p38MAPK对猪MODS时内皮祖细胞的调控作用

目的探讨猪MODS时p38MAPK介导的TNF-α对内皮祖细胞(EPC)的调控作用,从EPC分化障碍的角度进一步研究创伤后MODS的发病机制。方法将20头家猪分为MODS组(M组)和对照组(C组),每组10头。采用"二次打击法"建立猪MODS模型,监测其各主要脏器功能的变化,体内用Western-blot法检测外周血单核细胞p38MAPK的磷酸化变化,用ELISA法测定外周血血浆TNF-α的浓度变化,流式细胞仪检测外周血EPC的数量变化。结果M组的MODS发生率明显高于C组,M组外周血单核细胞p38MAPK的磷酸化明显增强,TNF-α合成与分泌明显增加,外周血EPC数量与功能下降。结论外周血单核细胞p38MAPK的磷酸化使TNF-α的转录、合成增加,血浆TNF-α升高,使外周血中的EPC其数量与功能下降,炎症病理反应加重,可能是MODS的发病机制。

Biological Characteristics of Endothelial Progenitor Cell and Its Influence on Angiogenesis Improvement

Endothelial progenitor cell （EPC） is a term that refers to multiple cell types that play roles in the regeneration of the endothelial lining of blood vessels. The EPCs in bone marrow will participate in the internal circulation in a body sub- jected to the stimulation by external factors such as injury, ischemia or drug. EPCs regulate the angiogenic switch via paracrine secretion of proangiogenic growth factors and by direct luminal incorporation into sprouting nascent vessels. Therefore, this paper reviews the sources, isolation and culture of EPCs, the factors influencing the proliferation and activity of EPCs, and the roles of EPCs in angiogenesis.

Endothelial precursor cells promote angiogenesis in hepatocellular carcinoma

AIM:To investigate the role of bone marrow-derived endothelial progenitor cells(EPCs) in the angiogenesis of hepatocellular carcinoma(HCC).METHODS:The bone marrow of HCC mice was reconstructed by transplanting green fluorescent protein(GFP) + bone marrow cells.The concentration of circulating EPCs was determined by colony-forming assays and fluorescence-activated cell sorting.Serum and tissue levels of vascular endothelial growth factor(VEGF) and colony-stimulating factor(CSF) were quantified by enzyme-linked immunosorbent assay.The distribution of EPCs in tumor and tumor-free tissues was detected by immunohistochemistry and real-time polymerase chain reaction.The incorporation of EPCs into hepatic vessels was examined by immunofluorescence and immunohistochemistry.The proportion of EPCs in vessels was then calculated.RESULTS:The HCC model was successful established.The flow cytometry analysis showed the mean percentage of CD133CD34 and CD133VEGFR2 double positive cells in HCC mice was 0.45% ± 0.16% and 0.20% ± 0.09% respectively.These values are much higher than in the sham-operation group(0.11% ± 0.13%,0.05% ± 0.11%,n = 9) at 14 d after modeling.At 21 d,the mean percentage of circulating CD133CD34 and CD133VEGFR2 cells is 0.23% ± 0.19%,0.25% ± 0.15% in HCC model vs 0.05% ± 0.04%,0.12% ± 0.11% in control.Compared to the transient increase observed in controls,the higher level of circulating EPCs were induced by HCC.In addition,the level of serum VEGF and CSF increased gradually in HCC,reaching its peak 14 d after modeling,then slowly decreased.Consecutive sections stained for the CD133 and CD34 antigens showed that the CD133+ and CD34+ VEGFR2 cells were mostly recruited to HCC tissue and concentrated in tumor microvessels.Under fluorescence microscopy,the bone-marrow(BM)-derived cells labeled with GFP were concentrated in the same area.The relative levels of CD133 and CD34 gene expression were elevated in tumors,around 5.0 and 3.8 times that of the tumor free area.In frozen liver sections from HCC mice,cells co-expressing CD133 and VEGFR2 were identified by immunohistochemical staining using anti-CD133 and VEGFR2 antibodies.In tumor tissue,the double-positive cells were incorporated into vessel walls.In immunofluorescent staining.These CD31 and GFP double positive cells are direct evidence that tumor vascular endothelial cells(VECs) come partly from BM-derived EPCs.The proportion of GFP CD31 double positive VECs(out of all VECs) on day 21 was around 35.3% ± 21.2%.This is much higher than the value recorded on day 7 group(17.1% ± 8.9%).The expression of intercellular adhesion molecule 1,vascular adhesion molecule 1,and VEGF was higher in tumor areas than in tumor-free tissues.CONCLUSION:Mobilized EPCs were found to participate in tumor vasculogenesis of HCC.Inhibiting EPC mobilization or recruitment to tumor tissue may be an efficient strategy for treating HCC.

Erythropoietin Receptor Positive Circulating Progenitor Cells and Endothelial Progenitor Cells in Patients with Different Stages of Diabetic Retinopathy

Objective To investigate the possible involvement of erythropoietin （EPO）/erythropoietin receptor （EPOR） system in neovascularization and vascular regeneration in diabetic retinopathy （DR）. Methods EPOR positive circulating progenitor cells （CPCs： CD34^＋） and endothelial progenitor cells （EPCs： CD34^＋KDR^＋） were assessed by flow cytometry in type 2 diabetic patients with different stages of DR. The cohort consisted of age- and sex-matched control patients without diabetes （n=7）, non-prolif- erative DR （NPDR, n=7）, proliferative DR （PDR, n=8）, and PDR complicated with diabetic nephropathy （PDR-DN, n=7）. Results The numbers of EPOR^＋ CPCs and EPOR^＋ EPCs were reduced remarkably in NPDR corn pared with the control group （both P（0.01）, whereas rebounded in PDR and PDR-DN groups in varying degrees. Similar changes were observed in respect of the proportion of EPOR^＋ CPCs in CPCs （NPDR vs. control, P（0.01） and that of EPOR^＋ EPCs in EPCs （NPDR vs. control, P〈0.05）. Conclusion Exogenous EPO, mediated via the EPO/EPOR system of EPCs, may alleviate the impaired vascular regeneration in NPDR, whereas it might aggravate retinal neovascularization in PDR due to a rebound of EPOR^＋ EPCs associated with ischemia.

Sleep-disordered breathing is associated with depletion of circulating endothelial progenitor cells and elevation in pulmonary arterial pressure in patients with decompensated systolic heart failure

Background Sleep-disordered breathing （SDB） is known to occur frequently in and may predict worsening progression of patients with congestive heart failure （CHF）. SDB is also known to play an important role in the development of idiopathic pulmonary arterial hyper- tension （PAH） via inducing endothelial dysfunction and vascular remodeling, a pathological process that can be significantly influenced by factors such as osteoprotegerin （OPG） and endothelial progenitor cells （EPCs）. The objective of this study is to determine if CHF with SDB is associated with changes in OPG, EPCs, and PAIl. Methods EPCs were isolated, cultured, and quantified from CHF patients with SDB （n = 52）, or without SDB （n - 68）. OPG and N-terminal pro-brain natriuretic peptide （NT-proBNP） from each group was analyzed and cor- related with EPCs and the mean pulmonary artery pressure （mPAP） measured by right heart catheterization. Results A significant decrease in circulating EPCs （29.30 ± 9.01 vs. 45.17 ± 10.51 EPCs/x 200 field; P 〈 0.05） was found in CHF patients with SDB compared to those without SDB. Both OPG （789.83 ±89.38 vs. 551.29 ± 42.12 pg/mL; P 〈 0.05） and NT-proBNP （5946.50 ± 1434.50 vs. 3028.60 ± 811.90 ng/mL; P 〈 0.05） were also significantly elevated in SDB CHF patients who also had significantly elevated mPAP （50.2 ± 9.5 vs. 36.4 ± 4.1 mm Hg; P 〈 0.05）. EPC numbers correlated inversely with the episodes of apnea and hypopnea per hour （RDI, r = -0.45, P = 0.037） and blood level of OPG （r =-0.53, P = 0.011）. Although NT-proBNP was also increased significantly in patients with SDB, it had no correlation with either EPCs or RD1. Conclusions SDBdue to hypoxemia from decompensated CHF is associated with （1） OPG elevation, （2） EPC depletion, and （3） mPAP elevation. The inverse relationship of circulating OPG with EPCs suggests a likely mechanism for hypoxemia and OPG in the development of pulmonary vascular dysfunction via depleting EPCs, thus worsening prognosis of CHF.

A novel vWF A3-GPI modified EPCs to enhance its adhesion ability to collegen

Objective:To gain purified recombination protein rGPI-vWF A3 which can guide endothelial progenitor cells (EPCs) gathering to the internal surface of the injured vessels theoretically and analyze its bioactivity in vitro. Methods: The plasmid vector of vWF A3 fusion protein which contained GPI structure was constructed and recombination protein rGPI-vWF A3 was expressed and its bioactivity was analyzed by ELISA and FCM. Results: Fusion protein vWF A3-GPI was expressed and purified by immunoaffinity chromatography and it could stably anchored on the surface of EPCs and showed its biological activities of collagen conjunction. Conclusion: These findings represent a novel strategy in EPC transplantation treatment for vessel injury.

Effect of Intracoronary Infusion of Bone Marrow Mononuclear Cells or Peripheral Endothelial Progenitor Cells on Myocardial Ischemia-reperfusion Injury in Mini-swine

Objective To simulate and assess the clinical effect of intracoronary infusion of bone marrow mononuclear cells or peripheral endothelial progenitor cells on myocardial reperfusion injury in mini-swine model. Methods Twenty-three mini-swine with myocardial reperfusion injury were used as designed in the study protocol. About （3.54±0.90）×10^7 bone marrow mononuclear cells （MNC group, n=9） or （1.16± 1.07）× 10^7 endothelial progenitor cells （EPC group, n=7） was infused into the affected coronary segment of the swine. The other mini-swine were infused with phosphate buffered saline as control （n=7）. Echocardio- graphy and hemodynamic studies were performed before and 4 weeks after cell infusion. Myocardium infarc- tion size was calculated. Stem cell differentiation was analyzed under a transmission electromicroscope. Results Left ventricular ejection fraction dropped by 0% in EPC group, 2% in MNC group, and 10% in the control group 4 weeks after cell infusion, respectively （P〈0.05）. The systolic parameters increased in MNC and EPC groups but decreased in the control group. However, the diastolic parameters demonstrated no significant change in the three groups （P〉0.05）. EPC decreased total infarction size more than MNC did （1.60±0.26 cm2 vs. 3.71±1.38 cm2, P〈0.05）. Undermature endothelial cells and myocytes were found under transmission electromlcroscope. Conclusions Transplantation of either MNC or EPC may be beneficial to cardiac systolic function, but might not has obvious effect on diastolic function. Intracoronary infusion of EPC might be better than MNC in controlling infarction size. Both MNC and EPC may stimulate angiogenesis, inhibit flbrogenesis, and differentiate into myocardial cells.

HYPOXIA CONCOMITANT WITH SERUM DEPRIVATION INDUCES ENDOTHELIAL PROGENITOR CELL DEATH

Objective To evaluate the effects endothelial progenitor cells （EPCs） survival in vitro of serum deprivation （SD） and hypoxia on the bone marrow Methods Rat bone marrow EPCs were exposed for 48 h to 02 deprivation, serum deprivation （ SD ） , and prolonged （ 120 h） hypoxia concomitant with serum deprivation. Cell death was assessed by Live/Dead staining and image analysis. Reaulta The EPCs death rate seemed not affected by 48 h hypoxia （ P 〉 0. 05 ）, but affected by SD （ P 〈 0. 01 ）. Prolonged hypoxia concomitant with SD resulted in the death of nearly all EPCs from 72 h, but this rate was remarkably reduced when with 20% fetal bovine serum（ P 〈 0. 01 ）. Conclusion Our findings indicate that EPCs are sensitive to hypoxia/SD stimuli, while serum may be the more important factor for EPCs survival.

Notch signaling in blood vessels: from morphogenesis to homeostasis

Notch signaling is an evolutionarily conserved intercellular signaling pathway that plays numerous crucial roles in vascular development and physiology.Compelling evidence indicates that Notch signaling is vital for vascular morphogenesis including arterial and venous differentiation and endothelial tip and stalk cell specification during sprouting angiogenesis and also vessel maturation featured by mural cell differentiation and recruitment.Notch signaling is also required for vascular homeostasis in adults by keeping quiescent phalanx cells from re-entering cell cycle and by modulating the behavior of endothelial progenitor cells.We will summarize recent advances of Notch pathway in vascular biology with special emphasis on the underlying molecular mechanisms.

Adiponectin levels are associated with the number and activity of circulating endothelial progenitor cells in patients with coronary artery disease

Objective:To study the relationship between plasma adiponectin concentration and the functional activities of circulating endothelial progenitor cells(EPCs) in patients with coronary artery disease(CAD).Methods:Circulating EPCs were enumerated as AC133+/KDR+ cells via flow cytometry and identified by co-staining with DiI-acLDL and fluorescein isothiocy-anate(FITC)-conjugated lectin under a fluorescent microscope.The migratory capacity of EPCs was measured by modified Boyden chamber assay.Adhesion capacity was performed to count adherent cells after replating EPCs on six-well culture dishes coated with fibronectin.Results:The number of circulating EPCs(AC133+/KDR+ cells) decreased significantly in CAD patients,compared with control subjects [(74.2±12.3) vs(83.5±12.9) cells/ml blood,P<0.01].In addition,the number of EPCs also decreased in CAD patients after ex vivo cultivation [(54.4±8.6) vs(71.9±11.6) EPCs/field,P<0.01].Both circulating EPCs and differentiated EPCs were positively correlated with plasma adiponectin concentration.The functional activities of EPCs from CAD patients,such as migratory and adherent capacities,were also impaired,compared with control subjects,and positively correlated with plasma adiponectin concentration.Conclusion:The study demonstrates that the impairment of the number and functional activities of EPCs in CAD patients is correlated with their lower plasma adiponectin concentrations.

Circulating endothelial progenitor cells in traumatic brain injury： an emerging therapeutic target？

Traumatic brain injury （TBI） is a major cause of mortality and morbidity in the world. Recent clinical investigations and basic researches suggest that strategies to improve angiogenesis following TBI may provide promising opportunities to improve clinical outcomes and brain functional recovery. More and more evidences show that circulating endothelial progenitor cells （EPCs）, which have been identified in the peripheral blood, may play an important role in the pathologic and physiological anglo-genesis in adults. Moreover, impressive data demonstrate that EPCs are mobilized from bone marrow to blood circula- tion in response to traumatic or inflammatory stimulations. In this review, we discussed the role of EPCs in the repair of brain injury and the possible therapeutic implication for func- tional recovery of TBI in the future.

Involvement of endothelial progenitor cells in the formation of plexiform lesions in broiler chickens:possible role of local immune/inflammatory response

Plexiform lesions （PLs）, which are often accompanied by perivascular infiltrates of mononuclear cells, represent the hallmark lesions of pulmonary arteries in humans suffering from severe pulmonary arterial hypertension （PAH）. Endothelial progenitor cells （EPCs） have been recently implicated in the formation of PLs in human patients. PLs rarely develop in rodent animal models of PAH but can develop spontaneously in broiler chickens. The aim of the present study was to confirm the presence of EPCs in the PLs in broilers. The immune mechanisms involved in EPC dysfunction were also evaluated. Lungs were collected from commercial broilers at 1 to 4 weeks of age. The right/total ventricle ratios indicated normal pulmonary arterial pressures for all sampled birds. Immunohistochemistry was per- formed to determine the expressions of EPC markers （CD133 and VEGFR-2） and preangiogenic molecule hepatocyte growth factor （HGF） in the lung samples. An EPC/lymphocyte co-culture system was used to investigate the functional changes of EPCs under the challenge of immune cells. PLs with different cellular composition were detected in the lungs of broilers regardless of age, and they were commonly surrounded by moderate to dense perivascular mono- nuclear cell infiltrates. Immunohistochemical analyses revealed the presence of CD133＊ and VEGFR-2＊ cells in PLs. These structures also exhibited a strong expression of HGF. Lymphocyte co-culture enhanced EPC apoptosis and completely blocked HGF-stimulated EPC survival and in vitro tube formation. Taken together, this work provides evidence for the involvement of EPCs in the development of PLs in broilers. It is suggested that the local immune cell infiltrate might serve as a contributor to EPC dysfunction by inducing EPC death and limiting their response to angi- ogenic stimuli. Broiler chickens may be valuable for investigating reversibility of plexogenic arteriopathy using gene- modified inflammation-resistant EPCs.

Effects of cannabinoid receptor type 2 on endogenous myocardial regeneration by activating cardiac progenitor cells in mouse infarcted heart

Cannabinoid receptor type 2(CB2)activation is recently reported to promote proliferation of some types of resident stem cells(e.g.,hematopoietic stem/progenitor cell or neural progenitor cell).Resident cardiac progenitor cell(CPC)activation and proliferation are crucial for endogenous cardiac regeneration and cardiac repair after myocardial infarction(MI).This study aims to explore the role and possible mechanisms of CB2receptor activation in enhancing myocardial repair.Our results revealed that CB2receptor agonist AM1241 can significantly increase CPCs by c-kit and Runx1 staining in ischemic myocardium as well as improve cardiomyocyte proliferation.AM1241 also decreased serum levels of MDA,TNF-αand IL-6 after MI.In addition,AM1241 can ameliorate left ventricular ejection fraction and fractional shortening,and reduce fibrosis.Moreover,AM1241 treatment markedly increased p-Akt and HO-1 expression,and promoted Nrf-2 nuclear translocation.However,PI3K inhibitor wortmannin eliminated these cardioprotective roles of AM1241.In conclusion,AM1241 could induce myocardial regeneration and improve cardiac function,which might be associated with PI3K/Akt/Nrf2 signaling pathway activation.Our findings may provide a promising strategy for cardiac endogenous regeneration after MI.