Bone marrow mononuclear cells are recruited to the sites of VEGF-induced neovascularization but are not incorporated into the newly formed vessels by Lorena Zentilin, Sabrina Tafuro, Serena Zacchigna, Nikola Arsic, Lucia Pattarini, Milena Sinigaglia, and Mauro Giacca Blood Volume 107(9):3546-3554 May 1, 2006 ©2006 by American Society of Hematology

Mononuclear cells infiltrate the foci of VEGF-induced neovascularization. Mononuclear cells infiltrate the foci of VEGF-induced neovascularization. (A) Immunohistochemical time course analysis, by the visualization of small arteries using an antibody against smooth muscle actin (α-SMA), of the angiogenic response induced by AAV-mediated VEGF expression in normal skeletal muscle at different times after vector injection. Abundant mononuclear cell infiltrates are detectable in the perivascular spaces of the injected muscles (enlargements i-iii in panel B). Images were obtained using an Olympus CX40 microscope and 10 ×/0.30 NA and 40 × 0.75 UPlan FL objective lenses (Olympus, Tokyo, Japan). Images were captured using Olympus Camedia C-3030 digital camera and processed with Adobe Photoshop 7.0 software. (C) Quantification of the number of α-SMA–positive vessels over time. Shown are means and standard deviations of counts, expressed as number of α-SMA vessels per muscle fiber. The quantifications were carried out by 3 independent investigators who observed 10 different sections from 3 animals per time point. d indicates days; mo, months. (D) Quantification of the number of the infiltrating cells in the AAV-VEGF–treated muscles. Shown are means and standard deviations of counts, expressed as number of nuclei per muscle fiber. The quantifications were carried out by 3 independent investigators who observed 10 different sections from 3 animals per time point. Lorena Zentilin et al. Blood 2006;107:3546-3554 ©2006 by American Society of Hematology

Immunologic characterization of the mononuclear cells infiltrating the foci of VEGF-induced neovascularization. Immunologic characterization of the mononuclear cells infiltrating the foci of VEGF-induced neovascularization. Immunofluorescence staining was performed using CD31-, α-SMA–, Flk-1–, CD34-, Sca-1–, F4/80-, CD11b-, and c-Kit–specific antibodies on histologic sections of mouse tibialis anterior muscles perfused in vivo with FITC-lectin. The analysis was performed at 2 weeks after AAV-VEGF transduction. As shown in the 3 small subpanels, red indicates cells positive for the different antibodies; green, whole vasculature stained with FITC-lectin; and blue, nuclei stained with DAPI. The 3 stainings are merged in the large panels. Lorena Zentilin et al. Blood 2006;107:3546-3554 ©2006 by American Society of Hematology

Sex-mismatch bone marrow transplantation model. Sex-mismatch bone marrow transplantation model. (A) Flow chart of the experimental procedure. Unfractionated BM cells from male Balb/c donor mice were transplanted into lethally irradiated syngenic female recipients. After hematopoietic recovery, the efficiency of engraftment was evaluated by competitive PCR by quantifying the copies of donor-specific Y-chromosome sequences in the bone marrow of the animals that received a transplant, using β-globin as reference gene. (B) Schematic representation of the templates for quantitative PCR. Amplification of the Y-chromosome sequence and of the β-globin gene were obtained with primer pairs Y-F/Y-R and BG-F/BG-R, respectively. The multicompetitor used for competitive PCR contains a core sequence of 218 bp, corresponding to a 20-bp–deleted version of the mouse β-globin amplification fragment, flanked by Y-F and Y-R primer sequences. (C) Example of competitive PCR amplification. Fixed amounts of sample DNA from PBMCs were mixed with scalar amounts of the multicompetitor DNA and PCR amplified with the 2 primer pairs. After amplification, the gels were stained with ethidium bromide and the competitor (Comp), Y chromosome (Y chr), or β-globin DNA bands were quantified. According to the principles of competitive PCR, the ratio between the amplification products in each reaction is linearly correlated with the input DNA amounts for the 2 DNA species. Dashed red boxes indicate the point of equivalence. (D) Examples of FISH analysis on muscle sections from mice that received a transplant at 30 days after injection of AAV-VEGF, using a mouse-specific Y-chromosome probe labeled with FITC. The green dots, indicated by arrows in the enlargements, correspond to Y-chromosome–specific signals. Red indicates nuclei stained by propidium iodide; and M, muscle fibers. Images in panel D were obtained using a Zeiss LSM510 confocal microscope (Carl Zeiss, Göttingen, Germany), equipped with an Axiovert 100M reverse microscope and 40 ×/0.75 NA and 100 ×/1.30 NA oil objectives. LSM510 software 3.2 was used for image acquisition. Pictures were assembled with Adobe Photoshop 7.0 software. Lorena Zentilin et al. Blood 2006;107:3546-3554 ©2006 by American Society of Hematology

Immuno-FISH analysis of VEGF-treated muscles of mice that received a transplant at 15 days, 30 days, and 3 months after vector injection. Immuno-FISH analysis of VEGF-treated muscles of mice that received a transplant at 15 days, 30 days, and 3 months after vector injection. Immunofluorescence staining for endothelial (CD31; A) and smooth muscle (α-SMA; B) antigens was combined with Y-chromosome fluorescent in situ hybridization. The vast majority of cells expressing either of the 2 markers were not found to contain a Y chromosome (enlargements in panels A and B). Very rare cells (< 1% of total Y-chromosome–positive nuclei) were positive for CD31 and Y-chromosome markers (shown in the enlargements in panel C); cells of donor origin and positive for the α-SMA antigen were never found in the tunica media of vessels. Blue indicates nuclei stained with DAPI; red, (A) CD31- and (B) α-SMA–positive cells; and yellow, Y chromosome. Lorena Zentilin et al. Blood 2006;107:3546-3554 ©2006 by American Society of Hematology

FVB/TgN transgenic mouse bone marrow transplantation model. FVB/TgN transgenic mouse bone marrow transplantation model. (A) Flow chart of the experimental procedure. BM from FVB/TgN transgenic mice, expressing GFP under the transcriptional control of the endothelial specific Tie2 promoter, was transplanted into lethally irradiated FVB/NJ wild-type recipients 30 days before treatment with AAV-VEGF. (B) Immunostaining for the endothelial-specific CD31 and GFP markers in muscle sections of VEGF-treated mice at 1 month after vector injection. In the FVB/TgN transgenic control mouse, most of the endothelial cells also stained positive for GFP (i-iii). In contrast, no Tie2-GFP–positive cells were found within the cellular infiltrates or incorporated into the wall of the newly formed vessels in the FVB/NJ-BMT TgN chimeric mice (iv-ix). Red indicates CD31+ cells; green, GFP; and blue, nuclei stained with DAPI. Lorena Zentilin et al. Blood 2006;107:3546-3554 ©2006 by American Society of Hematology

Erythropoietin (EPO) mobilizes bone marrow cells with an EPC phenotype that are recruited to the sites of neovascularization but are not incorporated in the neovessels. Erythropoietin (EPO) mobilizes bone marrow cells with an EPC phenotype that are recruited to the sites of neovascularization but are not incorporated in the neovessels. (A) Flow chart of the experimental procedure. Sex-mismatched AAV-VEGF–treated mice that received a transplant were treated with EPO in order to mobilize endothelial progenitor cells from the bone marrow. (B) Flow cytometry analysis of mononuclear cells from peripheral blood and spleen. Animals treated with EPO (but not those injected with AAV-VEGF or AAV-LacZ) showed a significant increase of Flk-1+ and Flk-1+/CD34+ cell populations, indicative of the mobilization of endothelial progenitors. Data (mean ± SD from 10 animals per group) represent the percentage of positive cells in the population of light scatter dot plots of monocyte-sized cells (see also Figure S2 and Asahara et al8). NS indicates not significant. (C) Immuno-FISH analysis of VEGF-treated muscle sections of animals treated with EPO. Despite the mobilization of EPCs, there was no increase in the number of Y-chromosome–positive cells expressing the CD31 endothelial markers at the sites of VEGF-induced neovascularization. Red indicates CD31+ cells; green, Y chromosome; and blue, nuclei stained with DAPI. Lorena Zentilin et al. Blood 2006;107:3546-3554 ©2006 by American Society of Hematology

In vitro–cultured bone marrow cells with EPC phenotype home at sites of VEGF-induced angiogenesis but are not incorporated into the vessel wall.(A) Experimental design. In vitro–cultured bone marrow cells with EPC phenotype home at sites of VEGF-induced angiogenesis but are not incorporated into the vessel wall.(A) Experimental design. Balb/c mice adherent bone marrow cells were cultured in vitro for 15 days in differentiating (D) and nondifferentiating medium (ND; see text) in order to enrich for endothelial progenitor cells. After 15 days, the EPCs were labeled with the PKH26 red fluorescent die and administered intravenously to syngenic recipient mice previously injected with AAV-VEGF in the right tibialis anterior muscle. (B) Flow cytometry phenotypic profile of the cell populations before injection into the animals (mean ± SD of 3 different experiments). (C) Number of PKH16-positive cells infiltrating the sites of VEGF-induced neovascularization at day 15 after cell injection. The cells expanded ex vivo using the ND medium were recruited approximately 4 times more efficiently than those cultivated in differentiating medium (mean ± SD of 3 different experiments). (D) Immunofluorescence analysis for the visualization of CD31 (green) and PKH26 (red). Colocalization of the 2 markers in the same cells was highly infrequent. (E) Immunofluorescence analysis for the visualization of CD11b (green) and PKH26 (red); arrows indicate 2 in vitro–labeled recruited cells. Most of the recruited cells were positive for the myelocytic/monocytic marker CD11b. Lorena Zentilin et al. Blood 2006;107:3546-3554 ©2006 by American Society of Hematology