Zhu Jh

Effects of nicotine on the number and activity of circulating endothelial progenitor cells.

Recently, some studies have shown that nicotine increased neovascularization, which involves endothelial progenitor cells (EPCs). The effects of nicotine on EPCs are still unclear at present. Therefore, the authors investigated whether nicotine had influences on EPC number and activity. The EPCs were stimulated with nicotine (to make a series of final concentrations: 10−12 mol/L, 10−10 mol/L, 10−8 mol/L, 10−6 mol/L, 10−4 mol/L) or vehicle control for the respective time points(12, 18, 24, 32, and 48 hours). The EPCs were characterized as adherent cells double positive for DiLDL uptake and lectin binding by direct fluorescent staining under a laser‐scanning confocal microscope. They were further documented by demonstrating the expression of KDR, VEGFR‐2, and AC133 with flow cytometry. The EPC proliferation, migration, and in vitro vasculogenesis activity were assayed with the 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide assay; the modified Boyden chamber assay; and the in vitro vasculogenesis kit, respectively. The EPC adhesion assay was performed by replating those on fibronectin‐coated dishes and then counting the adherent cells. As a result, nicotine dose dependently increased the EPC number and the proliferative, migratory, adhesive, and in vitro vasculogenesis capacity at nicotine concentrations of 10−12 to 10−8 mol/L. The peak effects on EPCs were observed at concentrations of nicotine 10−8 mol/L, similar to those in the blood of smokers. In addition, nicotine (10−8 mol/L) time dependently increased the EPC number and activity. However, cytotoxicity was seen at higher nicotine concentrations (> 10−6 mol/L). In conclusion, nicotine had complex effects on EPCs: nicotine might induce the augmentation of EPCs with enhanced functional activity at relatively low concentrations. However, cytotoxicity was seen at higher nicotine concentrations.

Effects of ox-LDL on number and activity of circulating endothelial progenitor cells.

Backgrounds: Endothelial dysfunction is thought to play a crucial role in the pathogenesis of atherosclerosis induced by ox‐LDL. Recently, a variety of evidence suggested that endothelial progenitor cells (EPCs) participated in neovascularization and reendothelialization. However, effects of ox‐LDL on EPCs number and activity are ill understood. Methods: Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin‐coated culture dishes. After 7 days culture, attached cells were stimulated with ox‐LDL (to make a series of final concentrations: 25 µg/mL, 50 µg/mL, 100 µg/mL, 200 µg/mL), native LDL (100 µg/mL) or vehicle control for the respective time points (6 h, 12 h, 24 h and 48 h). EPCs were characterized as adherent cells double positive for DiLDL‐uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. EPCs were further documented by demonstrating the expression of KDR, VEGFR‐2 and AC133 with flow cytometry. Proliferation, migration and in vitro vasculogenesis activity of EPCs were assayed by MTT assay, modified Boyden chamber assay and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating those on fibronectin‐coated dishes, and then counting adherent cells. Results: Incubation of isolated human EPCs with ox‐LDL decreased the number of EPCs in concentration‐dependent manner, maximum at 200 µg/mL (approximately 70% reduction, P < 0.001). In time‐course experiments performed with an ox‐LDL concentration of 100 µg/mL, decrease of EPCs number became apparent at 12 hours and reached the maximum at 24 hours (approximately 50% reduction, P < 0.01). In addition, ox‐LDL dose and time dependently impaired EPC proliferative, migratory, adhesive and in vitro vasculogenesis capacity. Conclusion: The results of the present study defined a novel mechanism of action of ox‐LDL: the reduction of EPCs with decreased functional activity.

Serum levels of calcitonin gene-related peptide and substance P are decreased in patients with diabetes mellitus and coronary artery disease.

OBJECTIVE: This study evaluated serum levels of the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP) in coronary artery disease (CAD) patients with and without a history of diabetes mellitus (DM). METHODS: Patients undergoing coronary angiography for suspected myocardial ischaemia were divided into four groups depending on their clinical status: control group (no CAD or DM; n = 44), DM group (DM without CAD; n = 46), CAD group (stable CAD without DM; n = 44) and DM + CAD group (stable CAD with DM; n = 50). Serum levels of CGRP and SP were determined using radioimmunoassays. RESULTS: CGRP and SP levels in the DM and CAD groups were significantly lower than in the control group. The lowest levels of CGRP and SP were observed in the DM + CAD group. There were no significant differences in CGRP and SP levels between the DM group and the CAD group. CONCLUSION: CGRP and SP may have a role in the pathogenesis of CAD in patients with diabetes.

Resveratrol attenuates apoptosis of pulmonary microvascular endothelial cells induced by high shear stress and proinflammatory factors

Endothelial injury usually underlies the initial pathologic step of cardiovascular diseases. Primary endothelial cell (EC) apoptosis and secondary hyperproliferation both contribute to the development of atherosclerosis and luminal occlusion. In order to investigate the effects of resveratrol (RSV) on EC apoptosis, we applied high shear stress (HSS) with proinflammatory factors [tumor necrosis factor alpha (TNF-α) plus cycloheximide] to human pulmonary microvascular ECs (PMVECs) through an artificial capillary system. Intracellular reactive oxygen species (ROS) was measured by spectrofluorometry using dihydrorhodamine 123 fluorescent probe. Apoptosis and proliferation was determined by flow cytometric analysis. Protein expression was examined by Western blot. HSS plus inflammation significantly raised the ROS and the apoptosis level of PMVECs, which could be diminished by RSV pretreatment. In a 7-days incubation assay, RSV effectively inhibited the initial increase in apoptosis and thereby prevented subsequent PMVEC hyperproliferation induced by HSS plus inflammation. Mercaptosuccinate, a glutathione peroxidase (GPx-1) inhibitor or nicotinamide, a silent information regulator 2/sirtuin 1 (SIRT1) inhibitor could attenuate the antiapoptotic action of RSV on PMVECs; and RSV treatment upregulated GPx-1 and SIRT1 expression in PMVECs. In conclusion, RSV, probably by activating SIRT1 signaling pathway, inhibits the oxidative-stress-dependent phenotypical shift of ECs induced by HSS and proinflammatory factors in vitro.

Effects of diltiazem on platelet activation and cytosolic calcium during percutaneous transluminal coronary angioplasty

Aims: To evaluate effects of diltiazem on platelet hyper-reactivity in situations associated with endothelial injury and their possible relationship to cytosolic calcium concentration. Methods: Blood samples were collected at seven time points from 35 patients undergoing percutaneous transluminal coronary angioplasty (PTCA) who received combined diltiazem and aspirin/ticlopidine therapy or aspirin/ticlopidine therapy alone. Platelet expression of glycoprotein IIb/IIIa and P-selectin, production of thromboxane B2, and cytosolic calcium concentration were measured, respectively, by whole blood flow cytometry, radioimmunoassay, and fluorospectrophotometry. The effects of diltiazem of different concentrations on expression of glycoprotein IIb/IIIa and P-selectin were also studied in vitro in blood samples from patients with chronic stable angina. Results: Of the two treatments, aspirin/ticlopidine therapy did not prevent an acute increase of expression of glycoprotein IIb/IIIa and P-selectin and plasma thromboxane B2 five minutes and 10 minutes after first inflation and 10 minutes after PTCA, whereas combined diltiazem and aspirin/ticlopidine therapy had a significant inhibitory effect. In the group receiving aspirin/ticlopidine therapy, there was a short term increase of platelet [Ca2+]i immediately after PTCA which was significantly reduced by diltiazem treatment. Expression of glycoprotein IIb/IIIa and P-selectin was significantly inhibited in vitro by diltiazem in the concentration of 200 ng/ml or higher, but not 50 ng/ml. Conclusions: Combined diltiazem and aspirin/ticlopidine therapy significantly inhibited platelet activation that continued in the presence of conventional aspirin/ticlopidine treatment. Antiplatelet effects of diltiazem were probably a consequence of reduction of platelet [Ca2+]i and may only be achieved in higher than therapeutic concentrations.

Up-Regulation of CXC Chemokine Receptor 4 Expression in Chronic Atrial Fibrillation Patients with Mitral Valve Disease May Be Attenuated by Renin–Angiotensin System Blockers

This study characterized CXC chemokine receptor 4 (CXCR4) expression in patients with mitral valve disease and chronic atrial fibrillation (AF). Forty-eight patients with chronic AF formed two groups based on whether they were treated with or without renin–angiotensin system (RAS) blockers (AF + RAS group; n = 25, or AF – RAS group; n = 23). The controls comprised 17 mitral valve disease patients with sinus rhythm (SR group). CXCR4 mRNA and protein levels in the left atria were significantly higher in the AF – RAS and AF + RAS groups than in the SR group. CXCR4 expression was significantly lower in the AF + RAS group than the AF – RAS group. More CD34+ cells expressed CXCR4 in the AF – RAS and AF + RAS groups than in the SR group. Angiotensin II, collagen I and left atrial diameter significantly positively correlated with CXCR4 expression in the AF – RAS group. These results suggest that CXCR4 expression is up-regulated in chronic AF patients with mitral valve disease, is associated with atrial remodelling, and that these effects are attenuated by RAS blockers.