BackgroundWe have previously reported stromal upregulation of the endogenous angiogenesis inhibitor thrombospondin-2 (TSP-2) during multistep carcinogenesis, and we found accelerated and enhanced skin angiogenesis and carcinogenesis in TSP-2 deficient mice.GoalsTo investigate whether enhanced levels of TSP-2 might protect from skin cancer development.MethodsWe established transgenic mice with targeted overexpression of TSP-2 in the skin and subjected hemizygous TSP-2 transgenic mice and their wild-type littermates to a chemical skin carcinogenesis regimen.ResultsTSP-2 transgenic mice showed a significantly delayed onset of tumor formation compared to wild-type mice, whereas the ratio of malignant conversion to squamous cell carcinomas was comparable in both genotypes. Computer-assisted morphometric analysis of blood vessels revealed pronounced tumor angiogenesis already in the early stages of carcinogenesis in wild type mice. TSP-2 overexpression significantly reduced tumor blood vessel density in transgenic mice but had no overt effect on LYVE-1 positive lymphatic vessels. The percentage of desmin surrounded, mature tumor-associated blood vessels and the degree of epithelial differentiation remained unaffected. The antiangiogenic effect of transgenic TSP-2 was accompanied by a significantly increased number of apoptotic tumor cells in transgenic mice.ConclusionOur results demonstrate that enhanced levels of TSP-2 in the skin result in reduced susceptibility to chemically-induced skin carcinogenesis and identify TSP-2 as a new target for the prevention of skin cancer.

Introduction: Chemically-induced skin carcinogenesis is a well established cancer model in mice that allows a detailed evaluation of distinct stages of carcinogenesis, and that has also provided a more in-depth understanding of human epithelial cancer development. We have recently reported stromal upregulation of the endogenous angiogenesis inhibitor thrombospondin-2 (TSP-2) during multistage skin carcinogenesis, and we found accelerated and enhanced tumor development and angiogenesis in TSP-2 deficient mice. Aim: To investigate whether enhanced levels of TSP-2 might protect from cancer development. Methods: We generated transgenic FVB mice overexpressing TSP-2 under the control of the keratin K14 promoter. TSP-2 transgenic mice and wild-type littermates were subjected to a standard two-step skin carcinogenesis regimen. For initiation, mice were treated once with 50µg DMBA, applied to the dorsal skin, followed by 20 weekly topical applications of 5µg of the tumor promoter PMA. Results: TSP-2 transgenic mice showed a delayed onset of tumor formation with an average latency period of 18 weeks, as compared to 14 weeks in wild-type mice. At 18 weeks after initiation of PMA promotion, the percentage of tumor-bearing mice was 100% in wild-type, as compared to 52% in TSP-2 transgenic mice. Moreover, the number of benign papillomas and malignant SCCs was significantly reduced in TSP-2 transgenic mice when compared to wild-type littermates. However, the ratio of malignant conversion papillomas to SCC was comparable in both genotypes. Quantitative analysis of tumor-associated blood vessels revealed that both vessel density and vessel size were decreased in TSP-2 transgenic mice. Conclusion: These results demonstrate that enhanced levels of TSP-2 result in reduced susceptibility to chemically-induced skin carcinogenesis, and they identify TSP-2 as a potential new target for the prevention of cancer development and progression.

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 OTSR) and expression of just the second type-1 repeat containing the transforming growth factor (TGF)-beta activating sequence KRFK (TSR2 + IURFK) 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 endogenons TSP-1 involves regulation of both active and total TGF-beta and the sequences KRFK and WSHWSPW in the second type-1 repeat.

Die Neubildung von Blutgefäßen ist für das Tumorwachstum unerlässlich. Die unterschiedlichen Funktionen von Angiopoietin-1 (Ang1) und Ang2, die Gefäßstabilität regulierende, gegensätzlich wirkende Liganden am endothel-spezifischen Tie2-Rezeptor, bei der Entwicklung und Progression von Tumoren ist noch weitestgehend ungeklärt. Wir analysierten mittels in situ Hybridisierung die Expression von Ang1 und Ang2 während der chemisch induzierten Karzinogenese in der Epidermis von Mäusen, einem Modell, das bekanntermaßen die Grundlage für das Konzept der mehrstufigen Entstehung maligner Tumoren bildet. Wir untersuchten außerdem in einem Xenotransplantationsmodell das Wachstum und die Angiogenese von A431-Plattenepithelkarzinomen, die nach stabiler Transfektion Ang1 oder Ang2 überexprimierten. Während der aufeinander folgenden Karzinogenesestadien war die Expression von Ang2, jedoch nicht die von Ang1, in den Tumorgefäßen deutlich hochreguliert. In den Ang1 überexprimierenden Tumoren fand sich eine gesteigerte Tie2-Rezeptorphosphorylierung, die mit einem signifikant verminderten Tumorwachstum einherging. Im Gegensatz dazu führte die Überexpression von Ang2 zu keiner gesteigerten Tie2-Rezeptoraktivierung und hatte weder Einfluss auf das Wachstum noch auf die Angiogenese der A431-Tumoren. Im Vergleich zu den Kontrollen wurden in den Ang1 überexprimierenden Tumoren keine wesentlichen Veränderungen der Tumorgefäßdichte beobachtet. Der Anteil, der von periendothelialen, alpha-smooth-muscle-actin positiven Zellen umgebenden Tumorgefäße war jedoch in den Ang1 exprimierenden Tumoren deutlich erhöht, was auf einen reiferen Status der Tumorgefäße hinweist. Zusammenfassend belegen unsere Daten eine wachstumshemmende Funktion der durch Ang1 vermittelten Tie2-Rezeptoraktivierung in Plattenepithelkarzinomen und lassen zudem eine wichtige Funktion des Reifestatuts von Tumorgefäßen für die Tumorprogression vermuten.

We have previously shown that stable overexpression of the thrombospondin-2 (TSP-2) gene inhibited the tumor growth and angiogenesis of human squamous cell carcinoma xenotransplants. To investigate the potential antitumoral efficacy of systemic TSP-2 therapy, we expressed a recombinant 80 kDa fragment of human TSP-2 (TSP-2/NTF), encompassing the N-terminal globular region through the three type 1 repeats, in human kidney 293 EBNA cells, using a modified pCEP4 expression vector. Daily intraperitoneal injections of TSP-2/NTF resulted in a significant inhibition of the growth of human A431 squamous cell carcinomas in vivo and in reduced tumor vascularization. To further investigate possible mechanisms of the antiangiogenic activity of TSP-2/NTF, several in vitro angiogenesis assays were performed in human dermal microvascular endothelial cells. TSP-2/NTF inhibited vascular endothelial growth factor induced migration of human dermal microvascular endothelial cells and inhibited tube formation on Matrigel in vitro. TSP-2/NTF also inhibited vascular endothelial growth factor induced angiogenesis in an in vivo Matrigel assay. Moreover, TSP-2/NTF potently induced human dermal microvascular endothelial cell apoptosis in vitro but did not affect A431 tumor cell proliferation or apoptosis. These findings identify TSP-2/NTF as a potent systemic inhibitor of tumor growth and angiogenesis, acting by direct inhibition of several endothelial cell functions involved in neovascularization.

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.

Recent studies indicate that continuous administration improves the antitumoral efficacy of angiogenesis inhibitors, as compared with intermittent dosing, suggesting a potential role of gene therapy in antiangiogenic tumor therapy. We established a tissue-engineered implant system for the continuous in vivo production of thrombospondin-2 (TSP-2), a potent endogenous inhibitor of tumor growth and angiogenesis. Fibroblasts were retrovirally transduced to overexpress TSP-2 and were seeded onto biodegradable polymer scaffolds. After transplantation into the peritoneal cavity of nude mice, bioimplants maintained high levels of TSP-2 secretion over extended time periods, resulting in increased levels of circulating TSP-2. Bioimplant-generated TSP-2 potently inhibited tumor growth and angiogenesis of human squamous cell carcinomas, malignant melanomas, and Lewis lung carcinomas that were implanted at a distant site. These results provide the first proof-of-principle for the feasibility and therapeutic efficiency of systemic, cell-based antiangiogenic gene therapy using biodegradable polymer grafts for the treatment of cancer.

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.

Inhibition of tumor angiogenesis represents a promising new approach for the treatment of human cancers. It has remained unclear, however, whether inhibition of tumor angiogenesis may also result in impaired wound healing, a process thought to be angiogenesis dependent. To determine the effects of the angiogenesis inhibitor vasostatin, a 180 amino acid calreticulin fragment, on wound healing at tumor inhibiting doses, full-thickness wounds were generated on the back of nude mice that were also injected intradermally with CA46 Burkitt lymphoma cells. Mice were treated with daily injections of vasostatin or vehicle control at a site between the wounds and the transplanted tumor cells over 14 d. Vasostatin potently inhibited tumor growth and significantly reduced tumor angiogenesis, as measured by computer-assisted image analysis of CD31-stained tumor sections. Moreover, vasostatin treatment resulted in an increased fraction of mature tumor-associated blood vessels. In contrast, no impairment of wound healing was observed in vasostatin-treated mice, despite a significantly reduced vascularity of the wound granulation tissue. Our results reveal a different sensitivity of malignant tumor growth and physiologic wound healing to inhibition of angiogenesis, and they suggest that therapeutic inhibition of tumor angiogenesis may be achieved without impairment of tissue repair.

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.

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.

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.

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 Fc ere 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.

Recent evidence suggests a potential role for thrombospondin-2 (TSP-2), a matricellular glycoprotein, in the regulation of primary angiogenesis. To directly examine the biological effect of TSP-2 expression on tumor growth and angiogenesis, human A431 squamous cell carcinoma cells, which do not express TSP-2, were stably transfected with a murine TSP-2 expression vector or with vector alone. A431 cells expressing TSP-2 did not show an altered growth rate, colony-forming ability, or susceptibility to induction of apoptosis in vitro. However, injection of TSP-2-transfected clones into the dermis of nude mice resulted in pronounced inhibition of tumor growth that was significantly stronger than the inhibition observed in A431 clones stably transfected with a thrombospondin-1 (TSP-1) expression vector, and combined overexpression of TSP-1 and TSP-2 completely prevented tumor formation. Extensive areas of necrosis were observed in TSP-2-expressing tumors, and both the density and the size of tumor vessels were significantly reduced, although tumor cell expression of the major tumor angiogenesis factor, vascular endothelial growth factor, was maintained at high levels. These findings establish TSP-2 as a potent endogenous inhibitor of tumor growth and angiogenesis.