An Important Role of Lymphatic Vessels in the Control of UVB-Induced Edema Formation and Inflammation

Abstract

lymphatic endothelial hyaluronan receptor-1 vascular endothelial growth factor vascular endothelial growth factor receptor Exposure of human skin to UVB-irradiation results in epidermal hyperplasia, erythema, vascular hyperpermeability, and edema formation. Previous studies have revealed that pronounced angiogenesis is induced by acute UVB-irradiation, and that several angiogenesis factors – including vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factor and interleukin 8 – are upregulated after acute UVB-irradiation (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; 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). Recently, we have shown that targeted overexpression of VEGF-A enhances sensitivity to UVB-induced cutaneous photodamage (Hirakawa et al., 2005Hirakawa S. Fujii S. Kajiya K. Yano K. Detmar M. Vascular endothelial growth factor promotes sensitivity to ultraviolet B-induced cutaneous photodamage.Blood. 2005; 105: 2392-2399Crossref PubMed Scopus (52) Google Scholar), whereas transgenic overexpression of the angiogenesis inhibitor thrombospondin-1 in the epidermis completely prevented UVB-induced damage (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). Together, these findings indicate that the cutaneous blood vasculature plays a critical role in the mediation of photodamage. In contrast, the role of lymphatic vessels in the response to UVB-irradiation has remained unknown. The lymphatic vascular system is composed of a dense network of thin-walled capillaries that drain protein-rich lymph from the extracellular spaces. Its major functions are the maintenance of tissue fluid homeostasis and the mediation of the afferent immune response (Witte et al., 2001Witte M.H. Bernas M.J. Martin C.P. Witte C.L. Lymphangiogenesis and lymphangiodysplasia: from molecular to clinical lymphology.Microsc Res Technol. 2001; 55: 122-145Crossref PubMed Scopus (169) Google Scholar; Oliver and Detmar, 2002Oliver G. Detmar M. The rediscovery of the lymphatic system: old and new insights into the development and biological function of the lymphatic vasculature.Genes Dev. 2002; 16: 773-783Crossref PubMed Scopus (296) Google Scholar). Studies of the role of lymphatic vessels in disease have been hampered by the lack of specific markers that distinguish blood and lymphatic vessels, and by the lack of knowledge about specific growth factors for lymphatic endothelium. Recent studies have identified VEGF-C and VEGF-D as specific lymphangiogenesis factors, acting via activation of the VEGF receptor-3 (VEGFR-3), which is specifically expressed on lymphatic endothelial cells (Jussila and Alitalo, 2002Jussila L. Alitalo K. Vascular growth factors and lymphangiogenesis.Physiol Rev. 2002; 82: 673-700Crossref PubMed Scopus (338) Google Scholar). Transgenic overexpression of VEGF-C in the epidermis promotes lymphatic vessel formation in the skin (Jeltsch et al., 1997Jeltsch M. Kaipainen A. Joukov V. Meng X. Lakso M. Rauvala H. et al.Hyperplasia of lymphatic vessels in VEGF-C transgenic mice.Science. 1997; 276: 1423-1425Crossref PubMed Scopus (1060) Google Scholar), whereas targeted overexpression of a soluble VEGFR-3 – which prevents VEGF-C and VEGF-D from reaching their receptor on lymphatic endothelium – in the epidermis of transgenic mice leads to lymphedema (Makinen et al., 2001Makinen T. Jussila L. Veikkola T. Karpanen T. Kettunen M.I. Pulkkanen K.J. et al.Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3.Nat Med. 2001; 7: 199-205Crossref PubMed Scopus (592) Google Scholar). In fact, missense mutations of VEGFR-3 have been found in several families affected by congenital lymphedema (Karkkainen et al., 2000Karkkainen M.J. Ferrell R.E. Lawrence E.C. Kimak M.A. Levinson K.L. McTigue M.A. et al.Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema.Nat Genet. 2000; 25: 153-159Crossref PubMed Scopus (505) Google Scholar). Moreover, several specific lymphatic markers have been recently identified, including the homeobox transcription factor Prox1, the lymphatic endothelial hyaluronan receptor (LYVE-1), and the mucin-type transmembrane glycoprotein podoplanin (Oliver and Detmar, 2002Oliver G. Detmar M. The rediscovery of the lymphatic system: old and new insights into the development and biological function of the lymphatic vasculature.Genes Dev. 2002; 16: 773-783Crossref PubMed Scopus (296) Google Scholar). As one of the major functions of lymphatic vessels is drainage of tissue fluid from normal and inflamed tissues (Kunstfeld et al., 2004Kunstfeld R. Hirakawa S. Hong Y.K. Schacht V. Lange-Asschenfeldt B. Velasco P. et al.Induction of cutaneous delayed-type hypersensitivity reactions in VEGF-A transgenic mice results in chronic skin inflammation associated with persistent lymphatic hyperplasia.Blood. 2004; 104: 1048-1057Crossref PubMed Scopus (250) Google Scholar), we hypothesized that they might also play a functional role in the cutaneous response to UVB-induced skin damage. Previously established transgenic mice which overexpress VEGF-A under the control of the K14 promoter (Detmar et al., 1998Detmar M. Brown L.F. Schon M.P. Elicker B.M. Velasco P. Richard L. 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; Hirakawa et al., 2005Hirakawa S. Fujii S. Kajiya K. Yano K. Detmar M. Vascular endothelial growth factor promotes sensitivity to ultraviolet B-induced cutaneous photodamage.Blood. 2005; 105: 2392-2399Crossref PubMed Scopus (52) Google Scholar) (males, 7 weeks old) were treated intraperitoneally with 700 μg of a blocking antibody against mouse VEGFR-3 (kindly provided by Dr Bronek Pytowski, Imclone Systems Inc., New York, NY; n=5) or with 700 μg of control rat IgG (R&D Systems, Minneapolis, MN) every other day (five injections total). One day after the first antibody injection, mice were exposed to a single dose of 200 mJ/cm2 UVB (day 0) and ear thickness was measured daily until day 9 after UVB-irradiation. All experiments were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care. UVB-irradiation induced inflammation and edema of the ear skin as early as 1 day after irradiation (Figure 1a), with a maximum ear swelling observed after 4 days (Figure 1a). Whereas no differences between the treatment groups were observed during the first 4 days after irradiation, the ear thickness of the control-IgG-treated mice went back to normal levels after day 6, whereas anti-VEGFR-3-treated mice showed prolonged ear swelling until day 9 (P<0.05 at days 6 and 9; P<0.01 at days 7 and 8). As VEGF-A does not bind to VEGFR-3, the effect observed was specifically due to inhibition of lymphatic function. Hematoxylin–eosin stains of frozen sections at day 9 after the UVB-irradiation revealed that the ear skin of anti-VEGFR-3-treated mice still displayed the characteristic features of acute photodamage, including epidermal hyperplasia, marked dermal edema, vascular dilation, and inflammatory cell infiltration in the dermis (Figure 1c). In contrast, the ear skin of control-IgG-treated mice already resembled histologically the non-UVB-irradiated skin (Figure 1b). Immunohistochemistry using an antibody against mouse CD11b (BD Pharmingen, San Diego, CA) revealed increased macrophage infiltration in the dermis of anti-VFGFR-3-treated mice, as compared with mice treated with control-IgG (Figure 1d and e). These surprising findings indicate that the enlarged lymphatic vessels in anti-VEGFR-3-treated mice were functionally impaired with regard to fluid and cell transport. To elucidate whether inhibition of the VEGF-C/VEGFR-3 pathway prevented lymphatic vessel activation, we next stained ear skin sections – by immunofluorescence – with antibodies against the lymphatic vessel-specific marker LYVE-1 (kindly provided by Dr D. Jackson, Oxford, UK) and against the panvascular marker CD31 (BD Pharmingen) at day 9 after the UVB-irradiation. We found that LYVE-1-positive lymphatic vessels were greatly enlarged in the ear skin of anti-VEGFR-3-treated mice (Figure 2b), whereas lymphatic vessels in mice treated with control-IgG were collapsed and were indistinguishable from lymphatic vessels in non-irradiated ear skin (Figure 2a). In contrast, CD31-positive/LYVE-1-negative blood vessels were comparable in both treatment groups (Figure 2a and b). Morphometric analysis, using the IP-LAB software (Scanalytics, Fairfax, VA) as described (Kajiya et al., 2005Kajiya K. Hirakawa S. Ma B. Drinnenberg I. Detmar M. Hepatocyte growth factor promotes lymphatic vessel formation and function.EMBO J. 2005; 24: 2885-2895Crossref PubMed Scopus (244) Google Scholar), revealed that the average skin area covered by lymphatic vessels was significantly larger in anti-VEGFR-3-treated mice than in control-IgG-treated mice (P<0.05) (Figure 2c). Moreover, the average size of lymphatic vessels was significantly larger in the ear skin of anti-VEGFR-3-treated mice than in control-treated mice (P<0.001) (Figure 2d). The number of lymphatic vessels was similar in both groups (Figure 2e). Future studies are needed to evaluate the distinct effects of specific blockade of VEGFR-2 versus VEGFR-3 on lymphatic function and UVB-induced cutaneous photodamage in wild-type mice. Our results provide the first evidence that the lymphatic vascular system plays a functional role in the cutaneous response to UVB damage. Our findings that inhibition of lymphatic function via blockade of VEGFR-3 prolonged UVB-induced edema formation and inflammation indicate that promotion of lymphatic function might represent a new strategy to reduce or prevent UVB-induced skin damage. This work was supported by National Institutes of Health Grants CA69184, CA86410 and CA92644 (M.D.), American Cancer Society Research Project Grant 99-23901 (M.D.), and by the Cutaneous Biology Research Center through the Massachusetts General Hospital/Shiseido Co. Ltd Agreement (to M.D.).