Thrombospondin-2 overexpression in the skin of transgenic mice reduces the susceptibility to chemically induced multistep skin carcinogenesis.

BACKGROUND: We 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. GOALS: To investigate whether enhanced levels of TSP-2 might protect from skin cancer development. METHODS: We 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. RESULTS: TSP-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. CONCLUSION: Our 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.

RhoB differentially controls Akt function in tumor cells and stromal endothelial cells during breast tumorigenesis.

Tumors are composed of cancer cells but also a larger number of diverse stromal cells in the tumor microenvironment. Stromal cells provide essential supports to tumor pathophysiology but the distinct characteristics of their signaling networks are not usually considered in developing drugs to target tumors. This oversight potentially confounds proof-of-concept studies and increases drug development risks. Here, we show in established murine and human models of breast cancer how differential regulation of Akt by the small GTPase RhoB in cancer cells or stromal endothelial cells determines their dormancy versus outgrowth when angiogenesis becomes critical. In cancer cells in vitro or in vivo, RhoB functions as a tumor suppressor that restricts EGF receptor (EGFR) cell surface occupancy as well as Akt signaling. However, after activation of the angiogenic switch, RhoB functions as a tumor promoter by sustaining endothelial Akt signaling, growth, and survival of stromal endothelial cells that mediate tumor neoangiogenesis. Altogether, the positive impact of RhoB on angiogenesis and progression supercedes its negative impact in cancer cells themselves. Our findings elucidate the dominant positive role of RhoB in cancer. More generally, they illustrate how differential gene function effects on signaling pathways in the tumor stromal component can complicate the challenge of developing therapeutics to target cancer pathophysiology.

Proteolytic cleavage of versican and involvement of ADAMTS-1 in VEGF-A/VPF-induced pathological angiogenesis.

Malignant tumors and chronic inflammatory diseases induce angiogenesis by overexpressing vascular endothelial growth factor A (VEGF-A/VPF). VEGF-A-induced pathological angiogenesis can be mimicked in immunoincompetent mice with an adenoviral vector expressing VEGF-A(164) (Ad-VEGF-A(164)). The initial step is generation of greatly enlarged "mother" vessels (MV) from preexisting normal venules by a process involving degradation of their rigid basement membranes. Immunohistochemical and Western blot analyses revealed that versican, an extracellular matrix component in the basement membranes of venules, is degraded early in the course of MV formation, resulting in the appearance of a versican N-terminal DPEAAE fragment associated with MV endothelial cells. The protease ADAMTS-1, known to cleave versican near its N terminus to generate DPEAAE, is also upregulated by VEGF-A in parallel with MV formation and localizes to the endothelium of the developing MV. The authors also show that MMP-15 (MT-2 MMP), a protease that activates ADAMTS-1, is upregulated by VEGF-A in endothelial cells in vitro and in vivo. These data suggest VEGF-A initiates MV formation, in part, by inducing the expression of endothelial cell proteases such as ADAMTS-1 and MMP-15 that act in concert to degrade venular basement membrane versican. Thus, versican is actively processed during the early course of VEGF-A-induced pathological angiogenesis.

The L6 protein TM4SF1 is critical for endothelial cell function and tumor angiogenesis.

Transmembrane-4-L-six-family-1 (TM4SF1) was originally described as a cancer cell protein. Here, we show that it is highly expressed in the vascular endothelium of human cancers and in a banded pattern in the filopodia of cultured endothelial cells (EC). TM4SF1 knockdown prevented filopodia formation, inhibited cell mobility, blocked cytokinesis, and rendered EC senescent. Integrin-alpha5 and integrin-beta1 subunits gave a similar staining pattern and interacted constitutively with TM4SF1, whereas integrin subunits often associated with angiogenesis (alphaV, beta3, beta5) interacted with TM4SF1 only after vascular endothelial growth factor (VEGF)-A or thrombin stimulation. TM4SF1 knockdown substantially inhibited maturation of VEGF-A(164)-induced angiogenesis. Thus, TM4SF1 is a key regulator of EC function in vitro and of pathologic angiogenesis in vivo and is potentially an attractive target for antiangiogenesis therapy.

Role of vascular endothelial growth factor A in children with acquired airway stenosis.

OBJECTIVES: Vascular endothelial growth factor A (VEGF-A) is important in the angiogenic response for wound healing. This study investigated whether VEGF-A may play a role in the pathogenesis of acquired airway stenosis. METHODS: Eight lesions from 5 pediatric patients with subglottic stenosis after airway reconstruction (N = 4) or prolonged intubation (N = 1) and normal laryngeal tissue from 5 autopsy patients were included. Formalin-fixed sections of subglottic tissue from each patient were examined by in situ hybridization for the presence of messenger RNA (mRNA) for VEGF-A, vascular endothelial growth factor receptor 1 (VEGFR-1), and vascular endothelial growth factor receptor 2 (VEGFR-2). RESULTS: Strong expression of VEGF-A mRNA was noted in hyperplastic squamous epithelium overlying granulation tissue. Strong expression of VEGFR-1 and VEGFR-2 was noted in the endothelial cells within granulation tissue. No strong labeling of VEGF-A mRNA or its receptors was noted in 2 specimens with mature scar tissue or in the control specimens. CONCLUSIONS: The angiogenic growth factor VEGF-A is strongly expressed in hyperplastic epithelium overlying granulation tissue in airway stenosis. Also, VEGFR-1 and VEGFR-2 mRNAs are strongly expressed in the endothelial cells of granulation tissue. This finding suggests an important role of VEGF-A in the pathogenesis of airway scar formation and stenosis.

VEGF-A produced by chronically inflamed tissue induces lymphangiogenesis in draining lymph nodes.

Lymphangiogenesis is involved in tumor cell metastasis and plays a major role in chronic inflammatory disorders. To investigate the role of lymphangiogenesis in inflammation, we induced and maintained delayed-type hypersensitivity (DTH) reactions in the ears of mice and then analyzed the resulting lymphangiogenesis in the inflamed tissue and draining lymph nodes (LNs) by quantitative fluorescence-activated cell sorting (FACS) and by immunofluorescence. Long-lasting inflammation induced a significant increase in the number of lymphatic endothelial cells, not only in the inflamed ears but also in the ear-draining auricular LNs. Inflammation-induced lymphangiogenesis was potently blocked by systemic administration of a vascular endothelial growth factor (VEGF)-A neutralizing antibody. Surprisingly, tissue inflammation specifically induced LN lymphangiogenesis but not LN angiogenesis. These findings were explained by analysis of both VEGF-A protein and mRNA levels, which revealed that VEGF-A was expressed at high mRNA and protein levels in inflamed ears but that expression was increased only at the protein level in activated LNs. Inflammation-induced lymphangiogenesis in LNs was independent of the presence of nodal B lymphocytes, as shown in B cell-deficient mice. Our data reveal that chronic inflammation actively induces lymphangiogenesis in LNs, which is controlled remotely, by lymphangiogenic factors produced at the site of inflammation.

Upregulation of hypoxia-inducible factors in normal and psoriatic skin.

Angiogenesis induced by vascular endothelial growth factor (VEGF) plays an important role in psoriasis. Hypoxic adaptation is conferred through hypoxia-inducible transcription factors (HIFs). VEGF and its receptor Flt-1 are HIF target genes. Growth factors and inflammatory cytokines activate the phosphoinositol-3 kinase pathway, and via activated protein kinase B (phospho-Akt) augment HIF activity. Here, we demonstrate that the major oxygen-dependent HIF isoforms are strongly upregulated in psoriatic skin: HIF-1alpha mainly in the epidermis, in an expression pattern similar to VEGF mRNA; HIF-2alpha in both the epidermis and in capillary endothelial cells of the dermis. In contrast, normal human skin shows low expression of HIF-alpha proteins, with the exception of hair follicles, and glands, which strongly express HIF-1alpha. In normal human skin, phospho-Akt appeared in the basal epidermal layer, in hair follicles, and in dermal glands. In contrast, in psoriasis, phospho-Akt expression was low in the epidermis, but markedly enhanced in the dermal capillaries and in surrounding interstitial/inflammatory cells. Our data suggest that hypoxia initiates a potentially self-perpetuating cycle involving HIF, VEGF, and Akt activation, which could drive physiologic growth of hair follicles and skin glands. Furthermore, such a cycle may exist in psoriasis in dermal capillaries and contribute to disease progression.

VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumor metastasis to distant sites.

The mechanisms by which tumors metastasize to sentinel and distant lymph nodes, and beyond, are poorly understood. We developed transgenic mice that overexpress vascular endothelial growth factor-C (VEGF-C) and green fluorescent protein specifically in the skin and studied the effects of chemically-induced skin carcinogenesis in this model. We found that in contrast to VEGF-A, VEGF-C does not increase the growth of primary tumors, but instead induces expansion of lymphatic networks within sentinel lymph nodes, even before the onset of metastasis. Once the metastatic cells arrived at the sentinel lymph nodes, the extent of lymphangiogenesis at these sites increased. Of importance, in mice with metastasis-containing sentinel lymph nodes, tumors that expressed VEGF-C were more likely to metastasize to additional organs, such as distal lymph nodes and lungs. No metastases were observed in distant organs in the absence of lymph node metastases. These findings indicate an important role of VEGF-C-induced lymph node lymphangiogenesis in the promotion of cancer metastasis beyond the sentinel lymph nodes. VEGF-C is therefore a good target to slow or even prevent the onset of metastasis.

Down syndrome candidate region 1 isoform 1 mediates angiogenesis through the calcineurin-NFAT pathway.

Down syndrome candidate region 1 (DSCR1) is one of more than 50 genes located in a region of chromosome 21 that has been implicated in Down syndrome. DSCR1 can be expressed as four isoforms, one of which, isoform 4 (DSCR1-4), has recently been found to be strongly induced by vascular endothelial growth factor A (VEGF-A(165)) and to provide a negative feedback loop that inhibits VEGF-A(165)-induced endothelial cell proliferation in vitro and angiogenesis in vivo. We report here that another DSCR1 isoform, DSCR1-1L, is also up-regulated by VEGF-A(165) in cultured endothelial cells and is strongly expressed in several types of pathologic angiogenesis in vivo. In contrast to DSCR1-4, the overexpression of DSCR1-1L induced the proliferation and activation of the transcription factor NFAT in cultured endothelial cells and promoted angiogenesis in Matrigel assays in vivo, even in the absence of VEGF-A. Similarly, small interfering RNAs specific for DSCR1-1L and DSCR1-4 had opposing inhibitory and stimulatory effects, respectively, on these same functions. DSCR1-4 is thought to inhibit angiogenesis by inactivating calcineurin, thereby preventing activation and nuclear translocation of NFAT, a key transcription factor. In contrast, DSCR1-1L, regulated by a different promoter than DSCR1-4, activates NFAT and its proangiogenic activity is inhibited by cyclosporin, an inhibitor of calcineurin. In sum, DSCR1-1L, unlike DSCR1-4, potently activates angiogenesis and could be an attractive target for antiangiogenesis therapy.

Pharmacologic blockade of angiopoietin-2 is efficacious against model hemangiomas in mice.

Hemangioma of infancy is the most common neoplasm of childhood. While hemangiomas are classic examples of angiogenesis, the angiogenic factors responsible for hemangiomas are not fully understood. Previously, we demonstrated that malignant endothelial tumors arise in the setting of autocrine loops involving vascular endothelial growth factor (VEGF) and its major mitogenic receptor vascular endothelial growth factor receptor 2. Hemangiomas of infancy differ from malignant endothelial tumors in that they usually regress, or can be induced to regress by pharmacologic means, suggesting that angiogenesis in hemangiomas differs fundamentally from that of malignant endothelial tumors. Here, we demonstrate constitutive activation of the endothelial tie-2 receptor in human hemangioma of infancy and, using a murine model of hemangioma, bEnd.3 cells; we show that bEnd.3 hemangiomas produce both angiopoietin-2 (ang-2) and its receptor, tie-2, in vivo. We also demonstrate that inhibition of tie-2 signaling with a soluble tie-2 receptor decreases bEnd.3 hemangioma growth in vivo. The efficacy of tie-2 blockade suggests that either tie-2 activation or ang-2 may be required for in vivo growth. To address this issue, we used tie-2-deficient bEnd.3 hemangioma cells, which, surprisingly, were fully proficient in in vivo growth. Previous studies from our laboratory and others have implicated reactive oxygen-generating nox enzymes in the angiogenic switch, so we examined the effect of nox inhibitors on ang-2 production in vitro and on bEnd.3 tumor growth in vivo. We then inhibited ang-2 production pharmacologically using novel inhibitors of nox enzymes and found that this treatment nearly abolished bEnd.3 hemangioma growth in vivo. Signal-transduction blockade targeting ang-2 production may be useful in the treatment of human hemangiomas in vivo.

Orphan nuclear receptor TR3/Nur77 regulates VEGF-A-induced angiogenesis through its transcriptional activity.

Vascular endothelial growth factor (VEGF)-A has essential roles in vasculogenesis and angiogenesis, but the downstream steps and mechanisms by which human VEGF-A acts are incompletely understood. We report here that human VEGF-A exerts much of its angiogenic activity by up-regulating the expression of TR3 (mouse homologue Nur77), an immediate-early response gene and orphan nuclear receptor transcription factor previously implicated in tumor cell, lymphocyte, and neuronal growth and apoptosis. Overexpression of TR3 in human umbilical vein endothelial cells (HUVECs) resulted in VEGF-A-independent proliferation, survival, and induction of several cell cycle genes, whereas expression of antisense TR3 abrogated the response to VEGF-A in these assays and also inhibited tube formation. Nur77 was highly expressed in several types of VEGF-A-dependent pathological angiogenesis in vivo. Also, using a novel endothelial cell-selective retroviral targeting system, overexpression of Nur77 DNA potently induced angiogenesis in the absence of exogenous VEGF-A, whereas Nur77 antisense strongly inhibited VEGF-A-induced angiogenesis. B16F1 melanoma growth and angiogenesis were greatly inhibited in Nur77-/- mice. Mechanistic studies with TR3/Nur77 mutants revealed that TR3/Nur77 exerted most of its effects on cultured HUVECs and its pro-angiogenic effects in vivo, through its transactivation and DNA binding domains (i.e., through transcriptional activity).

Role of vascular endothelial growth factor-A in recurrent respiratory papillomatosis.

Vascular endothelial growth factor-A (VEGF-A) is known to play an important role in the angiogenic response essential for tumor growth in a variety of human and experimental tumors. This study was designed to investigate whether VEGF-A may play a role in the pathogenesis of recurrent respiratory papillomatosis (RRP). A retrospective study with institutional review board approval was performed at a tertiary care medical center on 12 patients with a history of laryngeal RRP. Their ages at the time of initial diagnosis ranged from 19 to 96 months (mean, 56 months). All patients had involvement of right and left true vocal cords. All patients required multiple endoscopic procedures (range, 4 to 66; mean, 12). Normal pediatric larynx samples from 5 autopsy patients were used as controls. Formalin-fixed, paraffin-embedded sections of laryngeal squamous papillomas from the 12 patients with a diagnosis of RRP and the 5 control patients were examined by in situ hybridization for the presence of messenger RNA (mRNA) for VEGF-A and vascular endothelial growth factor receptor 1 (VEGFR-1) and vascular endothelial growth factor receptor 2 (VEGFR-2). The biopsy specimens were from the true vocal cord (N = 10) or subglottis (N = 2) in the patients with RRP and consisted of large sections of larynx including the true vocal cord in the control patients (N = 5). Strong expression of VEGF-A mRNA was noted in the squamous epithelium of papillomas of all 12 patients. Strong expression of VEGFR-1 and VEGFR-2 was noted in the endothelial cells of the underlying vessels in all 12 patients. Neither strong labeling of VEGF-A mRNA nor labeling of its receptors wasnoted in the control patients. We conclude that the angiogenic growth factor VEGF-A is strongly expressed in the epithelium of squamous papillomas in RRP. Also, VEGFR-1 and VEGFR-2 mRNAs are strongly expressed by underlying vascular endothelial cells, suggesting an important role in the pathogenesis of RRP.

VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and promotes lymphatic metastasis.

The mechanisms of tumor metastasis to the sentinel lymph nodes are poorly understood. Vascular endothelial growth factor (VEGF)-A plays a principle role in tumor progression and angiogenesis; however, its role in tumor-associated lymphangiogenesis and lymphatic metastasis has remained unclear. We created transgenic mice that overexpress VEGF-A and green fluorescent protein specifically in the skin, and subjected them to a standard chemically-induced skin carcinogenesis regimen. We found that VEGF-A not only strongly promotes multistep skin carcinogenesis, but also induces active proliferation of VEGF receptor-2-expressing tumor-associated lymphatic vessels as well as tumor metastasis to the sentinel and distant lymph nodes. The lymphangiogenic activity of VEGF-A-expressing tumor cells was maintained within metastasis-containing lymph nodes. The most surprising finding of our study was that even before metastasizing, VEGF-A-overexpressing primary tumors induced sentinel lymph node lymphangiogenesis. This suggests that primary tumors might begin preparing their future metastatic site by producing lymphangiogenic factors that mediate their efficient transport to sentinel lymph nodes. This newly identified mechanism of inducing lymph node lymphangiogenesis likely contributes to tumor metastasis, and therefore, represents a new therapeutic target for advanced cancer and/or for the prevention of metastasis.

VEGF-A promotes tissue repair-associated lymphatic vessel formation via VEGFR-2 and the alpha1beta1 and alpha2beta1 integrins.

Vascular endothelial growth factor-A (VEGF-A) is strongly up-regulated in wounded cutaneous tissue and promotes repair-associated angiogenesis. However, little is known about its role in lymphatic regeneration of the healing skin. We studied wound healing in transgenic mice that overexpress VEGF-A specifically in the epidermis and in wild-type mice in the absence or presence of inhibitors of VEGF-A signaling. Surprisingly, transgenic overexpression of VEGF-A in the skin promoted lymphangiogenesis at the wound healing site, whereas systemic blockade of VEGFR-2 prevented lymphatic vessel formation. Studies in cultured lymphatic endothelial cells revealed that VEGF-A induced expression of the alpha1 and alpha2 integrins, which promoted their in vitro tube formation and their haptotactic migration toward type I collagen. VEGF-A-induced lymphatic endothelial cord formation and haptotactic migration were suppressed by anti-alpha1 and anti-alpha2 integrin blocking antibodies, and systemic blockade of the alpha1 and alpha2 integrins inhibited VEGF-A-driven lymphangiogenesis in vivo. We propose that VEGF-A promotes lymphatic vasculature formation via activation of VEGFR-2 and that lineage-specific differences of integrin receptor expression contribute to the distinct dynamics of wound-associated angiogenesis and lymphangiogenesis.

Keloids demonstrate high-level epidermal expression of vascular endothelial growth factor.

BACKGROUND: Keloids are a major cause of morbidity, and arise after operation, injury, or cutaneous infection. Clinically, keloids differ from hypertrophic scars in that they grow beyond the original borders of the injury. Keloids occur most commonly for patients of African and Asian descent, and treatment options are multiple, indicating that there is no entirely satisfactory treatment for keloids. Angiogenesis inhibition has been shown to be effective in treatment of malignancy in both animal models and human beings. OBJECTIVE: We sought to determine whether keloids produce the potent angiogenic factor vascular endothelial growth factor (VEGF). METHODS: We performed in situ hybridization for VEGF on keloid tissue and normal skin. RESULTS: Our study demonstrated abundant production of VEGF in keloids and, surprisingly, the major source of VEGF was the overlying epidermis. CONCLUSIONS: Our results suggest that the overlying epidermis is the major source of keloid angiogenesis. These findings demonstrate that keloids are angiogenic lesions. Topical antiangiogenic therapy, directed at either down-regulating epidermal VEGF or inhibiting keratinocyte-derived VEGF activity on its endothelial receptors, may be useful in the treatment of keloids.

Alpha-chemokine-mediated signal transduction in human Kaposi's sarcoma spindle cells.

The role of chemokines and their receptors in HIV biology and Kaposi's sarcoma (KS) pathogenesis has recently gained considerable attention. It has been shown that KS-associated human herpes virus type 8 (KSHV/HHV-8) encodes functional homologues of certain chemokines and chemokine receptors. This suggests that chemokines may contribute to the growth and spread of KS seen in AIDS. We found the expression of CXCR4 in primary KS tissue by using in situ hybridization (ISH). Recently, alpha-chemokine receptors CXCR1 and CXCR2 have also been shown to be expressed by KS tissues. We further characterized the expression of these chemokines as well as the signaling events induced upon binding to their respective cognate ligands in the KS 38 spindle cell line. These cells express authentic characteristics of primary KS spindle cells and provide a useful in vitro model for these studies. We observed using RT-PCR that KS 38 cells express mRNA for the alpha-chemokine receptors CXCR1, CXCR2, and CXCR4. We also confirmed the cell surface protein expression by FACS analysis. Characterization of signaling pathways revealed that the alpha-chemokines, IL-8 and stromal cell-derived factor 1alpha (SDF1alpha/CXCL12), activated members of the mitogen-activated protein (MAP) kinase family, including Erk kinase, c-Jun amino terminal kinase (JNK)/stress-activated protein kinase (SAPK) and the p38 MAP kinase. Furthermore, using DNA protein-binding experiments, we have shown that IL-8 increased AP-1 and NF Kappa B activity in these cells. IL-8 also enhanced the chemotaxis of KS cells. These results reveal that chemokine-induced signaling pathways may mediate cell growth, transcriptional activation and cell migration in KS.

Infectious angiogenesis: Bartonella bacilliformis infection results in endothelial production of angiopoetin-2 and epidermal production of vascular endothelial growth factor.

Pathological angiogenesis, the development of a microvasculature by neoplastic processes, is a critical component of the development of tumors. The role of oncogenes in the induction of angiogenesis has been extensively studied in benign and malignant tumors. However, the role of infection in inducing angiogenesis is not well understood. Verruga peruana is a clinical syndrome caused by the bacterium Bartonella bacilliformis, and is characterized by the development of hemangioma-like lesions, in which bacteria colonize endothelial cells. To gain insight into how this bacteria induces angiogenesis in vivo, we performed in situ hybridization of clinical specimens of verruga peruana for the angiogenesis factors vascular endothelial growth factor (VEGF), its receptors VEGFR1 and VEGFR2, and angiopoietin-2. High-level expression of angiopoietin-2 and VEGF receptors was observed in the endothelium of verruga peruana. Surprisingly, the major source of VEGF production in verruga peruana is the overlying epidermis. Infection of cultured endothelium with B. bacilliformis also resulted in induction of angiopoetin-2 in vitro. These findings imply a collaboration between infected endothelium and overlying epidermis to induce angiogenesis.

Tumor lymphangiogenesis: a novel prognostic indicator for cutaneous melanoma metastasis and survival.

Malignant melanomas of the skin are distinguished by their propensity for early metastatic spread via lymphatic vessels to regional lymph nodes, and lymph node metastasis is a major determinant for the staging and clinical management of melanoma. However, the importance of tumor-induced lymphangiogenesis for lymphatic melanoma spread has remained unclear. We investigated whether tumor lymphangiogenesis occurs in human malignant melanomas of the skin and whether the extent of tumor lymphangiogenesis may be related to the risk for lymph node metastasis and to patient survival, using double immunostains for the novel lymphatic endothelial marker LYVE-1 and for the panvascular marker CD31. Tumor samples were obtained from clinically and histologically closely matched cases of primary melanomas with early lymph node metastasis (n = 18) and from nonmetastatic melanomas (n = 19). Hot spots of proliferating intratumoral and peritumoral lymphatic vessels were detected in a large number of melanomas. The incidence of intratumoral lymphatics was significantly higher in metastatic melanomas and correlated with poor disease-free survival. Metastatic melanomas had significantly more and larger tumor-associated lymphatic vessels, and a relative lymphatic vessel area of >1.5% was significantly associated with poor disease-free and overall survival. In contrast, no differences in the density of tumor-associated blood vessels were found. Vascular endothelial growth factor and vascular endothelial growth factor-C expression was equally detected in a minority of cases in both groups. Our results reveal tumor lymphangiogenesis as a novel prognostic indicator for the risk of lymph node metastasis in cutaneous melanoma.

High-level expression of vascular endothelial growth factor and its receptors in an aphthous ulcer.

BACKGROUND: Aphthous ulcers are an extremely common disorder of unknown etiology. These ulcers cause significant morbidity through pain and interference with eating. Thalidomide, an angiogenesis inhibitor, is efficacious for the treatment of aphthous ulcers. METHODS: In situ hybridization was performed on an idiopathic aphthous ulcer using probes specific for the angiogenesis factor vascular endothelial growth factor, and its receptors, in order to determine whether these ulcers are highly angiogenic. CONCLUSIONS: Aphthous ulcers are highly angiogenic. Thalidomide may act to heal aphthous ulcers by inhibiting angiogenesis and promoting reepithelialization. Excess angiogenesis may inhibit reepithelialization in certain types of ulcers, and angiogenesis inhibitors may actually promote wound healing if ulcers are caused by excess angiogenesis.