Objective To examine whether the increase of carbon monoxide (CO) induced by oral methylene chloride (MC) administration in recipients before heart transplantation would protect heart grafts against isehemia/reperfusion (I/R) injury associated with transplantation and to explore the possible mechanism.Methods Inbred male Balb/c mice were used as donors and recipients to establish cervical heart transplantation model Recipients were treated with either MC (100 mg/kg or 500 mg/kg,per os)(group MC 100 mg,n=10;group MC 500 mg,n=12) or olive oil(0.15 ml,per os.group olive,n=10) 3 h prior to anesthesia.Age-matched norwlal mice served as controls (group N,n=5).The serum COHb and the CO content of myocardial tissue were measured at 0,1,3,6,12,24 h after oral MC administration.Half of recipients were killed at 3 and 24h after transplantation for senum or cardiac graft samples.The serum cTnI levels,the mRNA levels of TNF-α,IL-10,Bcl-2,Bax.the protein levels of NF-κB and the ultrastructures of myocardium were examined.Results As tompared with group olive.the serum COHb and tissue CO were increased significantly and peaked within 3 h in group MC 100 mg and group MC 500 mg.The serum cTnI levels in group MC 100 mg and group MC 500 mg were significantly decreased (P<0. 01 ), especially in group MC 500 mg. The increase of CO in recipients of group MC100 mg and group MC 500 mg significantly inhibited the proinflammatory gene expression of TNF-α mRNA and the pro-apoptotic gene expression of Bax mRNA (P<0. 01), and increased the anti-apoptotic gene expression of Bcl-2 mRNA (P<0. 01), but did not increase the anti-inflammatory gene expression of IL-10 mRNA (P>0. 05) in the heart grafts. As compared with group N, the myocardial NF-κB activation was increased significantly in group olive,group MC 100 mg and group MC 500 mg (P<0. 01 ), but there was no significant difference among the later three groups (P>0. 05). The myocardial ultrastructure was also alleviated significantly in group MC 100 mg and group MC 500 mg as compared with group N. Conclusion The increase of CO induced by MC in recipients suppresses pro-inflammatory and pro-apoptotic gene expression and efficiently ameliorates transplant-induced heart I/R injury. The possible mechanism does not seem to be associated with down-regulation of the NF-κB signaling pathway.

AIM: To explore the ex vivo expansion characteristics of the endothelial progenitor cells (EPCs). METHODS: CD34+ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded at the same conditions as that for total MNC, coincubation of CD34+ and CD34- from the same donation for EPCs. In addition, we tested the effect of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis. EPCs were determined and quantified by immunocytochemistry and flow cytometry. RESULTS: Coculture of CD34+ and CD34-,total MNC led to a significant increase in the expansion of CD34+ cells compared with CD34 enrichment (P0.05). These differentiated EPCs were stained positive for CD34+, von Willebrand factor (vWF), KDR, CD31 and incorporate acetylated low-density lipoprotein (LDL). CD34+ and AC133+cells accounted for 68.2%?6.3% (n=6) and 57.2%?9.8% (n=6) of attaching (AT) cells at day 7 of culture, respectively. CONCLUSIONS: Coculture of CD34+ and CD34- or culture of MNC enhances ex vivo expansion of EPCs. Early passage decreases apoptosis rate, VEGF has no significant effect on ex vivo expansion of EPCs.

AIM: To determine the combined effect of transmuscle laser revascularization (TMR) and endothelial progenitor cells(EPCs) treatment on ischemic hindlimb of nude rats.METHODS: Mononuclear cells (MNCs) isolated from human umbilical cord-blood (HUCB) by density gradient centrifugation were expanded in vitro. Immunocytochemistry and flow cytometry studies were performed. EPCs were labeled with 1, 1'- dioctadecyl-1 to 3, 3, 3', 3'- tetramethyl-indocarbocyanine perchlorate (DiI) before injected into the laser induced channels or ischemic region. Acute ischemic limb was created in 4 groups of nude rats by ligating right external iliac artery. All animals were divided randomly into the following four groups: TMR+EPCs group: local transplantation of EPCs into laser channels; TMR group: transmuscular channels were created without EPCs; EPCs group: EPCs were injected into ischemic hindlimb; control group: ischemic model without TMR or EPCs. All rats underwent femoral artery ultrasonic blood flow measurements of the ischemic and nonischemic limbs to obtained a flow ratio [femoral artery flow index (FAFI): right femoral artery flow /left femoral artery flow] at baseline (after ligating artery immediately) and 28 days postoperation, and then the samples of ischemic limb muscle underwent histochemical and immunohistologic analysis. RESULTS: The attached cells expressed endothelial cell (ECs) markers (KDR, CD34, CD31, AC133 and von Willebrand factor) and exhibited function similar to that of ECs judged by Ac-LDL incorporation. Flow cytometric analysis disclosed that AT cells were positive for CD34 (62%?7%) and AC133 (57.2%?9.8%) at day 7 of culture. 28 days after therapy, FAFI was significantly higher in the TMR +EPCs (0.66?0.09, P0.05). FAFI in the control group was unchanged and no difference was found between TMR group and control group. TMR+EPCs (5.66?0.77), TMR (4.96?0.31) as well as EPCs (4.68?0.44) treatment resulted in an increased number of capillaries in the treated regional area compared with control group (2.60?0.31, P

To develop a more efficient antithrombotic way after coronary artery bypass grafting (CABG), the anticoagulant effects were compared of human tissue factor pathway inhibitor (TFPI) gene transfection and aspirin oral administration (traditional method) on vein grafts. An eukaryotic expression plasmid pCMV-(Kozak) TFPI was prepared. Animal model of carotid artery bypass grafting was constructed. In operation, endothelial cells of vein grafts in TFPI group and empty plasmid control group were transfected with pCMV-(Kozak) TFPI and empty plasmid pCMV respectively, while no transfection was conducted in aspirin control group. After operation, aspirin (2 mg·kg-1·d-1) was administered (I.g.) in aspirin control group. Three days later, grafts (n=10) were harvested for RT-PCR, Western blotting and immunohistochemical analyses of exogenous gone expression and for pathological, scanning electron microscopic observation of thrombus. Thirty days later, the patency rates of remnant grafts (n=10) were recorded by vessel Doppler ultrasonography. Human TFPI gene products were detected in gene transferred vein grafts. Three days later, thrombi were found in 7 animals of aspirin control group and in 8 animals of empty plasmid control group, but in only 1 of TFPI group (P<0.01). Thirty days later, 5 grafts were occluded in empty plasmid control group, but none of grafts was occluded in the other groups (P<0.05). The endothelial surfaces of grafts in both of the control groups were covered with aggregated erythrocytes and platelets, and it were not seen in TFPI group. R was suggested that the anticoagulant effects on vein grafts of human TFPI gene trans- fection are better than those of aspirin.