Journal: Blood

Article Title: Tissue type plasminogen activator regulates myeloid-cell dependent neoangiogenesis during tissue regeneration

doi: 10.1182/blood-2009-08-236851

Figure Lengend Snippet: tPA administration accelerates revascularization in a HL ischemia model. (A) tPA or vehicle was injected intraperitoneally daily from day 0 to day 2 into tPA+/+ and tPA−/− mice after HL ischemia induction. The frequency of circulating CD45+CD11b+ cells was measured (n = 3 per group). The single dagger (†) symbol denotes a significant difference (P < .05) between day 0 and day 2 in HL tPA+/+ mice. (B) C57BL/6 mice were treated with tPA or vehicle. Macroscopic pictures of the lower limb region were taken on day 15 after HL induction. Images were acquired with 0.5× and 2.5×/0.1 numeric aperture (NA) lenses (corresponding to 5× and 25× magnification), an Olympus SZX7 camera, and Adobe Photoshop CS3. (C) The ischemic/normal blood flow ratio, determined by analysis of the whole cross-section of the limb by laser Doppler perfusion imaging analysis, is shown as a function of time. (D-G) Evaluation of necrotic areas (D-E, after 15 days; bar = 200 μm) and capillary density in sections (F-G, after 28 days; bar = 50 μm) of HL-induced mice treated with or without tPA (n = 6 per group). Panels D and F were acquired with a 20×/0.50 NA and a Carl Zeiss KS400 camera, and analyzed using Axio Vision (Carl Zeiss). (H-I) Quantification of VEGF-A+ cells (arrows) in ischemic tissues (n = 6 per group, after 15 days; bar = 20 μm). Images were acquired with a 40×/0.75 NA and a Carl Zeiss KS400 camera, and analyzed using Axio Vision (Carl Zeiss). (J) Immunohistochemical staining of VEGFR-1 and VEGF-A of ischemic muscle tissues derived from vehicle- or tPA-treated HL ischemic mice and nonischemic mice. Arrows indicate VEGFR-1+VEGF-A+ cells (bar = 20 μm). Images were acquired with a 40×/0.75 NA and a Carl Zeiss KS400 camera, and analyzed using Axio Vision (Carl Zeiss). (K-L) tPA or vehicle was injected intraperitoneally daily into tPA (K) or Plg (L) wild-type and deficient mice after induction of HL ischemia. Functional perfusion measurements of the collateral region were performed (n = 3 per group). A significant difference between tPA+/+ and tPA−/− mice treated with BSA-PBS is shown. If not otherwise mentioned, asterisks denote significant differences between BSA-PBS– and tPA-treated groups. *P < .05; **P < .001; values represent the mean ± SEM.

Article Snippet: Images were acquired with 20×/0.50 NA and a Carl Zeiss KS400 camera, and analyzed using Axio Vision (Carl Zeiss). (D-E) Within ischemic muscle tissues GFP + donor cells (arrows) were quantified.

Techniques: Injection, Imaging, Immunohistochemical staining, Staining, Derivative Assay, Functional Assay

Journal: Blood

Article Title: Tissue type plasminogen activator regulates myeloid-cell dependent neoangiogenesis during tissue regeneration

doi: 10.1182/blood-2009-08-236851

Figure Lengend Snippet: Transplantation of tPA-treated CD11b+ and VEGFR-1+ myeloid cells improve neoangiogenesis. (A-F) tPA- or BSA-PBS–mobilized PBMCs from GFP mice or carrier (no cells) were injected intravenously into HL ischemia–induced recipients. (A) Blood flow was determined. (B-F) Ischemic tissues of lower limbs isolated 15 days after injection and HL ischemia induction were stained with (B) H&E and (C) antibodies against CD31 for capillary density evaluation (n = 3 per group). Images were acquired with 20×/0.50 NA and a Carl Zeiss KS400 camera, and analyzed using Axio Vision (Carl Zeiss). (D-E) Within ischemic muscle tissues GFP+ donor cells (arrows) were quantified. Inserts depict a magnified view. Images were acquired with a 20×/0.50 NA and a 63×/1.3 oil objective using a Carl Zeiss KS400 camera. (E) Quantification of the number of GFP+ cells and GFP+ cells coexpressing CD11b in ischemic tissues (n = 8 per group). (F) CD11b (Mac-1) staining of lower limb ischemic tissue from mice receiving carrier injections, from mice that received a transplant with PBMCs, or from BSA-PBS–treated or tPA-treated donor GFP mice. Arrows indicate transplanted GFP+CD11b+ cells in ischemic tissues (bar = 20 μm). Images were acquired with 40×/0.75 NA and a Carl Zeiss KS400 camera, and analyzed using Axio Vision (Carl Zeiss). (G-H) PB-derived CD11b+ or CD11b− cells isolated from tPA- or BSA-PBS–treated donors were transplanted into HL ischemia–induced recipients for 3 days (n = 4 per group). (G) Blood flow was determined. (H) Capillary density was evaluated. (I-J) HL ischemic C57BL/6 mice were treated with tPA and coinjected with anti-CD11b or control monoclonal antibodies. (I) Blood flow was analyzed by laser Doppler analysis. (J) Capillary density was evaluated. (K) PB-derived VEGFR-1+ or VEGFR-1− cells, isolated from tPA- or BSA-PBS–treated donors, were injected into HL ischemia–induced recipients, and blood flow was examined (n = 4 per group). Single asterisk (P < .05) and double asterisk (P < .001) indicate a significant difference between the groups. Values represent mean ± SEM.

Article Snippet: Images were acquired with 20×/0.50 NA and a Carl Zeiss KS400 camera, and analyzed using Axio Vision (Carl Zeiss). (D-E) Within ischemic muscle tissues GFP + donor cells (arrows) were quantified.

Techniques: Transplantation Assay, Injection, Isolation, Staining, Derivative Assay

Journal: Blood

Article Title: Tissue type plasminogen activator regulates myeloid-cell dependent neoangiogenesis during tissue regeneration

doi: 10.1182/blood-2009-08-236851

Figure Lengend Snippet: Administration of recombinant VEGF-A partially rescues the angiogenic defect observed in Plg- and MMP-9–deficient mice. (A-B) Plasma VEGF-A levels were analyzed in tPA-treated and untreated HL-induced C57BL/6 (A) and chimeric (B) mice (n = 3 per group). Chimerism was achieved as follows: Lethally irradiated Plg+/+ or Plg−/− mice received a transplant with Plg+/+ or Plg−/− donor BM cells. Six weeks later, HL ischemia–induced chimeric mice were treated intraperitoneally with/without tPA. Asterisks and single dagger indicate a significant difference between tPA-treated and BSA-PBS–treated groups. (C) Immunohistochemical staining for CD11b and VEGF-A of ischemic muscle tissue derived from tPA-treated and vehicle-treated HL ischemic mice. Arrows indicate CD11b+ VEGF-A+ cells (bar = 25 μm). Images were acquired with 40×/0.75 NA and a Carl Zeiss KS400 camera, and analyzed using Axio Vision (Carl Zeiss). (D-H) HL-induced wild-type control (D-E,H), Plg−/− (F,H), and MMP-9−/− (G,H) mice were injected intraperitoneally daily with tPA, mVEGF-A, or BSA-PBS from day 0 until day 2. (D) Functional perfusion measurements of the collateral region with the use of a laser scan at the indicated time points in wild-type animals. Representative laser Doppler perfusion images are shown of the regions analyzed. Blood perfusion (red to yellow) was augmented in tPA-treated mice compared with vehicle-injected controls (green to blue = reduced perfusion signal). (E-G) Blood flow recovery was determined by laser Doppler perfusion imaging in the ischemic limbs (n = 3; *P < .05, **P < .001). (H) Capillary density was evaluated on CD31-stained sections 15 days after ligation (n = 3; **P < .001). Asterisks indicate a significant difference between the indicated groups. Error bars represent SEM.

Article Snippet: Images were acquired with 20×/0.50 NA and a Carl Zeiss KS400 camera, and analyzed using Axio Vision (Carl Zeiss). (D-E) Within ischemic muscle tissues GFP + donor cells (arrows) were quantified.

Techniques: Recombinant, Irradiation, Immunohistochemical staining, Staining, Derivative Assay, Injection, Functional Assay, Imaging, Ligation