Fibronectin Potentiates Topical Erythropoietin-Induced Wound Repair in Diabetic Mice  Saher Hamed, Yehuda Ullmann, Dana Egozi, Essam Daod, Elias Hellou, Manal Ashkar, Amos Gilhar, Luc Teot  Journal of Investigative Dermatology  Volume 131, Issue 6, Pages 1365-1374 (June 2011) DOI: 10.1038/jid.2011.15 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Wound closure. (a) Comparison between the time course of wound healing of healthy and diabetic mice treated with vehicle and (b) diabetic mice treated with vehicle, EPO, FN, or EPO/FN. (c) Typical healthy and diabetic wounds after 12 days of treatment. (d) The black area represents the surface of the wound area. (e) Time course of wound healing in diabetic mice that were treated with vehicle and (f) with EPO/FN. *P<0.05 and **P<0.001; vehicle-treated wounds of the healthy mice versus diabetic mice, and for EPO-, FN-, or EPO/FN-treated wounds versus vehicle-treated wounds of diabetic mice. †P<0.05; for EPO- versus FN-treated wounds. Abbreviations: D, diabetic mice; d, day; EPO, erythropoietin; EPO/FN, combination of erythropoietin and fibronectin; FN, fibronectin; H, healthy mice. Journal of Investigative Dermatology 2011 131, 1365-1374DOI: (10.1038/jid.2011.15) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Microvascular density (MVD), vascular endothelial growth factor (VEGF), and collagen formation in the wound bed. Representative micrographs of (a) newly formed skin of a wound in a healthy mouse (upper) and a diabetic mouse (lower) after 6 days of topical treatment with the vehicle-containing cream, and (b) of wounds in diabetic mice treated with creams containing erythropoietin (EPO; upper), fibronectin (FN; middle), and a combination of erythropoietin and fibronectin (EPO/FN; lower). Each arrow points to a newly formed blood vessel. Scale bar=200μm. (c) MVD, (d) VEGF content, and (e) hydroxyproline (HP) content in the wound beds after 6 days of topical treatment. *P<0.05, **P<0.001; EPO/FN versus vehicle, ††P<0.001; EPO/FN versus EPO or FN, •P<0.001; FN versus EPO, and °P<0.001; FN versus vehicle in diabetic mice. Journal of Investigative Dermatology 2011 131, 1365-1374DOI: (10.1038/jid.2011.15) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Inflammatory response in the wound bed. Representative micrographs of (a) newly formed skin in a healthy mouse (left) and a diabetic mouse (right) after 6 days of topical treatment with the vehicle-containing cream, and (b) of wounds in diabetic mice treated with creams containing erythropoietin (EPO; left), fibronectin (FN; middle), and a combination of erythropoietin and fibronectin (EPO/FN; right). Black arrows point to newly formed blood vessels and white arrows point to CD68-positive cells. Scale bar=400μm. The effect of topical treatments on (c) the number of CD68-positive cells (macrophages) in wound tissues after 6 days and (d) proinflammatory cytokines. Each bar represents the mean pooled level of each proinflammatory cytokine in wound tissues. **P<0.01; EPO or EPO/FN versus vehicle or FN in the diabetic mice. Journal of Investigative Dermatology 2011 131, 1365-1374DOI: (10.1038/jid.2011.15) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Effect of treatment on apoptosis. (a–d) Levels of eNOS, β1-integrin, casp-3, and cyt-c determined by western analysis from samples of excised wound tissues from diabetic mice after 6 days of treatment. Bars represent the mean pooled level of each protein±SD. casp-3, caspase 3; cyt-c, cytochrome c; EPO, erythropoietin; EPO/FN, combination of erythropoietin and fibronectin; eNOS, endothelial nitric oxide synthase; FN, fibronectin. **P<0.001; EPO or EPO/FN versus vehicle, †P<0.05, ††P<0.001; EPO or EPO/FN versus FN, •P<0.05; EPO/FN versus EPO. (e) Representative micrographs of apoptotic cells in newly formed skin after 6 days of treatment with vehicle (upper panel) in a healthy mouse (left) and a diabetic mouse (right), and (lower panel) in diabetic mice treated with EPO (left), FN (middle), and EPO/FN (right). Arrows point to apoptotic cells. Scale bar=200μm. (f) Mean percentage of apoptotic cells±SD in each group. *P<0.05, **P<0.001. Journal of Investigative Dermatology 2011 131, 1365-1374DOI: (10.1038/jid.2011.15) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Effect of erythropoietin (EPO) and fibronectin (FN) on human dermal microvascular endothelial cells (HDMECs) and neonatal human foreskin cells (NHFCs) under different conditions. Effect of high-glucose (HG) treatment on (a) proliferation of nontreated (white bars) and EPO-treated (black bars) HDMECs, and (b) NHFCs, **P<0.001. (c) Assessment of NHFC adherence to fibrin, (d) the levels of hydroxyproline (HP) produced by NHFCs in conditioned media, (e) the score of HDMEC tube formation on fibrin, (f) representative micrographs of tube formation on matrigel under different treatments. Scale bar=100μm. Each bar represents the mean±SD from three independent experiments performed in duplicates.*P<0.05 and **P<0.001; for treatment versus control. †P<0.001; for the difference in each treatment between cells cultured in the presence of fibrin (+Fibrin) and cells cultured without fibrin (-Fibrin). HDFBCs, human dermal fibroblasts; NG, normal glucose; RGD, arg-gly-asp. Journal of Investigative Dermatology 2011 131, 1365-1374DOI: (10.1038/jid.2011.15) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions