Gene transfer as a tool to induce therapeutic vascular growth in plastic surgery.

Sufficient tissue blood perfusion is critical for wound healing and for reconstructions in plastic surgery. There is considerable interest in the use of therapeutic angiogenesis to improve survival of ischemic flaps and to improve wound healing. Several growth factors have been tested in experimental flap models. Clinically, angiogenic therapy has been extensively studied in cardiovascular ischemic diseases and several clinical studies are ongoing in human patients. Results from these cardiovascular trials will benefit the planning of clinical trials in the field of plastic surgery. Furthermore, the development of strategies for local and controlled induction of lymphangiogenesis will hopefully improve the treatment of edema.

Vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 expression in squamous cell carcinomas of the head and neck.

BACKGROUND AND METHODS: VEGF proteins and their receptors are involved in tumor vessel neoformation. The third VEGF receptor, VEGFR3 (flt-4) is important during both blood vessel development and lymphatic vessel formation. Because HNSCC preferentially metastasizes to regional lymph nodes, we investigated the expression of VEGFR3 and its ligand VEGF-C in head and neck squamous cell carcinomas by semiquantitative RT-PCR (4 HNSCC cells lines and 6 HNSCC specimens) and by immunohistochemistry (18 HNSCC specimens). VEGFR3 protein expression was confirmed by Western blotting in four HNSCC cell lines and six HNSCC specimens. RESULTS: Semiquantitative mRNA analysis showed VEGF-C mRNA expression in three (SCC9, SCC25, LFFR) of four HNSCC cell lines and all six HNSCC specimens. VEGFR3 mRNA was found in two HNSCC cell lines (JPPA and SCC25) and only weakly detected in the other two HNSCC cell lines (SCC9 and LFFR). High amounts of VEGFR3 mRNA were shown in all six patients' tumor specimens. VEGFR3 Western blot analysis yielded a distinct band at the predicted size of 210 kD in JPPA and SCC9 and hardly detectable bands in SCC25 and LFFR cell lines. All six HNSCC specimens displayed strong VEGFR3 protein bands. Immunohistochemistry in 18 HNSCC specimens assigned strong to mediate VEGF-C IR and minor VEGFR3 IR to tumor cells and strong VEGF-C and VEGFR3 IR to tumor surrounding vessels. In addition, intense VEGF-C immunostaining was observed on perivascular and mononuclear cells in the tumor surrounding stroma. Subtyping of VEGFR3+ microvascular tumor vessels revealed partially double immunolabeling with CD34 and flk-1, indicating a common origin of blood and lymphatic vessels. The expression of VEGF-C on tumor cells could be correlated with recurrences, and larger primary tumors had more VEGF-C-positive vessels. CONCLUSIONS: The broad expression of VEGF C and VEGFR3 in HNSCC suggests involvement in tumor lymph angiogenesis and vascular angiogenesis, promoting tumor growth and propagation of cancer cells. This implies that inhibitors of lymph angiogenesis could become effective therapeutic options similar to classical angiogenesis inhibitors.

A model for gene therapy of human hereditary lymphedema.

Primary human lymphedema (Milroy's disease), characterized by a chronic and disfiguring swelling of the extremities, is associated with heterozygous inactivating missense mutations of the gene encoding vascular endothelial growth factor C/D receptor (VEGFR-3). Here, we describe a mouse model and a possible treatment for primary lymphedema. Like the human patients, the lymphedema (Chy) mice have an inactivating Vegfr3 mutation in their germ line, and swelling of the limbs because of hypoplastic cutaneous, but not visceral, lymphatic vessels. Neuropilin (NRP)-2 bound VEGF-C and was expressed in the visceral, but not in the cutaneous, lymphatic endothelia, suggesting that it may participate in the pathogenesis of lymphedema. By using virus-mediated VEGF-C gene therapy, we were able to generate functional lymphatic vessels in the lymphedema mice. Our results suggest that growth factor gene therapy is applicable to human lymphedema and provide a paradigm for other diseases associated with mutant receptors.

Macrophage-derived chemokine (MDC/CCL22) and CCR4 are involved in the formation of T lymphocyte-dendritic cell clusters in human inflamed skin and secondary lymphoid tissue.

Our previous study demonstrated formation of T cell-dendritic cell (DC) clusters in inflamed dermis of intraorally autotransplanted skin flaps. Such T cell-DC clusters are supposed to be important for close interactions between T cells and DCs including the specific antigen presentation. Here we show the involvement of the macrophage-derived chemokine (MDC/CCL22) and its specific receptor CC chemokine receptor 4 (CCR4) in the formation of T cell-DC clusters. Reverse transcriptase-polymerase chain reaction analysis revealed high levels of mRNA expression for MDC and CCR4 in inflamed skin and neck lymph nodes (LNs), but not in normal skin. Immunohistochemically, MDC(+) cells and CCR4(+) cells were mainly located within the T cell-DC clusters both in the dermis of inflamed skin and the T cell area of LNs. MDC(+) cells were identified to be DCs both in inflamed skin and LNs. The majority of CCR4(+) cells were CD4(+) T cells, accounting for approximately one-third of total CD4(+) T cells in the inflamed skin. Our data suggest that the MDC-CCR4 system plays an important role in the formation of T cell-DC clusters both in inflamed skin and LNs.

VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissues.

Recently, vascular endothelial growth factor receptor 3 (VEGFR-3) has been shown to provide a specific marker for lymphatic endothelia in certain human tissues. In this study, we have investigated the expression of VEGFR-3 and its ligands VEGF-C and VEGF-D in fetal and adult tissues. VEGFR-3 was consistently detected in the endothelium of lymphatic vessels such as the thoracic duct, but fenestrated capillaries of several organs including the bone marrow, splenic and hepatic sinusoids, kidney glomeruli and endocrine glands also expressed this receptor. VEGF-C and VEGF-D, which bind both VEGFR-2 and VEGFR-3 were expressed in vascular smooth muscle cells. In addition, intense cytoplasmic staining for VEGF-C was observed in neuroendocrine cells such as the alpha cells of the islets of Langerhans, prolactin secreting cells of the anterior pituitary, adrenal medullary cells, and dispersed neuroendocrine cells of the gastrointestinal tract. VEGF-D was observed in the innermost zone of the adrenal cortex and in certain dispersed neuroendocrine cells. These results suggest that VEGF-C and VEGF-D have a paracrine function and perhaps a role in peptide release from secretory granules of certain neuroendocrine cells to surrounding capillaries.

Splenic angiosarcoma: a clinicopathologic and immunophenotypic study of 28 cases.

Primary angiosarcoma of the spleen is a rare neoplasm that has not been well characterized. We describe the clinical, morphologic, and immunophenotypic findings of 28 cases of primary splenic angiosarcoma, including one case that shares features of lymphangioma/lymphangiosarcoma. The patients included 16 men and 12 women, aged 29 to 85 years, with a mean of 59 years and median of 63 years. The majority of patients (75%) complained of abdominal pain, and 25% presented with splenic rupture. The most common physical finding was splenomegaly (71%). Seventeen of 21 patients were reported to have anemia. Macroscopic examination showed splenomegaly in 85% cases. Sectioning revealed discrete lesions in 88% of cases, ranging from well-circumscribed firm nodules to poorly delineated foci of necrosis and hemorrhage associated with cystic spaces. Microscopically, the tumors were heterogenous; however, all cases demonstrated at least a focal vasoformative component lined by atypical endothelial cells. Solid sarcomatous, papillary, and epithelioid growth patterns were observed. The solid sarcomatous component resembled fibrosarcoma in two cases and malignant fibroushistiocytoma in one case. Hemorrhage, necrosis, hemosiderin, extramedullary hematopoiesis, and intracytoplasmic hyaline globules were frequently identified. A panel of immunohistochemical studies revealed that the majority of tumors were immunoreactive for at least two markers of vascular differentiation (CD34, FVIIIRAg, VEGFR3, and CD31) and at least one marker of histiocytic differentiation (CD68 and/or lysozyme). Metastases developed in 100% of patients during the course of their disease. Twenty-six patients died of disease despite aggressive therapy, whereas only two patients are alive at last follow-up, one with disease at 8 years and the other without disease at 10 years. In conclusion, primary splenic angiosarcoma is an extremely aggressive neoplasm that is almost universally fatal. The majority of splenic angiosarcomas coexpress histiocytic and endothelial markers by immunohistochemical analysis, which suggest that some tumors may originate from splenic lining cells.

Vascular endothelial growth factor-C and its receptor VEGFR-3 in the nasal mucosa and in nasopharyngeal tumors.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are important regulators of blood and lymphatic vessel growth and vascular permeability. Both blood and lymphatic vessels of the upper respiratory tract play important roles in pathological conditions, such as infections and tumors. Here we have studied the expression of VEGF-C and its receptor VEGFR-3 in the upper respiratory system by Northern blot analysis and immunohistochemistry of human tissues, and in situ mRNA hybridization of developing mouse embryos and beta-galactosidase staining of mouse embryos having a LacZ marker gene in the VEGFR-3 gene locus. The results demonstrate expression of VEGF-C and VEGFR-3 in the developing and adult nasal respiratory epithelium and in the nasal vascular plexus, respectively. Unlike in most other tissues, in the nasal mucosa VEGFR-3 is expressed in both blood and lymphatic vessels. Expression of VEGF-C was also detected in nasal and nasopharyngeal tumor islands, which were surrounded by VEGFR-3-positive angiogenic blood vessels. These results suggest that VEGF-C and VEGFR-3 have a role in the development of the nasal submucosal vascular plexus and in its normal function and that they are associated with angiogenesis in nasal and nasopharyngeal tumors.

Immunological activation of dermal Langerhans cells in contact with lymphocytes in a model of human inflamed skin.

Langerhans cells play an important role in the skin's immune system. Little is known, however, about the antigen-presenting capacity of Langerhans cells in the context of skin inflammation. By immunohistochemistry we investigated the phenotypic characteristics of epidermal and dermal Langerhans cells and their spatial relationship with infiltrating lymphocytes. We studied skin flaps autotransplanted to the oral cavity to fill a defect after maxillofacial cancer surgery. In 15 of 21 cases sampled for the present study, the skin flaps were severely inflamed by Candida albicans infection. In contrast to the normal skin, such inflamed skin showed a marked increase in CD1a(+) dermal Langerhans cells. Double immunohistochemistry revealed that dermal Langerhans cells abundantly expressed B7-2 (CD86), a representative costimulatory molecule, and CD83, a marker of mature dendritic cells. Furthermore, these dermal Langerhans cells were in close contact with CD4(+)/CD45RO(+) lymphocytes. This cell-to-cell contact was further visualized by immunoelectron microscopy. Langerhans cells were also observed within lymphatic vessels that were identified by the expression of vascular endothelial growth factor receptor-3. Ki-67 labeling indices were 4.2% in CD4(+) T cells and 0.8% in CD8(+) T cells within the dermis. Factor XIIIa(+) dermal dendrocytes were distributed outside the clusters of lymphocytes and were not in contact with them. Our observations indicate that dermal Langerhans cells in the inflamed skin are activated to express common phenotypes to mature dendritic cells so that they could stimulate neighboring memory CD4(+) T cells.

Mechanisms of angiogenesis and their use in the inhibition of tumor growth and metastasis.

There is a constant requirement for vascular supply in solid tumors. Tumor-associated neovascularization allows the tumor cells to express their critical growth advantage. Axillary lymph node status is the most important prognostic factor in operable breast cancer, and experimental and clinical evidence suggests that the process of metastasis is also angiogenesis-dependent. Various angiogenic growth factors and cytokines induce neovascularization in tumors, namely members of the vascular endothelial growth factor (VEGF) and angiopoietin (Ang) gene families. A strong correlation has been found between VEGF expression and increased tumor microvasculature, malignancy, and metastasis in breast cancer. Anti-angiogenic therapy approaches offer a new promising anti-cancer strategy and a remarkably diverse group of over 20 such drugs is currently undergoing evaluation in clinical trials.