VEGF-C and VEGF-C156S in the pro-lymphangiogenic growth factor therapy of lymphedema: a large animal study.

INTRODUCTION: VEGF-C156S, a lymphangiogenesis-specific form of vascular endothelial growth factor C (VEGF-C), has been considered as a promising candidate for the experimental pro-lymphangiogenic treatment, as it lacks potential angiogenic effects. As a precursor to future clinical trials, the therapeutic efficacy and blood vascular side effects of VEGF-C and VEGF-C156S were compared in a large animal model of secondary lymphedema. Combination of lymphatic growth factor treatment and autologous lymph node transfer was used to normalize the lymphatic anatomy after surgical excision of lymphatic tissue. METHODS: Lymph vessels around the inguinal lymph node of female domestic pigs were destroyed in order to impair the normal lymphatic drainage from the hind limb. Local injections of adenoviruses (Ad) encoding VEGF-C or VEGF-C156S were used to enhance the regrowth of the lymphatic vasculature. AdLacZ (ß-galactosidase) and saline injections served as controls. RESULTS: Both VEGF-C and VEGF-C156S induced growth of new lymphatic vessels in the area of excision, although lymphangiogenesis was notably stronger after VEGF-C treatment. Also the transferred lymph nodes were best-preserved in the VEGF-C-treated pigs. Despite the enlargement of blood vessels following the VEGF-C therapy, no signs of sprouting angiogenesis or increased blood vascular permeability in the form of increased wound exudate volumes were observed. CONCLUSIONS: Our results show that VEGF-C provides the preferred alternative for growth factor therapy of lymphedema when compared to VEGF-C156S, due to the superior lymphangiogenic response and minor blood vessel effects. Furthermore, these observations suggest that activation of both VEGFR-2 and VEGFR-3 might be needed for efficient lymphangiogenesis.

Growth factor therapy and lymph node graft for lymphedema.

BACKGROUND: Lymphedema still remains an unsolved problem. Secondary lymphedema often develops after cancer operations or radiation therapy, especially in breast cancer patients. Using a mouse model, we show here that the lymphatic network can be regenerated using lymphatic vascular growth factor therapy in combination with lymph node transfer. MATERIALS AND METHODS: We have compared the therapeutic effects of different vascular endothelial growth factors (VEGF-C, VEGF-D, VEGF-C156S, and VEGF-A), in combination with lymph node transfer in mouse axilla. The lymphangiogenic effects of the growth factor therapy were examined at 3 mo postoperatively. RESULTS: VEGF therapy with VEGF-C and VEGF-D induced growth of new lymphatic vessels in the defect area, and VEGF-C also improved lymphatic vessel function compared with that of controls. VEGF-C156S induced moderate lymphangiogenesis, but the effect remained statistically nonsignificant. Prolymphangiogenic growth factors (VEGF-C, -D, and -C156S) also improved lymph node survival as compared with those of the VEGF-A and control group. VEGF-C, which activates both vascular endothelial growth factor receptor 2 and vascular endothelial growth factor receptor 3, gave the best therapeutic effect in this experimental lymphedema model. CONCLUSIONS: These results support our goal to treat secondary lymphedema by combining lymph node transfer with the growth factor therapy. VEGF-C provides the preferred alternative for growth factor therapy of lymphedema when compared with other VEGF-family growth factors, due to the superior lymphangiogenic response and minor blood vascular effects.

Lymph node transfer and perinodal lymphatic growth factor treatment for lymphedema.

OBJECTIVE: Our objective was to define the optimal growth factor treatment to be used in combination with lymph node transfer to normalize lymphatic vascular anatomy. BACKGROUND: In the lymph node transfer method, lymphatic anastomoses are expected to form spontaneously. However, lymphangiogenic growth factor therapies have shown promising results in preclinical models of lymphedema. METHODS: The inguinal lymphatic vasculature of pigs was surgically destroyed around the inguinal lymph node. To enhance the regrowth of the lymphatic network in the defected area, adenoviral vascular endothelial growth factor C (VEGF-C) was administered intranodally or perinodally. Control animals received injections of saline or control vector. The lymphangiogenic effect of the growth factor therapy and any potential adverse effects associated with the 2 alternative delivery routes were examined 2 months postoperatively. RESULTS: Both routes of growth factor administration induced robust growth of lymphatic vessels and helped to preserve the structure of the transferred lymph nodes in comparison with the controls. The lymph nodes of the control treated animals regressed in size and their nodal structure was partly replaced by fibro-fatty scar tissue. Intranodally injected adenoviral VEGF-C and adenoviral vector encoding control gene LacZ induced macrophage accumulation inside the node, whereas perinodal administration of VEGF-C did not have this adverse effect. CONCLUSIONS: Lymphangiogenic growth factors improve lymphatic vessel regeneration and lymph node function after lymph node transfer. The perinodal route of delivery provides a basis for future clinical trials in lymphedema patients.

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.

Fat Grafting Can Induce Browning of White Adipose Tissue.

BACKGROUND: Fat grafting is commonly used when treating soft-tissue defects. However, much of the basic biology behind fat transfer is still uncovered. Adipocytes can be divided into energy storing white and energy burning brown adipose cells. It is now well known, that also adult humans have metabolically active brown adipose tissue (BAT) within white adipose tissue (WAT). Previously our group showed that transfer of metabolically inactive WAT into a new environment increased the metabolic activity of the fat grafts to resemble the activity in the recipient site and that different WAT depots have variation in the metabolic activity. This led us to speculate, whether the metabolic increase of the graft is a result of "browning" of the transferred WAT toward beige adipose tissue. METHODS: We investigated the metabolic and histological characteristics and BAT marker Ucp1 gene expression in different types of WAT grafts placed either in subcutaneous or muscle tissue in mice. Metabolic activity of the grafts was investigated by FDG-PET/CT at 4- and 12-week time-points. RESULTS: The glucose uptake of all transferred fat types was increased when compared with respective control WAT regardless of transfer location. Ucp1 gene and protein expression was increased in 4 of 15 intramuscularly placed fat graft samples and showed histological resemblance to BAT with multilocular cells. CONCLUSIONS: Grafting of metabolically inactive fat intramuscularly may induce browning of fat grafts toward more active beige adipose tissue. This opens up new research areas in exploiting fat grafting in metabolic diseases.

Analysis of fat graft metabolic adaptation and vascularization using positron emission tomography-computed tomographic imaging.

BACKGROUND: Fat tissue transfer is commonly used for different soft-tissue defects in surgery. The immediate result of these operations is often good, but the long-term result is unfortunately unpredictable. The authors used an experimental model to evaluate the vascularization, survival, and metabolic changes after free fat transfer and the impact of proangiogenic therapy on these processes. METHODS: Fat was collected from the mouse epididymal region and placed into the subcutaneous tissue of the forehead. Fat grafts were treated with proangiogenic vascular endothelial growth factor (VEGF)-A (n = 9) or the control vector (n = 9). Metabolic activity and fat graft volume were investigated by positron emission tomography-computed tomography at 4 weeks and at 12 weeks. Histologic analysis was performed at 12 weeks. RESULTS: The glucose metabolism (fluorodeoxyglucose uptake) of the transferred epididymal fat was higher than in the epididymal fat before transplantation in both study groups (VEGF-A and control) and resembled that of normal subcutaneous fat. VEGF-A therapy enhanced the survival and capillary density of the transferred fat after surgery. CONCLUSIONS: Transfer of the metabolically inactive (epididymal) fat into a new environment modulated the metabolic activity of the fat grafts to resemble the situation in the recipient site. These novel findings support the clinical use of free fat grafts in various anatomical regions and tissue types. Proangiogenic VEGF-A therapy enhanced the vascularization and survival of the free fat grafts.