Michael Streit

Delayed wound repair and impaired angiogenesis in mice lacking syndecan-4

The syndecans make up a family of transmembrane heparan sulfate proteoglycans that act as coreceptors with integrins and growth factor tyrosine kinase receptors. Syndecan-4 is upregulated in skin dermis after wounding, and, in cultured fibroblasts adherent to the ECM protein fibronectin, this proteoglycan signals cooperatively with beta1 integrins. In this study, we generated mice in which the syndecan-4 gene was disrupted by homologous recombination in embryonic stem cells to test the hypothesis that syndecan-4 contributes to wound repair. Mice heterozygous or homozygous for the disrupted syndecan-4 gene are viable, fertile, and macroscopically indistinguishable from wild-type littermates. Compared with wild-type littermates, mice heterozygous or homozygous for the disrupted gene have statistically significant delayed healing of skin wounds and impaired angiogenesis in the granulation tissue. These results indicate that syndecan-4 is an important cell-surface receptor in wound healing and angiogenesis and that syndecan-4 is haplo-insufficient in these processes.

The α1β1 and α2β1 Integrins Provide Critical Support for Vascular Endothelial Growth Factor Signaling, Endothelial Cell Migration, and Tumor Angiogenesis

Angiogenesis is a complex process, involving functional cooperativity between cytokines and endothelial cell (EC) surface integrins. In this study, we investigated the mechanisms through which the α1β1 and α2β1 integrins support angiogenesis driven by vascular endothelial growth factor (VEGF). Dermal microvascular EC attachment through either α1β1 or α2β1 supported robust VEGF activation of the Erk1/Erk2 (p44/42) mitogen-activated protein kinase signal transduction pathway that drives EC proliferation. Haptotactic EC migration toward collagen I was dependent on α1β1 and α2β1 as was VEGF-stimulated chemotaxis of ECs in a uniform collagen matrix. Consistent with the functions of α1β1 and α2β1 in supporting signal transduction and EC migration, antibody antagonism of either integrin resulted in potent inhibition of VEGF-driven angiogenesis in mouse skin. Moreover, combined antagonism of α1β1 and α2β1 substantially reduced tumor growth and angiogenesis of human squamous cell carcinoma xenografts. Collectively, these studies identify critical collaborative functions for the α1β1 and α2β1 integrins in supporting VEGF signal transduction, EC migration, and tumor angiogenesis.

Overexpression of Thrombospondin-1 Decreases Angiogenesis and Inhibits the Growth of Human Cutaneous Squamous Cell Carcinomas

The function of the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in epithelial tumor development has remained controversial. We studied the in vitro growth characteristics and the in vivo tumor xenograft growth of the human squamous cell carcinoma cell lines A431 and SCC-13, stably transfected to overexpress human TSP-1. Overexpression of TSP-1 inhibited tumor growth of A431 xenotransplants, and completely abolished tumor formation by SCC-13 cells. TSP-1 overexpressing A431 tumors were characterized by extensive areas of necrosis and by decreased tumor vessel number and size. The effects of TSP-1 on tumor cell growth were indirect since tumor cell proliferation rates in vivo and in vitro, anchorage-dependent and -independent growth in vitro, and susceptibility to induction of apoptosis by serum withdrawal were unchanged in TSP-1 overexpressing tumor cells. However, TSP-1 overexpression up-regulated the TSP-1 receptor CD36, leading to enhanced adhesion of A431 cells to TSP-1. These findings establish TSP-1 as a potent inhibitor of angiogenesis and tumor growth in carcinomas of the skin.

Angiogenesis, lymphangiogenesis, and melanoma metastasis

The induction of angiogenesis is a critical point in the development of most human tumors – including melanomas. Some of the earliest studies in the field of tumor angiogenesis showed that transplantation of human melanoma fragments into the hamster cheek pouch stimulated blood vessel growth. Since then, numerous studies have demonstrated that human melanomas also induce angiogenesis. The prognostic importance of the degree of melanoma vascularization, however, has remained controversial. Elevated expression of several angiogenic factors, including vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8, has been detected in primary cutaneous melanomas, and the importance of these mediators in promoting melanoma angiogenesis and metastasis has been confirmed in tumor xenotransplant models. Based upon these findings, several clinical trials of angiogenesis inhibitors have been initiated in human melanoma patients and are currently underway. Recent experimental evidence indicates that tumor-associated lymphangiogenesis also plays an important role in mediating tumor spread to regional lymph nodes. These observations have important implications for prognosis and treatment of human melanomas.

Thrombospondin‐1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice

The function of the endogenous angiogenesis inhibitor thrombospondin‐1 (TSP‐1) in tissue repair has remained controversial. We established transgenic mice with targeted overexpression of TSP‐1 in the skin, using a keratin 14 expression cassette. TSP‐1 transgenic mice were healthy and fertile, and did not show any major abnormalities of normal skin vascularity, cutaneous vascular architecture, or microvascular permeability. However, healing of full‐thickness skin wounds was greatly delayed in TSP‐1 transgenic mice and was associated with reduced granulation tissue formation and highly diminished wound angiogenesis. Moreover, TSP‐1 potently inhibited fibroblast migration in vivo and in vitro. These findings demonstrate that TSP‐1 preferentially interfered with wound healing‐associated angiogenesis, rather than with the angiogenesis associated with normal development and skin homeostasis, and suggest that therapeutic application of angiogenesis inhibitors might potentially be associated with impaired wound vascularization and tissue repair.

Activation of the tie2 receptor by angiopoietin-1 enhances tumor vessel maturation and impairs squamous cell carcinoma growth.

The distinct roles of angiopoietin (Ang)-1 and Ang2, counteracting ligands for the endothelium-specific Tie2 receptor, in tumor development and progression have remained poorly understood. We investigated the expression of Ang1 and Ang2 during multistep mouse skin carcinogenesis and in human squamous cell carcinoma (SCC) xenografts. Expression of Ang2, but not of Ang1, was up-regulated in angiogenic tumor vessels already in early stages of skin carcinogenesis and was also strongly increased in SCCs. Stable overexpression of Ang1 in human A431 SCCs resulted in a more than 70% inhibition of tumor growth, associated with enhanced Tie2 phosphorylation levels, as compared with low levels in control transfected tumors. No major changes in the vascular density, vascular endothelial growth factor mRNA and protein expression, and vascular endothelial growth factor receptor-2 phosphorylation levels were observed in Ang1-expressing tumors. However, the fraction of tumor blood vessels with coverage by alpha-smooth muscle actin-positive periendothelial cells was significantly increased, indicative of an increased vascular maturation status. These findings identify an inhibitory role of Ang1/Tie2 receptor-mediated vessel maturation in SCC growth and suggest that up-regulation of its antagonist, Ang2, during early-stage epithelial tumorigenesis contributes to the angiogenic switch by counteracting specific vessel-stabilizing effects of Ang1.

Thrombospondin-2 plays a protective role in multistep carcinogenesis: a novel host anti-tumor defense mechanism.

The angiogenic switch during tumorigenesis is thought to be induced by a change in the balance of pro‐ angiogenic and anti‐angiogenic factors. To elucidate the biological role of the endogenous angiogenesis inhibitor thrombospondin‐2 (TSP‐2) during multistep carcinogenesis, we subjected TSP‐2‐deficient and wild‐type mice to a chemical skin carcinogenesis regimen. Surprisingly, TSP‐2 expression was strongly upregulated in the mesenchymal stroma of wild‐type mice throughout the consecutive stages of tumorigenesis whereas the angiogenesis factor, vascular endothelial growth factor, was induced predominantly in tumor cells. TSP‐2 deficiency dramatically enhanced susceptibility to skin carcinogenesis and resulted in accelerated and increased tumor formation. The angiogenic switch occurred in early stages of pre‐malignant tumor formation, and tumor angiogenesis was significantly enhanced in TSP‐2‐deficient mice. While TSP‐2 deficiency did not affect tumor differentiation or proliferation, tumor cell apoptosis was signific antly reduced. These results reveal upregulation of an endogenous angiogenesis inhibitor during multi step tumorigenesis and identify enhanced stromal TSP‐2 expression as a novel host anti‐tumor defense mechanism.

Thrombospondin-1 selectively inhibits early-stage carcinogenesis and angiogenesis but not tumor lymphangiogenesis and lymphatic metastasis in transgenic mice

The roles played by the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in the early stages of multi-step carcinogenesis and in the control of hematogenous versus lymphatic metastasis are unknown. To investigate these issues we compared tumor development in normal mice and in transgenic mice with targeted overexpression of TSP-1 in the epidermis following a standard two-step chemical skin carcinogenesis regimen. Overexpression of TSP-1 resulted in delayed and reduced development of premalignant epithelial hyperplasias, but did not inhibit the malignant conversion to squamous cell carcinomas. TSP-1 overexpression also suppressed tumor angiogenesis and distant organ metastasis, but failed to inhibit tumor-associated lymphangiogenesis or lymphatic tumor spread to regional lymph nodes. Concomitant with these results, we found that the endothelial TSP-1 receptor CD36 was mostly absent from cutaneous lymphatic vessels. Our findings indicate the potential use of TSP-1 for the prevention of premalignant stages of tumorigenesis and are likely to have implications for the further development of anti-angiogenic cancer therapies.

Expression of Vascular Endothelial Growth Factor Induces an Invasive Phenotype in Human Squamous Cell Carcinomas

Inhibition of the vascular endothelial growth factor (VEGF) receptor Flk-1 has been shown to prevent invasion of experimental squamous cell carcinomas (SCC). To directly investigate the role of VEGF in tumor invasion, we stably transfected human SCC-13 cells, which are characterized by a noninvasive phenotype in vivo, with expression vectors containing murine VEGF(164) in sense (SCC/VEGF+) or antisense (SCC/VEGF-) orientation or with vector alone (SCC/vec). SCC/vec cells formed slowly growing, well-differentiated tumors with well-defined borders between tumor and stroma, after intradermal or subcutaneous injection. In contrast, SCC/VEGF+ tumors were characterized by rapid tumor growth, with small cell groups and single cells invading into the surrounding tissue, and by admixture of blood vessels and tumor cells in areas of tumor invasion. We detected an increase in tumor vessel density and size in VEGF-overexpressing tumors, resulting in a more than fourfold increase in total vascular areas. In contrast, SCC/VEGF- clones formed noninvasive, sharply circumscribed tumors with reduced vascular density. These findings demonstrate that selective VEGF overexpression was sufficient to induce tumor invasiveness, and they provide further evidence for an active role of the tumor stroma in cancer progression.

Expression of the Type-1 Repeats of Thrombospondin-1 Inhibits Tumor Growth Through Activation of Transforming Growth Factor-β

In the present study, the type-1 repeats of thrombospondin-1 (TSP-1) were transfected into A431 cells. Expression of all three type-1 repeats (3TSR) and expression of just the second type-1 repeat containing the transforming growth factor (TGF)-beta activating sequence KRFK (TSR2 + KRFK) significantly inhibited in vivo tumor angiogenesis and growth in nude mice. These tumors expressed increased levels of both active and total TGF-beta. A431 cells expressing the second type-1 repeat without the KRFK sequence (TSR2 - KRFK) produced tumors that were slightly larger than the 3TSR and TSR2 + KRFK tumors. These tumors expressed elevated levels of active TGF-beta but levels of total TGF-beta were not different from control tumors. Injection of the peptide, LSKL, which blocks TSP-1 activation of TGF-beta, reversed the growth inhibition observed with cells expressing TSR2 + KRFK to a level comparable to controls. Various residues in the WSHWSPW region and the VTCG sequence of both TSR2+/- KRFK were mutated. Although mutation of the VTCG sequence had no significant effect on tumor growth, mutation of the WSHWSPW sequence reduced inhibition of tumor growth. These findings suggest that the inhibition of tumor angiogenesis and growth by endogenous TSP-1 involves regulation of both active and total TGF-beta and the sequences KRFK and WSHWSPW in the second type-1 repeat.