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

In order to investigate the promoting effect of low-intensity treadmill exercise on rat dorsal wound healing and the mechanism, 20 Sprague-Dawley rats were randomly divided into two groups: exercise group (Ex) and non-exercise group (non-ex). The rats in Ex group were given treadmill exercise for one month, and those in non-ex group raised on the same conditions without treadmill exercise. Both groups received dorsal wound operation with free access to food and water. By two-week continuous observation and recording of the wound area, the healing rate was analyzed. The blood sample was collected at day 14 post-operation via cardiac puncture for determination of the number of endothelial progenitor cells (EPCs) by flow cytometry, and the concentrations of relevant cytokines such as basic fibroblast growth factor (bFGF), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) were measured by ELISA. The skin tissue around the wound was dissected to observe the vascular density under the microscope after HE staining, to detect the mRNA level of VEGFR2 and angiopoietin-1 (Ang-1) receptor using RT-qPCR, and protein expression of a-smooth muscle actin (αSMA) and type III collagen (ColIII) using Western blotting. It was found that the wound area in Ex group was smaller at the same time point than in non-ex group. The number of circulating EPCs was greater and the concentrations of vasoactive factors such as VEGF, eNOS and bFGF were higher in Ex group than in non-ex group. HE staining displayed a higher vessel density in Ex group than in non-ex group. Moreover, the mRNA expression of VEGFR2 and Ang-1 detected in the wound tissue in Ex group was higher than in non-ex group. Meanwhile, the protein expression of αSMA and ColIII was more abundant in Ex group than in non-ex group. Conclusively, the above results demonstrate Ex rats had a higher wound healing rate, suggesting low-intensity treadmill exercise accelerates wound healing. The present work may provide some hint for future study of treating refractory wound.
Actins ; metabolism ; Animals ; Collagen Type III ; metabolism ; Cytokines ; blood ; Endothelial Progenitor Cells ; cytology ; Male ; Nitric Oxide Synthase Type III ; blood ; Physical Exertion ; RNA, Messenger ; blood ; Rats ; Rats, Sprague-Dawley ; Receptor, TIE-1 ; metabolism ; Running ; Vascular Endothelial Growth Factor A ; blood ; Vascular Endothelial Growth Factor Receptor-2 ; blood ; Wound Healing

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

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

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 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

The purpose of this study was to devise an expanded ischemic flap model and to investigate the role of AMD-3100 (Plerixafor, chemokine receptor 4 inhibitor) in this model by confirming its effect on mobilization of stem cells from the bone marrow. Male Sprague-Dawley rats were used as an animal research model. The mobilization of stem cells from the bone marrow was confirmed in the AMD-3100-treated group. The fractions of endothelial progenitor cells (EPC) and the vascular endothelial growth factor receptor (VEGFR) 2+ cells in the peripheral blood were increased in groups treated with AMD-3100. The expression of vascular endothelial growth factor (VEGF) was increased in response to expansion or AMD injection. The expression of stromal cell derived factor (SDF)-1 and VEGFR2 were increased only in unexpanded flap treated with AMD-3100. Treatment with AMD-3100 increased both the number and area of blood vessels. However, there were no statistically significant differences in the survival area or physiologic microcirculation in rats from the other groups. This endogenous neovascularization induced by AMD-3100 may be a result of the increase in both the area and number of vessels, as well as paracrine augmentation of the expression of VEGF and EPCs. However, the presence of a tissue expander under the flap could block the neovascularization between the flap and the recipient regardless of AMD-3100 treatment and expansion.
Animals ; Anti-HIV Agents/pharmacology ; Bone Marrow Cells/cytology ; Chemokine CXCL12/biosynthesis ; Endothelial Progenitor Cells/*cytology ; Hematopoietic Stem Cells/*cytology ; Heterocyclic Compounds/*pharmacology ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism ; Male ; Neovascularization, Physiologic ; Nitric Oxide Synthase Type III/metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, CXCR4/antagonists & inhibitors ; Surgical Flaps/*blood supply/surgery ; Tissue Expansion/*methods ; Vascular Endothelial Growth Factor A/biosynthesis ; Vascular Endothelial Growth Factor Receptor-2/biosynthesis/metabolism

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

BACKGROUNDCoronary artery damage from Kawasaki disease (KD) is closely linked to the dysfunction of the endothelial progenitor cells (EPCs). The aim of the present study was to evaluate the modulatory effect of granulocyte colony-stimulating factor (G-CSF) on EPCs and elastin breakdown of coronary arteries in a KD mouse model.

METHODSA Lactobacillus casei cell wall extract (LCWE)-induced KD model was established in C57BL/6 mice that were subsequently administrated with recombinant human G-CSF (rhG-CSF). Nω-nitro-L-arginine methyl ester (L-NAME) was administrated for the negative intervention. Evaluations included coronary artery lesions, EPC number and functions, and the plasma concentration of nitric oxide (NO).

RESULTSElastin breakdown was found in the coronary arteries of model mice 56 days after injection of LCWE. The number of circulating EPCs, plasma concentration of NO, and functions of bone marrow EPCs, including proliferation, adhesion, and migration abilities, were all lower in the KD model group compared with those in the control group. After administration of rhG-CSF, the number of circulating EPCs and plasma concentration of NO were increased significantly compared with those in the KD model group. There were also increases in the functional indexes of EPCs. Furthermore, rhG-CSF administration improved the elastin breakdown effectively. However, these protective effects of rhG-CSF on coronary arteries were attenuated by L-NAME.

CONCLUSIONThe present study indicated that the administration of G-CSF prevents elastin breakdown of the coronary arteries by enhancing the number and functions of EPCs via the NO system, and then accelerates the repair of coronary artery lesions in the KD.

Animals ; Coronary Vessels ; cytology ; drug effects ; metabolism ; Disease Models, Animal ; Elastin ; metabolism ; Endothelial Progenitor Cells ; cytology ; Granulocyte Colony-Stimulating Factor ; therapeutic use ; Male ; Mice ; Mice, Inbred C57BL ; Mucocutaneous Lymph Node Syndrome ; blood ; drug therapy ; metabolism ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitrogen Oxides ; blood

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