In vivo evaluation of antitumoral and antiangiogenic effect of imiquimod-loaded polymeric nanoparticles.

The chemotherapeutic agent imiquimod (Imq) is used to treat skin cancers, the most common type of human cancer. However, the high incidence of local and systemic side effects associated with its use as well as its low skin permeation impair patient compliance and therapeutic effectiveness To overcome these limitations, nanostructured systems such as nanoparticles can be a promising alternative. Nanoparticles are submicron particles (size less than 1000 nm) with high surface area that facilitates the interaction and cellular uptake by biological membranes. Therefore, the aim of the present work is to evaluate antiangiogenic effect and antitumoral activity of imiquimod-loaded nanoparticles compared to market Imq formulation. Polymeric nanoparticles containing Imq were obtained by the technique of precipitation of preformed polymer. Antiangiogenic activity of the formulations was determined in chicken embryo chorioallantoic membrane (CAM) and its chemopreventive potential was evaluate during multistage DMBA and croton oil model of skin carcinogenesis in mice. Nanoparticles containing Imq presented antiangiogenic activity superior than negative control, placebo dispersion and market Imq (p < 0.05) in the CAM model and also significantly reduced the number and size of papillomas compared to all other groups. These results suggest, therefore, that the obtained delivery system can be an alternative to treat diseases related to vessels formation and also potentially increase cutaneous permeation and efficacy of poor soluble drugs normally used to treat cutaneous diseases.

Identification of Feeder Vessels in Ocular Surface Neoplasia Using Indocyanine Green Angiography.

PURPOSE: To evaluate indocyanine green angiography (ICGA) for the identification and characterization of afferent (feeding) and efferent (draining) vessels in patients with ocular surface neoplasia. MATERIALS AND METHODS: Consecutive patients with biopsy-proven benign, pre-invasive, or invasive ocular surface tumors of the bulbar conjunctiva were included. Patients underwent anterior segment optical coherence tomography, ICGA, and color photography for the evaluation of the thickness, location, number, and diameter of afferent and efferent vessels of the lesions. RESULTS: Twenty-two eyes of 22 patients with papillomas (n = 4), intra-epithelial neoplasia lesion (n = 2) in situ or invasive carcinomas (n = 6), nevus (n = 5), conjunctival melanocytic intra-epithelial neoplasia lesion (n = 1), and in situ or invasive melanomas (n = 4) were investigated. Afferent (feeder) vessels were identified in all lesions. There were fewer afferent (3.1 ± 1.6) than efferent (7.5 ± 3.5) vessels per lesion (p < 0.001) and the mean diameter was smaller for afferent (101 ± 62 µm, 28-281) than efferent vessels (137 ± 51 µm, 31-652; p = 0.017). The number of afferent and efferent vessels was associated with the thickness of the lesion (p = 0.037, p < 0.01). Lesion filling times differed between benign and invasive or pre-invasive lesions (p = 0.018). CONCLUSIONS: ICGA is a useful adjunctive in vivo imaging method for the assessment of the vasculature in patients with suspected ocular surface neoplasia.

Incomplete Dll4/Notch signaling inhibition promotes functional angiogenesis supporting the growth of skin papillomas.

BACKGROUND: In invasive malignancies, Dll4/Notch signaling inhibition enhances non-functional vessel proliferation and limits tumor growth by reducing its blood perfusion. METHODS: To assess the effects of targeted Dll4 allelic deletion in the incipient stages of tumor pathogenesis, we chemically induced skin papillomas in wild-type and Dll4 (+/-) littermates, and compared tumor growth, their histological features, vascularization and the expression of angiogenesis-related molecules. RESULTS: We observed that Dll4 down-regulation promotes productive angiogenesis, although with less mature vessels, in chemically-induced pre-cancerous skin papillomas stimulating their growth. The increase in endothelial activation was associated with an increase in the VEGFR2 to VEGFR1 ratio, which neutralized the tumor-suppressive effect of VEGFR-targeting sorafenib. Thus, in early papillomas, lower levels of Dll4 increase vascularization through raised VEGFR2 levels, enhancing sensitivity to endogenous levels of VEGF, promoting functional angiogenesis and tumor growth. CONCLUSION: Tumor promoting effect of low-dosage inhibition needs to be considered when implementing Dll4 targeting therapies.

Dual-energy contrast-enhanced digital mammography in routine clinical practice in 2013.

To date, analysis of the vascularisation of breast lesions mainly relies on MR imaging. However, the accessibility of MRI is sometimes limited and has led to the development of new means of imaging, such as dual-energy contrast-enhanced mammography, which provides data on the vascularisation of the breast along with the usual morphological information. The purpose of this paper is to present this new imaging technique as well as the recent references, illustrated by clinical reports derived from our everyday practice to focus on the advantages and disadvantages of this new breast exploration. Dual-energy contrast-enhanced mammography is a recent, seemingly promising technique, in the management of breast cancer. The main advantages consist of its easy installation, the good tolerance and the comfort in the interpretation of difficult to read mammograms. However, the indications and the role of dual-energy contrast-enhanced mammography still have to be determined within the diagnostic strategy of breast tumours. New studies are expected, especially to compare dual-energy contrast-enhanced mammography with breast MRI.

Mast cells are dispensable for normal and activin-promoted wound healing and skin carcinogenesis.

The growth and differentiation factor activin A is a key regulator of tissue repair, inflammation, fibrosis, and tumorigenesis. However, the cellular targets, which mediate the different activin functions, are still largely unknown. In this study, we show that activin increases the number of mature mast cells in mouse skin in vivo. To determine the relevance of this finding for wound healing and skin carcinogenesis, we mated activin transgenic mice with CreMaster mice, which are characterized by Cre recombinase-mediated mast cell eradication. Using single- and double-mutant mice, we show that loss of mast cells neither affected the stimulatory effect of overexpressed activin on granulation tissue formation and reepithelialization of skin wounds nor its protumorigenic activity in a model of chemically induced skin carcinogenesis. Furthermore, mast cell deficiency did not alter wounding-induced inflammation and new tissue formation or chemically induced angiogenesis and tumorigenesis in mice with normal activin levels. These findings reveal that mast cells are not major targets of activin during wound healing and skin cancer development and also argue against nonredundant functions of mast cells in wound healing and skin carcinogenesis in general.

A novel function of p38-regulated/activated kinase in endothelial cell migration and tumor angiogenesis.

The p38 mitogen-activated protein kinase (MAPK) pathway has been implicated in both suppression and promotion of tumorigenesis. It remains unclear how these 2 opposite functions of p38 operate in vivo to impact cancer development. We previously reported that a p38 downstream kinase, p38-regulated/activated kinase (PRAK), suppresses tumor initiation and promotion by mediating oncogene-induced senescence in a murine skin carcinogenesis model. Here, using the same model, we show that once the tumors are formed, PRAK promotes the growth and progression of skin tumors. Further studies identify PRAK as a novel host factor essential for tumor angiogenesis. In response to tumor-secreted proangiogenic factors, PRAK is activated by p38 via a vascular endothelial growth factor receptor 2 (VEGFR2)-dependent mechanism in host endothelial cells, where it mediates cell migration toward tumors and incorporation of these cells into tumor vasculature, at least partly by regulating the phosphorylation and activation of focal adhesion kinase (FAK) and cytoskeletal reorganization. These findings have uncovered a novel signaling circuit essential for endothelial cell motility and tumor angiogenesis. Moreover, we demonstrate that the tumor-suppressing and tumor-promoting functions of the p38-PRAK pathway are temporally and spatially separated during cancer development in vivo, relying on the stimulus, and the tissue type and the stage of cancer development in which it is activated.

Further pharmacological and genetic evidence for the efficacy of PlGF inhibition in cancer and eye disease.

Our findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies.

Reduction in IkappaB kinase alpha expression promotes the development of skin papillomas and carcinomas.

We reported recently a marked reduction in IkappaB kinase alpha (IKKalpha) expression in a large proportion of human poorly differentiated squamous cell carcinomas (SCC) and the occurrence of Ikkalpha mutations in human SCCs. In addition, overexpression of IKKalpha in the epidermis inhibited the development of skin carcinomas and metastases in mice. However, whether a reduction in IKKalpha expression promotes skin tumor development is currently unknown. Here, we assessed the susceptibility of Ikkalpha hemizygotes to chemical carcinogen-induced skin carcinogenesis. Ikkalpha+/- mice developed 2 times more papillomas and 11 times more carcinomas than did Ikkalpha+/+ mice. The tumors were larger in Ikkalpha+/- than in Ikkalpha+/+ mice, but tumor latency was shorter in Ikkalpha+/- than in Ikkalpha+/+ mice. Some of the Ikkalpha+/- papillomas and most Ikkalpha+/- carcinomas lost the remaining Ikkalpha wild-type allele. Somatic Ikkalpha mutations were detected in carcinomas and papillomas. The chemical carcinogen-induced H-Ras mutations were detected in all the tumors. The phorbol ester tumor promoter induced higher mitogenic and angiogenic activities in Ikkalpha+/- than in Ikkalpha+/+ skin. These elevated activities were intrinsic to keratinocytes, suggesting that a reduction in IKKalpha expression provided a selective growth advantage, which cooperated with H-Ras mutations to promote papilloma formation. Furthermore, excessive extracellular signal-regulated kinase and IKK kinase activities were observed in carcinomas compared with those in papillomas. Thus, the combined mitogenic, angiogenic, and IKK activities might contribute to malignant conversion. Our findings provide evidence that a reduction in IKKalpha expression promotes the development of papillomas and carcinomas and that the integrity of the Ikkalpha gene is required for suppressing skin carcinogenesis.

Angiogenesis in preinvasive, early invasive bronchial lesions and micropapillomatosis and correlation with EGFR expression.

AIMS: To study the association between morphological changes of the bronchial epithelium and its angiogenic status evaluated by microvessel count (MVC), in order to gain a better understanding of bronchial carcinogenesis. Also, to correlate MVC with epidermal growth factor receptor (EGFR) expression. METHODS AND RESULTS: Eighty-three biopsy specimens were assessed for MVC: four normal bronchial epithelia, 23 hyperplasias, 26 metaplasias, two mild dysplasias, five moderate dysplasias, nine severe dysplasias, three carcinomas in situ, six early invasive squamous cell carcinomas (EIC) and five cases of micropapillomatosis. We observed a statistically significant difference in terms of MVC between EIC and all other subgroups and between micropapillomatosis and all other subgroups. There was also a statistically significant difference between micropapillomatosis and EIC. We did not observe any difference in MVC between normal mucosa, metaplasias, hyperplasias, dysplasias or carcinoma in situ. EGFR expression was higher in severe dysplasia, carcinoma in situ and EIC, whereas it was very low in micropapillomatosis. A statistically significant difference was observed in the expression profile of EGFR vs. MVC. EGFR expression was increased in severe dysplasia, whereas an increase in MVC occurred only in EIC. CONCLUSION: During bronchial carcinogenesis, except for micropapillomatosis, EGFR expression appears to be a prerequisite for neoangiogenesis in bronchial carcinogenesis.

c-Myc interacts with hypoxia to induce angiogenesis in vivo by a vascular endothelial growth factor-dependent mechanism.

The proto-oncogene c-myc is involved in the regulation of cell proliferation, differentiation, and apoptosis. In this study, we used an inducible transgenic mouse model in which c-Myc was targeted to the epidermis and, after activation, gave rise to hyperplastic and dysplastic skin lesions and to dermal angiogenesis, involving both vascular endothelial growth factor (VEGF) receptor-1 and VEGF receptor-2. After c-Myc activation, VEGF mRNA was expressed in postmitotic keratinocytes where it colocalized with transgene expression and areas of tissue hypoxia, suggesting a role of hypoxia in VEGF induction. In vitro, c-Myc activation alone was able to induce VEGF protein release and in conjunction with hypoxia, c-Myc activation further increased VEGF protein. Blocking VEGF signaling in vivo significantly reduced dermal angiogenesis, demonstrating the importance of VEGF as a mediating factor for the c-Myc-induced angiogenic phenotype.

Loss of vascular endothelial growth factor a activity in murine epidermal keratinocytes delays wound healing and inhibits tumor formation.

The angiogenic cytokine vascular endothelial growth factor (VEGF)-A plays a central role in both wound healing and tumor growth. In the skin, epidermal keratinocytes are a major source of this growth factor. To study the contribution of keratinocyte-derived VEGF-A to these angiogenesis-dependent processes, we generated mice in which this cytokine was inactivated specifically in keratin 5-expressing tissues. The mutant mice were macroscopically normal, and the skin capillary system was well established, demonstrating that keratinocyte-derived VEGF-A is not essential for angiogenesis in the skin during embryonic development. However, healing of full-thickness wounds in adult animals was appreciably delayed compared with controls, with retarded crust shedding and the appearance of a blood vessel-free zone underneath the newly formed epidermis. When 9,12-dimethyl 1,2-benzanthracene was applied as both tumor initiator and promoter, a total of 143 papillomas developed in 20 of 23 (87%) of control mice. In contrast, only three papillomas arose in 2 of 17 (12%) of the mutant mice, whereas the rest merely displayed epidermal thickening and parakeratosis. Mutant mice also developed only 2 squamous cell carcinomas, whereas 11 carcinomas were found in seven of the control animals. These data demonstrate that whereas keratinocyte-derived VEGF-A is dispensable for skin vascularization under physiological conditions, it plays an important albeit nonessential role during epidermal wound healing and is crucial for the development of 9,12-dimethyl 1,2-benzanthracene-induced epithelial skin tumors.

Modulation of the angiogenesis response through Ha-ras control, placenta growth factor, and angiopoietin expression in mouse skin carcinogenesis.

Tumor angiogenesis is governed by a complex balance of positive and negative angiogenic factors. Development of chemically-induced mouse skin tumors appears to be highly dependent on an early burst of neovascularization. We have previously shown that Ha-ras-driven vascular endothelial growth factor (VEGF) expression plays a pivotal role in this process. However, the status of other critical positive and negative angiogenic factors throughout skin tumorigenesis has not been studied to the same extent. In the present study, we show that another VEGF family member, placenta growth factor (PlGF), was highly upregulated at all tumor stages in a ras-dependent manner. The study of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), ligands of receptor tyrosine kinase 2 (Tie-2), showed that while stroma-derived Ang-2 was increased, epidermal Ang-1 expression was completely abolished at early papilloma formation. Studies using epidermal tumor cell lines suggest that the disappearance of Ang-1 also depends on ras activation, extending the plethora of events controlled by this oncogene in mouse skin carcinogenesis. Our results indicated that tumor development occurred in a strong angiogenesis-prone scenario in which PlGF and Ang-2 acted cooperatively with VEGF, whereas the negative or stabilizing effect of Ang-1 was abrogated. A time-course sequence of expression of angiogenic factors expressed throughout tumor growth, as well as the identification of key signaling molecules triggering the angiogenic response, may contribute to the development and testing of antiangiogenic therapeutic strategies with this in vivo tumor model.

Free radical scavenging, antitumor and anticarcinogenic activity of gossypin.

Antioxidant, antitumor and anticarcinogenic activity of gossypin (3,5,8,3',4'-pentahydroxy-7-O glucosyl flavone) was carried out. The compound needed for 50% inhibition of superoxide, hydroxyl and nitric oxide radicals was 3 microg/ml, 41 microg/ml and 12 microg/ml, respectively. Gossypin also impart 50% inhibition at concentrations 37 microg/ml and 43 microg/ml, respectively for in vitro lipid peroxidation. The compound shows IC50 of 30 microM, 42.5 microM and 45.1 microM concentrations in L 929, HT 29 and K 562 cell lines in 72 hrs MTT assay. The compound shows a zone of inhibition of 8 mm in topo I and topo II inhibition assay in Saccharomyces ceriviseae mutant cultures. It reduces the tumor burden in solid tumor harboring animals (p < 0.001) and effectively inhibits the formation of new blood vessels on tumor mass. 20 mg/kg b.wt of gossypin increase the life span of ascites tumor harboring animals (100%) and (164.7%) respectively by oral and intraperitoneal administration of the drug. Gossypin reduced the incidence of papilloma formation and papilloma/mouse in DMBA/ croton oil induced skin papilloma.

A critical role for ras-mediated, epidermal growth factor receptor-dependent angiogenesis in mouse skin carcinogenesis.

Epidermal growth factor receptor (EGFR) plays a critical role in epidermal biology. Abnormal EGFR function has been described in epithelial tumors including those induced by two-stage chemical carcinogenesis in mouse skin. A large body of evidence indicates that in this model, activation of Ha-ras is the critical event in papilloma formation, a process that involves epidermal proliferation and stroma remodeling, which includes angiogenesis. This study reports that activated Ha-ras results in a dramatic induction of EGFR in epidermal tumor cells and provides experimental evidence that EGFR signaling is responsible for Ha-ras-dependent vascular endothelial growth factor (VEGF) induction, as well as for the repression of other angiogenic factors such as angiopoietin 1. The pivotal role of functional EGFR in throwing the angiogenic switch necessary for tumor growth was confirmed by s.c. injection of immunodeficient mice with epidermal tumor cells carrying a dominant negative (dn) EGFR and by in vivo chemical skin carcinogenesis assays in transgenic mice expressing the same dn EGFR form in the epidermis. Immunohistochemical analysis of the tumors obtained by both ex vivo and in vivo approaches showed that dn EGFR expression abolished the changes in blood vessels that occurred during tumor progression. A strong reduction of VEGF expression in dn EGFR tumors appears to be the key event responsible for angiogenesis and tumor growth suppression. The apoptotic rate was increased, and Akt activity was decreased, suggesting that impaired nutrient and oxygen supply might contribute to diminished cell survival in dn EGFR tumors. Support for this mechanism is provided by the fact that the ectopic expression of VEGF in dn EGFR-expressing tumor cell lines restored tumor growth capacity. Although ras activation might suffice for epidermal transformation and the stroma-remodeling events of tumor induction, such effects may not be operative without a functional upstream EGFR. It is tempting to speculate that EGFR family members may function as angiogenic regulators in other epithelial tumors such as those of the colon, breast, and prostate, reinforcing their value as targets for therapeutic intervention.

Inducible expression of transforming growth factor beta1 in papillomas causes rapid metastasis.

Transforming growth factor beta1 (TGF-beta1) acts as a tumor suppressor at early stages of carcinogenesis, however, it has also been suggested to promote tumor progression at late stages. To determine at which stage and by what mechanisms this functional switch occurs, we have generated gene-switch-TGF-beta1 mice in which TGF-beta1 transgene expression can be induced in skin tumors at specific stages. These mice were exposed to a chemical carcinogenesis protocol, which allows tumorigenesis to develop in progressive stages from benign papillomas to malignant carcinomas. Remarkably, TGF-beta1 transgene induction in papillomas rapidly induced metastasis. This function is in sharp contrast to its tumor suppressive effect when TGF-beta1 transgene expression was induced early in the protocol. Transgenic papillomas exhibited down-regulation of TGF-beta receptors and their signal transducer, the Smads, and loss of the invasion suppressor E-cadherin/catenin complex in the cell membrane. These molecules were lost only in malignant carcinomas in control mice at a much later stage. Furthermore, transgenic papillomas exhibited elevated expression of matrix metalloproteinases and increased angiogenesis. Our study suggests that TGF-beta1 overexpression may directly induce tumor metastasis by initiating events necessary for invasion. Down-regulation of TGF-beta signaling components in tumor epithelia selectively abolishes growth inhibition, thus, switching the role of TGF-beta1 to a metastasis promoter.

Ethylnitrosourea induces neoplasia in zebrafish (Danio rerio).

The zebrafish (Danio rerio) has been successfully used to discover hundreds of genes involved in development and organogenesis. To address the potential of zebrafish as a cancer model, it is important to determine the susceptibility of zebrafish to tumors. Germ line mutations are most commonly induced for zebrafish mutant screens by exposing adult male zebrafish to the alkylating agent, ethylnitrosourea (ENU). To determine whether ENU induces tumors, we compared the incidence of tumors in ENU-treated fish with untreated controls. Interestingly, 18 of 18 (100%) fish mutagenized with either 2.5 or 3.0 mM ENU developed epidermal papillomas, which numbered 1 to 22 per fish, within 1 year of treatment. The induced epidermal lesions included epidermal hyperplasia, flat papillomas (0.2 to 1.2 mm), and pedunculated papillomas (1.2 to 8 mm in greatest dimension), but no skin cancers. Angiogenesis was evident in papillomas larger than approximately 1 mm. All but two papillomas contained the three cell types (keratinocytes, club, and mucous cells) of normal zebrafish epidermis; histologic variants lacked either club cells or mucous cells. Two cavernous hemangiomas and a single malignant peripheral nerve sheath tumor were also found in the treated fish. None of five untreated controls developed tumors. These studies establish the feasibility of the zebrafish as an experimental model for the study of skin tumors.

Blocking transforming growth factor beta signaling in transgenic epidermis accelerates chemical carcinogenesis: a mechanism associated with increased angiogenesis.

Mutations in the transforming growth factor beta type II receptor (TGF-betaRII) have been identified in human cancers, which suggests a causal role for the loss of TGF-betaRII in cancer development. To directly test this in vivo, we have generated transgenic mice expressing a dominant negative TGF-betaRII (delta betaRII) in the epidermis, using a truncated mouse loricrin promoter (ML). ML.delta betaRII transgenic mice exhibited a thickened skin due to epidermal hyperproliferation. When these mice were subjected to a standard two-stage chemical carcinogenesis protocol, they exhibited an increased sensitivity, with an earlier appearance and a 2-fold greater number of papillomas than control mice. In addition, papillomas in control mice regressed after termination of 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment; whereas ML.delta betaRII papillomas progressed to carcinomas. Furthermore, TPA promotion alone induced papilloma formation in ML.delta betaRII mice, which suggests an initiating role for delta betaRII in skin carcinogenesis. ML.delta betaRII tumors also exhibited increased neovascularization and progressed to metastases, although the primary tumors were still classified as carcinoma in situ or well-differentiated carcinomas. Increased expression of vascular endothelial growth factor, an angiogenesis factor, and decreased expression of thrombospondin-1, an angiogenesis inhibitor, were also observed in ML.delta betaRII tumors. The increased angiogenesis correlated with elevated endogenous TGF-beta1 in ML.delta betaRII tumors. These data provide in vivo evidence that inactivation of TGF-betaRII accelerates skin carcinogenesis at both earlier and later stages, and increased angiogenesis is one of the important mechanisms of accelerated tumor growth and metastasis.

Analysis of centrosome abnormalities and angiogenesis in epidermal-targeted p53172H mutant and p53-knockout mice after chemical carcinogenesis: evidence for a gain of function.

We previously developed a transgenic mouse model that expresses in the epidermis a murine p53172R-->H mutant (p53m) under the control of a human keratin-1-based vector (HK1.p53m). In contrast to mice with wild-type p53 and p53-knockout mice, HK1.p53m mice exhibit increased susceptibility to chemical carcinogenesis, with greatly accelerated benign papilloma formation, malignant conversion, and metastasis. In the study presented here, we examined the expression pattern of several differentiation markers and observed that p53m tumors exhibited a less differentiated phenotype than tumors elicited in non-transgenic mice. Metastasis in p53m tumors was also associated with a poorly differentiated phenotype. To determine whether genomic instability was associated with a putative gain-of-function role for this p53m, in situ examination of centrosomes was performed in HK1.p53m and equivalent p53-null papillomas. In contrast to HK1.p53m papillomas, which had centrosome abnormalities at high frequencies (75% of cells contained more than three centrosomes/cell), p53-null tumors exhibited few abnormal centrosomes (4% of cells contained more than three centrosomes/cell). To determine whether angiogenesis played a role in the rapid progression of p53m tumors, the expression of vascular endothelial growth factor, a promoter of angiogenesis, and thrombospondin-1, an inhibitor of angiogenesis, was examined in tumors derived from either p53m or p53-knockout mice. Regardless of their p53 status (wild type, p53m, p53-/-), all of the papillomas exhibited similar levels of vascular endothelial growth factor expression and decreased expression of thrombospondin-1 as did normal epidermis. In addition, tumors from different p53 genotypes showed a similar density of blood vessels. Because p53 status did not appear to play an overt role in angiogenesis, these data suggest that p53m accelerates tumorigenesis primarily by exerting a gain of function associated with genomic instability.

VEGF/VPF overexpression in skin of transgenic mice induces angiogenesis, vascular hyperpermeability and accelerated tumor development.

Upregulation of keratinocyte-derived VEGF-A expression has recently been established in non-neoplastic processes of skin such as wound healing, blistering diseases and psoriasis, as well as in skin neoplasia. To further characterize the effects of VEGF-A in skin in vivo, we have developed transgenic mice expressing the mouse VEGF120 under the control of a 2.4 kb 5' fragment of keratin K6 gene regulatory sequences that confers transgene inducibility upon hyperproliferative stimuli. As expected from the inducible nature of the transgene, two of the three founder mice obtained (V27 and V208), showed no apparent phenotype. However, one founder (V2), mosaic for transgene integration, developed scattered red spots throughout the skin at birth. The transgenic offspring derived from this founder developed a striking phenotype characterized by swelling and erythema, resulting in early postnatal lethality. Histological examination of the skin of these transgenics demonstrated highly increased vascularization and edema leading to disruption of skin architecture. Expression of the transgene was silent in adult animals of lines derived from founders V27 and V208. Phorbol ester-induced hyperplasia resulted in transgene induction and increased cutaneous vascularization in adult transgenic mice of these lines. Skin carcinogenesis experiments performed on hemizygous crosses of V208 mice with activated H-ras-carrying transgenic mice (TG.AC) resulted in accelerated papilloma development and increased tumor burden. Previous results from our laboratory showed that VEGF upregulation is a major angiogenic stimulus in mouse epidermal carcinogenesis. By overexpressing VEGF in the skin of transgenic mice we now move a step further toward showing that VEGF-mediated angiogenesis is a rate-limiting step in the genesis of premalignant lesions, such as mouse skin papilloma. Our transgenic mice constitute an interesting model system for in vivo study of the cutaneous angiogenic process and its relevance in tumorigenesis and other skin diseases.