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The role of VEGF and thrombospondins in skin angiogenesis
Journal of Dermatological Science 24 Suppl. 1 (2000) S78 – S84 www.elsevier.com/locate/jdermsci
The role of VEGF and thrombospondins in skin angiogenesis Michael Detmar * Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital and Har6ard Medical School, Charlestown, MA 02129, USA
Abstract The vasculature in adult skin remains normally quiescent, due to the dominant influence of endogenous angiogenesis inhibitors over angiogenic stimuli. However, skin retains the capacity for brisk initiation of angiogenesis, the growth of new blood vessels from preexisting vessels, during tissue repair and in numerous diseases, including inflammatory skin diseases such as psoriasis and skin cancers such as cutaneous squamous cell carcinomas. Moreover, cyclic vascular expansion occurs during the growth phase of the hair follicle. Recent evidence suggests vascular endothelial growth factor as the major skin angiogenesis factor. During skin angiogenesis, expression of vascular endothelial growth factor is induced in epidermal keratinocytes by several stimuli including transforming growth factor-a and hypoxia, leading to increased vascularization of the dermis. In contrast, vascular endothelial growth factor-C induces skin lymphangiogenesis. Thrombospondin-1 and thrombospondin-2 are endogenous inhibitors of angiogenesis that are expressed in normal skin, maintaining the quiescence of cutaneous vessels. Both inhibitors potently inhibit skin cancer growth via inhibition of tumor angiogenesis. Targeting cutaneous blood vessels represents a promising new therapeutic approach for the treatment of a variety of skin diseases. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: VEGF; TSP-1; TSP-2; Skin
1. Introduction Blood vessels are essential to supply sufficient oxygen and nutrients to the skin, in order to maintain normal tissue homeostasis and function.
Abbre6iations: TSP, thrombospondin; VEGF, vascular endothelial growth factor. * Tel.: +1-617-7241170; fax: + 1-617-7264453. E-mail address: [email protected] (M. Detmar).
During embryogenesis, blood vessels are formed through vasculogenesis, the differentiation of undifferentiated angioblasts into endothelial cells that form a primitive vascular network. Angiogenesis then occurs as either: (1) the sprouting of new vessels from preexisting postcapillary venules or (2) as remodeling of preexisting blood vessels through enlargement and/or division of preexisting blood vessels by vascular cell pillars (intussusception) [1]. Angiogenesis is a complex, multi-step process (Table 1) involving as a first step the induction of microvascular hyperpermeability, e.g.
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M. Detmar / Journal of Dermatological Science 24 (2000) S78–S84
after tissue damage, leading to extravasation of plasma proteins such as fibrinogen and prothrombin which serve either as a provisional matrix for migrating endothelial cells or as initiators of the extrinsic coagulation cascade, leading to cleavage and activation of several matrix proteins, thereby providing an angiogenic stroma. As a next step, the vascular basement membrane is degraded by the concerted action of activated matrix metalloproteinases, including MMP-2. This is essential for angiogenesis to occur since vascular endothelial cells in healthy adult skin are ‘trapped’ within the barrier of the anti-angiogenic vascular basement membrane that contains extracellular matrix molecules such as collagen type IV, laminin, and thrombospondin-1. Integrin receptors on endothelial cells, in particular the a1b1, a2b1, and avb3 integrins on the surface of endothelial cells then interact with either native or degraded matrix molecules (e.g. collagen type I, fibronectin) in order to mediate endothelial cell migration. The migration of endothelial cells is directed towards a gradient of angiogenesis factors which are most commonly produced by epithelial organs/tissues. Endothelial cells then undergo a series of cell divisions, promoted by potent angiogenesis factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor. Finally, the migrated endothelial cells form a vascular lumen, and the vessels are gradually transformed into mature new blood vessels. Whereas angiogenesis is a common feature of developing skin during embryogenesis, the blood vessels in healthy adults are predominantly quiescent, with the exception of the cyclic angiogenesis occurring in the uterus and the ovaries during the female reproductive cycle. Another exception is the hair cycle that we have found to be characterized by dramatic cyclic expansion and involution of perifollicular blood vessels. However, adult skin retains the capacity for brisk initiation of angiogenesis during tissue repair and in numerous diseases, including inflammatory skin diseases such as psoriasis and various types of dermatitis, several blistering diseases, cutaneous neoplasias including squamous cell carcinomas, malignant melanomas, and Kaposi’s sarcomas, and prolifer-
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Table 1 The multi-step process of angiogenesis 1. Induction of microvascular hyperpermeability 2. Enzymatic degradation of vascular basement membrane and interstitial matrix 3. Endothelial cell migration 4. Endothelial cell proliferation 5. Formation of mature blood vessels
ative hemangiomas of childhood (Table 2). Moreover, several other diseases are characterized by macroscopically visible, prominent blood vessels, including rosacea and basal cell carcinoma. Recent advances in angiogenesis research have identified some of the key molecules that control vascular growth, and have led to the concept that in normal skin, vascular quiescence is maintained by the dominant influence of potent endogenous angiogenesis inhibitors over angiogenic stimuli, whereas angiogenesis is induced by increased secretion of angiogenic factors and/or by downregulation of angiogenesis inhibitors, leading to a ‘pro-angiogenic imbalance’ (Fig. 1). This overview Table 2 Conditions and diseases associated with skin angiogenesis Hair growth and cycling Wound healing Skin neoplasis Squamous cell carcinoma Basal cell carcinoma Malignant melanoma Malignant cutaneous lymphomas Vascular tumors Angiosarcoma Kaposi’s sarcoma Proliferating hemangiomas Inflammatory dermatoses Psoriasis Dermatitis (atopic, contact) Bullous diseases Bullous pemphigoid Erythema multiforme Other diseases Viral warts Rosacea UV-damage
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Fig. 1. In normal skin the influence of endogenous angiogenesis inhibitors dominates over pro-angiogenic factors, ensuring quiescence of the vasculature. During angiogenesis, the expression of angiogenesis factors is upregulated, shifting the balance towards a pro-angiogenic environment.
intends to summarize our current knowledge about some of the major molecules involved in the control of skin angiogenesis and to identify skin diseases that are characterized by angiogenesis and that might be targets for therapeutical inhibition of blood vessel growth.
2. Vascular endothelial growth factor (VEGF) Vascular endothelial growth factor (VEGF) was originally discovered as vascular permeability factor (VPF), due to the activity of tumor cellconditioned media to induce accumulation of ascites fluid [2] . VEGF is a homodimeric, heparin-binding glycoprotein occurring in at least four isoforms of 121, 165, 189 and 201 amino acids, due to alternative splicing [3]. VEGF binds to two type III tyrosine kinase receptors that are expressed predominantly on vascular endothelial cells, Flt-1/VEGF receptor-1 (VEGFR-1) and KDR/Flk-1/VEGFR-2 (for a review see Ref. [1] ). In addition, VEGF165 also binds to the neuropilin receptor on endothelial and other cells [4]. In vitro, VEGF acts as a specific mitogen for human dermal microvascular endothelial cells [5] and induces endothelial cell migration towards several extracellular matrices, partly through upregulation of the fibronectin receptor aVb3 and of
the collagen receptors a1b1 and a2b1 on endothelial cells [6,7]. In vivo, VEGF enhances microvascular permeability [2] and angiogenesis [8]. Over the last several years we and others have found that VEGF is expressed at low levels in normal skin, whereas skin diseases associated with angiogenesis show prominent upregulation of VEGF expression by epidermal keratinocytes. These diseases include psoriasis, contact dermatitis, several bullous diseases, viral papillomas, and squamous cell carcinoma (for a review see Ref. [9]). Recently, we have confirmed the in vivo biological importance of epidermis-derived VEGF for cutaneous angiogenesis in a transgenic mouse model, using the keratin 14 promoter to selectively target expression of murine VEGF164 to basal epidermal keratinocytes and to follicular keratinocytes of the outer root sheath of the hair follicle [10]. VEGF transgenic mice were characterized by a significant increase in the density of dermal blood vessels, predominantly of capillaries that were tortuous and displayed increased branching. Moreover, blood vessels in the skin of VEGF transgenic mice were hyperpermeable for circulating plasma proteins, as shown by the enhanced leakage of radioactively labeled fibrinogen after intravenous injection [10]. It is of interest that VEGF overexpressing mice were also characterized by greatly enhanced rolling and adhesion of peripheral blood mononuclear cells on cutaneous postcapillary venules, suggesting that VEGF, in addition to its pro-angiogenic activity, might also contribute to the recruitment of leukocytes to inflamed skin. These effects were mediated through activation of specific endothelial cell adhesion molecules, since systemic application of blocking antibodies to E-selectin and P-selectin abrogated VEGF’s effect on leukocyte rolling, and antibodies to ICAM-1 and VCAM-1 completely inhibited VEGF’s proadhesive activity [10]. As a surprising finding, VEGF transgenic mice also showed a significantly increased density of cutaneous mast cells, mostly located around dermal blood vessels. These findings suggest that VEGF might act indirectly on mast cells by inducing the release of mast cell-activating factors by endothelial cells and are in accordance with the previously reported association of angiogenesis with mast cell accumulation.
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Upregulation of VEGF expression in epidermal keratinocytes is induced by distinct molecular pathways: (1) In psoriasis, healing wounds, and squamous cell carcinomas, transforming growth factor-a and other ligands of the epidermal growth factor receptor are upregulated on suprabasal keratinocytes. In an autocrine loop, these growth factors induce hyperplasia of the epidermis. Simultaneously, they induce VEGF gene expression and protein secretion by epidermal keratinocytes [11] which leads, in a paracrine way, to the induction of angiogenesis through interaction with specific receptors on cutaneous microvessels (Fig. 2). This mechanism automatically links epidermal hyperplasia with increased vascularization, thereby providing enhanced vascular support to meet the enhanced nutritional needs of proliferating keratinocytes. More recently, several other growth factors that stimulate keratinocyte proliferation, including keratinocyte growth factor, have joined the group of VEGF-inducing molecules. (2) Skin hypoxia directly leads to upregulation of VEGF expression by epidermal keratinocytes, dermal fibroblasts and dermal endothelial cells [12]. Hypoxia also induces upregulation of the VEGF receptor Flt1/VEGFR-1 on microvessels, suggesting the existence of an autocrine pro-angiogenic loop. Recently we have shown that the hypoxia-induced upregulation of VEGF expression is mediated both by activation of VEGF gene transcription and by enhanced
Fig. 2. Diagrammatic representation of the molecular regulation of VEGF-mediated angiogenesis in the skin. KC, epidermal keratinocytes.
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VEGF mRNA stability, mediated, at least in part, by specific interactions between a defined mRNA stability sequence in the 3% untranslated region and distinct mRNA-binding proteins [13]. This mechanism is likely important in the induction of epidermal VEGF expression during wound healing and in areas of skin cancers adjacent to tumor necroses. The majority of human cancers studied thus far are characterized by overexpression of VEGF by tumor cells and by overexpression of VEGF receptors on tumor-associated blood vessels, and blocking of VEGF function inhibits angiogenesis and suppresses tumor growth in vivo (for a review see Ref. [14]). Importantly, VEGF also appears to affect the very early tumor development and progression of squamous cell carcinomas of the skin, since antibody inhibition of the VEGF receptor Flk-1/VEGFR-2 completely prevented squamous cell carcinoma invasion [15] and selective overexpression of VEGF in a non-invasive squamous cell carcinoma cell line resulted in a more malignant phenotype with the occurrence of single-cell tumor invasion [16].
3. Vascular endothelial growth factor-C Several new members of the VEGF family of angiogenesis factors have been identified during the last few years (Fig. 3). Thus far, VEGF-C has been characterized in most detail, and there is convincing evidence that VEGF-C acts on lymphatic endothelium through interaction with the Flt4/VEGFR-3 receptor as well as with KDR/ VEGFR-2. Overexpression of VEGF-C in the skin of transgenic mice, under control of the keratin 14 promoter, resulted in hyperplasia of cutaneous lymphatic vessels, but not of blood vessels [17]. Recently, we have identified VEGF-C as a growth factor for HIV-associated Kaposi’s sarcomas, inducing proliferation of cultured KS cells in vitro [18]. VEGF-C is expressed within Kaposi’s sarcomas, and the tumor cells strongly express Flt4/VEGFR-3, demonstrating the lymphatic origin and/or differentiation of this vascular tumor [18,19]. Importantly, VEGF-C expression in vascular endothelial cells is induced by
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Fig. 3. Overlapping binding patterns of members of the VEGF family of angiogenesis factors to endothelial cell VEGF receptors (VEGFR). PlGF-2 and VEGF165 bind to the neuropilin receptor.
VEGF, suggesting a possible molecular link between angiogenesis and lymphangiogenesis. The direct biological roles of VEGF-C for lymphangiogenesis and lymphatic tumor metastasis are currently under investigation. Similar to VEGFC, VEGF-D also binds to the Flt4/VEGFR-3 and to KDR/VEGFR-2; however, the biological importance of VEGF-D for lymphangiogenesis remains to be established.
4. Thrombospondin-1 (TSP-1) TSP-1 belongs to a family of matricellular proteins (TSP-1 to TSP-5) that mediate interactions between extracellular matrix molecules such as collagen and cellular integrin receptors such as avb3 [20]. TSP-1 is a 450 kDa modular, homotrimeric glycoprotein, containing a procollagen homology region, three properdin-like type I repeats, three epidermal growth factor-like repeats, and seven Ca2 + -binding repeats (Fig. 4). TSP-1 is involved in a large number of biological processes, including cell proliferation, migration and differentiation of various cell types; moreover, TSP-1 inhibits endothelial cell proliferation and migration and has been shown to inhibit angiogenesis in vivo [21]. In normal human skin, TSP-1 is expressed by basal epidermal keratinocytes and is deposited in the dermo-epidermal basement membrane zone, contributing to the barrier that
prevents ingrowth of blood vessels into the dermis. In contrast, TSP-1 expression is downregulated in squamous cell carcinomas of the skin [22]. We recently demonstrated that reintroduction of the TSP-1 gene into A431 and SCC-13 squamous cell carcinomas significantly inhibited tumor growth after intradermal transplantation onto immunodeficient mice [22] . The antitumoral effect of TSP-1 was mainly due to its inhibition of tumor angiogenesis, leading to enhanced tumor cell necrosis. Three potential mechanisms have been suggested to mediate the anti-angiogenic and anti-tumoral effects of TSP-1: (1) Two CSVTCG sequences that are contained within the second and third type I repeats interact with the CD36 receptor on endothelial cells to induce endothelial
Fig. 4. Modular structure of the two matricellular, endogenous angiogenesis inhibitors, TSP-1 and TSP-2. PC, procollagen homology region. Both molecules contain three properdin-like type I repeats, three EGF-like type II repeats, and seven Ca2 + -binding type III repeats.
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cell apoptosis [23]. (2) TSP-1 contains a RFK sequence within the second type I repeat that activates TGF-b. Indeed, the phenotype of TSP-1deficient mice could partially be reversed by systemic application of the synthetic peptide LSKL that inhibits TSP-1 mediated TGF-b activation [24]. (3) Distinct heparin-binding sequences within the TSP-1 type I repeats have been shown to inhibit endothelial cell migration in vitro and might also play a role in the antitumoral activity [25]. More recently, we have shown that overexpression of TSP-1 in the skin of transgenic mice results in impaired wound healing, mainly through inhibition of early granulation tissue formation [26].
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Furthermore, our recent evidence, using a chemical skin carcinogenesis protocol in genetically altered mice, suggests that the expression of TSP-2 in normal skin protects from skin cancer development. Although the TSP-2 molecule does not contain the TGF-b activating sequence RFK, TSP-2 is a more potent inhibitor of skin angiogenesis than TSP-1 since: (1) TSP-2 deficient mice show a more dramatic increase in cutaneous blood vessels than TSP-1 deficient mice, and (2) the antitumoral effect of TSP-2 was stronger than the effect of TSP-1 [30]. Currently, we are investigating the molecular mechanisms by which TSP-2 exerts its potent anti-angiogenic effects, using recombinant human TSP-2 and TSP-2 fragments and synthetic peptides derived from distinct TSP2 sequences.
5. Thrombospondin-2 (TSP-2) Similar to TSP-1, TSP-2 is a 450 kDa modular glycoprotein (Fig. 4), which is secreted as a disulfide-bonded homotrimer [27] that binds to a variety of several cell surface receptors, including the integrin avb3, low-density lipoprotein-related receptor protein, and heparan sulfate proteoglycans. However, TSP-2 expression during embryonic development and in adult tissues is spatially and temporally different from TSP-1 expression. Moreover, the regulation of TSP-2 gene expression by growth factors is distinct from that of TSP-1. Importantly, mice that are deficient in TSP-2 show increased numbers of blood vessels in the skin, suggesting that TSP-2 functions as an endogenous inhibitor of angiogenesis [28]. Our recent data show that TSP-2 mRNA and protein are expressed in basal epidermal keratinocytes and are downregulated in squamous cell carcinomas of the skin, suggesting that, similar to TSP-1, TSP-2 also contributes to the anti-angiogenic barrier function that prevents vascularization of the epidermis [29]. Using human A431 squamous cell carcinoma cells stably transfected with TSP-2, we recently identified TSP-2 as a novel, potent endogenous inhibitor of tumor growth and angiogenesis [30]. Importantly, co-expression of both TSP-2 and TSP-1 completely prevented any tumor formation, demonstrating synergistic antitumoral effects of both molecules.
References [1] Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med 2000;6:389 – 95. [2] Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 1983;219:983 – 5. [3] Tischer E, Mitchell R, Hartman T, et al. The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem 1991;266:11947– 54. [4] Soker S, Takashima S, Miao HQ, Neufeld G, Klagsbrun M. Neuropilin-1 is expressed by endothelial cells and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 1998;92:735 – 45. [5] Detmar M, Yeo K-T, Nagy JA, et al. Keratinocytederived vascular permeability factor (vascular endothelial growth factor) is a potent mitogen for dermal microvascular endothelial cells. J Invest Dermatol 1995;105:44 – 50. [6] Senger DR, Ledbetter SR, Claffey KP, PapadopoulosSergiou A, Perruzzi CA, Detmar M. Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the avb3 integrin, osteopontin, and thrombin. Am J Pathol 1996;149:293 – 305. [7] Senger DR, Claffey KP, Benes JE, Perruzzi CA, Sergiou AP, Detmar M. Angiogenesis promoted by vascular endothelial growth factor: regulation through a1b1, and a2b1integrins. Proc Natl Acad Sci USA 1997;94:13612– 7. [8] Connolly DT, Heuvelman DM, Nelson R, et al. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest 1989;84:1470 – 8.
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[9] Detmar M. Molecular regulation of angiogenesis in the skin. J Invest Dermatol 1996;106:207–8. [10] Detmar M, Brown LF, Scho¨n MP, et al. Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice. J Invest Dermatol 1998;111:1–6. [11] Detmar M, Brown LF, Claffey KP, et al. Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis. J Exp Med 1994;180:1141– 6. [12] Detmar M, Brown LF, Berse B, et al. Hypoxia regulates the expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) and its receptors in human skin. J Invest Dermatol 1997;108:263–8. [13] Claffey KP, Shih SC, Mullen A, et al. Identification of a human VPF/VEGF 3% untranslated region mediating hypoxia-induced mRNA stability. Mol Biol Cell 1998;9:469 – 81. [14] Dvorak HF, Brown LF, Detmar M, Dvorak AM. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol 1995;146:1029–39. [15] Skobe M, Rockwell P, Goldstein N, Vosseler S, Fusenig NE. Halting angiogenesis suppresses carcinoma cell invasion. Nat Med 1997;3:1222–7. [16] Detmar M, Velasco P, Richard L, et al. Expression of vascular endothelial growth factor induces an invasive phenotype in human squamous cell carcinomas. Am J Pathol 2000;156:159 –67. [17] Jeltsch M, Kaipainen A, Joukov V, et al. Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science 1997;276:1423– 5. [18] Skobe M, Brown LF, Tognazzi K, et al. Vascular endothelial growth factor-C (VEGF-C) and its receptors KDR and flt-4 are expressed in AIDS-associated Kaposi’s sarcoma. J Invest Dermatol 1999;113:1047–53. [19] Jussila L, Valtola R, Partanen TA, et al. Lymphatic endothelium and Kaposi’s sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor receptor-3. Cancer Res 1998;58:1599–604.
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[20] Bornstein P. Diversity of function is inherent in matricellular proteins: an appraisal of thrombospondin 1. J Cell Biol 1995;130:503 – 6. [21] Tolsma SS, Volpert OV, Good DJ, Frazier WA, Polverini PJ, Bouck N. Peptides derived from two separate domains of the matrix protein thrombospondin-1 have antiangiogenic activity. J Cell Biol 1993;122:497 – 511. [22] Streit M, Velasco P, Brown LF, et al. Overexpression of thrombospondin-1 decreases angiogenesis and inhibits the growth of human squamous cell carcinomas. Am J Pathol 1999;155:441 – 52. [23] Dawson DW, Pearce SF, Zhong R, Silverstein RL, Frazier WA, Bouck NP. CD36 mediates the In vitro inhibitory effects of thrombospondin-1 on endothelial cells. J Cell Biol 1997;138:707 – 17. [24] Crawford S, Stellmach V, Murphy-Ullrich J, et al. Thrombospondin-1 is a major activator of TGF-b1 in vivo. Cell 1998;93:1159 – 70. [25] Iruela-Arispe L, Lombardo M, Krutzsch HC, Lawler J, Roberts DD. Inhibition of angiogenesis by thrombospondin-1 is mediated by 2 independent regions within the type 1 repeats. Circulation 1999;100:1423 – 31. [26] Streit M, Velasco P, Riccardi, et al. Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice. EMBO J 2000;19:3272 – 82. [27] Bornstein P. Thrombospondins: structure and regulation of expression. FASEB J 1992;6:3290 – 9. [28] Kyriakides TR, Zhu YH, Smith LT, et al. Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, and a bleeding diathesis. J Cell Biol 1998;140:419 – 30. [29] M. Detmar, Tumor angiogenesis, J. Invest. Dermatol. Symp. Proc. 2000 (in press). [30] Streit M, Riccardi L, Velasco P, et al. Thrombospondin2: a potent endogenous inhibitor of tumor growth and angiogenesis. Proc Natl Acad Sci USA 1999;96:14888– 93.
The role of VEGF and thrombospondins in skin angiogenesis Michael Detmar * Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital and Har6ard Medical School, Charlestown, MA 02129, USA
Abstract The vasculature in adult skin remains normally quiescent, due to the dominant influence of endogenous angiogenesis inhibitors over angiogenic stimuli. However, skin retains the capacity for brisk initiation of angiogenesis, the growth of new blood vessels from preexisting vessels, during tissue repair and in numerous diseases, including inflammatory skin diseases such as psoriasis and skin cancers such as cutaneous squamous cell carcinomas. Moreover, cyclic vascular expansion occurs during the growth phase of the hair follicle. Recent evidence suggests vascular endothelial growth factor as the major skin angiogenesis factor. During skin angiogenesis, expression of vascular endothelial growth factor is induced in epidermal keratinocytes by several stimuli including transforming growth factor-a and hypoxia, leading to increased vascularization of the dermis. In contrast, vascular endothelial growth factor-C induces skin lymphangiogenesis. Thrombospondin-1 and thrombospondin-2 are endogenous inhibitors of angiogenesis that are expressed in normal skin, maintaining the quiescence of cutaneous vessels. Both inhibitors potently inhibit skin cancer growth via inhibition of tumor angiogenesis. Targeting cutaneous blood vessels represents a promising new therapeutic approach for the treatment of a variety of skin diseases. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: VEGF; TSP-1; TSP-2; Skin
1. Introduction Blood vessels are essential to supply sufficient oxygen and nutrients to the skin, in order to maintain normal tissue homeostasis and function.
Abbre6iations: TSP, thrombospondin; VEGF, vascular endothelial growth factor. * Tel.: +1-617-7241170; fax: + 1-617-7264453. E-mail address: [email protected] (M. Detmar).
During embryogenesis, blood vessels are formed through vasculogenesis, the differentiation of undifferentiated angioblasts into endothelial cells that form a primitive vascular network. Angiogenesis then occurs as either: (1) the sprouting of new vessels from preexisting postcapillary venules or (2) as remodeling of preexisting blood vessels through enlargement and/or division of preexisting blood vessels by vascular cell pillars (intussusception) [1]. Angiogenesis is a complex, multi-step process (Table 1) involving as a first step the induction of microvascular hyperpermeability, e.g.
0923-1811/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 9 2 3 - 1 8 1 1 ( 0 0 ) 0 0 1 4 5 - 6
M. Detmar / Journal of Dermatological Science 24 (2000) S78–S84
after tissue damage, leading to extravasation of plasma proteins such as fibrinogen and prothrombin which serve either as a provisional matrix for migrating endothelial cells or as initiators of the extrinsic coagulation cascade, leading to cleavage and activation of several matrix proteins, thereby providing an angiogenic stroma. As a next step, the vascular basement membrane is degraded by the concerted action of activated matrix metalloproteinases, including MMP-2. This is essential for angiogenesis to occur since vascular endothelial cells in healthy adult skin are ‘trapped’ within the barrier of the anti-angiogenic vascular basement membrane that contains extracellular matrix molecules such as collagen type IV, laminin, and thrombospondin-1. Integrin receptors on endothelial cells, in particular the a1b1, a2b1, and avb3 integrins on the surface of endothelial cells then interact with either native or degraded matrix molecules (e.g. collagen type I, fibronectin) in order to mediate endothelial cell migration. The migration of endothelial cells is directed towards a gradient of angiogenesis factors which are most commonly produced by epithelial organs/tissues. Endothelial cells then undergo a series of cell divisions, promoted by potent angiogenesis factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor. Finally, the migrated endothelial cells form a vascular lumen, and the vessels are gradually transformed into mature new blood vessels. Whereas angiogenesis is a common feature of developing skin during embryogenesis, the blood vessels in healthy adults are predominantly quiescent, with the exception of the cyclic angiogenesis occurring in the uterus and the ovaries during the female reproductive cycle. Another exception is the hair cycle that we have found to be characterized by dramatic cyclic expansion and involution of perifollicular blood vessels. However, adult skin retains the capacity for brisk initiation of angiogenesis during tissue repair and in numerous diseases, including inflammatory skin diseases such as psoriasis and various types of dermatitis, several blistering diseases, cutaneous neoplasias including squamous cell carcinomas, malignant melanomas, and Kaposi’s sarcomas, and prolifer-
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Table 1 The multi-step process of angiogenesis 1. Induction of microvascular hyperpermeability 2. Enzymatic degradation of vascular basement membrane and interstitial matrix 3. Endothelial cell migration 4. Endothelial cell proliferation 5. Formation of mature blood vessels
ative hemangiomas of childhood (Table 2). Moreover, several other diseases are characterized by macroscopically visible, prominent blood vessels, including rosacea and basal cell carcinoma. Recent advances in angiogenesis research have identified some of the key molecules that control vascular growth, and have led to the concept that in normal skin, vascular quiescence is maintained by the dominant influence of potent endogenous angiogenesis inhibitors over angiogenic stimuli, whereas angiogenesis is induced by increased secretion of angiogenic factors and/or by downregulation of angiogenesis inhibitors, leading to a ‘pro-angiogenic imbalance’ (Fig. 1). This overview Table 2 Conditions and diseases associated with skin angiogenesis Hair growth and cycling Wound healing Skin neoplasis Squamous cell carcinoma Basal cell carcinoma Malignant melanoma Malignant cutaneous lymphomas Vascular tumors Angiosarcoma Kaposi’s sarcoma Proliferating hemangiomas Inflammatory dermatoses Psoriasis Dermatitis (atopic, contact) Bullous diseases Bullous pemphigoid Erythema multiforme Other diseases Viral warts Rosacea UV-damage
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Fig. 1. In normal skin the influence of endogenous angiogenesis inhibitors dominates over pro-angiogenic factors, ensuring quiescence of the vasculature. During angiogenesis, the expression of angiogenesis factors is upregulated, shifting the balance towards a pro-angiogenic environment.
intends to summarize our current knowledge about some of the major molecules involved in the control of skin angiogenesis and to identify skin diseases that are characterized by angiogenesis and that might be targets for therapeutical inhibition of blood vessel growth.
2. Vascular endothelial growth factor (VEGF) Vascular endothelial growth factor (VEGF) was originally discovered as vascular permeability factor (VPF), due to the activity of tumor cellconditioned media to induce accumulation of ascites fluid [2] . VEGF is a homodimeric, heparin-binding glycoprotein occurring in at least four isoforms of 121, 165, 189 and 201 amino acids, due to alternative splicing [3]. VEGF binds to two type III tyrosine kinase receptors that are expressed predominantly on vascular endothelial cells, Flt-1/VEGF receptor-1 (VEGFR-1) and KDR/Flk-1/VEGFR-2 (for a review see Ref. [1] ). In addition, VEGF165 also binds to the neuropilin receptor on endothelial and other cells [4]. In vitro, VEGF acts as a specific mitogen for human dermal microvascular endothelial cells [5] and induces endothelial cell migration towards several extracellular matrices, partly through upregulation of the fibronectin receptor aVb3 and of
the collagen receptors a1b1 and a2b1 on endothelial cells [6,7]. In vivo, VEGF enhances microvascular permeability [2] and angiogenesis [8]. Over the last several years we and others have found that VEGF is expressed at low levels in normal skin, whereas skin diseases associated with angiogenesis show prominent upregulation of VEGF expression by epidermal keratinocytes. These diseases include psoriasis, contact dermatitis, several bullous diseases, viral papillomas, and squamous cell carcinoma (for a review see Ref. [9]). Recently, we have confirmed the in vivo biological importance of epidermis-derived VEGF for cutaneous angiogenesis in a transgenic mouse model, using the keratin 14 promoter to selectively target expression of murine VEGF164 to basal epidermal keratinocytes and to follicular keratinocytes of the outer root sheath of the hair follicle [10]. VEGF transgenic mice were characterized by a significant increase in the density of dermal blood vessels, predominantly of capillaries that were tortuous and displayed increased branching. Moreover, blood vessels in the skin of VEGF transgenic mice were hyperpermeable for circulating plasma proteins, as shown by the enhanced leakage of radioactively labeled fibrinogen after intravenous injection [10]. It is of interest that VEGF overexpressing mice were also characterized by greatly enhanced rolling and adhesion of peripheral blood mononuclear cells on cutaneous postcapillary venules, suggesting that VEGF, in addition to its pro-angiogenic activity, might also contribute to the recruitment of leukocytes to inflamed skin. These effects were mediated through activation of specific endothelial cell adhesion molecules, since systemic application of blocking antibodies to E-selectin and P-selectin abrogated VEGF’s effect on leukocyte rolling, and antibodies to ICAM-1 and VCAM-1 completely inhibited VEGF’s proadhesive activity [10]. As a surprising finding, VEGF transgenic mice also showed a significantly increased density of cutaneous mast cells, mostly located around dermal blood vessels. These findings suggest that VEGF might act indirectly on mast cells by inducing the release of mast cell-activating factors by endothelial cells and are in accordance with the previously reported association of angiogenesis with mast cell accumulation.
M. Detmar / Journal of Dermatological Science 24 (2000) S78–S84
Upregulation of VEGF expression in epidermal keratinocytes is induced by distinct molecular pathways: (1) In psoriasis, healing wounds, and squamous cell carcinomas, transforming growth factor-a and other ligands of the epidermal growth factor receptor are upregulated on suprabasal keratinocytes. In an autocrine loop, these growth factors induce hyperplasia of the epidermis. Simultaneously, they induce VEGF gene expression and protein secretion by epidermal keratinocytes [11] which leads, in a paracrine way, to the induction of angiogenesis through interaction with specific receptors on cutaneous microvessels (Fig. 2). This mechanism automatically links epidermal hyperplasia with increased vascularization, thereby providing enhanced vascular support to meet the enhanced nutritional needs of proliferating keratinocytes. More recently, several other growth factors that stimulate keratinocyte proliferation, including keratinocyte growth factor, have joined the group of VEGF-inducing molecules. (2) Skin hypoxia directly leads to upregulation of VEGF expression by epidermal keratinocytes, dermal fibroblasts and dermal endothelial cells [12]. Hypoxia also induces upregulation of the VEGF receptor Flt1/VEGFR-1 on microvessels, suggesting the existence of an autocrine pro-angiogenic loop. Recently we have shown that the hypoxia-induced upregulation of VEGF expression is mediated both by activation of VEGF gene transcription and by enhanced
Fig. 2. Diagrammatic representation of the molecular regulation of VEGF-mediated angiogenesis in the skin. KC, epidermal keratinocytes.
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VEGF mRNA stability, mediated, at least in part, by specific interactions between a defined mRNA stability sequence in the 3% untranslated region and distinct mRNA-binding proteins [13]. This mechanism is likely important in the induction of epidermal VEGF expression during wound healing and in areas of skin cancers adjacent to tumor necroses. The majority of human cancers studied thus far are characterized by overexpression of VEGF by tumor cells and by overexpression of VEGF receptors on tumor-associated blood vessels, and blocking of VEGF function inhibits angiogenesis and suppresses tumor growth in vivo (for a review see Ref. [14]). Importantly, VEGF also appears to affect the very early tumor development and progression of squamous cell carcinomas of the skin, since antibody inhibition of the VEGF receptor Flk-1/VEGFR-2 completely prevented squamous cell carcinoma invasion [15] and selective overexpression of VEGF in a non-invasive squamous cell carcinoma cell line resulted in a more malignant phenotype with the occurrence of single-cell tumor invasion [16].
3. Vascular endothelial growth factor-C Several new members of the VEGF family of angiogenesis factors have been identified during the last few years (Fig. 3). Thus far, VEGF-C has been characterized in most detail, and there is convincing evidence that VEGF-C acts on lymphatic endothelium through interaction with the Flt4/VEGFR-3 receptor as well as with KDR/ VEGFR-2. Overexpression of VEGF-C in the skin of transgenic mice, under control of the keratin 14 promoter, resulted in hyperplasia of cutaneous lymphatic vessels, but not of blood vessels [17]. Recently, we have identified VEGF-C as a growth factor for HIV-associated Kaposi’s sarcomas, inducing proliferation of cultured KS cells in vitro [18]. VEGF-C is expressed within Kaposi’s sarcomas, and the tumor cells strongly express Flt4/VEGFR-3, demonstrating the lymphatic origin and/or differentiation of this vascular tumor [18,19]. Importantly, VEGF-C expression in vascular endothelial cells is induced by
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Fig. 3. Overlapping binding patterns of members of the VEGF family of angiogenesis factors to endothelial cell VEGF receptors (VEGFR). PlGF-2 and VEGF165 bind to the neuropilin receptor.
VEGF, suggesting a possible molecular link between angiogenesis and lymphangiogenesis. The direct biological roles of VEGF-C for lymphangiogenesis and lymphatic tumor metastasis are currently under investigation. Similar to VEGFC, VEGF-D also binds to the Flt4/VEGFR-3 and to KDR/VEGFR-2; however, the biological importance of VEGF-D for lymphangiogenesis remains to be established.
4. Thrombospondin-1 (TSP-1) TSP-1 belongs to a family of matricellular proteins (TSP-1 to TSP-5) that mediate interactions between extracellular matrix molecules such as collagen and cellular integrin receptors such as avb3 [20]. TSP-1 is a 450 kDa modular, homotrimeric glycoprotein, containing a procollagen homology region, three properdin-like type I repeats, three epidermal growth factor-like repeats, and seven Ca2 + -binding repeats (Fig. 4). TSP-1 is involved in a large number of biological processes, including cell proliferation, migration and differentiation of various cell types; moreover, TSP-1 inhibits endothelial cell proliferation and migration and has been shown to inhibit angiogenesis in vivo [21]. In normal human skin, TSP-1 is expressed by basal epidermal keratinocytes and is deposited in the dermo-epidermal basement membrane zone, contributing to the barrier that
prevents ingrowth of blood vessels into the dermis. In contrast, TSP-1 expression is downregulated in squamous cell carcinomas of the skin [22]. We recently demonstrated that reintroduction of the TSP-1 gene into A431 and SCC-13 squamous cell carcinomas significantly inhibited tumor growth after intradermal transplantation onto immunodeficient mice [22] . The antitumoral effect of TSP-1 was mainly due to its inhibition of tumor angiogenesis, leading to enhanced tumor cell necrosis. Three potential mechanisms have been suggested to mediate the anti-angiogenic and anti-tumoral effects of TSP-1: (1) Two CSVTCG sequences that are contained within the second and third type I repeats interact with the CD36 receptor on endothelial cells to induce endothelial
Fig. 4. Modular structure of the two matricellular, endogenous angiogenesis inhibitors, TSP-1 and TSP-2. PC, procollagen homology region. Both molecules contain three properdin-like type I repeats, three EGF-like type II repeats, and seven Ca2 + -binding type III repeats.
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cell apoptosis [23]. (2) TSP-1 contains a RFK sequence within the second type I repeat that activates TGF-b. Indeed, the phenotype of TSP-1deficient mice could partially be reversed by systemic application of the synthetic peptide LSKL that inhibits TSP-1 mediated TGF-b activation [24]. (3) Distinct heparin-binding sequences within the TSP-1 type I repeats have been shown to inhibit endothelial cell migration in vitro and might also play a role in the antitumoral activity [25]. More recently, we have shown that overexpression of TSP-1 in the skin of transgenic mice results in impaired wound healing, mainly through inhibition of early granulation tissue formation [26].
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Furthermore, our recent evidence, using a chemical skin carcinogenesis protocol in genetically altered mice, suggests that the expression of TSP-2 in normal skin protects from skin cancer development. Although the TSP-2 molecule does not contain the TGF-b activating sequence RFK, TSP-2 is a more potent inhibitor of skin angiogenesis than TSP-1 since: (1) TSP-2 deficient mice show a more dramatic increase in cutaneous blood vessels than TSP-1 deficient mice, and (2) the antitumoral effect of TSP-2 was stronger than the effect of TSP-1 [30]. Currently, we are investigating the molecular mechanisms by which TSP-2 exerts its potent anti-angiogenic effects, using recombinant human TSP-2 and TSP-2 fragments and synthetic peptides derived from distinct TSP2 sequences.
5. Thrombospondin-2 (TSP-2) Similar to TSP-1, TSP-2 is a 450 kDa modular glycoprotein (Fig. 4), which is secreted as a disulfide-bonded homotrimer [27] that binds to a variety of several cell surface receptors, including the integrin avb3, low-density lipoprotein-related receptor protein, and heparan sulfate proteoglycans. However, TSP-2 expression during embryonic development and in adult tissues is spatially and temporally different from TSP-1 expression. Moreover, the regulation of TSP-2 gene expression by growth factors is distinct from that of TSP-1. Importantly, mice that are deficient in TSP-2 show increased numbers of blood vessels in the skin, suggesting that TSP-2 functions as an endogenous inhibitor of angiogenesis [28]. Our recent data show that TSP-2 mRNA and protein are expressed in basal epidermal keratinocytes and are downregulated in squamous cell carcinomas of the skin, suggesting that, similar to TSP-1, TSP-2 also contributes to the anti-angiogenic barrier function that prevents vascularization of the epidermis [29]. Using human A431 squamous cell carcinoma cells stably transfected with TSP-2, we recently identified TSP-2 as a novel, potent endogenous inhibitor of tumor growth and angiogenesis [30]. Importantly, co-expression of both TSP-2 and TSP-1 completely prevented any tumor formation, demonstrating synergistic antitumoral effects of both molecules.
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