-mice fed with a high-fat diet. Five macrophage populations were found in the atherosclerotic plaques. It is worth noting that these subset populations of macrophages seem to be endowed with different functions in lipid metabolism and catabolism. A total of 100 differentially expressed mRNAs were selected for these subset populations. Importantly, these macrophage populations were also present in human advanced atherosclerosis. To clarify the specific functions and the regulatory mechanism of these macrophage populations, we extracted exosome RNAs from the plasma of patients with chronic coronary artery disease (CAD) and performed RNA sequencing analysis. Compared with the healthy control, a total of 14 miRNAs were significantly expressed in these patients. A total of 5,248 potential mRNAs were predicted by the bioinformatics platform. Next, we determined the outcome of the intersection of these predicted mRNAs with 100 mRNAs expressed in the above-mentioned five macrophage populations. Based on the screening of miRNA-mRNA pairs, a co-expression network was drawn to find out the key RNAs. Three down-regulated miRNAs and five up-regulated mRNAs were selected for validation by real-time RT-PCR. The results showed that the expression of miR-4498 in plasma exosomes was lower than that in the healthy control, and the expressions of Ctss, Ccr2 and Trem2 mRNA in peripheral blood mononuclear cells isolated from CAD patients were higher. In order to clarify the regulatory mechanism, we established a co-culture system in vitro. Studies have shown that the uptake of exosomes from CAD patients can up-regulate the expression of Ctss, Trem2, and Ccr2 mRNA in THP-1 cells induced by lipopolysaccharide. Our findings revealed a unique relationship between the transcriptional signature and the phenotypic heterogeneity of macrophage in the atherosclerotic microenvironment.

Relationship between adenosine A2a receptor polymorphism rs5751876 and fractional flow reserve during percutaneous coronary intervention.

Fractional flow reserve (FFR) assessed during adenosine-induced maximal hyperemia has emerged as a useful tool for the guidance of percutaneous coronary interventions (PCI). However, interindividual variability in the response to adenosine has been claimed as a major limitation to the use of adenosine for the measurement of FFR, carrying the risk of underestimating the severity of coronary stenoses, with potential negative prognostic consequences. Genetic variants of the adenosine receptor A2a (ADORA2A gene), located in the coronary circulation, have been involved in the modulation of the hyperemic response to adenosine. However, no study has so far evaluated the impact of the single nucleotide polymorphism rs5751876 of ADORA2A on the measurement of FFR in patients undergoing percutaneous coronary intervention that was, therefore, the aim of our study. We included patients undergoing coronary angiography and FFR assessment for intermediate (40-70%) coronary lesions. FFR measurement was performed by pressure-recording guidewire (Prime Wire, Volcano), after induction of hyperemia with intracoronary boli of adenosine (from 60 to 1440 µg, with dose doubling at each step). Restriction fragment length polymorphism (RFLP) analysis was performed to assess the presence of rs5751876 C>T polymorphism of ADORA2a receptor. We included 204 patients undergoing FFR measurement of 231 coronary lesions. A total of 134 patients carried the polymorphism (T allele), of whom 41 (30.6%) in homozygosis (T/T).Main clinical and angiographic features did not differ according to ADORA2A genotype. The rs5751876 C>T polymorphism did not affect mean FFR values (p = 0.91), the percentage of positive FFR (p = 0.54) and the duration of maximal hyperemia. However, the time to recovery to baseline FFR values was more prolonged among the T-allele carriers as compared to wild-type patients (p = 0.04). Based on these results, in patients with intermediate coronary stenoses undergoing FFR assessment with adenosine, the polymorphism rs5751876 of ADORA2A does not affect the peak hyperemic response to adenosine and the results of FFR. However, a more prolonged effect of adenosine was observed in T-carriers.

Polygenic risk for coronary heart disease acts through atherosclerosis in type 2 diabetes.

BACKGROUND: Type 2 diabetes increases the risk of coronary heart disease (CHD), yet the mechanisms involved remain poorly described. Polygenic risk scores (PRS) provide an opportunity to understand risk factors since they reflect etiologic pathways from the entire genome. We therefore tested whether a PRS for CHD influenced risk of CHD in individuals with type 2 diabetes and which risk factors were associated with this PRS. METHODS: We tested the association of a CHD PRS with CHD and its traditional clinical risk factors amongst individuals with type 2 diabetes in UK Biobank (N = 21,102). We next tested the association of the CHD PRS with atherosclerotic burden in a cohort of 352 genome-wide genotyped participants with type 2 diabetes who had undergone coronary angiograms. RESULTS: In the UK Biobank we found that the CHD PRS was strongly associated with CHD amongst individuals with type 2 diabetes (OR per standard deviation increase = 1.50; p = 1.5 × 10- 59). But this CHD PRS was, at best, only weakly associated with traditional clinical risk factors, such as hypertension, hyperlipidemia, glycemic control, obesity and smoking. Conversely, in the angiographic cohort, the CHD PRS was strongly associated with multivessel stenosis (OR = 1.65; p = 4.9 × 10- 4) and increased number of major stenotic lesions (OR = 1.35; p = 9.4 × 10- 3). CONCLUSIONS: Polygenic predisposition to CHD is strongly associated with atherosclerotic burden in individuals with type 2 diabetes and this effect is largely independent of traditional clinical risk factors. This suggests that genetic risk for CHD acts through atherosclerosis with little effect on most traditional risk factors, providing the opportunity to explore new biological pathways.

Potential of circulating pro-angiogenic microRNA expressions as biomarkers for rapid angiographic stenotic progression and restenosis risks in coronary artery disease patients underwent percutaneous coronary intervention.

BACKGROUND: This study aimed to investigate the correlation of pro-angiogenic microRNA (miRNA) expressions with rapid angiographic stenotic progression (RASP) and restenosis risks in coronary artery disease (CAD) patients underwent percutaneous coronary intervention (PCI) with drug-eluting stents (DES). METHODS: A total of 286 CAD patients underwent PCI with DES were consecutively recruited in this study. Plasma samples were collected before PCI operation, and 14 pro-angiogenic miRNAs were measured by real-time quantitative reverse transcription-polymerase chain reaction. Rapid angiographic stenotic progression at nontarget lesions and restenosis at stented lesions were evaluated by quantitative coronary angiography at 12 months after PCI operation. RESULTS: The occurrence rates of RASP and restenosis were 39.5% and 22.4%, respectively. Let-7f, miR-19a, miR-19b-1, miR-92a, miR-126, miR-210, and miR-296 were decreased in RASP patients than non-RASP patients, among which let-7f, miR-19a, miR-126, miR-210, and miR-296 independently correlated with lower RASP occurrence by multivariate analysis, followed by receiver-operating characteristic (ROC) curve exhibited that these five miRNAs showed great value in predicting RASP risk with area under curve (AUC) 0.879 (95% CI: 0.841-0.917). Besides, let-7f, miR-19a, miR-92a, miR-126, miR-130a, and miR-210 were reduced in restenosis patients than non-restenosis patients, among them miR-19a, miR-126, miR-210, and miR-378 independently correlated with lower restenosis occurrence by multivariate analysis, followed by ROC curve disclosed that these four miRNAs had good value in predicting restenosis risk with AUC 0.776 (95% CI: 0.722-0.831). CONCLUSIONS: Circulating let-7f, miR-19a, miR-126, miR-210, and miR-296 independently correlate with reduced RASP risk, while miR-19a, miR-126, miR-210, and miR-378 independently correlate with decreased restenosis risk in CAD patients underwent PCI with DES.

Smooth muscle cell-specific fibronectin-EDA mediates phenotypic switching and neointimal hyperplasia.

Fibronectin-splice variant containing extra domain A (Fn-EDA) is associated with smooth muscle cells (SMCs) following vascular injury. The role of SMC-derived Fn-EDA in SMC phenotypic switching or its implication in neointimal hyperplasia remains unclear. Herein, using human coronary artery sections with a bare metal stent, we demonstrate the expression of Fn-EDA in the vicinity of SMC-rich neointima and peri-strut areas. In mice, Fn-EDA colocalizes with SMCs in the neointima of injured carotid arteries and promotes neointima formation in the comorbid condition of hyperlipidemia by potentiating SMC proliferation and migration. No sex-based differences were observed. Mechanistic studies suggested that Fn-EDA mediates integrin- and TLR4-dependent proliferation and migration through activation of FAK/Src and Akt1/mTOR signaling, respectively. Specific deletion of Fn-EDA in SMCs, but not in endothelial cells, reduced intimal hyperplasia and suppressed the SMC synthetic phenotype concomitant with decreased Akt1/mTOR signaling. Targeting Fn-EDA in human aortic SMCs suppressed the synthetic phenotype and downregulated Akt1/mTOR signaling. These results reveal that SMC-derived Fn-EDA potentiates phenotypic switching in human and mouse aortic SMCs and neointimal hyperplasia in the mouse. We suggest that targeting Fn-EDA could be explored as a potential therapeutic strategy to reduce neointimal hyperplasia.

Differences in phenotype, genotype and cardiovascular events between patients with probable and definite heterozygous familial hypercholesterolemia.

Aim: To investigated the potential differences between probable and definite heterozygous familial hypercholesterolemia (HeFH) patients diagnosed by Dutch Lipid Clinic Network criteria. Methods: Clinical characteristics, lipid profile, severity of coronary artery stenosis and gene mutations were compared. Kaplan-Meier curve was performed to evaluate the cardiovascular events. Results: Overall, 325 participants were included and divided into two groups: probable (n = 233) and definite HeFH (n = 92). Definite HeFH patients had higher low-density lipoprotein cholesterol (LDL-C), oxidized-LDL and proprotein convertase subtilisin/kexin 9 levels, and higher prevalence of tendon xanthomas. The incidence of genetic mutations was statistically higher in definite HeFH than probable HeFH patients. The coronary stenosis calculated by Gensini score was statistically severer in definite HeFH patients. The best LDL-C threshold for predicting mutations was 5.14 mmol/l. Definite HeFH had lower event-free survival rates. Conclusion: Definite HeFH patients had higher severity of phenotype and genotype, and higher risk of cardiovascular events.

Identification of a blood-based 12-gene signature that predicts the severity of coronary artery stenosis: An integrative approach based on gene network construction, Support Vector Machine algorithm, and multi-cohort validation.

BACKGROUND AND AIMS: We aimed to identify a blood-based gene expression score (GES) to predict the severity of coronary artery stenosis in patients with known or suspected coronary artery disease (CAD) by integrative use of gene network construction, Support Vector Machine (SVM) algorithm, and multi-cohort validation. METHODS: In the discovery phase, a public blood-based microarray dataset of 110 patients with known CAD was analyzed by weighted gene coexpression network analysis and protein-protein interaction network analysis to identify candidate hub genes. In the training set with 151 CAD patients, bioinformatically identified hub genes were experimentally verified by real-time polymerase chain reaction, and statistically filtered with the SVM algorithm to develop a GES. Internal and external validation of GES was performed in patients with suspected CAD from two validation cohorts (n = 209 and 206). RESULTS: The discovery phase screened 15 network-centric hub genes significantly correlated with the Duke CAD Severity Index. In the training cohort, 12 of 15 hub genes were filtered to construct a blood-based GES12, which showed good discrimination for higher modified Gensini scores (AUC: 0.798 and 0.812), higher Sullivan Extent scores (AUC: 0.776 and 0.778), and the presence of obstructive CAD (AUC: 0.834 and 0.792) in two validation cohorts. A nomogram comprising GES12, smoking status, hypertension status, low density lipoprotein cholesterol level, and body mass index further improved performance, with respect to discrimination, risk classification, and clinical utility, for prediction of coronary stenosis severity. CONCLUSIONS: GES12 is useful in predicting the severity of coronary artery stenosis in patients with known or suspected CAD.

Expression pattern of miR-21, miR-25 and PTEN in peripheral blood mononuclear cells of patients with significant or insignificant coronary stenosis.

Coronary artery disease (CAD) is primarily caused by atherosclerosis, which is a series of chronic inflammatory processes leading to the initiation and progression of vascular endothelial cell injury enhancing plaque formation. As critical components of the immune system, peripheral blood mononuclear cells (PBMCs) actively cross-talk with pathophysiological conditions induced by endothelial cell injury, reflecting in altered PBMC expression pattern. This study explored PBMC expression levels of miR-21, miR-25 and PTEN in patients with angiographically proven significant coronary stenosis (the CAD group), patients with insignificant coronary stenosis (the ICAD group) and healthy subjects, and assessed potentials of PBMC expressions in discriminating groups of study subjects. In-silico analysis was also performed to obtain insights into CAD-related pathways and biological processes that may be influenced by altered miRNA expressions. A reduced level of PBMC miR-21 was observed in the ICAD group compared to the CAD group (P: 0.004) or healthy controls (P: 0.0001). PBMC miR-21 level was negatively correlated with the PTEN expression (Spearman r: -0.43, P: 3.9e-09). The PTEN expression was increased in the CAD or ICAD group compared to the control group (CAD vs. controls P: 0.0003, ICAD vs. controls P: 0.03). A stepwise increase in PBMC miR-25 levels was observed from healthy controls to ICADs and CAD patients (Kruskal-Wallis P: 7.68e-12). PBMC gene expressions had reasonable power to discriminate between pairs of study groups. PBMC miR-21 levels were able to discriminate ICADs from both CADs and controls and miR-25 levels had potentials to differentiate among all pairs of study groups (i.e. CADs-ICADs, CADs-controls, CADs-all other subjects, ICADs-controls). PBMC PTEN expression was able to discriminate patients with CAD or ICAD from control subjects. Overrepresentation enrichment analysis of experimentally validated targets of miR-21 and miR-25 highlighted key biological processes and pathways, such as "angiogenesis" and "leukocyte cell-cell adhesion", that may be influenced by dysregulation of PBMC miR-21 and miR-25. In conclusion, these findings suggest that patients with insignificant coronary stenosis may have a distinct PBMC miRNA expression profile than those with significant stenosis or healthy controls.

Long noncoding RNA H19 promotes vascular remodeling by sponging let-7a to upregulate the expression of cyclin D1.

Vascular remodeling is mainly caused by excessive proliferation of vascular smooth muscle cells (VSMCs). Noncoding RNAs (ncRNAs) have emerged as important regulators in diverse pathological processes. Previous work has shown the functions and mechanisms of long noncoding RNA H19 (LncRNA H19) on VSMCs. As long noncoding RNAs (lncRNAs) are complex in their mechanisms of action, the aim of the study is to identify if there are any other molecular mechanisms of LncRNA H19 on VSMCs. In vivo studies demonstrated that cyclin D1 was overexpressed in neointima of balloon-injured artery. In vitro studies identified that the overexpression of LncRNA H19 promoted VSMCs proliferation and cyclin D1 upregulation. On the contrary, cellular proliferation and expression of cyclin D1 were inhibited in VSMCs after infection with let-7a. Furthermore, luciferase reporter assays and RNA pull-down assays were used to explore the regulatory mechanism, we found that LncRNA H19 functioned as a competing endogenous RNA (ceRNA) by sponging let-7a to promote the expression of the target gene cyclin D1. In conclusion, LncRNA H19 positively regulated cyclin D1 expression through directly binding to let-7a in VSMCs. Our findings provide new insight into the mechanism of LncRNA H19 in VSMCs proliferation and vascular remodeling, and further indicate the implications of LncRNA H19 in the diagnosis and treatment of vascular proliferative diseases.