OBJECTIVETo establish a method for detecting circulating brain microvascular endothelial cells (cBMECs), a novel biomarker of blood-brain barrier (BBB) injury.

METHODSBlood samples were collected from 33 patients with AIDS encephalitis and 13 healthy subjects for detection of cBMECs, cECs and EPCs using magnetic affinity isolation and immune identification technology.

RESULTSThe numbers of cBMECs, cECs and EPCs were significantly higher in the AIDS patients than in the control subjects (t=4.298, P<0.01; t=4.886, P<0.01; t=4.889, P<0.01). An significant association was also noted between HIV load and cBMEC number (r=0.928, P<0.01).

CONCLUSIONWe have successfully established a method for detecting peripheral blood cBMECs, which can be of important value in non-invasive assessment of BBB injury.

Acquired Immunodeficiency Syndrome ; physiopathology ; Biomarkers ; Blood-Brain Barrier ; pathology ; Cell Separation ; methods ; Cells, Cultured ; Endothelial Progenitor Cells ; cytology ; Humans

OBJECTIVETo investigate the effects of intercellular adhesion molecule-1(ICAM-1) on the adherence between mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC).

METHODSMSC and EPC were isolated, cultured and expanded from the 6-8 weeks aged C57BL/6 murine bone marrow by in vitro. Immuno-fluorescence was used to detect the expression of ICAM-1 in MSC group, EPC group and co-cultured MSC and EPC group. The mRNA and protein levels of ICAM-1 were detected by RT-PCR and Western blot respectively, then, the ICAM-1 adherence between MSC and EPC was observed by adding different concentration of neutralizing antibody.

RESULTSThe expression of ICAM-1 on surface of MSC and EPC could be detected by cell immunofluorescence method. According to results of the semiquantitative fluorescene detection, the fluorescence strength of MSC+EPC co-cultured group (89.02 ± 24.52) was higher than that of MSC group (31.25 ± 2.95) and EPC group (34.32 ± 5.02), and there was statistical difference between them (P < 0.01), but there was no obvious difference between MSC group and EPC group (P > 0.05). RT-PCR detection showed that the expression levels of ICAM-1 in MSC+EPC co-cultured group were higher than that in MSC group and that in EPC group (P < 0.01), and expression level of ICAM in EPC group was higher than that in MSC group (P < 0.01). Western blot detection showed that the expression level of ICAM-1 protein in MSC+EPC co-cultured group (0.33 ± 0.4) was higher than that in MSC group (0.11 ± 0.01) (P < 0.05) and than that in EPC group (0.19+0.02) (P < 0.05), However, the expression level of ICAM-1 protein in EPC group was higher than that in MSC group (P < 0.05). The test of different concentrations against neutralizing antibody showed that with the increasing of concentration of ICAM-1 neutralizing antibody, the adhesion capability of MSC and EPC was gradually decreasing.

CONCLUSIONThe ICAM-1 can mediate the adherence process between MSC and EPC.

Animals ; Bone Marrow ; Cell Adhesion ; Coculture Techniques ; Endothelial Progenitor Cells ; cytology ; Intercellular Adhesion Molecule-1 ; metabolism ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred C57BL

To improve the function of endothelial progenitor cells (EPCs) is one of the goals in Chinese traditional therapy to treat various cardio-celebrovascular diseases. In the past decades, scholars in the field of traditional Chinese medicine (TCM) have found fifteen active compounds to regulate the function of EPC. These metabolites are extracted from thirteen, plant-based Chinese medicine, with majority of them as potent reductive or oxidative hydrophilic molecules containing phenyl groups. These active compounds either enhance the mobilization of EPC, or inhibit their apoptosis through different signaling pathways. In this review, the molecular structure, biophysical properties, and the plant sources of these active ingredients and their regulatory effects on the function of EPC are summarized, aiming to reveal the modern basis of Chinese medicine for promoting blood circulation and removing blood stasis at the progenitor cell level.
Animals ; Apoptosis ; drug effects ; Cell Movement ; drug effects ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Progenitor Cells ; cytology ; drug effects ; metabolism ; Humans ; Signal Transduction ; drug effects

Coprecipitation method was used to prepare triiron tetroxide magnetic nanoparticles enclosed in L-DOPA, and then EDC was used to activate the carboxyl group of L-DOPA after the nanoparticles were synthesized. The carboxyl group of L-DOPA formed amide bond with specific amino on the aptamer by dehydration condensation reaction. The surfaces of magnetic nanoparticles were modified with aptamer and L-DOPA. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), nanoparticle size analysis (SEM), magnetic measurement (VSM) and other testing methods were used to detect the magnetic nanoparticles in different stages. The endothelial progeni-tor cells (EPCs) were cocultured with the surface modified magnetic nanoparticles to evaluate cell compatibility and the combination effect of nanoparticles on EPCs in a short period of time. Directional guide of the surface-modified magnetic nanoparticles to endothelial progenitor cells (EPCs) was evaluated under an applied magnetic field and simulated dynamic blood flow condition. The results showed that the prepared magnetic nanoparticles had good magnetic response, good cell compatibility within a certain range of the nanoparticle concentrations. The surface modified nanoparticles could combine with EPCs effectively in a short time, and those nanoparticles combined EPCs can be directionally guided on to a stent surface under the magnetic field in the dynamic flow environment.
Endothelial Progenitor Cells ; cytology ; drug effects ; Ferrosoferric Oxide ; chemistry ; Humans ; Levodopa ; chemistry ; Magnetite Nanoparticles ; chemistry ; Spectroscopy, Fourier Transform Infrared ; X-Ray Diffraction

BACKGROUNDEndothelial cell damage is an important pathophysiological step of restenosis after angioplasty and stenting. Cell transplantation has great therapeutic potential for endothelial recovery. We investigated the effect of transplanting endothelial progenitor cells (EPCs) derived from human early fetal aortas in rat injured arteries.

METHODSThe carotid arterial endothelium of Sprague-Dawley rats was damaged by dilatation with a 1.5 F balloon catheter, and then EPCs derived from human early fetal aortas (<14 weeks) were injected into the lumen of the injured artery in transplanted rats, with an equal volume of normal saline injected into control rats. Rats were sacrificed at 2 and 4 weeks after treatment and transplanted cells were identified by immunohistochemical staining with anti-human CD31 and anti-human mitochondria antibodies. Arterial cross-sections were analyzed by pathology, immunohistochemistry, and morphometry.

RESULTSGreen fluorescence-labeled EPCs could be seen in the endovascular surface of balloon-injured vessels after transplantation. The intimal area and intimal/medial area ratio were significantly smaller in the transplanted group than in the control (P < 0.05) and the residual lumen area was larger (P < 0.05). After EPC transplantation, a complete vascular endothelial layer was formed, which was positive for human von Willebrand factor after immunohistochemical staining, and immunohistochemical staining revealed many CD31- and mitochondria-positive cells in the re-endothelialized endothelium with EPC transplantation but not control treatment.

CONCLUSIONEPCs derived from human early fetal aorta were successfully transplanted into injured vessels and might inhibit neointimal hyperplasia after vascular injury.

Animals ; Carotid Arteries ; pathology ; Cell Adhesion ; physiology ; Cell Survival ; physiology ; Cell Transplantation ; Endothelial Progenitor Cells ; cytology ; physiology ; Humans ; Immunohistochemistry ; Microscopy, Fluorescence ; Neointima ; therapy ; Rats ; Rats, Sprague-Dawley

The present study was designed to investigate the effects of various extracellular matrix (ECM) proteins on the biological characteristics of late endothelial progenitor cells (EPCs). Density gradient centrifugation-isolated rat bone marrow mononuclear cells were cultured in complete M199 medium, which contained 15% fetal calf serum, 10 μg/L vascular endothelial growth factor (VEGF) and 5 μg/L basic fibroblast growth factor (bFGF). EPCs were plated on substrates containing fibronectin (Fn), laminin (Ln) or rat tail tendon collagen (Col), and the corresponding cells were defined as Fn, Ln and Col groups. The 3rd generation EPCs, namely late EPCs, were harvested. The proliferation, adhesion, migration and the ability of forming tubes were assayed using CCK-8, adhesion test, wound healing assay and Matrigel, respectively. The mRNA expressions of endothelial cell differentiation markers, vWF and CD31, were analyzed by real time RT-PCR. The apoptosis was assayed by flow cytometry (FCM). The results showed that cell proliferation ability of Fn and Col groups were higher than that of Ln group; Fn group showed increased adhesion compared to Col and Ln groups (P < 0.01); The migration ability of Fn and Col groups were higher than that of Ln group. Moreover, Fn group showed increased tube formation abilities compared to Col and Ln groups (P < 0.05). Although 24-hour free-serum-induced apoptosis in Ln group was the highest, there was no difference of auto-apoptosis among the three groups. Furthermore, the mRNA expressions of vWF and CD31 exhibited no difference among the three groups. These results suggest the ECM affects the biological functions of late EPCs, which would have a high probability of providing new directions that lead to the development of artificial heart and blood vessels.
Animals ; Cell Proliferation ; Cells, Cultured ; Collagen ; chemistry ; Endothelial Progenitor Cells ; cytology ; Extracellular Matrix ; physiology ; Extracellular Matrix Proteins ; chemistry ; Fibroblast Growth Factor 2 ; chemistry ; Fibronectins ; chemistry ; Rats ; Vascular Endothelial Growth Factor A ; chemistry

The aim of this study was to investigate whether shear stress could promote function of endothelial progenitor cells (EPCs) with Shexiang Baoxin Pill (SBP) treatment in vitro, and to study whether shear stress contributed to vascular injury repair by EPCs. EPCs were isolated and characterized; EPCs' proliferation, migration, adhesion, tube formation and eNOS protein level in vitro were investigated by culturing confluent EPCs in 4 mg/mL SBP under physiological shear stress (15 dyne/cm2) for up to 24 hours. Afterwards, EPCs were transfused into rats after wire-induced carotid artery injury augmented re-endothelialization. The results showed that, compared to the SBP group, the shear stress+SBP group obviously enhanced EPCs proliferation, migration, adhesion, tube formation and eNOS protein expression in vitro (P<0.01). After one week, immunofluorescence staining showed that endothelial regeneration rate obviously enhanced in shear stress+SBP group (P<0.01). The present study demonstrates that shear stress can promote function of endothelial progenitor cells treated with SBP, which improves the vascular injury repair potentials of EPCs.
Animals ; Cell Adhesion ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Progenitor Cells ; cytology ; drug effects ; Endothelium, Vascular ; Nitric Oxide Synthase Type III ; metabolism ; Rats ; Regeneration ; Stress, Mechanical

This study was purpose to examine the effect of dimethyl sulfoxide (DMSO) and Tween 80 on the growth and viability of stromal cells (BMSC), colony-forming units for granulocytes and macrophages (CFU-GM) and bone marrow endothelial cell line (BMEC) from murine bone marrow in vitro, and to analyze the concentration-effect relationship. The colony yields of colony-forming units fibroblastic (CFU-F) and CFU-GM were assessed in the murine bone marrow cell cultures at various concentrations of DMSO or Tween 80 and in the control groups. The MTT assay and trypan blue exclusion were used to determine the cell viability and percentage of survival in BMSC and BMEC cultures with or without either of these organic solvents. The results showed that the colony yields of both CFU-F and CFU-GM were decreased significantly (p<0.05 or <0.01) at the concentrations (v/v final) of 2% DMSO or 0.005%-0.01% Tween 80 respectively, as compared with control. The cell viability and percentage of survival of BMSC and BMEC cultures were significantly reduced (p<0.05 or <0.01) at 0.5%-1.0% DMSO or 0.002%-0.005% Tween 80, as compared with control. With the increase of volume fractions of these solvents, the decreased percentages of corresponding measurements were increased by degrees. It is concluded that when the concentration of DMSO or Tween 80 goes to a certain level in cell culture medium, either of the organic solvents has an inhibitory action or/and cytotoxicity on the growth and viability of BMSCs, CFU-GM and BMECs. The growth inhibition and cytotoxic response are more significant at higher concentrations of these solvents.
Animals ; Bone Marrow Cells ; cytology ; Cell Line ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Dimethyl Sulfoxide ; pharmacology ; Dose-Response Relationship, Drug ; Endothelial Cells ; cytology ; Female ; Granulocyte-Macrophage Progenitor Cells ; cytology ; Male ; Mice ; Polysorbates ; pharmacology ; Solvents ; pharmacology ; Stromal Cells ; cytology

OBJECTIVETo observe the effect of Tongguan Capsule (TC) on the number of endothelial progenitor cells (EPCs) in the peripheral blood of patients with coronary artery disease (CAD) after percutaneous coronary intervention (PCI).

METHODSRecruited were 60 CAD patients undergoing PCI who were admitted and treated at ICU and the Heart Center of Guangdong Provincial Hospital of Traditional Chinese Medicine from March to October 2010. They were assigned to the treatment group (treated by TC) and the control group (treated by placebos) according to the random digit table, 30 cases in each group. They took TC or placebos from the day of PCI, three pills each time, three times a day, for three consecutive months. The numbers of peripheral blood CD34 and vascular endothelial growth factor receptor-2 (VEGFR2) positive cells were detected before PCI and 3 months after PCI respectively. The echocardiography was performed before PCI and 3 months after PCI respectively to determinate the left ventricular end diastolic volume (LVEDV), left ventricular end systolic volume (LVESV), stroke volume (SV), cardiac output (CO), and left ventricular ejection fraction (LVEF). The wall motion score index (WMSI) was assessed in the two groups.

RESULTSThere was no statistical difference in the number of EPCs, LVEF,WMSI, or SV in the two groups before PCI (P > 0.05). The number of EPCs increased in both the two groups after 1 month of PCI (P < 0.05). It was obviously higher in the treatment group than in the control group (P < 0.05). The LVEF both increased in the two groups 3 months after PCI (P < 0.05, P < 0.01). The WMSI decreased and SV increased in the treatment group (P < 0.05). The improvement of LVEF and WMSI was better in the treatment group than in the control group (P < 0.05).

CONCLUSIONTC could up-regulate the number of EPCs in the peripheral blood of CAD patients after PCI, and improve their cardiac functions.

Adult ; Aged ; Aged, 80 and over ; Blood Cell Count ; Coronary Artery Disease ; drug therapy ; therapy ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Endothelial Progenitor Cells ; cytology ; drug effects ; Female ; Humans ; Male ; Middle Aged ; Percutaneous Coronary Intervention ; Phytotherapy

To investigate the effects of adenosine triphosphate (ATP) on expression of inflammatory factors induced by lipopolysaccharide (LPS) in endothelial progenitor cells (EPCs), and to elucidate the possible mechanisms.
 Methods: Mononuclear cells were isolated from human umbilical cord blood by density gradient centrifugation, RT-PCR was performed to detect the expression of inflammatory factors induced by LPS (1 mg/mL) in EPCs, the effect of low concentration (5 μmol/L) of ATP on expression of IL-1β, MCP-1 and ICAM-1, and the effect of different concentrations (5, 50 μmol/L) of ATP on the expression of Toll-like receptor (TLR) 4, myeloid differentiation primary response protein 88 (MyD88) and CD14. Western blot was performed to detect expression of TLR4 regulated proteins MyD88 and CD14 or to detect the low concentration (1, 5 μmol/L) of ATP on the expression of TLR4, MyD88 and CD14 and the NF-κB signaling pathway.
 Results: EPCs highly expressed TLR4, and its ligand LPS (1 mg/mL) significantly upregulated mRNA expression of IL-1β, MCP-1 and ICAM-1 and protein expression of MyD88 and CD14 in a time-dependent manner (P<0.01), accompanied by activation of ERK and NF-κB signal pathway. ATP at low concentration (5 μmol/L) significantly inhibited LPS-induced mRNA expression of IL-1β, MCP-1 and ICAM-1(P<0.05), downregulated the LPS-induced protein expression of TLR4, MyD88 and CD14 in EPCs (P<0.05), and suppressed LPS-induced activation of NF-κB signaling pathway (P<0.05).
 Conclusion: ATP at low concentration may suppress LPS-induced expression of inflammatory factors in EPCs through negative regulation of the TLR4 signaling pathway.
Adenosine Triphosphate ; pharmacology ; Endothelial Progenitor Cells ; drug effects ; Gene Expression Regulation ; drug effects ; Humans ; Leukocytes, Mononuclear ; cytology ; Lipopolysaccharide Receptors ; genetics ; Lipopolysaccharides ; pharmacology ; Myeloid Differentiation Factor 88 ; genetics ; NF-kappa B ; metabolism ; Signal Transduction ; drug effects ; Toll-Like Receptor 4 ; genetics