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• W2075346153 abstract "We have previously shown that targeted overexpression of the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in the epidermis prevented chronic ultraviolet B (UVB)-induced angiogenesis and cutaneous photodamage in mice, suggesting that angiogenesis plays a critical role in the mediation of UVB effects on the skin. Nevertheless, the molecular regulation of angiogenesis factors and inhibitors by acute UVB irradiation still remains to be elucidated. We performed quantitative analyses of cutaneous vascularity and of vascular endothelial growth factor (VEGF) and TSP-1 expression after acute UVB irradiation of mouse skin. Skin vascularity in the upper dermis was greatly increased until day 8 after a single dose of UVB irradiation (200 mJ per cm2) and associated with upregulation of VEGF mRNA expression, with downregulation of TSP-1 mRNA, and with protein expression in the hyperplastic epidermis. After 13 days, skin vascularity was normalized with downregulation of VEGF mRNA expression and upregulation of TSP-1 mRNA expression to the levels observed in non-UVB-irradiated skin. In contrast, the angiogenic UVB response was prolonged in TSP-1-deficient mice. We found a pronounced induction of the TSP-1 receptor CD36 in CD31-positive vessels on day 8 after UVB irradiation, associated with vascular endothelial cell apoptosis. These results demonstrate that acute UVB irradiation leads to a shift toward a proangiogenic environment and they suggest that the balance between VEGF and TSP-1 plays a critical role in the control of angiogenesis and vascular regression induced by acute UVB irradiation. We have previously shown that targeted overexpression of the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in the epidermis prevented chronic ultraviolet B (UVB)-induced angiogenesis and cutaneous photodamage in mice, suggesting that angiogenesis plays a critical role in the mediation of UVB effects on the skin. Nevertheless, the molecular regulation of angiogenesis factors and inhibitors by acute UVB irradiation still remains to be elucidated. We performed quantitative analyses of cutaneous vascularity and of vascular endothelial growth factor (VEGF) and TSP-1 expression after acute UVB irradiation of mouse skin. Skin vascularity in the upper dermis was greatly increased until day 8 after a single dose of UVB irradiation (200 mJ per cm2) and associated with upregulation of VEGF mRNA expression, with downregulation of TSP-1 mRNA, and with protein expression in the hyperplastic epidermis. After 13 days, skin vascularity was normalized with downregulation of VEGF mRNA expression and upregulation of TSP-1 mRNA expression to the levels observed in non-UVB-irradiated skin. In contrast, the angiogenic UVB response was prolonged in TSP-1-deficient mice. We found a pronounced induction of the TSP-1 receptor CD36 in CD31-positive vessels on day 8 after UVB irradiation, associated with vascular endothelial cell apoptosis. These results demonstrate that acute UVB irradiation leads to a shift toward a proangiogenic environment and they suggest that the balance between VEGF and TSP-1 plays a critical role in the control of angiogenesis and vascular regression induced by acute UVB irradiation. bromodeoxyuridine thrombospondin-1 ultraviolet B Chronic sun exposure of human skin results in wrinkle formation, elastosis, and degradation of matrix macromolecules (Kligman, 1989Kligman L.H. The ultraviolet-irradiated hairless mouse: A model for photoaging.J Am Acad Dermatol. 1989; 21: 623-631Abstract Full Text PDF PubMed Scopus (90) Google Scholar;Leyden et al., 1989Leyden J.J. Grove G.L. Grove M.J. Thorne E.G. Lufrano L. Treatment of photodamaged facial skin with topical tretinoin.J Am Acad Dermatol. 1989; 21: 638-644Abstract Full Text PDF PubMed Scopus (129) Google Scholar) and leads to an enhanced risk for the development of epithelial skin cancers (Kripke, 1994Kripke M.L. Ultraviolet radiation and immunology: Something new under the sun—Presidential address.Cancer Res. 1994; 54: 6102-6105PubMed Google Scholar). Skin alterations observed after a single exposure to ultraviolet B (UVB) irradiation (290- to 320-nm wavelength) include erythema (Cox et al., 1992Cox N.H. Diffey B.L. Farr P.M. The relationship between chronological age and the erythemal response to ultraviolet B radiation.Br J Dermatol. 1992; 126: 315-319Crossref PubMed Scopus (37) Google Scholar;Kripke, 1994Kripke M.L. Ultraviolet radiation and immunology: Something new under the sun—Presidential address.Cancer Res. 1994; 54: 6102-6105PubMed Google Scholar), vascular hyperpermeability, dilation of dermal blood vessels, and epidermal hyperplasia (Pearse et al., 1987Pearse A.D. Gaskell S.A. Marks R. Epidermal changes in human skin following irradiation with either UVB or UVA.J Invest Dermatol. 1987; 88: 83-87Abstract Full Text PDF PubMed Google Scholar;Berton et al., 1997Berton T.R. Mitchell D.L. Fischer S.M. Locniskar M.F. Epidermal proliferation but not quantity of DNA photodamage is correlated with UV-induced mouse skin carcinogenesis.J Invest Dermatol. 1997; 109: 340-347Crossref PubMed Scopus (68) Google Scholar). The pronounced vascular changes observed after acute UVB irradiation suggest that the cutaneous vasculature plays an important role in the mediation of acute photodamage. Several angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor, and interleukin-8 (Bielenberg et al., 1998Bielenberg D.R. Bucana C.D. Sanchez R. Donawho C.K. Kripke M.L. Fidler I.J. Molecular regulation of UVB-induced cutaneous angiogenesis.J Invest Dermatol. 1998; 111: 864-872Crossref PubMed Scopus (120) Google Scholar;Kramer et al., 1993Kramer M. Sachsenmaier C. Herrlich P. Rahmsdorf H.J. UV irradiation-induced interleukin-1 and basic fibroblast growth factor synthesis and release mediate part of the UV response.J Biol Chem. 1993; 268: 6734-6741Abstract Full Text PDF PubMed Google Scholar;Strickland et al., 1997Strickland I. Rhodes L.E. Flanagan B.F. Friedmann P.S. TNF-alpha and IL-8 are upregulated in the epidermis of normal human skin after UVB exposure: Correlation with neutrophil accumulation and E-selectin expression.J Invest Dermatol. 1997; 108: 763-768Crossref PubMed Scopus (158) Google Scholar), have been found to be upregulated after UVB irradiation of the skin, whereas a decreased expression of interferon-β, a cytokine with antiangiogenic activity, has been reported (Bielenberg et al., 1998Bielenberg D.R. Bucana C.D. Sanchez R. Donawho C.K. Kripke M.L. Fidler I.J. Molecular regulation of UVB-induced cutaneous angiogenesis.J Invest Dermatol. 1998; 111: 864-872Crossref PubMed Scopus (120) Google Scholar). Nevertheless, the regulation of endogenous inhibitors of angiogenesis by acute UVB irradiation has remained unknown. Angiogenesis, the formation of new blood vessels from existing vessels, involves increased microvascular permeability, degradation of extracellular matrix molecules, and proliferation and migration of endothelial cells, leading to the formation of new capillaries (Dvorak et al., 1995Dvorak H.F. Brown L.F. Detmar M. Dvorak A.M. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis.Am J Pathol. 1995; 146: 1029-1039PubMed Google Scholar). In normal skin, angiogenesis is restricted to the perifollicular vasculature during the growth phase of the hair follicle (Yano et al., 2001Yano K. Brown L.F. Detmar M. Control of hair growth and follicle size by VEGF-mediated angiogenesis.J Clin Invest. 2001; 107: 409-417Crossref PubMed Scopus (384) Google Scholar). Nevertheless, the skin can initiate a rapid angiogenic response during wound healing and inflammation. We have previously identified VEGF, released by epidermal keratinocytes, as a major skin angiogenesis factor (Detmar et al., 1995Detmar M. Yeo K.T. Nagy J.A. et al.Keratinocyte-derived vascular permeability factor (vascular endothelial growth factor) is a potent mitogen for dermal microvascular endothelial cells.J Invest Dermatol. 1995; 105: 44-50Crossref PubMed Scopus (219) Google Scholar). VEGF expression is upregulated in the hyperplastic epidermis of psoriasis (Detmar et al., 1994Detmar M. Brown L.F. Claffey K.P. et al.Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis.J Exp Med. 1994; 180: 1141-1146Crossref PubMed Scopus (626) Google Scholar), in healing wounds (Brown et al., 1992Brown L.F. Yeo K.T. Berse B. Yeo T.K. Senger D.R. Dvorak H.F. Van De Water L. Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing.J Exp Med. 1992; 176: 1375-1379Crossref PubMed Scopus (749) Google Scholar), and in other skin diseases characterized by enhanced angiogenesis (Brown et al., 1995aBrown L.F. Harrist T.J. Yeo K.T. et al.Increased expression of vascular permeability factor (vascular endothelial growth factor) in bullous pemphigoid, dermatitis herpetiformis, and erythema multiforme.J Invest Dermatol. 1995; 104: 744-749Crossref PubMed Scopus (134) Google Scholar). Moreover, targeted overexpression of VEGF in the epidermis of transgenic mice resulted in enhanced skin vascularization with increased numbers of tortuous and hyperpermeable blood vessels (Detmar et al., 1998Detmar M. Brown L.F. Schön M.P. et al.Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice.J Invest Dermatol. 1998; 111: 1-6Crossref PubMed Scopus (441) Google Scholar). Pronounced upregulation of VEGF mRNA expression was also detected in murine skin after chronic UVB irradiation (Yano et al., 2002Yano K. Oura H. Detmar M. Targeted overexpression of the angiogenesis inhibitor thrombospondin-1 in the epidermis of transgenic mice prevents ultraviolet-B-induced angiogenesis and cutaneous photo-damage.J Invest Dermatol. 2002; 118: 800-805Crossref PubMed Scopus (79) Google Scholar). Thrombospondin-1 (TSP-1) is a 450-kDa matricellular protein that inhibits proliferation and migration of endothelial cells in vivo and potently diminishes squamous cell carcinoma growth and angiogenesis in vivo (Tolsma et al., 1993Tolsma S.S. Volpert O.V. Good D.J. Frazier W.A. Polverini P.J. Bouck N. Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity.J Cell Biol. 1993; 122: 497-511Crossref PubMed Scopus (503) Google Scholar;Bleuel et al., 1999Bleuel K. Popp S. Fusenig N.E. Stanbridge E.J. Boukamp P. Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization.Proc Natl Acad Sci USA. 1999; 96: 2065-2070Crossref PubMed Scopus (105) Google Scholar;Streit et al., 1999Streit M. Velasco P. Brown L.F. et al.Overexpression of thrombospondin-1 decreases angiogenesis and inhibits the growth of human cutaneous squamous cell carcinomas.Am J Pathol. 1999; 155: 441-452Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). TSP-1 mediates inhibition of proliferation and migration of endothelial cells by specific interaction of distinct sequences within its type I repeats with the CD36 receptor on endothelial cells (Jimenez et al., 2000Jimenez B. Volpert O.V. Crawford S.E. Febbraio M. Silverstein R.L. Bouck N. Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1.Nat Med. 2000; 6: 41-48Crossref PubMed Scopus (816) Google Scholar). In human skin, TSP-1 mRNA is produced by epidermal keratinocytes, contributing to the normal antiangiogenic barrier that separates the avascular epidermis from the vascularized dermis (Detmar, 2000Detmar M. The role of VEGF and thrombospondins in skin angiogenesis.J Dermatol Sci. 2000; 24: S78-S84Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). We have previously reported that mice with skin-specific overexpression of TSP-1 showed diminished skin damage and angiogenesis after chronic UVB irradiation (Yano et al., 2002Yano K. Oura H. Detmar M. Targeted overexpression of the angiogenesis inhibitor thrombospondin-1 in the epidermis of transgenic mice prevents ultraviolet-B-induced angiogenesis and cutaneous photo-damage.J Invest Dermatol. 2002; 118: 800-805Crossref PubMed Scopus (79) Google Scholar), suggesting that inhibition of angiogenesis might prevent UVB-mediated photodamage. The regulation of TSP-1 expression by UVB, however, has remained unclear. Here, we report that enhanced cutaneous vascularity after UVB irradiation is associated with an angiogenic switch. UVB irradiation induced upregulation of VEGF mRNA expression and downregulation of TSP-1 mRNA expression in the epidermis, resulting in a shift toward a proangiogenic environment. Skin vascularity was reduced to normal levels at approximately 10 d after UVB irradiation, associated with endothelial cell apoptosis, upregulation of epidermal TSP-1 expression, and induction of the TSP-1 receptor CD36 on blood vessels. Moreover, the angiogenic UVB response was prolonged in TSP-1-deficient mice. These results suggest that the balance between VEGF and TSP-1 expression plays a critical role in the mediation of angiogenesis and vascular regression in UVB-irradiated skin. To investigate the effects of acute UVB irradiation on angiogenesis in the skin, we exposed 30 female HR-1 hairless mice to a single dose of UVB irradiation (200 mJ per cm2). Histologic analysis showed thickening of the epidermis Figure 1a, b, c, d, e and the dermis (data not shown) in UVB-treated mice, associated with recruitment of inflammatory cells to the upper dermis (data not shown). Immunohistochemistry for the endothelial junction molecule CD31 revealed a progressive increase in the number of enlarged blood vessels in UVB irradiated skin until day 8 Figure 1f, g, h, i. Skin vascularity returned to levels before irradiation on day 13 Figure 1j after UVB irradiation. Computer-assisted morphometric analysis of CD31-stained cutaneous vessels revealed a significant increase in vascular density on day 4 (+23%; p<0.01) and day 8 (+30%; p<0.001) after UVB irradiation, compared with nonirradiated skin Figure 1q. Moreover, the average vessel size was significantly increased on day 2 (+21%; p<0.05), day 4 (+115%; p<0.001), and day 8 (149%; p<0.001) after UVB irradiation Figure 1p. Accordingly, the cutaneous area covered by blood vessels was increased on day 2 (+23%; p<0.001), day 4 (+101%; p<0.001), and day 8 (+195%; p<0.001) Figure 1r, compared with nonirradiated skin. Importantly, on day 13, all vascular parameters examined were comparable to the levels of non-UVB-irradiated skin Figure 1p, q, r. Double immunofluorescence stains for CD31 and BrdU revealed increased proliferation of epidermal keratinocytes beginning on day 2 Figure 1l after UVB irradiation, resulting in prominent epidermal hyperplasia from day 4 (Figure 1c, m). Proliferation of) to day 8 Figure 1d, n. Proliferation of endothelial cells was detected on day 4 Figure 1m and, more prominently, on day 8 Figure 1n. No proliferating endothelial cells were detected in non-UVB-irradiated skin Figure 1k on day 13 after UVB irradiation Figure 1o. We next examined the effects of acute UVB irradiation on epidermal VEGF and TSP-1 mRNA expression levels. Using quantitative real-time RT-PCR analysis of total RNA isolated from the epidermis of mouse skin, we found that VEGF mRNA expression levels were significantly upregulated on day 2 (+289%; p<0.05), day 4 (+420%; p<0.01), and day 6 (+298%; p<0.05) after UVB treatment, compared with nonirradiated skin. This was followed by gradual downregulation on days 8 and 13 to the levels observed in normal skin Figure 2a. All three major VEGF isoforms, VEGF 120, VEGF 164, and VEGF188, were comparably upregulated (data not shown). In contrast, TSP-1 mRNA expression was potently downregulated on day 2 (–92%; p<0.01), day 4 (–70%; p<0.05), and day 6 (–49%; n.s.) after UVB treatment, followed by normalization of expression levels on days 8 and 13 to the levels observed in nonirradiated skin Figure 2a. Importantly, the ratio of VEGF to TSP-1 expression levels was 0.3 in normal skin in accordance with the normal vascular quiescence, whereas the VEGF-to-TSP-1 ratio was dramatically increased on days 2 and 4 after UVB irradiation Figure 2b, indicative of a proangiogenic switch induced by UVB irradiation. Immunohistochemical stains for VEGF and TSP-1 confirmed these findings. Whereas no or little VEGF was detected in normal skin Figure 3a, its expression was strongly increased in the epidermis and in endothelial cells from day 2 to day 8 Figure 3b, c, d after UVB irradiation. VEGF expression on day 13 Figure 3e was comparable to the levels observed in normal skin. In contrast, high levels of TSP-1 expression were observed in the epidermis and in endothelial cells in normal skin Figure 3f, with pronounced downregulation 2 and 4 d after UVB irradiation Figure 3g, h. Importantly, strong TSP-1 expression was again detected on days 8 and 13 Figure 3i, j after UVB irradiation, comparable with the levels in normal skin Figure 3f. In situ hybridization for TSP-1 confirmed the results seen by immunostains. TSP-1 mRNA was constitutively and highly expressed throughout the normal epidermis, predominantly within the suprabasal keratinocyte layer Figure 3k. In addition, moderate TSP-1 mRNA expression was detected in endothelial cells and in some fibroblasts in normal skin Figure 3k. In contrast, a pronounced downregulation of TSP-1 mRNA expression was detected on day 2 Figure 3l and day 4 Figure 3m after UVB irradiation. In accordance with the immunohistochemical data, TSP-1 mRNA expression returned to normal levels on day 8 Figure 3n and day 13 Figure 3o after UVB irradiation. CD36 is an endothelial cell receptor for TSP-1 that has been implicated in the induction of endothelial cell apoptosis (Jimenez et al., 2000Jimenez B. Volpert O.V. Crawford S.E. Febbraio M. Silverstein R.L. Bouck N. Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1.Nat Med. 2000; 6: 41-48Crossref PubMed Scopus (816) Google Scholar). Double immunofluorescence stains for CD31 and CD36 revealed induction of CD36 expression in vascular endothelial cells in the upper dermis on day 8 after UVB irradiation Figure 4b, compared with absent or low-level expression in non-UVB-irradiated skin Figure 4a. Double immunofluorescence stains for CD31 and TUNEL revealed several apoptotic endothelial cells in the upper dermis 8 d after UVB irradiation Figure 4d, whereas no apoptotic endothelial cells were detected in normal skin Figure 4c. To investigate whether the observed upregulation of TSP-1 expression was necessary to mediate the vascular regression during the late phase of the UVB response, we next subjected TSP-1 deficient mice (TSP-1–/–) and their wild-type controls to the same UVB irradiation regimen and determined skin vascularity on day 0 (before UVB irradiation) and on day 13. Whereas no major differences of cutaneous vascularity were detected in the nonirradiated skin of both genotypes Figure 5a, b, enhanced vascularity—as visualized by CD31 immunofluorescence stains—was still detected in the skin of TSP-1-deficient mice on day 13 after UVB irradiation Figure 5d. In contrast, the cutaneous vascularity had largely returned to baseline levels in wild-type mice at this time point Figure 5c, e. Acute photodamage of the skin is characterized by epidermal hyperplasia and by erythema formation. Previously, endothelial cell proliferation has been found after acute UVB irradiation of the skin (Bielenberg et al., 1998Bielenberg D.R. Bucana C.D. Sanchez R. Donawho C.K. Kripke M.L. Fidler I.J. Molecular regulation of UVB-induced cutaneous angiogenesis.J Invest Dermatol. 1998; 111: 864-872Crossref PubMed Scopus (120) Google Scholar). Nevertheless, the distinct regulation of the major skin angiogenesis factor VEGF and the endogenous angiogenesis inhibitor TSP-1 by UVB have remained unclear. Our results identify the induction of a proangiogenic switch by acute UVB irradiation. TSP-1 expression dominated over VEGF expression in normal skin, in accordance with the normal quiescence of the cutaneous vasculature. In contrast, acute UVB irradiation resulted in pronounced upregulation of VEGF and in downregulation of TSP-1 expression, leading to a shift toward a proangiogenic environment. Normalization of VEGF and TSP-1 expression and induction of the endothelial TSP-1 receptor CD36 on day 8 after UVB irradiation resulted in apoptosis-driven vascular regression. Quantitative morphometric analysis of tissue sections stained for the endothelial junction molecules CD31 (Dejana et al., 1995Dejana E. Corada M. Lampugnani M.G. Endothelial cell-to-cell junctions.FASEB J. 1995; 9: 910-918Crossref PubMed Scopus (383) Google Scholar) revealed a marked induction of skin angiogenesis after acute UVB irradiation, with a significant increase of both vessel density and vessel size that was accompanied by increased proliferation of endothelial cells. These findings were comparable to the angiogenic changes that occur in chronically photodamaged skin (Yano et al., 2002Yano K. Oura H. Detmar M. Targeted overexpression of the angiogenesis inhibitor thrombospondin-1 in the epidermis of transgenic mice prevents ultraviolet-B-induced angiogenesis and cutaneous photo-damage.J Invest Dermatol. 2002; 118: 800-805Crossref PubMed Scopus (79) Google Scholar) and in cutaneous wound healing where both sprouting from existing blood vessels and vessel enlargement contribute to the formation of a vessel-rich granulation tissue (Streit et al., 2000Streit M. Velasco P. Riccardi L. et al.Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice.EMBO J. 2000; 19: 3272-3282Crossref PubMed Scopus (172) Google Scholar). In contrast, chronic inflammatory skin diseases such as psoriasis predominantly show vascular remodeling with elongation and enlargement of cutaneous microvessels without the formation of new blood vessels (Braverman and Keh-Yen, 1986Braverman I.M. Keh-Yen A. Three dimensional reconstruction of endothelial cell gaps in psoriatic vessels and their morphologic identity with gaps produced by the intradermal injection of histamine.J Invest Dermatol. 1986; 86: 577-581Crossref PubMed Scopus (39) Google Scholar;Detmar et al., 1994Detmar M. Brown L.F. Claffey K.P. et al.Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis.J Exp Med. 1994; 180: 1141-1146Crossref PubMed Scopus (626) Google Scholar). Our findings indicate that acute UVB irradiation of the skin results in a tissue-repair-like reaction and they suggest that angiogenesis might play a important role in the mediation of acute UVB-induced skin damage. It is of interest that we detected a significant increase in the average vessel size, but not in vessel density 2 d after acute UVB irradiation, whereas both vessel size and density were increased from day 4 to day 8. These findings indicate a biphasic regulation of the vascular response to acute UVB irradiation, with an initial phase of vasodilation, followed by a second phase of sprouting angiogenesis and endothelial cell proliferation. A previous study found prolonged angiogenesis over at least 4 wk after a single dose of UVB irradiation of ear skin (Bielenberg et al., 1998Bielenberg D.R. Bucana C.D. Sanchez R. Donawho C.K. Kripke M.L. Fidler I.J. Molecular regulation of UVB-induced cutaneous angiogenesis.J Invest Dermatol. 1998; 111: 864-872Crossref PubMed Scopus (120) Google Scholar), whereas in our study, angiogenic vessels regressed within 2 wk after UVB irradiation of the dorsal body skin. This difference in the time course of the vascular response is likely due to different UVB sensitivity of dorsal skin and of ear skin that is more richly vascularized. VEGF is a major keratinocyte-derived skin angiogenesis factor (Detmar et al., 1995Detmar M. Yeo K.T. Nagy J.A. et al.Keratinocyte-derived vascular permeability factor (vascular endothelial growth factor) is a potent mitogen for dermal microvascular endothelial cells.J Invest Dermatol. 1995; 105: 44-50Crossref PubMed Scopus (219) Google Scholar). Its expression is upregulated by the hyperplasic epidermis in chronic UVB-irradiated skin (Yano et al., 2002Yano K. Oura H. Detmar M. Targeted overexpression of the angiogenesis inhibitor thrombospondin-1 in the epidermis of transgenic mice prevents ultraviolet-B-induced angiogenesis and cutaneous photo-damage.J Invest Dermatol. 2002; 118: 800-805Crossref PubMed Scopus (79) Google Scholar), in psoriatic lesions (Detmar et al., 1994Detmar M. Brown L.F. Claffey K.P. et al.Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis.J Exp Med. 1994; 180: 1141-1146Crossref PubMed Scopus (626) Google Scholar), and in other skin diseases associated with dermal angiogenesis (Brown et al., 1995aBrown L.F. Harrist T.J. Yeo K.T. et al.Increased expression of vascular permeability factor (vascular endothelial growth factor) in bullous pemphigoid, dermatitis herpetiformis, and erythema multiforme.J Invest Dermatol. 1995; 104: 744-749Crossref PubMed Scopus (134) Google Scholar;Brown et al., 1995bBrown L.F. Olbricht S.M. Berse B. et al.Overexpression of vascular permeability factor (VPF/VEGF) and its endothelial cell receptors in delayed hypersensitivity skin reactions.J Immunol. 1995; 154: 2801-2807PubMed Google Scholar), as well as in the neoepidermis of healing wounds (Brown et al., 1992Brown L.F. Yeo K.T. Berse B. Yeo T.K. Senger D.R. Dvorak H.F. Van De Water L. Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing.J Exp Med. 1992; 176: 1375-1379Crossref PubMed Scopus (749) Google Scholar;Kishimoto et al., 2000Kishimoto J. Ehama R. Ge Y. Kobayashi T. Nishiyama T. Detmar M. Burgeson R.E. In vivo detection of human vascular endothelial growth factor promoter activity in transgenic mouse skin.Am J Pathol. 2000; 157: 103-110Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In general, VEGF has been found to be upregulated in most, if not all, skin conditions that are characterized by epidermal hyperplasia and dermal angiogenesis. In contrast, the endogenous angiogenesis inhibitor TSP-1 is expressed by epidermal keratinocytes in normal skin (Wight et al., 1985Wight T.N. Raugi G.J. Mumby S.M. Bornstein P. Light microscopic immunolocation of thrombospondin in human tissues.J Histochem Cytochem. 1985; 33: 295-302Crossref PubMed Scopus (200) Google Scholar) and is thought to contribute to the quiescence of the cutaneous vasculature in normal human skin (Detmar, 2000Detmar M. The role of VEGF and thrombospondins in skin angiogenesis.J Dermatol Sci. 2000; 24: S78-S84Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). We detected constitutive TSP-1 mRNA and protein expression in the entire epidermis of normal skin, predominantly in the upper epidermal layers. In a previous report by Wight et al (1995), the authors found—using an affinity-purified rabbit anti-TSP-1 antibody on frozen sections of human skin—selective staining for TSP-1 in the basement membrane region below the epidermis, likely representing secreted TSP-1 that was bound to the matrix. These findings suggest that the antibody used in that study only recognized the secreted TSP-1 protein or a protein fragment of TSP-1. In contrast, in our study we have used a monoclonal rat anti-TSP-1 antibody that recognizes the 450-kDa TSP-1 protein also within the cytoplasm of basal and suprabasal keratinocytes and other cells. Importantly, the in situ hybridization results confirmed that both basal and suprabasal keratinocytes produced TSP-1, as evidenced by strong mRNA expression by these cells. Taken together, cellular production of TSP-1 by epidermal keratinocytes, leading to secretion and deposition in the dermal matrix and the basement membrane zone, likely contribute to the antiangiogenic barrier that prevents vessel ingrowth into the epidermis. We have previously reported that transgenic mice with selective overexpression of TSP-1 in basal epidermal keratinocytes and in outer root sheath follicular keratinocytes showed reduced dermal damage, wrinkle formation, and angiogenesis after chronic UVB irradiation (Yano et al., 2002Yano K. Oura H. Detmar M. Targeted overexpression of the angiogenesis inhibitor thrombospondin-1 in the epidermis of transgenic mice prevents ultraviolet-B-induced angiogenesis and cutaneous photo-damage.J Invest Dermatol. 2002; 118: 800-805Crossref PubMed Scopus (79) Google Scholar). Based on these findings, we hypothesized that the balance of VEGF and TSP-1 expression might play a critical role in the shift from vascular quiescent to a proangiogenic environment. This study confirmed a predominant expression of TSP-1 but low levels of VEGF in normal skin. Importantly, UVB irradiation led to an upregulation of VEGF and downregulation of TSP-1 in the epidermis during the first 6 d after UVB irradiation, suggesting that the increased VEGF-to-TSP-1 ratio contributed to the molecular induction of the angiogenic switch. In situ hybridization for TSP-1 confirmed the results of quantitative PCR that pronounced downregulation of TSP-1 mRNA expression was detected on days 2, 4, and 6 after acute UVB irradiation, followed by upregulation of expression on day 8 and day 13 to the levels found in non-UVB-treated skin. These findings are in accordance with previous reports that acute or chronic UVB irradiation induced VEGF expression by human epidermal keratinocytes in vitro (Brauchle et al., 1996Brauchle M. Funk J.O. Kind P. Werner S. Ultraviolet B and H2O2 are potent inducers of vascular endothelial growth factor expression in cultured keratinocytes.J Biol Chem. 1996; 271: 21793-21797Crossref PubMed Scopus (156) Google Scholar;Mildner et al., 1999Mildner M. Weninger W. Trautinger F. Ban J. Tschachler E. UVA and UVB radiation differentially regulate vascular endothelial growth factor expression in keratinocyte-derived cell lines and in human keratinocytes.Photochem Photobiol. 1999; 70: 674-679Crossref PubMed Scopus (55) Google Scholar;Blaudschun et al., 2002Blaudschun R. Sunderkotter C. Brenneisen P. et al.Vascular endothelial growth factor causally contributes to the angiogenic response upon ultraviolet B irradiation in vivo.Br J Dermatol. 2002; 146: 581-587Crossref PubMed Scopus (34) Google Scholar) and by murine keratinocytes in vivo (Bielenberg et al., 1998Bielenberg D.R. Bucana C.D. Sanchez R. Donawho C.K. Kripke M.L. Fidler I.J. Molecular regulation of UVB-induced cutaneous angiogenesis.J Invest Dermatol. 1998; 111: 864-872Crossref PubMed Scopus (120) Google Scholar;Yano et al., 2002Yano K. Oura H. Detmar M. Targeted overexpression of the angiogenesis inhibitor thrombospondin-1 in the epidermis of transgenic mice prevents ultraviolet-B-induced angiogenesis and cutaneous photo-damage.J Invest Dermatol. 2002; 118: 800-805Crossref PubMed Scopus (79) Google Scholar). Interestingly, upregulation of another angiogenic factor, basic fibroblast growth factor, and downregulation of interferon-β, a cytokine with antiangiogenic activity, has been previously found after a single dose of UVB irradiation of mouse ear skin (Bielenberg et al., 1998Bielenberg D.R. Bucana C.D. Sanchez R. Donawho C.K. Kripke M.L. Fidler I.J. Molecular regulation of UVB-induced cutaneous angiogenesis.J Invest Dermatol. 1998; 111: 864-872Crossref PubMed Scopus (120) Google Scholar), suggesting that additional factors contribute to the angiogenic UVB response. Moreover, a similar switch in the expression levels of VEGF and TSP-1 has recently been observed during the progressive phase of multistep epithelial carcinogenesis that is associated with pronounced tumor angiogenesis (Hawighorst et al., 2002Hawighorst T. Oura H. Streit M. et al.Thrombospondin-1 selectively inhibits early-stage carcinogenesis and angiogenesis but not tumor lymphangiogenesis and lymphatic metastasis in transgenic mice.Oncogene. 2002; 21: 7945-7956Crossref PubMed Scopus (80) Google Scholar). It will be of interest to investigate, in future studies, the biologic importance of other endogenous angiogenesis inhibitors for the cutaneous UVB response. Preliminary results indicate that the related molecule thrombospondin-2 is also downregulated after UVB irradiation of mouse skin, although less potently than TSP-1 (data not shown). We observed that the shift from the proangiogenic environment to vascular quiescence at 13 d after UVB irradiation was associated with upregulation of TSP-1 and downregulation of VEGF expression to the levels of non-UVB-treated skin. These results further corroborate the close correlation between TSP-1 and VEGF expression levels and cutaneous vascularization, and they implicate TSP-1 as a key factor in the mediation of vascular regression during the late stages of the cutaneous UVB response. Indeed, we found that the angiogenic UVB response was prolonged in mice deficient for TSP-1, demonstrating that TSP-1 upregulation is necessary for the timely vascular regression during the late phase of the UVB response. It has been recently reported that TSP-1 mediates inhibition of angiogenesis by specific interactions of distinct sequences with the type I repeats with the CD36 receptor on the endothelial cells, resulting in enhanced endothelial cell apoptosis rates (Jimenez et al., 2000Jimenez B. Volpert O.V. Crawford S.E. Febbraio M. Silverstein R.L. Bouck N. Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1.Nat Med. 2000; 6: 41-48Crossref PubMed Scopus (816) Google Scholar). We found concomitant induction of endothelial CD36 expression and endothelial apoptosis in cutaneous vessels 8 d after acute UVB irradiation, temporally associated with upregulation of TSP-1 expression to the levels detected in normal skin. These results suggest that the vascular regression observed during the late stages of the UVB response was, at least partially, mediated via the TSP-1–CD36 signal transduction pathway. Future studies in CD36-deficient mice will likely provide important information regarding the relative contribution of the CD36 to the angiogenic activity of TSP-1. Taken together, our findings suggest that changes in the normal balance between VEGF and TSP-1 expression in the skin play an important role in the mediation of UVB-induced cutaneous photodamage with potential implications for the development of novel strategies to prevent UVB-induced skin-aging. A total of 30 female albino hairless HOS:HR-1 mice (Hoshino Laboratory Animal Co., Ltd, Japan) were exposed to a single dose of UVB irradiation (200 mJ per cm2) at 8 wk of age using 10 Toshiba FL-20 SE fluorescent lamps that deliver energy in the UVB (280–340 nm) wavelength range, with a maximum energy at a wavelength of 305 nm (Naganumaa et al., 2001Naganumaa M. Yagi E. Fukuda M. Delayed induction of pigmented spots on UVB-irradiated hairless mice.J Dermatol Sci. 2001; 25: 29-35Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). During irradiation, the height of the lamps was adjusted to deliver 0.3 mW per cm2 at the dorsal skin surface of the anesthetized mice. The mice were euthanized on days 0, 2, 4, 6 (only for PCR analysis), 8, and 13 (n=5 per each time point) after UVB irradiation. In an additional experiment, TSP-1-deficient mice (Lawler et al., 1998Lawler J. Sunday M. Thibert V. Duquette M. George E.L. Rayburn H. Hynes R.O. Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia.J Clin Invest. 1998; 101: 982-992Crossref PubMed Scopus (369) Google Scholar) and wild-type FVB control mice were subjected to a single dose of UVB as described above (200 mJ per cm2) and were euthanized on days 0 and 13 (n=5 per group). Dorsal skin samples were obtained and were snap-frozen, fixed in 10% formalin, and fixed in cold acetone to process for histologic analysis as described (Sato et al., 1986Sato Y. Mukai K. Watanabe S. Goto M. Shimosato Y. The AMeX method: A simplified technique of tissue processing and paraffin embedding with improved preservation of antigens for immunostaining.Am J Pathol. 1986; 125: 431-435PubMed Google Scholar). All animal studies were approved by the Shiseido Life Science Research Center Subcommittee on Research Animal Care and the Massachusetts General Hospital Subcommittee on Research Animal Care. Immunohistochemical stainings were performed on 5-μm frozen sections as described (Yano et al., 2002Yano K. Oura H. Detmar M. Targeted overexpression of the angiogenesis inhibitor thrombospondin-1 in the epidermis of transgenic mice prevents ultraviolet-B-induced angiogenesis and cutaneous photo-damage.J Invest Dermatol. 2002; 118: 800-805Crossref PubMed Scopus (79) Google Scholar), using a monoclonal rat anti-mouse CD31 antibody (PharMingen, San Diego, CA). Representative sections were obtained from five UVB-irradiated mice and from five age-matched, non-UVB-irradiated control mice for each time point and were analyzed using an Olympus Vanox AHBT-3 microscope (Olympus, Japan). Images were captured with a Spot digital camera (Diagnostic Instruments, Sterling Heights, MI), and morphometric analyses were performed using the IP-LAB software (Scanalytics Inc, Fairfax, VA) as described (Yano et al., 2002Yano K. Oura H. Detmar M. Targeted overexpression of the angiogenesis inhibitor thrombospondin-1 in the epidermis of transgenic mice prevents ultraviolet-B-induced angiogenesis and cutaneous photo-damage.J Invest Dermatol. 2002; 118: 800-805Crossref PubMed Scopus (79) Google Scholar). Three different fields of each section were examined at 60× magnification, and the number of vessels per square millimeter, the average vessel size, and the relative area occupied by blood vessels were determined in the dermis, in an area within 100 μm distance from the epidermal–dermal junction. The two-sided unpaired Student's t test was used to analyze differences in microvessel density and vascular size. In addition, paraffin sections were obtained from the skin of the same mice, and routine hematoxylin-eosin stains were performed as described (Prophet et al., 1992Prophet E. Mills B. Arrington J. Sobin L. Laboratory Methods in Histotechnology. American Registry of Pathology, Washington (DC)1992: 236-237Google Scholar). Immunohistochemical stainings for VEGF and TSP-1 were performed on 5-μm AMeX-fixed sections using a polyclonal rabbit anti-VEGF antibody (Ab-1; Neomarkers, Fremont, CA) or a monoclonal rat antihuman TSP-1 antibody (Immunotech, France). Double immunofluorescence stains for CD31 and CD36 were performed using a rat monoclonal antimouse CD31 (PharMingen) and a rabbit polyclonal anti-human CD36 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Anti-rat IgG antibodies conjugated with FITC and anti-mouse IgG antibodies conjugated with Texas red (Jackson ImmunoResearch Laboratories, West Grove, PA) were used as secondary antibodies. Images were captured with a Spot digital camera. In situ hybridization was performed on 5-μm 10% formalin-fixed skin sections using a signal amplification method based on the deposition of biotinylated tyramine as previously described (Kerstens et al., 1995Kerstens H.M. Poddighe P.J. Hanselaar A.G. A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine.J Histochem Cytochem. 1995; 43: 347-352Crossref PubMed Scopus (273) Google Scholar). Mouse TSP-1 cDNA (nucleotides 1060–1360; GenBank Accession No. NM_011580) was amplified by reverse transcription–PCR. Digoxigenin-labeled antisense and sense RNA probes were prepared by in vitro transcription with T7 RNA polymerase, using mouse TSP-1 cDNA as a template. Total RNA was isolated from the dorsal skin of mice using Isogen (Nippon Gene, Japan), digested with DNase I (Qiagen, Germany), and reverse-transcribed using the Superscript first-strand synthesis system (Gibco BRL). VEGF, TSP-1, and G3PDH genes were amplified using Platinum Taq DNA polymerase for 30 cycles at 58°C. The PCR products were fractionated by gel electrophoresis. To quantify the gene expression, primers and probes for VEGF, TSP-1, and G3PDH were designed with Primer-Express software (Applied Biosystems) as follows: VEGF forward, 5′-CAACATCACCATGCAGAT-3′; VEGF reverse, 5′-TCACCGCCTTGGCTTGTCAC-3′; VEGF detection probe, 5′-FAM-AGAAAGCATTTGTTTGTCCAA-GATCCGCAG-TAMRA-3′; TSP-1 forward, 5′-TCCCATCATGCCCTG-CT-3′; TSP-1 reverse, 5′-ATCCTTGTGATCACACC-3′; TSP-1 detection probe, 5′-CACATGTGGCAATGGAATTCAGCAACG-3′; G3PDH forward, 5′-GAAGGTGAAGGTCGGAGTC-3′, G3PDH reverse, 5′-GAAGATGGTGATGGGATTTC-3′; and G3PDH detection probe, 5′-FAM-AGGCTGAGAACGGGAAGCTTGT-TAMRA-3′. The PCR mix contained 1 μL cDNA template; 1× TaqMan buffer A; 3.5 mM MgCl2; 200 μM each of dATP, dCTP, dGTP, and dUTP; 1.25 units Amplitaq Gold DNA polymerase; 300 nM of each primer; and 300 nM detection probe in a total volume of 50 μL. Standard reactions were performed using a PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA). Identical primers were used for both RT-PCR and Taqman PCR. All experiments were carried out in triplicate. To analyze endothelial cell proliferation, 0.2 mL of 65 mM bromodeoxyuridine (BrdU) (Sigma, St. Louis, MO) in PBS was injected 2 h before sampling skin as described (Yano et al., 2001Yano K. Brown L.F. Detmar M. Control of hair growth and follicle size by VEGF-mediated angiogenesis.J Clin Invest. 2001; 107: 409-417Crossref PubMed Scopus (384) Google Scholar). Double immunofluorescence stains for the endothelial cell marker CD31 and for the proliferation marker BrdU were performed on 5-μm frozen sections, using a monoclonal rat anti-mouse CD31 antibody and a mouse anti-BrdU monoclonal antibody (Novocastra Laboratories, Burlingame, CA). Anti-rat IgG conjugated with FITC and anti-mouse IgG conjugated with Texas red were used as secondary antibodies. Representative sections were obtained from five mice for each experimental group and were analyzed using an Olympus Vanox AHBT-3 microscope. Digital images of CD31 (red) and BrdU (green) stains were obtained in identical fields and were combined to reveal proliferating endothelial cells (yellow). Apoptotic endothelial cells were detected by double immunofluorescence, using the Fluorescence-FragEL DNA fragmentation detection kit (Oncogene, Cambridge, MA) and an anti-mouse CD31 antibody together with an anti-rat IgG conjugated with Texas red as described previously (Yano et al., 2002Yano K. Oura H. Detmar M. Targeted overexpression of the angiogenesis inhibitor thrombospondin-1 in the epidermis of transgenic mice prevents ultraviolet-B-induced angiogenesis and cutaneous photo-damage.J Invest Dermatol. 2002; 118: 800-805Crossref PubMed Scopus (79) Google Scholar). The authors thank S. Fujii, S. Hirakawa, and J. Bertoncini for technical assistance and J. Lawler for providing TSP-1-deficient mice. This work was supported in part by American Cancer Society Research Project Grant 99-23901 and by NIH/NCI Grants CA91861, CA69184, and CA92644 (to M.D.)." @default.
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• W2075346153 title "Ultraviolet B-Induced Skin Angiogenesis Is Associated with a Switch in the Balance of Vascular Endothelial Growth Factor and Thrombospondin-1 Expression" @default.
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