Pravastatin Limits Radiation-Induced Vascular Dysfunction in the Skin Valerie Holler, Valerie Buard, Marie-Helene Gaugler, Olivier Guipaud, Cedric Baudelin, Amandine Sache, Maria del R. Perez, Claire Squiban, Radia Tamarat, Fabien Milliat, Marc Benderitter  Journal of Investigative Dermatology  Volume 129, Issue 5, Pages 1280-1291 (May 2009) DOI: 10.1038/jid.2008.360 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Characterization of the cutaneous radiation model. (a) The skin reaction scoring system comprised 15 levels, from 0.5 to 3.5 adapted from Douglas and Fowler (1976). (b) Dorsal skin reaction scores of Wt Balb/c mice at various times following irradiation. Arrows indicated the skin reaction score corresponding to the representative images of Wt mice in (c) 21 days after exposure at the indicated doses. Journal of Investigative Dermatology 2009 129, 1280-1291DOI: (10.1038/jid.2008.360) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Irradiation increases leukocyte–endothelium interactions. Visualization of leukocyte–endothelium interactions in the dorsal skin vessels by intravital videomicroscopy after intravenous administration of rhodamine 6G, 28 days after radiation exposure: (a) no irradiation and (b) 45Gy irradiation. Blood vessels are delineated by dashed lines (scale bar: 40μm). The rolling velocity (c), the number of rolling leukocytes (R) per minute per 0.01mm2 (d), and the number of firmly adherent leukocytes (e) were determined by intravital microscopy until 90 days after a 45Gy radiation exposure. Data are expressed as the mean±SEM of at least three animals per group. **P<0.001 relative to nonirradiated group. Journal of Investigative Dermatology 2009 129, 1280-1291DOI: (10.1038/jid.2008.360) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Pravastatin improves radiation-induced skin reaction: clinical benefit. (a) Skin-reaction scores of the dorsal skin of mice following 45Gy irradiation and pravastatin treatment. (b) H&E-stained section of 45Gy irradiated mice dorsal skin (1) without and (2) with pravastatin, 28 days after exposure (bar: 1mm). The skin between the two arrows corresponds to the irradiated area. (c) Weight is expressed as a relative percentage calculated from the weight at day 0 for each of the eight Wt mice per group. Journal of Investigative Dermatology 2009 129, 1280-1291DOI: (10.1038/jid.2008.360) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Pravastatin limits radiation-induced vascular functional activation. Quantification of leukocyte–endothelium interactions in the dorsal skin vessels by intravital videomicroscopy 28 days after a 45Gy radiation exposure and after pravastatin treatment: (a) the rolling velocity, (b) the number of rolling leukocytes (R) per minute per 0.01mm2, and (c) the number of firmly adherent leukocytes. Data are expressed as the mean±SEM of six Wt animals per group. NS: nonsignificant. Journal of Investigative Dermatology 2009 129, 1280-1291DOI: (10.1038/jid.2008.360) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Pravastatin limits the radiation-induced increased production of inflammatory mediators.In vivo: Effect of pravastatin on CCL2 (a) and CXCL1 (b) expression in plasma and serum of 0 and 45Gy irradiated Wt mice, 28 days after exposure. (e) Effect of pravastatin on inflammatory cells infiltrated in tissue. Data are expressed as the mean±SEM. **P<0.01 and ***P<0.001 relative to nonirradiated group. NS: nonsignificant. In vitro: Effect of pravastatin on MCP-1 and IL-8 production by HMVEC-D cells 3, 7, and 14 days after 10Gy exposure (c, d). Histograms represent the mean±SEM of three separate experiments. *P<0.05; **P<0.01, and ***P<0.001 relative to nonirradiated group. NS: nonsignificant. Journal of Investigative Dermatology 2009 129, 1280-1291DOI: (10.1038/jid.2008.360) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effect of pravastatin on the expression of ICAM-1, E-selectin, and eNOS in tissue sections of mouse dorsal skin vessels. Representative immunohistochemical localization of ICAM-1 (a–c), E-selectin (d–f), and eNOS (g–i) visualized in blood vessels, 28 days after a 45Gy irradiation, is indicated by arrows (scale bar=100μm). Small windows represent enlarging of a blood vessel where labeling of EC can be visualized. Journal of Investigative Dermatology 2009 129, 1280-1291DOI: (10.1038/jid.2008.360) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Pravastatin limits radiation-induced endothelium activation in vitro and increases the expression of eNOS in vivo. Effect of pravastatin on ICAM-1 expression by HMVEC-D cells 3, 7, and 14 days after 10Gy exposure (a). ICAM-1 expression was determined using cell ELISA on paraformaldehyde-fixed HMVEC-D cells, as described in ‘Materials and Methods’ section. Histograms represent the mean±SEM of three separate experiments performed in triplicate. ***P<0.001 relative to nonirradiated group. NS: nonsignificant. Effect of pravastatin on western blot analysis of eNOS and GAPDH expression in HMVEC-D, respectively, at 1, 3, 7, and 14 days after 10Gy exposure (b). Journal of Investigative Dermatology 2009 129, 1280-1291DOI: (10.1038/jid.2008.360) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Pravastatin has no effect on the radiation-induced skin reaction of eNOS-/- mice. Skin-reaction scores of the dorsal skin of eNOS-/- mice following a 45Gy irradiation or a 45Gy irradiation+pravastatin treatment (a). Arrows indicated the skin reaction score corresponding to the representative images of eNOS-/- mice 14 days after exposure (b). Journal of Investigative Dermatology 2009 129, 1280-1291DOI: (10.1038/jid.2008.360) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions