Erythropoietin Improves the Survival of Fat Tissue after Its Transplantation in Nude Mice

Background: Autologous transplanted fat has a high resorption rate, providing a clinical challenge for the means to reduce it. Erythropoietin (EPO) has non-hematopoietic targets, and we hypothesized that EPO may improve long-term fat graft survival because it has both pro-angiogenic and anti-apoptotic properties. We aimed to determine the effect of EPO on the survival of human fat tissue after its transplantation in nude mice. Methodology/Principal Findings: Human fat tissue was injected subcutaneously into immunologically-compromised nude mice, and the grafts were then treated with either 20 IU or 100 IU EPO. At the end of the 15-week study period, the extent of angiogenesis, apoptosis, and histology were assessed in the fat grafts. The results were compared to vascular endothelial growth factor (VEGF)-treated and phosphate-buffered saline (PBS)-treated fat grafts. The weight and volume of the EPOtreated grafts were higher than those of the PBS-treated grafts, whose weights and volumes were not different from those of the VEGF-treated grafts. EPO treatment also increased the expression of angiogenic factors and microvascular density, and reduced inflammation and apoptosis in a dose-dependent manner in the fat grafts. Conclusions/Significance: Our data suggest that stimulation of angiogenesis by a cluster of angiogenic factors and decreased fat cell apoptosis account for potential mechanisms that underlie the improved long-term survival of fa

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 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

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