Effect of EPO treatment on fat graft weight and volume in all treatment groups in the two experiments.

<p><b><u>Footnotes</u></b></p><p>Values are presented as mean ± SD.</p><p>n  =  number of mice.</p><p>EPO  =  erythropoietin.</p><p>VEGF  =  vascular endothelial growth factor.</p><p>**P<0.01, ***P<0.001, for the difference between either the low-dose- or the high-dose EPO-treated fat grafts and the PBS-treated grafts.</p

Effect of EPO on the extent of apoptosis in the fat grafts.

<p>PBS (100 µl), 20 IU EPO/100 µl PBS (low-dose), or 100 IU EPO/100 µl PBS (high-dose) were injected into fat grafts in three different groups of mice on the day of the fat injection and then repeated every three days for 18 days. (A) The extent of apoptosis was measured by the TUNEL assay, and is expressed as a percentage of the presence of apoptosis in the PBS-treated fat grafts. Each bar represents the mean extent of apoptosis ± SD in the fat graft in each treatment group at the end of the 15-week study period. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 for the difference between either the low-dose- or the high-dose EPO-treated fat grafts and the PBS-treated grafts. (B) Representative western blots of the expression levels of caspase 3 (Casp 3) and cytochrome c (Cyt c) in the PBS- and EPO-treated fat grafts at the end of the 15-week study period.</p

Histological analysis of the dissected fat grafts in all treatment groups in the two experiments.

<p><b><u>Footnotes</u></b></p><p>Values are presented as mean ± SD.</p><p>n  =  number of mice.</p><p>EPO  =  erythropoietin.</p><p>VEGF  =  vascular endothelial growth factor.</p><p>*P<0.05, **P<0.01 for the difference between either the low-dose- or the high-dose EPO-treated fat grafts and the PBS-treated grafts.</p

Effect of EPO on the expression levels of angiogenic growth factors in the fat grafts.

<p>The fat grafts from the three different groups of mice were treated with either PBS (100 µl), 20 IU EPO/100 µl PBS (low-dose), or 100 IU EPO/100 µl PBS (high-dose) on the day of the fat injection, and the treatments were repeated every three days for 18 days. (A) Representative histological micrographs of PBS-, and low-dose- and high-dose-EPO treated fat grafts (left to right) presenting VEGF expression (upper panel), VEGFR-2 expression (middle panel), and EPOR expression (lower panel). (B) Representative western blots of the expression levels of the angiogenic factors in the PBS- and EPO-treated fat grafts at the end of the 15-week study period. bFGF: basic fibroblast growth factor; IGF-1: insulin-like growth factor-1; PDGF-BB: platelet-derived growth factor-BB; MMP-2: matrix metalloproteinase-2; PKB: protein kinase B; phosphoPKB: phosphorylated PKB. (C) Graphs representing the mean VEGF content (left), the mean VEGFR-2 expression (middle) and the mean EPOR expression (right) ± SD in the fat grafts in each treatment group. (D) The correlation between VEGF and MVD (left), and between mean VEGFR-2 (middle) and EPOR (right) expression and mean MVD in each group. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001for the difference between either the low-dose- or the high-dose EPO-treated fat grafts and the PBS-treated grafts. Scale bar: 200µm.</p

Effect of VEGF on MVD and the extent of apoptosis in the fat grafts.

<p>PBS (100 µl) or VEGF (200 ng VEGF/100 µl PBS) were injected into the fat grafts in two different groups of mice on the day of the fat injection and every three days for 18 days. (A) Each bar represents the mean MVD ± SD from five regions of interest in each slide that was prepared from the harvested fat grafts of each treatment group at the end of the 15-week study period. (B) Each bar represents the mean VEGF content ± SD in the harvested fat grafts in each treatment group at the end of the 15-week study period. (C) The extent of apoptosis was measured by the TUNEL assay, and is expressed as a percentage of the extent of apoptosis in the PBS-treated fat grafts. Each bar represents the mean extent of apoptosis ± SD in the fat graft in each treatment group at the end of the 15-week study period. **<i>P</i><0.01for the difference between the VEGF-treated fat grafts and the PBS-treated grafts. (D) Representative western blots of the expression levels of caspase 3 (Casp 3) and cytochrome c (Cyt c) in the PBS- and VEGF-treated fat grafts at the end of the 15-week study period.</p

Effect of EPO treatment on body weight, hematology, and plasma and tissue EPO concentrations in the three experimental groups.

<p><b><u>Footnotes</u></b></p><p>Values are presented as mean ± SD; n  =  number of mice; conc.  =  concentrations; RBC  =  red blood cells; EPO  =  erythropoietin; VEGF  =  vascular endothelial growth factor. *P<0.05, **P<0.01, ***P<0.001 for the difference between either the low-dose- or the high-dose-treated EPO grafts and the PBS-treated grafts.</p

Histological sections of fat grafts that were removed from the PBS-treated, low-dose, and high-dose EPO treated mice 15 week after fat transplantation.

<p>The fat grafts from three different groups of mice were treated with either PBS (100 µl), 20 IU EPO/100 µl PBS (low-dose), or 100 IU EPO/100 µl PBS (high-dose) on the day of the fat injection, and the treatments were repeated every three days for 18 days. After harvesting, sections were stained with hematoxylin and eosin or prepared for assessing inflammatory cell infiltration and MVD, and then examined under a light microscope for (a) the extent of integration, as evidenced by the extent of organization of intact and nucleated fat cells in the grafted fat tissue architecture, (b) the extent of fibrosis, as evidenced by the amount of collagen and elastic fibrils, (c) the presence of cysts and vacuoles, (d) the intensity of the inflammatory response, as evidenced by the extent of macrophage infiltration, and (e) MVD, as evidenced by the number of blood vessels in the fat grafts. Representative histological micrographs of PBS-, and low-dose- and high-dose-EPO treated fat grafts (left to right). (A) fat cell integration in fat grafts, (B) inflammation as evidenced by infiltration of CD68-positive cells (macrophages) in fat grafts, and (C) vascularization in the fat grafts as evidenced by MVD which was quantified by counting CD31-positive vessels. The arrows are pointing to dark-stained CD68-positive macrophages and to red-stained CD31-positive endothelial cells. (D) EPO treatment decreases inflammation (left) and increases MVD (middle) in a dose-dependent manner that correlates negatively (right). Each bar represents the mean CD68-positive cells or MVD ± SD from five regions of interest in each fat graft from each treatment group at the end of the 15-week study period. *<i>P</i><0.05, ***<i>P</i><0.001, for the difference between either the low-dose- or the high-dose EPO-treated fat grafts and the PBS-treated grafts. Scale bar for A: 200µm, scale bar for B or C: 400µm.</p

Photograph of five representative mice with fat grafts at the end of the 15-week study period.

<p>(A) Five PBS-treated fat grafts with small lumps that vary in size in the scalps. (B) Five high-dose erythropoietin (100 IU EPO)-treated fat grafts with large lumps that are similar in size in the scalps. (C) Fat grafts were dissected from the mice 15 weeks after transplantation. From left to right, a representative small fat graft from a PBS-treated fat graft, an intermediate-size low-dose EPO-treated fat graft, and a large high-dose EPO-treated fat graft respectively. Scale bar: 10 mm.</p