Figure 4.
Loss of VEGF-C from the BM microenvironment delays vascular and HSC regeneration after irradiation. (A) Experimental setup for evaluating Vegfc expression level in the BM after irradiation, by using qPCR. Relative Vegfc mRNA level in WBM, in sorted CD45−Ter119− stromal cells, and in CD45+Ter119+ hematopoietic cells 24 hours after 10-Gy radiation (normalized to untreated WBM; n = 2 mice). (B) Experimental setup for evaluating the efficiency of engraftment of WT WBM in lethally irradiated VciΔR26 and Vcfl/fl mice. (C) Transplantation of WT WBM (CD45.1) into lethally irradiated VciΔR26 mice and their Vcfl/fl littermate hosts. Representative flow cytometry graph of total WBCs from peripheral blood 12 weeks after transplantation (left). The kinetics of multilineage donor chimerism from peripheral blood after transplantation (right; n = 6-8 mice per group). (D) Quantification of BM LKS cells in lethally irradiated VciΔR26 mice and their Vcfl/fl littermate recipients 16 weeks after transplantation (n = 6-8 mice per group). (E) Representative confocal immunofluorescence images and quantification. Femur sections from VciΔR26 mice and their littermate controls stained for ECs and basement membranes (endomucin, green; laminin, white) and LepR cells (red) (n = 4-5 mice per group). Bars represent 50 μm. (F) Experimental setup for serial transplantation. WBM (CD45.1) from lethally irradiated primary VciΔR26 and Vcfl/fl recipients was transplanted competitively with CD45.2 WBM into lethally irradiated secondary VciΔR26 and Vcfl/fl recipients. (G) Multilineage donor chimerism from peripheral blood after secondary competitive transplantation (2 independent transplants with 3 recipients per condition per transplant). (H) Quantification of BM LKS numbers in secondary VciΔR26 mice and their Vcfl/fl littermate controls 16 weeks after transplantation. Reported values are mean ± SD. Statistical significance was determined using the 2-tailed, unpaired Student t test. *P < .05; **P < .01.