Plasma Of Coronary Artery Disease Patients Research Articles

Knockdown of lncRNA H19 alleviates ox-LDL-induced HCAECs inflammation and injury by mediating miR-20a-5p/HDAC4 axis.

Coronary artery disease (CAD) seriously disturbs the life of people. LncRNA H19 is reported to promote the progression of CAD; Nevertheless, the detailed mechanism by which H19 modulates CAD development is unclear. Clinical samples of CAD patients were collected, meanwhile we established in vitro and in vivo models of CAD by treating HCAECs with ox-LDL and feeding ApoE-/- mice with high fat diets (HFD). MTT assay was adopted to assess the cell viability. Transwell detection was applied to test the migration, and apoptosis was tested by flow cytometry. The levels of inflammatory cytokines were examined by ELISA. The relation among H19, miR-20a-5p and HDAC4 was explored by dual luciferase reporter and RIP assay. H19 and HDAC4 levels were elevated, while miR-20a-5p was reduced in plasma of CAD patients and ox-LDL-treated HCAECs. ox-LDL increased H19 level and induced apoptosis and inflammation in HCAECs, while silencing of H19 rescued this phenomenon. In addition, the level of H19 was negatively correlated with miR-20a-5p, and miR-20a-5p inhibitor restored the effect of H19 silencing on HCAECs function. HDAC4 was the downstream mRNA of miR-20a-5p, and miR-20a-5p upregulation reversed ox-LDL-induced HCAECs injury through targeting HDAC4. Furthermore, H19 silencing significantly alleviated the coronary atherosclerotic plaques and inhibited the inflammatory responses in vivo. We proved that knockdown of H19 alleviated ox-LDL-induced HCAECs injury via miR-20a-5p/HDAC4 axis, which might provide a new tacticsagainst CAD.

The association of plasma levels of miR-146a, miR-27a, miR-34a, and miR-149 with coronary artery disease.

Coronary artery disease (CAD) is considered to be one of the most pivotal causes of death in the world. Over the past two decades, significant changes occurred in the diagnosis, prognosis, and treatment of CAD, which has helped reduce mortality rates. microRNAs (miRs) are a class of more than 5000 non-encoding RNA molecules (21-25 nucleotides across the length) that regulate complex biological processes. Today, miRNAs are used to study cardiovascular diseases. In the present study, the expression of miR-146a،miR-27, miR-149, and miR-34a in plasma suffering from CAD and the control group were investigated. The present research was performed on 30 men with CAD and 30 healthy men as controls. The expression levels of miR-146a, miR-27a, miR-149, and miR-34a in the plasma of patients with CAD and the control group were measured using real-time PCR. Also, the correlation between the expression of circulating miRs levels and biochemical LDL-C, HDL-C, BMI, and total cholesterol was evaluated. The expression of miR-27a in the plasma of the CAD group was higher than in the control group (p = 0.020). The expression of miR-146a was downregulated in CAD patients compared to normal subjects (p = 0. 026). However, the expression of miR-34a, miR-149 in the plasma of CAD patients was not significantly different with the control group. In addition to, a direct correlation was found between the expression of miR-146a and HDL-c, the expression of miR-27a and LDL-C and the expression of miR-34a and total cholesterol. Also, the negative correlation between expressions of miR-149 with BMI was reported. The obtained results demonstrated that miRs were closely related to biochemical factors and it points out the fact that miRNAs can be applied as a potential strategy for diagnosis and treatment of CAD.

Endothelial miR-199a-3p regulating cell adhesion molecules by targeting mTOR signaling during inflammation

BackgroundAdherence of monocytes to endothelial cells is the initial stage for development of coronary artery disease (CAD). MiRNAs have been reported to participate in this process by regulating the expression of cell adhesion molecules. This study aimed to explore the function of miR-199a-3p in endothelial inflammation and adhesion. MethodsWe assessed the expression of miR-199a-3p in CAD patients and ApoE-/- mice. The relationship between miR-199a-3p level and endothelial inflammation and adhesion was examined. ELISA was used to test the level of IL-6 and IL-8. Dual luciferase reporter assay was used to evaluate the binding between miR-199a-3p and mTOR. ResultsA decreased expression of miR-199a-3p was observed in the PBMCs and plasma of CAD patients, aorta of ApoE-/- mice and inflammatory HUVECs. MiR-199a-3p significantly suppressed the expression levels of pro-inflammatory cytokine (IL-6, IL-8), endothelial adhesion molecules (ICAM-1, VCAM-1) and monocyte-endothelial cells interaction. MiR-199a-3p directly targeted and repressed mTOR, and its suppression effect on ICAM-1 and VCAM-1 was abolished by mTOR inhibitor rapamycin, and rescued by mTOR activator MHY1485. Overexpression of miR-199a-3p promoted autophagy in HUVECs and inhibiting autophagy by chloroquine attenuated the effect of miR-199a-3p on ICAM-1 and VCAM-1 expression. Inhibition of autophagy promoted endothelial adhesion molecule expression and monocyte-EC interaction. ConclusionsOur results suggested that miR-199a-3p suppressed endothelial inflammation and adhesion by targeting mTOR signaling and increasing autophagy. Our findings point to an important role for miR-199a-3p in the early stage of cardiovascular disease.

MiR-654-5p Suppresses Migration and Proliferation of Vascular Smooth Muscle Cells by Targeting ADAMTS-7

Coronary artery disease (CAD) is the first leading cause of death worldwide. Therefore, novel therapeutic strategies need to be explored. Numerous publications reported that microRNA-654-5p (miR-654-5p) had anti-cancer activities in various cancers, and it was proven to modulate cell migration, invasion, and proliferation, which played critical roles in CAD. However, its role in CAD is unknown. Thus, we aimed to evaluate the role of miR-654-5p in vascular smooth muscle cells (VSMCs) involved in CAD. A total of 25 CAD patients and 19 healthy individuals were enrolled to evaluate their circulating miR-654-5p levels. miR-654-5p mimic or inhibitor were transfected into human VSMCs to assess their role on cell migration and proliferation. Target genes of miR-654-5p were predicted using TargetScan 7.2 and confirmed by the dual-luciferase reporter assay. miR-654-5p was significantly downregulated in the plasma of CAD patients and tumor necrosis factor-a/platelet-derived growth factor (PDGF)-BB-stimulated VSMCs. miR-654-5p mimic inhibited the proliferation and migration of VSMCs, which could be promoted by miR-654-5p inhibitor. A disintegrin and metalloproteinase with thrombospondin motifs-7 (ADAMTS-7) was identified as the direct target of miR-654-5p, whose expression could be induced by miR-654-5p inhibitor and decreased by its mimic. In addition, ADAMTS-7 overexpression blocked the inhibitory effect of miR-654-5p on the migration and proliferation of VSMCs. In summary, miR-654-5p inhibits the migration and proliferation of VSMCs by directly targeting ADAMTS-7, and miR-654-5p might serve as a novel therapeutic target for the treatment of CAD.

Coagulation markers in patients with coronary artery disease

Coronary Artery Disease (CAD) is a major public health problem all over the world, and although diagnostic and treatment approaches are improving, the majority of individuals who suffer CAD will not survive. Therefore, it is a clinical demand to find out new biomarkers for identification of CAD in the earlier stage. The purpose of this study was to determine whether qualitative and quantitative alterations of plasma Soluble Fibrin Monomer Complexes (SFMCs) are associated with cardiovascular events. Additionally, some other well-known biomarkers of hypercoagulation state, such as fibrinogen, and prothrombin were analyzed in plasma of patients with different coronary artery pathologies. We found that plasma SFMC concentrations were remarkably increased in all tested CAD groups. Moreover, the development of CAD was accompanied by the changes in both quantity and quality of SFMC composition compared to that of healthy subjects. Our results also demonstrated the elevated level of high molecular weight Vitamin K-Dependent Protein (VKDP) complexes in plasma of CAD patients, which could be used as a potential biomarker of hypercoagulation state. More studies are clearly needed to investigate whether SFMCs or abnormal forms of VKDPs affect cardiovascular events under CAD development and progression.

Relationship between Expression of Plasma lncRNA-HEIH and Prognosis in Patients with Coronary Artery Disease.

Objective We aimed to investigate the expression of long noncoding RNA- (lncRNA-) HEIH in patients with coronary artery disease (CAD) and its impact on patients' prognosis. Patients and Methods. From July 2015 to December 2018, 250 patients who underwent coronary angiography, including 50 in the control group and 150 in the CAD group, were collected for detection of the expression of lncRNA-HEIH by real-time quantitative polymerase chain reaction (qPCR). The severity of CAD was evaluated through SYNTAX scoring system. In addition, these patients with CAD were followed up for 3 years, and the major cardiac adverse events such as myocardial infarction and revascularization were recorded. Results The expression of lncRNA-HEIH in plasma of patients with CAD was remarkably higher than that in the control subjects and was verified to be relevant to the severity of CAD. Meanwhile, it was found that CAD patients with high expression of lncRNA-HEIH had higher rates of dyslipidemia as well as CAD family history and higher overall incidence of major cardiac adverse events than those with low expression of lncRNA-HEIH. Conclusions lncRNA-HEIH expression is upregulated in the plasma of CAD patients, which is capable of affecting the prognosis of patients.

Hsa_circ_0054633 regulates PDGF-BB-induced proliferation, migration and oxidative stress of vascular smooth muscle cells through miR-107/TXNIP axis

IntroductionHsa_circ_0054633 has been found to be elevated in the blood of coronary artery disease (CAD) patients. However, the molecular mechanism and the role of hsa_circ_0054633 in the pathogenesis of CAD have not been reported in detail.Material and methodsThe expression of hsa_circ_0054633, microRNA (miR)-107 and thioredoxin-interacting protein (TXNIP) mRNA was measured using quantitative real-time polymerase chain reaction. Human artery vascular smooth muscle cell (HA-VSMC) proliferation, cell cycle, and migration were detected by cell counting kit-8 assay, flow cytometry and transwell assay, respectively. The generation of reactive oxygen species (ROS) was analyzed by dichlorofluorescein diacetate (DCFH-DA) assay. Western blot was utilized to determine the levels of proliferating cell nuclear antigen (PCNA), cyclin D1, matrix metallopeptidase 9 (MMP-9), Mn-superoxide dismutase (SOD2) and TXNIP protein. The interaction between miR-107 and hsa_circ_0054633 or TXNIP was confirmed by dual-luciferase reporter, RNA immunoprecipitation assay or pull-down assay.ResultsHsa_circ_0054633 was elevated in the plasma of CAD patients, and might be a potential blood biomarker for CAD prediction. Hsa_circ_0054633 silencing reversed PDGF-BB-induced promotion on HA-VSMC proliferation, cell cycle, migration and ROS production. MiR-107 directly interacted with hsa_circ_0054633 and TXNIP, and hsa_circ_0054633 regulated TXNIP expression by sponging miR-107. Besides, rescue assay indicated that the action of hsa_circ_0054633 silencing on PDGF-BB-treated HA-VSMCs could be attenuated by miR-107 inhibition or TXNIP overexpression, respectively.ConclusionsHsa_circ_0054633 knockdown protected HA-VSMCs against PDGF-BB-induced dysfunction through regulating miR-107/TXNIP axis, suggesting a potential therapeutic target for coronary atherosclerosis.

Circulating miR-133b and miR-21: potential non-invasive diagnostic biomarkers of coronary artery disease

Abstract Background While coronary artery disease (CAD) has become a major threat worldwide, the timely biomarker-based early diagnosis of CAD remains a major unmet clinical challenge. Purpose We aimed towards assessing the level of circulatory microRNAs as candidates of novel biomarkers in early diagnosis of CAD. Methods A total of 147 subjects were recruited from Delhi and surrounding regions which includes 78 subjects with angiographically proven CAD, 15 pre-atherosclerotic normal coronary artery (NCA) subjects and 54 healthy individuals. Quantitative real-time PCR assays were performed using total RNA isolated from plasma of CAD patients and healthy individuals in order to measure the relative expression profiles of circulating miR-133b and miR-21. The severity level of the disease in each individual was assessed on the basis of number of diseased vessels involved and extent of stenosis. Results MiR-133b was found to be significantly downregulated (p<0.05) by 4.6 fold whereas miR-21 was significantly upregulated (p<0.05) by ∼2 fold in plasma samples of CAD patients as compared to healthy individuals. Importantly, both the miRNAs showed an association with disease severity as miR-133b was downregulated by 8.45 fold in acute coronary syndrome (ACS), 3.38 fold in Stable angina (SA) and 2.08 fold in NCA. MiR-21 was upregulated by 2.46 fold in ACS, 1.90 fold in SA and 1.12 fold in NCA. Moreover, miR-133b could significantly differentiate subjects with ST-elevation myocardial infarction (STEMI) from Non-STEMI. Both the miRNAs exhibited a noteworthy diagnostic potential as translated through an upstanding sensitivity and specificity. MiR-133b showed an area under the curve (AUC): 0.80 with 75.6% sensitivity and 76% specificity at recommended optimal cut-off point of 14.0 whereas, miR-21 showed and AUC value of 0.79 with 72.2% sensitivity and 69.4% specificity at optimal cut-off point of 5.59 as assessed by receiver operating characteristics (ROC) curve analysis. Conclusions The reduced expression of miR-133b and elevated expression level of miR-21 in plasma suggest their significant role in the pathophysiology as well as their implication towards the individual's susceptibility in developing CAD. Thus, we conclude that these two circulating miRNAs could be possible candidates of non-invasive biomarkers in the early prediction and diagnosis of CAD. Funding Acknowledgement Type of funding source: Other. Main funding source(s): Department of Science and Technology, New Delhi, India; University Grant Commission (JRF/SRF Fellowship: Ref no. 22/12/2013(ii)EU-V

Single Endothelial Cell mRNA Sequencing Better Captures the Severity of Coronary Artery Disease Than Targeted Total Arterial Markers of Inflammation and Senescence

Endothelial dysfunction is the initial step towards atherosclerotic plaque development and coronary artery disease (CAD). Senescent endothelial cells (SnEC) have been linked to the atherosclerotic burden. In particular, the senescent‐associated secretory phenotype protein angiopoietin‐like 2 (ANGPTL2) was shown to be elevated in the plasma of CAD patients and related to the senescent cellular load in human internal mammary artery (IMA) segments discarded during coronary artery bypass grafting (CABG) surgery. We tested the hypothesis that the accumulation of vascular SnEC causes endothelial dysfunction and precedes the appearance of atherosclerotic lesions.Discarded atheroma‐free IMA segments from 12 patients (11 men and 1 woman, 69±3 years) were collected during consecutive elective CABG surgeries. Arterial rings of IMA segments were mounted in a wire myograph to record isometric changes in tension: arteries were pre‐contracted with U46619, a synthetic analogue of PGH2, and endothelium‐dependent relaxations to increasing concentrations of acetylcholine (ACh) were recorded. The maximal relaxation (Emax) and the concentration of ACh inducing 50% of relaxation (EC50) were calculated. Afterwards total mRNA was extracted from the IMA segments. Vascular gene expression of ANGPTL2 and p21 (senescence markers), and CD68 and PAI‐1 (inflammatory markers) were assessed by RT‐qPCR. In parallel, single‐cell RNA sequencing was performed in IMA segments from two age‐matched male patients obtained either during elective (stable CAD) or emergency (unstable CAD, with numerous risk factors) procedures, and differential gene expression was specifically analyzed in vascular EC.Endothelium‐dependent relaxations were characterized by Emax (56±7 %, [18–100%], n=12) and pD2 (‐log EC50: 6.9±0.1 [5.8–7.3], n=12). Patients were divided into 2 groups according to their Emax (< or > to 50%) to define low and high endothelial function; vascular gene expression of the 4 senescence and inflammatory markers were similar between the two groups, demonstrating that global arterial wall senescence and inflammation did not distinguish severity of endothelial dysfunction. We then used single‐cell mRNA sequencing and analysed the unbiased differential gene expression specifically in vascular EC. From the total IMA cellular counts, EC represented ~2%. Nine genes were identified that were differentially overexpressed in EC from unstable CAD patient that have been associated with senolytic drug targets and cardiovascular diseases (Table ), potentially attesting to the severity of endothelial dysfunction and CAD.In conclusion, unlike total arterial wall mRNA quantification, unbiased single‐cell mRNA sequencing identified differentially upregulated endothelial pathways that may contribute to the severity of the CAD by inducing precocious endothelial dysfunction and senescence. Top genes differentially expressed in endothelial cells between a stable and unstable CAD patient. Percentages represent the percent of endothelial cells expressing the gene in each patient dataset. P‐values are Bonferroni corrected. Stable CAD patient Unstable CAD patient p‐value Gene Associated CVD disorder or drug target 0.0% 41.7% 0.00061 TGFB2 Atrial fibrillation 0.0% 41.7% 0.00061 SQLE Statins 2.9% 58.3% 0.00090 RPPH1 Quercetin (senolytic drug) 7.1% 66.7% 0.00339 ARL14EP Systolic blood pressure 25.7% 100.0% 0.00914 RPL17 Myocardial ischemia 17.1% 83.3% 0.01556 IGFBP7 Ruptured aneurysm 0.0% 33.3% 0.01699 TMEM8B Heart valve disorders 0.0% 33.3% 0.01699 ZNF675 Congestive heart failure 8.6% 66.7% 0.04848 SELL Vaso‐occlusive crisis

Extracellular vesicles with ubiquitinated adenosine A2A receptor in plasma of patients with coronary artery disease.

Extracellular vesicles (EV) can transfer cellular molecules for specific intercellular communication with potential relevance in pathological conditions. We searched for the presence in plasma from coronary artery disease (CAD) patients of EV containing the adenosine A2A receptor (A2AR), a signalling receptor associated with myocardial ischaemia and whose expression is related to homocysteine (HCy) metabolism. Using protein organic solvent precipitation for plasma EV preparation and Western blotting for protein identification, we found that plasma from CAD patients contained various amounts of EV with ubiquitin bound to A2AR. Interestingly, the presence of ubiquitinated A2AR in EV from patients was dependent on hyperhomocysteinemia, the amount being inversely proportional to A2AR expression in peripheral mononuclear cells in patients with the highest levels of HCy. CEM, a human T cell line, was also found to released EV containing various amounts of ubiquitinated A2AR in stimulated conditions depending on the hypoxic status and HCy level of culture medium. Together, these data show that ubiquitinated A2AR‐containing EV circulate in the plasma of CAD patients and that this presence is related to hyperhomocysteinemia. A2AR in plasma EV could be a useful tool for diagnosis and a promising drug for the treatment of CAD.

Dysregulation of endothelial colony-forming cell function by a negative feedback loop of circulating miR-146a and -146b in cardiovascular disease patients.

Functional impairment of endothelial colony-forming cells (ECFCs), a specific cell lineage of endothelial progenitor cells (EPCs) is highly associated with the severity of coronary artery disease (CAD), the most common type of cardiovascular disease (CVD). Emerging evidence show that circulating microRNAs (miRNAs) in CAD patients’ body fluid hold a great potential as biomarkers. However, our knowledge of the role of circulating miRNA in regulating the function of ECFCs and the progression of CAD is still in its infancy. We showed that when ECFCs from healthy volunteers were incubated with conditioned medium or purified exosomes of cultured CAD ECFCs, the secretory factors from CAD ECFCs dysregulated migration and tube formation ability of healthy ECFCs. It is known that exosomes influence the physiology of recipient cells by introducing RNAs including miRNAs. By using small RNA sequencing (smRNA-seq), we deciphered the circulating miRNome in the plasma of healthy individual and CAD patients, and found that the plasma miRNA spectrum from CAD patients was significantly different from that of healthy control. Interestingly, smRNA-seq of both healthy and CAD ECFCs showed that twelve miRNAs that had a higher expression in the plasma of CAD patients also showed higher expression in CAD ECFCs when compared with healthy control. This result suggests that these miRNAs may be involved in the regulation of ECFC functions. For identification of potential mRNA targets of the differentially expressed miRNA in CAD patients, cDNA microarray analysis was performed to identify the angiogenesis-related genes that were down-regulated in CAD ECFCs and Pearson’s correlation were used to identify miRNAs that were negatively correlated with the identified angiogenesis-related genes. RT-qPCR analysis of the five miRNAs that negatively correlated with the down-regulated angiogenesis-related genes in plasma and ECFC of CAD patients showed miR-146a-5p and miR-146b-5p up-regulation compared to healthy control. Knockdown of miR-146a-5p or miR-146b-5p in CAD ECFCs enhanced migration and tube formation activity in diseased ECFCs. Contrarily, overexpression of miR-146a-5p or miR-146b-5p in healthy ECFC repressed migration and tube formation in ECFCs. TargetScan analysis showed that miR-146a-5p and miR-146b-5p target many of the angiogenesis-related genes that were down-regulated in CAD ECFCs. Knockdown of miR-146a-5p or miR-146b-5p restores CAV1 and RHOJ levels in CAD ECFCs. Reporter assays confirmed the direct binding and repression of miR-146a-5p and miR-146b-5p to the 3’-UTR of mRNA of RHOJ, a positive regulator of angiogenic potential in endothelial cells. Consistently, RHOJ knockdown inhibited the migration and tube formation ability in ECFCs. Collectively, we discovered the dysregulation of miR-146a-5p/RHOJ and miR-146b-5p/RHOJ axis in the plasma and ECFCs of CAD patients that could be used as biomarkers or therapeutic targets for CAD and other angiogenesis-related diseases.

MiR-206 Suppresses the Progression of Coronary Artery Disease by Modulating Vascular Endothelial Growth Factor (VEGF) Expression.

BackgroundWe investigated whether microRNA-206 (miR-206) is abnormally expressed in patients with coronary artery disease (CAD). The potential mechanism by which miR-206 may regulate CAD progression was also studied.Material/MethodsA total of 78 CAD patients in the case group and 65 subjects in the control group were enrolled in this study so that the correlation between miR-206 and CAD could be accurately determined. Serum total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides were detected using a biochemistry analyzer. MiR-206 and vascular endothelial growth factor (VEGF) expression levels were tested using either reverse transcription polymerase chain reaction or western blot. Associations between miR-206 expression and different clinicopathological features of CAD patients were also analyzed. CAD cells were transfected with miR-206 mimic (miR-206), its negative control (miR-NC), miR-206 inhibitor (anti-miR-206), and its negative control (anti-miR-NC), respectively. Flow cytometry was conducted to explore the function of miR-206 in CAD cell apoptosis after transfection. Moreover, transwell assay was carried out to study the migratory ability of endothelial progenitor cells (EPCs) in CAD patients.ResultsMiR-206 expression was enriched in both diseased EPCs and plasma of CAD patients. No significant correlation was found between decrease in miR-206 expression and different clinicopathological features. In addition, miR-206 significantly suppressed the viability and invasion of EPCs in CAD patients, and it promoted the apoptosis of their EPCs. Moreover, we found that miR-206 is able to inhibit VEGF expression.ConclusionsAs suggested by our study, MiR-206 can be a novel benign biomarker for CAD because it may regulate VEGF expression.

MiR-34a, miR-21 and miR-23a as potential biomarkers for coronary artery disease: a pilot microarray study and confirmation in a 32 patient cohort

The aim of this study was to investigate the expression of circulating microRNAs (miRNAs) in apolipoprotein E (apoE) knockout mice (apoE−/−) and to validate the role of these miRNAs in human coronary artery disease (CAD). Pooled plasma from 10 apoE−/− mice and 10 healthy C57BL/6 (B6) mice was used to perform the microarray analysis. The results showed that miR-34a, miR-21, miR-23a, miR-30a and miR-106b were differentially expressed in apoE−/− mice, and these expression changes were confirmed by real-time quantitative reverse-transcription PCR. Then, miR-34a, miR-21, miR-23a, miR-30a and miR-106b were detected in the plasma of 32 patients with CAD and of 20 healthy controls. Only miR-34a, miR-21 and miR-23a were significantly differentially expressed in the plasma of CAD patients (all P<0.01). In conclusion, miR-34a, miR-21 and miR-23a were elevated in CAD patients, which means that these miRNAs might serve as biomarkers of CAD development and progression.

Dysregulated miR-361-5p/VEGF axis in the plasma and endothelial progenitor cells of patients with coronary artery disease.

Dysfunction and reduction of circulating endothelial progenitor cell (EPC) is correlated with the onset of cardiovascular disorders including coronary artery disease (CAD). VEGF is a known mitogen for EPC to migrate out of bone marrow to possess angiogenic activities, and the plasma levels of VEGF are inversely correlated to the progression of CAD. Circulating microRNAs (miRNAs) in patient body fluids have recently been considered to hold the potential of being novel disease biomarkers and drug targets. However, how miRNAs and VEGF cooperate to regulate CAD progression is still unclear. Through the small RNA sequencing (smRNA-seq), we deciphered the miRNome patterns of EPCs with different angiogenic activities, hypothesizing that miRNAs targeting VEGF must be more abundant in EPCs with lower angiogenic activities. Candidates of anti-VEGF miRNAs, including miR-361-5p and miR-484, were enriched in not only diseased EPCs but also the plasma of CAD patients. However, we found out only miR-361-5p, but not miR-484, was able to suppress VEGF expression and EPC activities. Reporter assays confirmed the direct binding and repression of miR-361-5p to the 3′-UTR of VEGF mRNA. Knock down of miR-361-5p not only restored VEGF levels and angiogenic activities of diseased EPCs in vitro, but further promoted blood flow recovery in ischemic limbs of mice. Collectively, we discovered a miR-361-5p/VEGF-dependent regulation that could help to develop new therapeutic modalities not only for ischemia-related diseases but also for tumor angiogenesis.

Nitric Oxide Synthetic Pathway in Red Blood Cells Is Impaired in Coronary Artery Disease

BackgroundAll the enzymatic factors/cofactors involved in nitric oxide (NO) metabolism have been recently found in red blood cells. Increased oxidative stress impairs NO bioavailability and has been described in plasma of coronary artery disease (CAD) patients. The aim of the study was to highlight a potential dysfunction of the metabolic profile of NO in red blood cells and in plasma from CAD patients compared with healthy controls.MethodsWe determined L-arginine/NO pathway by liquid-chromatography tandem mass spectrometry and high performance liquid chromatography methods. The ratio of oxidized and reduced forms of glutathione, as index of oxidative stress, was measured by liquid-chromatography tandem mass spectrometry method. NO synthase expression and activity were evaluated by immunofluorescence staining and ex-vivo experiments of L-[15N2]arginine conversion to L-[15N]citrulline respectively.ResultsIncreased amounts of asymmetric and symmetric dimethylarginines were found both in red blood cells and in plasma of CAD patients in respect to controls. Interestingly NO synthase expression and activity were reduced in CAD red blood cells. In contrast, oxidized/reduced glutathione ratio was increased in CAD and was associated to arginase activity.ConclusionOur study analyzed for the first time the whole metabolic pathway of L-arginine/NO, both in red blood cells and in plasma, highlighting an impairment of NO pathway in erythrocytes from CAD patients, associated with decreased NO synthase expression/activity and increased oxidative stress.

Enhanced Expression of the Homeostatic Chemokines CCL19 and CCL21 in Clinical and Experimental Atherosclerosis

Based on their role in T-cell homing into nonlymphoid tissue, we examined the role of the homeostatic chemokines CCL19 and CCL21 and their common receptor CCR7 in coronary artery disease (CAD). We performed studies in patients with stable (n=40) and unstable (n=40) angina and healthy controls (n=20), in vitro studies in T-cells and macrophages, and studies in apolipoprotein-E-deficient (ApoE-/-) mice and human atherosclerotic carotid plaques. We found increased levels of CCL19 and CCL21 within the atherosclerotic lesions of the ApoE-/- mice, in human atherosclerotic carotid plaques, and in plasma of CAD patients. Whereas strong CCR7 expression was seen in T-cells from murine and human atherosclerotic plaques, circulating T-cells from angina patients showed decreased CCR7 expression. CCL19 and CCL21 promoted an inflammatory phenotype in T-cells and macrophages and increased matrix metalloproteinase (MMP) and tissue factor levels in the latter cell type. Although aggressive statin therapy increased CCR7 and decreased CCL19/CCL21 levels in peripheral blood from CAD patients, conventional therapy did not. The abnormal regulation of CCL19 and CCL21 and their common receptor in atherosclerosis could contribute to disease progression by recruiting T-cells and macrophages to the atherosclerotic lesions and by promoting inflammatory responses in these cells.