Xing-Xiang Wang

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.

Superparamagnetic iron oxide nanoparticles may affect endothelial progenitor cell migration ability and adhesion capacity

BACKGROUND AIMS Cell labeling with superparamagnetic iron oxide (SPIO) nanoparticles enables non-invasive tracking of transplanted cells. The aim of this study was to investigate whether SPIO nanoparticles have an effect on endothelial progenitor cell (EPC) functional activity and the feasibility of a protocol for labeling swine- and rat-origin EPC using SPIO nanoparticles at an optimized low dosage. METHODS EPC were isolated from the peripheral blood of swine and bone marrow of rat and characterized. After ex vivo cultivation, EPC were labeled with SPIO nanoparticles (to make a series of final concentrations, 50, 100, 200 and 400 microg/mL) or vehicle control. We also investigated the long-term effects of 200 microg/mL SPIO nanoparticles on EPC (4, 8, 12 and 16 days after labeling). The labeling efficiency was tested through Prussian blue (PB) staining and the intracellular iron uptake was also measured quantitatively and confirmed. EPC proliferation and migration were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and transwell chamber assay, respectively. An EPC adhesion assay was performed by replating the cells on fibronectin-coated dishes and then counting the adherent cells. EPC apoptosis was evaluated using an Annexin V-FITC apoptosis kit. RESULTS SPIO nanoparticles impaired EPC migration and promoted EPC adhesion. EPC proliferation and apoptosis were not affected. SPIO nanoparticles could label EPC efficiently at 200 microg/mL overnight without significantly affecting EPC functional activity. CONCLUSIONS SPIO nanoparticles impaired the EPC migration ability and promoted the EPC adhesion capacity. EPC could be labeled efficiently at an appropriate concentration (200 microg/mL) without significantly affecting their functional activity.

Endothelial progenitor cells may inhibit apoptosis of pulmonary microvascular endothelial cells: new insights into cell therapy for pulmonary arterial hypertension

BACKGROUND AIMS Endothelial apoptosis underlies the pathophysiology of pulmonary arterial hypertension (PAH). Some factors/cytokines released by endothelial progenitor cells (EPC) have been revealed as potent inhibitors of apoptosis. The aim of this study was to investigate the effects of EPC on pulmonary microvascular endothelial cell (PMVEC) survival with the PAH condition. METHODS PMVEC apoptosis was induced by high shear stress (HSS) with serum starvation or pro-inflammatory factors in an artificial capillary system. EPC were delivered into monocrotaline-induced PAH nude rats. RESULTS PMVEC apoptosis under HSS and serum starvation conditions was significantly inhibited by EPC conditioned medium (CM). It was attenuated by vascular endothelial growth factor (VEGF)-A or -B blocking. EPC CM promoted PMVEC proliferation, which was weakened by VEGF-A or interleukin (IL)-8 blocking. The EPC CM caused less apoptosis of PMVEC induced by HSS plus pro-inflammatory factors. The anti-apoptotic effect of EPC CM was attenuated by blockade of either vascular endothelial growth factor receptor (VEGFR)-1 or -2. However, the pro-proliferating effect appeared to be weakened only by VEGFR-2 blocking. Both Erk1/2 and Akt phosphorylation were enhanced by EPC CM. VEGFR-2 blockage led to significant inhibition of Erk1/2 and Akt activation; VEGFR-1 blockage only of Erk1/2 activation. Human-origin VEGF co-localized with incorporated EPC in small pulmonary arterioles, and EPC transplantation resulted in down-regulation of caspase-3 expression. CONCLUSIONS The VEGF-A/B-VEGFR-1/2-Erk1/2 signal pathway took major responsibility for the anti-apoptotic effects of EPC on PMVEC, and VEGF-A-VEGFR-2-Akt for pro-proliferating effects. Growth factors, secreted in a paracrine manner by transplanted EPC, inhibited cell apoptosis in PAH lung.

Proteomic analysis of the serum in patients with idiopathic pulmonary arterial hypertension.

Idiopathic pulmonary arterial hypertension (IPAH) is a rare disease of unknown etiology. The exact pathogenesis of pulmonary arterial hypertension is still not well known. In the past decades, many protein molecules have been found to be involved in the development of IPAH. With proteomic techniques, profiling of human plasma proteome becomes more feasible in searching for disease-related markers. In present study, we showed the protein expression profiles of the serum of IPAH and healthy controls after depleting a few high-abundant proteins in serum. Thirteen spots had changed significantly in IPAH compared with healthy controls and were identified by LC-MS/MS. Alpha-1-antitrypsin and vitronectin were down-regulated in IPAH and may be valuable candidates for further explorations of their roles in the development of IPAH.

Resveratrol Attenuates Adenosine Diphosphate-Induced Platelet Activation by Reducing Protein Kinase C Activity

Inappropriate platelet activation is the key point of thrombogenesis. The aim of the present study was to investigate the effects of resveratrol (RESV), a compound extracted from the Chinese medicinal herb Polygonum cuspidatum sieb et Zucc, on the platelet activation induced by adenosine diphosphate (ADP) and its possible mechanism. The percentage of platelet aggregation and surface P-selectin-positive platelets, and the activity of protein kinase C (PKC) of platelet were observed with platelet aggregometer, flow cytometry and phosphorimaging system, respectively. RESV at 25, 50 and 100 microM showed anti-platelet aggregation and inhibition of surface P-selectin-positive platelets in a concentration-dependent manner. RESV (50 microM) inhibited the activity of PKC in the membrane fraction of platelets and decreased the percentage of membrane associated PKC activity in total PKC activity. Moreover, DL-erythro-1,3-Dihydroxy-2-aminooctadecane, an elective protein kinase C inhibitor (PKCI), and RESV had additive effects of inhibiting the percentage of platelet aggregation and surface P-selectin-positive platelets. It is suggested that RESV may inhibit platelet aggregation, the percentage of surface P-selectin-positive platelets and subsequent thrombus formation. The mechanisms may be partly relative to the decrease of the activity of PKC of platelets.

Puerarin reduces endothelial progenitor cells senescence through augmentation of telomerase activity

Endothelial progenitor cells (EPCs) play an important role in both reendothelialization and neovascularization. Ex vivo expansion of EPCs might be useful for potential clinical cell therapy of ischemic diseases. However, ex vivo cultivation of EPCs leads to rapid onset of EPCs senescence, thereby severely limiting the proliferative capacity and clonal expansion potential. Therefore, we investigated whether puerarin might be able to prevent senescence of EPCs. EPCs were isolated from peripheral blood and characterized. After ex vivo cultivation, EPCs became senescent as determined by acidic beta-galactosidase staining. Puerarin dose dependently prevented the onset of EPCs senescence in culture. Moreover, puerarin increased proliferation of EPCs as assessed by BrdU incorporation assay and colony-forming capacity. To get further insights into the underlying mechanisms of these effects induced by puerarin, we measured telomerase activity and determined the phosphorylation of serine/threonine protein kinase Akt by using western blot. Puerarin significantly increased telomerase activity and phosphorylation of Akt, a downstream effector of phosphoinositide 3-kinase (PI-3K). Moreover, pretreatment with PI-3K blockers, either wortmannin or LY294002, significantly attenuated the puerarin puerarin-induced telomerase activity. Taken together, the results of the present study indicated that puerarin delayed the onset of EPCs senescence, which may be related to the activation of telomerase through the PI-3K/Akt pathway. The inhibition of EPCs senescence by puerarin in vitro may improve the functional activity of EPCs in a way that is important for potential cell therapy.

Impairment of Monocyte-derived Dendritic Cells in Idiopathic Pulmonary Arterial Hypertension

Background and AimWith the development of immunology, the role of immune inflammation in idiopathic pulmonary arterial hypertension (IPAH) has attracted interest. Recently, it was discovered that dendritic cells, which are key players in immune inflammation, are implicated in the pathogenesis of IPAH. To elucidate the role of dendritic cells in human IPAH, we compared the changes in the number and immunological function of monocyte-derived dendritic cells (MoDCs) from the peripheral blood of patients with IPAH and healthy controls.MethodsThe numbers of MoDC subsets (including plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells (mDCs)) in circulating peripheral blood mononuclear cells (PBMCs) was analyzed by flow cytometry, and the concentrations of interleukin (IL)-12, IL-10, and tumor necrosis factor-alpha were measured by enzyme-linked immunosorbent serologic assay kits. The morphology, phenotypic expression, and the ability to stimulate T cell proliferation of MoDCs, cultured from PBMCs in vitro with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4, was analyzed by microscopy, flow cytometry, and MTT assay.ResultsThe results of the study are as follows: (1) The number of circulating mDCs was lower in IPAH patients than in controls (0.07 ± 0.01% to 0.14 ± 0.02%; p < 0.05). (2) IL-12 levels were higher in IPAH patients than in controls (p < 0.05). (3) MoDCs showed higher expression of CD1a (53.34 ± 7.43% to 19.29 ± 7.37%; p < 0.05), and lower expression of costimulatory molecule CD86 (64.54 ± 5.93% to 87.04 ± 4.82%; p < 0.05), and less ability to simulate T cell proliferation (when the ratio is 1:10) compared to the controls.ConclusionsThe study shows that it is possible to obtain typical DCs by culturing PBMCs from patients with IPAH with GM-CSF and IL-4, and it demonstrates that patients with IPAH have a significant change in the number of mDC and a marked immune deficiency of MoDCs.

Tanshinone II A Attenuates TNF-α-Induced Expression of VCAM-1 and ICAM-1 in Endothelial Progenitor Cells by Blocking Activation of NF-κB

Background/Aims: Tanshinone IIA (Tan IIA) is effective in the treatment of inflammation and atherosclerosis. The adhesion of inflammatory cells to vascular endothelium plays important role in atherogenic processes. This study examined the effects of Tan IIA on expression of adhesion molecules in tumor necrosis factor-α (TNF-α)-induced endothelial progenitor cells (EPCs). Methods: EPCs were pretreated with Tan IIA and stimulated with TNF-α. Mononuclear cell (MNC) adhesion assay was performed to assess the effects of Tan IIA on TNF-α-induced MNC adhesion. Expression of vascular cell adhesion molecule-1 (VCAM-1)/intracellular adhesion molecule-1 (ICAM-1) and activation of Nuclear factor κB (NF-κB) signaling pathway were measured. Results: The results showed that the adhesion of MNCs to TNF-α-induced EPCs and expression of VCAM-1/ICAM-1 in EPCs were promoted by TNF-α, which were reduced by Tan IIA. TNF-α increased the amount of phosphorylation of NF-κB, IκB-α and IKKα/β in cytosolic fractions and NF-κB p65 in nucleus, while Tan IIA reduced its amount. Conclusion: This study demonstrated a novel mechanism for the anti-inflammatory/anti-atherosclerotic activity of Tan IIA, which may involve down-regulation of VCAM-1 and ICAM-1 through partial blockage of TNF-α-induced NF-κB activation and IκB-α phosphorylation by the inhibition of IKKα/β pathway in EPCs.

Single-nucleotide polymorphisms in SCN5A gene in Chinese Han population and their correlation with cardiac arrhythmias

Single-nucleotide polymorphisms in SCN5A gene in Chinese Han population and their correlation with cardiac arrhythmias