[Expression of alternatively spliced isoforms of platelet endothelial cell adhesion molecule-1 of embryonic stem cells during vasculogenesis and angiogenesis].

OBJECTIVE: To study the expression of alternatively spliced isoforms of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) of embryonic stem cells (ES cells) during vasculogenesis and angiogenesis. METHODS: Mouse ES cells of the line J1 were cultured. Another ES cells were cultured in differentiation medium to induce the formation of embryonic bodies (EBs). Then the ES cells with PECAM-1 and EBs were inoculated with methylcellulose into Petri dish, containing cell growth factor, VEGF, bFGF, EPO, and IL-6 and the ES cells cultured for 11 days were inoculated in the Petri dish with collagen for 72 hours so as to induce sprouting angiogenesis. Immunofluorescence analysis, RT-PCR, and flow cytometry were used to detect the expression of PECAM-1, Oct-4, and stage-specific embryonic antigen (SSEA)-1 in the undifferentiated ES cells, EBs, and EB sprouting. In order to delineate the alternatively spliced cytoplasmic domain isoforms of PECAM-1 specific primers were designed to span the exon-exon junctions in the regions of alternative splicing. In order to amplify the cytoplasmic domains of all possible PECAM-1 isoforms a sense primer spanning the border of exons 9 and 10 within the cytoplasmic domain and an antisense primer spanning the border of exon 16 and 3'-untranslated region were used. Then the PCR products of the cytoplasmic domain underwent subsequent sequencing to analyze the expression of the 8 known alternatively splice isoforms of PECAM-1. RESULTS: The ES cells expressed high level PECAM-1 that was mainly located at the cell-cell junctions. The SSEA-1 and Oct-4 levels rapidly decreased along with the differentiation of the ES cells. All 8 known alternatively splice isoforms of PECAM-1 were expressed in the ES cells and the EB sprouting, the expression of Delta14%15 and Delta12&14&15 being the highest. The expression level of Delta12&14&15 increased markedly and the expression of Delta15 decreased along with the differentiation of ES cells. CONCLUSION: PECAM-1 is a constitutive feature of undifferentiated ES cells. Its changes in splice form mark the differentiation and may participate in vasculogenesis and angiogenesis.

Enhancement of neovascularization with cord blood CD133+ cell-derived endothelial progenitor cell transplantation.

The endothelial progenitor cells (EPCs) are responsible for postnatal vasculogenesis in physiological and pathological neovascularization and have been used for attenuating ischemic diseases. However, EPCs from umbilical cord blood (CB) were not well understood and the homing mechanisms of EPCs remain unclear. To determine the potential application of CB-derived EPCs, we established a culture system to induce the differentiation of CB cells into EPCs. Purified CB CD133(+) cells proliferated and, after further vascular endothelial growth factor receptor 2 (VEGFR-2) antibody purification, differentiated into EPCs expressing endothelial markers, such as VE-cadherin, VEGFR-2, CD31, von Willebrand factor (vWF) and Weibel-Palade bodies. These cells could also take up acetylated lower density lipoprotein (Ac-LDL) and bind Ulex europaeus agglutinin-1 (UEA-1). When expanded EPCs were transplanted via tail vein into nude mice, they incorporated into capillary networks in ischemic hindlimb, augmented neovascularization, and improved ischemic limb salvage. In addition, in ischemic tissue, there were elevated expressions of VEGF and stromal derived factor 1 alpha (SDF-1 alpha), both of which had chemotactic effect on EPCs. Moreover, P-/E-selectins was found on mouse ischemic endothelium and P-selectin glycoprotein ligand-1 (PSGL-1) on CB-derived EPCs. Neutralizing antibody against PSGL-1 blocked the homing of EPCs to ischemic area by 61%. These results demonstrate that CB CD133(+) cell-derived EPCs can be applied for therapeutic neovascularization in ischemic diseases, and reveal important roles of chemoattractants and adhesive molecules in the homing of EPCs.

[Transplantation of cord blood endothelial progenitor cells ameliorates limb ischemia].

OBJECTIVE: To investigate the feasibility of transplanting cord blood CD133+ cells derived endothelial progenitor cells (EPC) in therapeutic vasculogenesis. METHODS: CD133+ cells from the cord blood of 52 neonates were cultured in fibronectin-coated flask in M199 medium supplemented with 10% fetal bovine serum, 50 ng/ml vascular endothelial growth factor (VEGF), 20 ng/ml interleukin-3 (IL-3) and 50 ng/ml stem cell factor (SCF). The cell markers of spindle-shaped adherent cells were determined with flow cytometry. The left femoral artery, great saphenous artery, iliac circumflex artery, and vein, and muscular branch of 22 Balb/c nude mice were cut to cause limb ischemia. One day after the unilateral ischemic limb surgery half million adherent cells were transplanted into 12 nude mice via tail vein (EPC group) and M199 was injected into the tail veins of 10 nude mice (M199 group). Fluorescence Dil, laser Doppler perfusion imaging (LDPI) and immunohistochemistry were Laser Doppler perfusion imaging (LDPI) was used to trace the transplanted cells and monitor the blood perfusion and capillary density of ischemic limbs. The ratio between the blood perfusion of the operated limb and of the non-operated opposite limb was recorded. Two to four mice in each group were killed 4, 7, 14, and 21 days after the operation and the gastrocnemius muscles of bilateral hind limbs were taken to count the number of capillaries. The VEGF mRNA levels of the ischemic and nonischemic limbs were examined with semi-quantitative RT-PCR. Seven days after the operation, fluorescein isothiocyanate (FITC)-binded ulex europaeus agglutinin-1 (UEA-1) was injected via tail vein to 3 EPC group mice. Thirty minutes later, the mice were killed. The heart, lung, liver, spleen and limb muscles were taken and examined with fluorescence microscopy. EPC were added into the upper chamber of Coster Transwell and chemotactic fluids of M199 with or without VEGE were added into the lower chamber. Four hours later the number of EPC in the lower chamber was counted so as to examine the chemotactic effect of VEGE. RESULTS: Numerous cell clusters, spindle-shaped adherent cells and cord-like structures, developed from the culture of cord blood CD133+ cells. These adherent cells expressed vascular endothelial growth factor receptor 2 (VEGFR-2), VE-cadherin, CD31, von Willebrand factor (vWF) and combined with ulex europaeus agglutinin-1 (UEA-1). Transplanted EPC survived and were incorporated into the capillary networks in the ischemic limbs of nude mice. The ratio between the blood perfusion of the ischemic limb and non-ischemic limbs was 19.1% +/- 3.1%. Two weeks after the transplantation, the ratio between the blood perfusion of the ischemic limb and non-ischemic limbs of the EPC group was 77.3% +/- 5.6%, significantly higher than that of the M199 group (40.6% +/- 3.4%, P<0.001). CD31 histochemical staining showed that the density of capillaries in the gastrocnemius muscles of ischemic hind limb was significantly higher 7, 14, and 21 days after operation in the EPC group than in the M199 group (P<0.05) RT-PCR showed obvious VEGF bands in the ischemic hind limb muscles, but not in the non-ischemic muscles. The number of EPC immigrating into the lower chamber of the Coster Transwell was 817 +/- 32.5, significantly higher than that of the control group (473.5 +/- 61.5, P<0.05). CONCLUSION: Cord blood CD133+ cells derived EPC is a robust cell source for therapeutic neovascularization. Upregulated expression of VEGF may account for the homing of transplanted EPC to ischemic tissue.

[Transplantation of autologous peripheral blood stem cells for the treatment of lower limb arteriosclerosis obliterans].

OBJECTIVE: To evaluate the clinical efficacy of mobilized autologous peripheral blood stem cells (PBSC) transplantation in a 48 years old patient with lower limb arteriosclerosis obliterans (ASO). METHODS: rhG-CSF 600 micro g/d for 5 days to mobilize stem cells. On the fifth day, PBSC were collected with a Version 4 blood-cells separator. Three hours late, the PBSC were intramuscularly injected into the ischemic areas of the two lower limbs (3 x 10(9) cells per limb). The clinical and laboratory findings were monitored every week for 3 months. Forty-four days after the implantation, left lower limb with severe ASO was given an additional implantation of the same number of cells as the first time. RESULTS: The peripheral blood CD(34)(+) cells were increased from 0.18% to 0.75% after 5 days of rhG-CSF mobilization. Three months after the first stem cell transplantation, severe pain lameness, local cool-feeling and ulcer were improved, and ABI increased from 0.49, 0.69 to 0.50, 0.85, the amplitude of blood flow and laser Doppler blood perfusion were also significantly improved (P < 0.01). At the same time, digital subtraction angiographic scores for new collateral vessel formation were showed as + 3(rich). No related complication or adverse effect were observed during the 3-month observation. CONCLUSION: Transplantation of mobilized autologous PBSC might be a simple, safe, and effective method for the treatment of ASO.