A novel minimally-invasive method to sample human endothelial cells for molecular profiling.

OBJECTIVE: The endothelium is a key mediator of vascular homeostasis and cardiovascular health. Molecular research on the human endothelium may provide insight into the mechanisms underlying cardiovascular disease. Prior methodology used to isolate human endothelial cells has suffered from poor yields and contamination with other cell types. We thus sought to develop a minimally invasive technique to obtain endothelial cells derived from human subjects with higher yields and purity. METHODS: Nine healthy volunteers underwent endothelial cell harvesting from antecubital veins using guidewires. Fluorescence-activated cell sorting (FACS) was subsequently used to purify endothelial cells from contaminating cells using endothelial surface markers (CD34/CD105/CD146) with the concomitant absence of leukocyte and platelet specific markers (CD11b/CD45). Endothelial lineage in the purified cell population was confirmed by expression of endothelial specific genes and microRNA using quantitative polymerase chain reaction (PCR). RESULTS: A median of 4,212 (IQR: 2161-6583) endothelial cells were isolated from each subject. Quantitative PCR demonstrated higher expression of von Willebrand Factor (vWF, P<0.001), nitric oxide synthase 3 (NOS3, P<0.001) and vascular cell adhesion molecule 1 (VCAM-1, P<0.003) in the endothelial population compared to similarly isolated leukocytes. Similarly, the level of endothelial specific microRNA-126 was higher in the purified endothelial cells (P<0.001). CONCLUSION: This state-of-the-art technique isolates human endothelial cells for molecular analysis in higher purity and greater numbers than previously possible. This approach will expedite research on the molecular mechanisms of human cardiovascular disease, elucidating its pathophysiology and potential therapeutic targets.

Receptor for advanced glycation end products (RAGE) and its inflammatory ligand EN-RAGE in non-diabetic subjects with pre-mature coronary artery disease.

OBJECTIVE: Inflammation participates in atherosclerosis from its inception onwards. RAGE (receptor for advanced glycation end products) and its natural pro-inflammatory ligand, EN-RAGE (extracellular newly identified RAGE-binding protein) have been implicated in various inflammatory diseases. In present study, we determined the expression of RAGE and EN-RAGE in peripheral blood mononuclear cells (PBMCs) of subjects with pre-mature coronary artery disease (CAD) for the first time. METHODS AND RESULTS: The study patients were angiographically proven non-diabetic patients with pre-mature CAD (Group I; N=100) and control group comprised of subjects with coronary risk factors and without coronary artery lesions (Group II; N=40). Semi-quantitative RT-PCR was performed to determine transcriptional expression of RAGE and EN-RAGE in PBMCs. Soluble RAGE (sRAGE) and C-reactive protein (hsCRP) levels were determined in serum of all study subjects using immunoassays. A significantly increased transcriptional expression of RAGE and EN-RAGE in PBMCs (p<0.01) of Group I patients was observed. Increased circulating hsCRP (p<0.01) levels and decreased sRAGE (p<0.01) levels were observed in Group I as compared with the Group II subjects. Severity of disease determined by Gensini score was found to be positively correlated with transcriptional expression of RAGE (r=0.530) and EN-RAGE (r=0.323). EN-RAGE expression revealed a strong association with RAGE (r=0.326), hsCRP (r=0.251) and a negative association with sRAGE (r=-0.222). CONCLUSIONS: Increased expression of RAGE and EN-RAGE in non-diabetic pre-mature CAD and various associations discussed may amplify several cellular perturbations and thus significantly contribute to the pathophysiology of CAD.

Correlation among soluble markers and severity of disease in non-diabetic subjects with pre-mature coronary artery disease.

Studies are lacking in literature, which demonstrate the cumulative impact of certain soluble markers in predicting the severity of CAD. Serum hsCRP, MMP-9, TIMP-1 and sRAGE levels were measured in non-diabetic 100 angiographically proven CAD patients (Group I) and 40 non-diabetic subjects with coronary risk factors and without any lesions (Group II). Increased levels of serum hsCRP, MMP-9, TIMP-1 and decreased levels of sRAGE were observed in Group I as compared to Group II. Gensini score, a measure for severity of CAD was found to be positively correlated with serum hsCRP, MMP-9, TIMP-1 and negatively with sRAGE. Multivariate analysis revealed serum MMP-9, hsCRP, sRAGE and family history as predictors of severity of CAD with a cumulative sensitivity and specificity of 92% and 82%, respectively. Cumulative impact of these soluble markers, in addition to the established markers will contribute to improve the predictive value for the assessment of disease severity.

Inhibitory effects of Terminalia arjuna on platelet activation in vitro in healthy subjects and patients with coronary artery disease.

Terminalia arjuna (TA) is a medicinal plant used as a cardiotonic in ayurveda. Besides others, scientific evidence dictates its strong hypolipidemic and antioxidant properties. However, anti-inflammatory and antiplatelet aggregatory properties of TA are not known. The present study demonstrates in vitro effects of its ethanolic bark extract (TAE) on platelet function indices. Twenty patients of angiographically proven coronary artery disease (CAD) were included in Group I and 20 age and sex-matched controls were included in Group II. Platelet activation was monitored by determining P-selectin (CD62P) expression, intracellular free calcium (Ca(2+)) release and platelet aggregation. In vitro effect of TA on platelets function indices was determined by incubating the platelets with TAE in a time and dose-dependent manner in presence/absence of ADP. TAE was able to significantly inhibit platelet aggregation both in patient and control groups. Significant attenuation in Ca(2+) release and expression of CD62P was also observed with TAE. Our data clearly demonstrates that the bark extract of TA decreases platelet activation and may possess antithrombotic properties. The possible mechanism of action could be by desensitizing platelets to the agonist by competing with platelet receptor or by interfering with signal transduction. Thus, TA can be exploited for its therapeutic potential in CAD and related cardiovascular disorders.