Figure 7
Figure 7. Angiogenic potential of murine BM-derived M2-macrophages depends on their TIMP-1-free proMMP-9. (A) Polarization of murine BM-derived macrophages. M0 macrophages were generated from WT mice (upper) or Mmp9-KO mice (lower) by incubation of BM cells in the presence of murine M-CSF. Following a 7-day maturation, M0 macrophages were incubated for 24 hours either in the mixture of IFNγ and LPS to induce the M1 phenotype or with IL-4 to induce the M2 phenotype. Phase contrast images were acquired using an Olympus CKX-41 microscope equipped with Olympus U-LS30-3 video camera and Infinity Capture software and processed using Adobe Photoshop. Objective lens, ×20. (B) Western blot analysis of MMP-9 and TIMP-1 produced by mature and polarized murine macrophages. Following maturation and polarization, murine macrophages were switched to SF medium. CM was collected 48 hours later and analyzed for production of MMP-9 and TIMP-1. Control proteins, murine recombinant proMMP-9 (105 kDa) and TIMP-1 (28 kDa), were loaded in the wells of the same gel to provide the means of quantification. After SDS-PAGE and transfer, the membrane was cut horizontally, and the upper potion was probed with anti-murine MMP-9 antibody, whereas the lower potion was probed with anti–TIMP-1 antibody. The position of molecular weight markers is indicated on the left. (C) Angiogenic capacity of murine M2 macrophages depends on expression of MMP-9. Intact M2 macrophages generated from BM of WT or Mmp9-KO mice were incorporated into 3D collagen onplants and analyzed in the CAM angiogenesis assay. SF medium was used as a negative no-cell (NC) control. The data are from 2 independent experiments, each involving from 4 to 6 embryos, each bearing from 5 to 6 onplants. Bar graph presents means ± SEM of fold changes in the levels of angiogenesis compared with NC control (1.0). *P < .05 and **P < .01 in comparison with angiogenesis levels induced by WT M2 macrophages. (D) Angiogenic capacity of murine M2 macrophages is sensitive to TIMP-1. M2 macrophages generated from BM of WT or Mmp9-KO mice were incubated in SF medium for 48 hours. CM was incorporated into 3D collagen onplants alone or with 4 ng of recombinant murine TIMP-1 per onplant. SF medium was used as a negative control. Data are from 2 independent CAM experiments, each involving from 4 to 6 embryos per variant (5-6 onplants per embryo). Bar graph presents means ± SEM of fold changes in the levels of angiogenesis compared with SF control (1.0). *P < .05, **P < .01, and ***P < .0001, all in comparison with angiogenesis levels induced by WT M2 macrophages.

Angiogenic potential of murine BM-derived M2-macrophages depends on their TIMP-1-free proMMP-9. (A) Polarization of murine BM-derived macrophages. M0 macrophages were generated from WT mice (upper) or Mmp9-KO mice (lower) by incubation of BM cells in the presence of murine M-CSF. Following a 7-day maturation, M0 macrophages were incubated for 24 hours either in the mixture of IFNγ and LPS to induce the M1 phenotype or with IL-4 to induce the M2 phenotype. Phase contrast images were acquired using an Olympus CKX-41 microscope equipped with Olympus U-LS30-3 video camera and Infinity Capture software and processed using Adobe Photoshop. Objective lens, ×20. (B) Western blot analysis of MMP-9 and TIMP-1 produced by mature and polarized murine macrophages. Following maturation and polarization, murine macrophages were switched to SF medium. CM was collected 48 hours later and analyzed for production of MMP-9 and TIMP-1. Control proteins, murine recombinant proMMP-9 (105 kDa) and TIMP-1 (28 kDa), were loaded in the wells of the same gel to provide the means of quantification. After SDS-PAGE and transfer, the membrane was cut horizontally, and the upper potion was probed with anti-murine MMP-9 antibody, whereas the lower potion was probed with anti–TIMP-1 antibody. The position of molecular weight markers is indicated on the left. (C) Angiogenic capacity of murine M2 macrophages depends on expression of MMP-9. Intact M2 macrophages generated from BM of WT or Mmp9-KO mice were incorporated into 3D collagen onplants and analyzed in the CAM angiogenesis assay. SF medium was used as a negative no-cell (NC) control. The data are from 2 independent experiments, each involving from 4 to 6 embryos, each bearing from 5 to 6 onplants. Bar graph presents means ± SEM of fold changes in the levels of angiogenesis compared with NC control (1.0). *P < .05 and **P < .01 in comparison with angiogenesis levels induced by WT M2 macrophages. (D) Angiogenic capacity of murine M2 macrophages is sensitive to TIMP-1. M2 macrophages generated from BM of WT or Mmp9-KO mice were incubated in SF medium for 48 hours. CM was incorporated into 3D collagen onplants alone or with 4 ng of recombinant murine TIMP-1 per onplant. SF medium was used as a negative control. Data are from 2 independent CAM experiments, each involving from 4 to 6 embryos per variant (5-6 onplants per embryo). Bar graph presents means ± SEM of fold changes in the levels of angiogenesis compared with SF control (1.0). *P < .05, **P < .01, and ***P < .0001, all in comparison with angiogenesis levels induced by WT M2 macrophages.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal