Anne Saaristo

A model for gene therapy of human hereditary lymphedema

Primary human lymphedema (Milroys 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.

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 α 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.—Partanen, T. A., Arola, J., Saaristo, A., Jussila, L., Ora, A., Miettinen, M., Stacker, S. A., Achen, M. G., Alitalo, K. VEGF‐C and VEGF‐D expression in neuroendocrine cells and their receptor, VEGFR‐3, in fenestrated blood vessels in human tissues. FASEB J. 14, 2087–2096 (2000)

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.

Therapeutic differentiation and maturation of lymphatic vessels after lymph node dissection and transplantation

Surgery or radiation therapy of metastatic cancer often damages lymph nodes, leading to secondary lymphedema. Here we show, using a newly established mouse model, that collecting lymphatic vessels can be regenerated and fused to lymph node transplants after lymph node removal. Treatment of lymph node–excised mice with adenovirally delivered vascular endothelial growth factor-C (VEGF-C) or VEGF-D induced robust growth of the lymphatic capillaries, which gradually underwent intrinsic remodeling, differentiation and maturation into functional collecting lymphatic vessels, including the formation of uniform endothelial cell-cell junctions and intraluminal valves. The vessels also reacquired pericyte contacts, which downregulated lymphatic capillary markers during vessel maturation. Growth factor therapy improved the outcome of lymph node transplantation, including functional reconstitution of the immunological barrier against tumor metastasis. These results show that growth factor–induced maturation of lymphatic vessels is possible in adult mice and provide a basis for future therapy of lymphedema.

Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are important regulators of blood and lymphatic vessel growth and vascular permeability. The VEGF‐C/VEGFR‐3 signaling pathway is crucial for lymphangiogenesis, and heterozygous inactivating missense mutations of the VEGFR‐3 gene are associated with hereditary lymphedema. However, VEGF‐C can have potent effects on blood vessels because its receptor VEGFR‐3 is expressed in certain blood vessels and because the fully processed form of VEGF‐C also binds to the VEGFR‐2 of blood vessels. To characterize the in vivo effects of VEGF‐C on blood and lymphatic vessels, we have overexpressed VEGF‐C via adenovirus‐and adeno‐associated virus‐mediated transfection in the skin and respiratory tract of athymic nude mice. This resulted in dose‐dependent enlargement and tortuosity of veins, which, along with the collecting lymphatic vessels were found to express VEGFR‐2. Expression of angiopoietin 1 blocked the increased leakiness of the blood vessels induced by VEGF‐C whereas vessel enlargement and lymphangiogenesis were not affected. However, angiogenic sprouting of new blood vessels was not observed in response to AdVEGF‐C or AAV‐VEGF‐C. These results show that virally produced VEGF‐C induces blood vessel changes, including vascular leak, but its angiogenic potency is much reduced compared with VEGF in normal skin.—Saaristo, A., Veikkola, T., Enholm, B. Hytönen, M., Arola, J., Pajusola, K., Turunen, P., Jeltsch, M., Karkkainen, M. J., Kerjaschki, D., Bueler, H., Ylä‐Herttuala, S., Alitalo, K. Adenoviral VEGF‐C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes. FASEB J. 16, 1041–1049 (2002)

Microvascular breast reconstruction and lymph node transfer for postmastectomy lymphedema patients.

Objective:Postoperative lymphedema after breast cancer surgery is a challenging problem. Recently, a novel microvascular lymph node transfer technique provided a fresh hope for patients with lymphedema. We aimed to combine this new method with the standard breast reconstruction. Methods:During 2008–2010, we performed free lower abdominal flap breast reconstruction in 87 patients. For all patients with lymphedema symptoms (n = 9), we used a modified lower abdominal reconstruction flap containing lymph nodes and lymphatic vessels surrounding the superficial circumflex vessel pedicle. Operation time, donor site morbidity, and postoperative recovery between the 2 groups (lymphedema breast reconstruction and breast reconstruction) were compared. The effect on the postoperative lymphatic vessel function was examined. Results:The average operation time was 426 minutes in the lymphedema breast reconstruction group and 391 minutes in the breast reconstruction group. The postoperative abdominal seroma formation was increased in patients with lymphedema. Postoperative lymphoscintigraphy demonstrated at least some improvement in lymphatic vessel function in 5 of 6 patients with lymphedema. The upper limb perimeter decreased in 7 of 9 patients. Physiotherapy and compression was no longer needed in 3 of 9 patients. Importantly, we found that human lymph nodes express high levels of endogenous lymphatic vessel growth factors. Transfer of the lymph nodes and the resulting endogenous growth factor expression may thereby induce the regrowth of lymphatic network in the axilla. No edema problems were detected in the lymph node donor area. Conclusion:Simultaneous breast and lymphatic reconstruction is an ideal option for patients who suffer from lymphedema after mastectomy and axillary dissection.

Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

Recent work from many laboratories has demonstrated that the vascular endothelial growth factor-C/VEGF-D/VEGFR-3 signaling pathway is crucial for lymphangiogenesis, and that mutations of the Vegfr3 gene are associated with hereditary lymphedema. Furthermore, VEGF-C gene transfer to the skin of mice with lymphedema induced a regeneration of the cutaneous lymphatic vessel network. However, as is the case with VEGF, high levels of VEGF-C cause blood vessel growth and leakiness, resulting in tissue edema. To avoid these blood vascular side effects of VEGF-C, we constructed a viral vector for a VEGFR-3–specific mutant form of VEGF-C (VEGF-C156S) for lymphedema gene therapy. We demonstrate that VEGF-C156S potently induces lymphangiogenesis in transgenic mouse embryos, and when applied via viral gene transfer, in normal and lymphedema mice. Importantly, adenoviral VEGF-C156S lacked the blood vascular side effects of VEGF and VEGF-C adenoviruses. In particular, in the lymphedema mice functional cutaneous lymphatic vessels of normal caliber and morphology were detected after long-term expression of VEGF-C156S via an adeno associated virus. These results have important implications for the development of gene therapy for human lymphedema.

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 fibrous-histiocytoma 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.

Lymphangiogenic growth factors, receptors and therapies

The lymphatic vasculature is essential for the maintenance of normal fluid balance and for the immune responses, but it is also involved in a variety of diseases. Hypoplasia or dysfunction of the lymphatic vessels can lead to lymphedema, whereas hyperplasia or abnormal growth of these vessels are associated with lymphangiomas and lymphangiosarcomas. Lymphatic vessels are also involved in lymph node and systemic metastasis of cancer cells. Recent novel findings on the molecular mechanisms involved in lymphatic vessel development and regulation allow the modulation of the lymphangiogenic process and specific targeting of the lymphatic endothelium. Recent results show that the homeodomain transcription factor Prox-1 is an important lymphatic endothelial cell (LEC) fate-determining factor which can induce LEC-specific gene transcription even in blood vascular endothelial cells (BECs). This suggests that the distinct phenotypes of cells in the adult vascular endothelium are plastic and sensitive to transcriptional reprogramming, which might be useful for future therapeutic applications involving endothelial cells. Vascular endothelial growth factor-C (VEGF-C) and VEGF-D are peptide growth factors capable of inducing the growth of new lymphatic vessels in vivo in a process called lymphangiogenesis. They belong to the larger family which also includes VEGF, placenta growth factor (PlGF) and VEGF-B, VEGF-C and VEGF-D are ligands for the endothelial cell specific tyrosine kinase receptors VEGFR-2 and VEGFR-3. In adult human as well as mouse tissues VEGFR-3 is expressed predominantly in lymphatic endothelial cells which line the inner surface of lymphatic vessels. While VEGFR-2 is thought to be the main mediator of angiogenesis, VEGFR-3 signaling is crucial for the development of the lymphatic vessels. Heterozygous inactivation of the VEGFR-3 tyrosine kinase leads to primary lymphedema due to defective lymphatic drainage in the limbs. Other factors that seem to be involved in lymphangiogenesis include the Tie/angiopoietin system, neuropilin-2 and integrin alpha 9. VEGF-C induces lymphatic vessel growth, but high levels of VEGF-C also resulted in blood vessel leakiness and growth. The VEGFR-3-specific mutant form of VEGF-C called VEGF-C156S lacks blood vascular side effects but is sufficient for therapeutic lymphangiogenesis in a mouse model of lymphedema. As VEGF-C156S is a specific lymphatic endothelial growth factor in the skin, it provides an attractive molecule for pro-lymphangiogenic therapy.

Angiopoietin-1 Protects Against the Development of Cardiac Allograft Arteriosclerosis

Background—Angiopoietin (Ang)–1 is an angiogenic growth factor that counteracts the permeability and proinflammatory effects of vascular endothelial growth factor and other proinflammatory cytokines. Recently, we demonstrated that vascular endothelial growth factor enhances cardiac allograft arteriosclerosis. Here, we studied the roles of Ang1, its natural antagonist Ang2, and their receptor Tie2 in rat cardiac allograft arteriosclerosis. Methods and Results—Heterotopic cardiac allografts and syngrafts were transplanted from Dark Agouti (DA) to Wistar-Furth rats and from DA to DA rats, respectively. Immunohistochemistry disclosed that only a few mesenchymal cells expressed Ang1 in normal hearts and syngrafts, whereas no immunoreactivity was found in cardiac allografts undergoing chronic rejection. Ang2 and Tie2 immunoreactivity was induced mainly in capillaries and postcapillary venules in chronic allografts when compared with syngeneic controls, but no immunoreactivity was found in arterial endothelium. Intracoronary perfusion of cardiac allografts with a clinical-grade adenoviral vector encoding human Ang1 (Ad.Ang1) protected against the development of allograft arteriosclerosis. Ad.Ang1 perfusion reduced Ang2 expression in microcirculation, the numbers of graft-infiltrating leukocytes, and the level of immunoactivation and interstitial fibrosis, as well as both the incidence and intensity of intimal lesions. Ad.Ang1 perfusion also increased CD34+ stem cell counts in peripheral blood. Conclusions—Our findings suggest that the antiinflammatory properties of Ang1 may offer an entirely new therapeutic approach to prevent cardiac allograft arteriosclerosis.