Sanjay V. Daluvoy

TGF-β1 is a negative regulator of lymphatic regeneration during wound repair

Although clinical studies have identified scarring/fibrosis as significant risk factors for lymphedema, the mechanisms by which lymphatic repair is impaired remain unknown. Transforming growth factor -beta1 (TGF-beta1) is a critical regulator of tissue fibrosis/scarring and may therefore also play a role in the regulation of lymphatic regeneration. The purpose of this study was therefore to assess the role of TGF-beta1 on scarring/fibrosis and lymphatic regeneration in a mouse tail model. Acute lymphedema was induced in mouse tails by full-thickness skin excision and lymphatic ligation. TGF-beta1 expression and scarring were modulated by repairing the wounds with or without a topical collagen gel. Lymphatic function and histological analyses were performed at various time points. Finally, the effects of TGF-beta1 on lymphatic endothelial cells (LECs) in vitro were evaluated. As a result, the wound repair with collagen gel significantly reduced the expression of TGF-beta1, decreased scarring/fibrosis, and significantly accelerated lymphatic regeneration. The addition of recombinant TGF-beta1 to the collagen gel negated these effects. The improved lymphatic regeneration secondary to TGF-beta1 inhibition was associated with increased infiltration and proliferation of LECs and macrophages. TGF-beta1 caused a dose-dependent significant decrease in cellular proliferation and tubule formation of isolated LECs without changes in the expression of VEGF-C/D. Finally, the increased expression of TGF-beta1 during wound repair resulted in lymphatic fibrosis and the coexpression of alpha-smooth muscle actin and lymphatic vessel endothelial receptor-1 in regenerated lymphatics. In conclusion, the inhibition of TGF-beta1 expression significantly accelerates lymphatic regeneration during wound healing. An increased TGF-beta1 expression inhibits LEC proliferation and function and promotes lymphatic fibrosis. These findings imply that the clinical interventions that diminish TGF-beta1 expression may be useful in promoting more rapid lymphatic regeneration.

Blockade of Transforming Growth Factor-β1 Accelerates Lymphatic Regeneration during Wound Repair

Lymphedema is a complication of cancer treatment occurring in approximately 50% of patients who undergo lymph node resection. Despite its prevalence, the etiology of this disorder remains unknown. In this study, we determined the effect of soft tissue fibrosis on lymphatic function and the role of transforming growth factor (TGF)-β1 in the regulation of this response. We determined TGF-β expression patterns in matched biopsy specimens collected from lymphedematous and normal limbs of patients with secondary lymphedema. To determine the role of TGF-β in regulating tissue fibrosis, we used a mouse model of lymphedema and inhibited TGF-β function either systemically with a monoclonal antibody or locally by using a soluble, defective TGF-β receptor. Lymphedematous tissue demonstrated a nearly threefold increase in the number of cells that stained for TGF-β1. TGF-β inhibition markedly decreased tissue fibrosis, increased lymphangiogenesis, and improved lymphatic function compared with controls. In addition, inhibition of TGF-β not only decreased TGF-β expression in lymphedematous tissues, but also diminished inflammation, migration of T-helper type 2 (Th2) cells, and expression of profibrotic Th2 cytokines. Similarly, systemic depletion of T-cells markedly decreased TGF-β expression in tail tissues. Inhibition of TGF-β function promoted lymphatic regeneration, decreased tissue fibrosis, decreased chronic inflammation and Th2 cell migration, and improved lymphatic function. The use of these strategies may represent a novel means of preventing lymphedema after lymph node resection.

Fibrosis Is a Key Inhibitor of Lymphatic Regeneration

Background: Lymphedema is a common debilitating sequela of lymph node dissection. Although numerous clinical studies suggest that factors that lead to fibrosis are associated with the development of lymphedema, this relationship has not been proven. The purpose of these experiments was therefore to evaluate lymphatic regeneration in the setting of variable soft-tissue fibrosis. Methods: A section of mouse tail skin including the capillary and collecting lymphatics was excised. Experimental animals (n = 20) were treated with topical collagen type I gel and a moist dressing, whereas control animals (n = 20) underwent excision followed by moist dressing alone. Fibrosis, acute lymphedema, lymphatic function, gene expression, lymphatic endothelial cell proliferation, and lymphatic fibrosis were evaluated at various time points. Results: Collagen gel treatment significantly decreased fibrosis, with an attendant decrease in acute lymphedema and improved lymphatic function. Tails treated with collagen gel demonstrated greater numbers of lymphatic vessels, more normal lymphatic architecture, and more proliferating lymphatic endothelial cells. These findings appeared to be independent of vascular endothelial growth factor C expression. Decreased fibrosis was associated with a significant decrease in the expression of extracellular matrix components. Finally, decreased soft-tissue fibrosis was associated with a significant decrease in lymphatic fibrosis as evidenced by the number of lymphatic endothelial cells that coexpressed lymphatic and fibroblast markers. Conclusions: Soft-tissue fibrosis is associated with impairment in lymphatic regeneration and lymphatic function. These defects occur as a consequence of impaired lymphatic endothelial cell proliferation, abnormal lymphatic microarchitecture, and lymphatic fibrosis. Inhibition of fibrosis using a simple topical dressing can markedly accelerate lymphatic repair and promote regeneration of normal capillary lymphatics.

Radiation therapy causes loss of dermal lymphatic vessels and interferes with lymphatic function by TGF-β1-mediated tissue fibrosis

Although radiation therapy is a major risk factor for the development of lymphedema following lymphadenectomy, the mechanisms responsible for this effect remain unknown. The purpose of this study was therefore to determine the effects of radiation on lymphatic endothelial cells (LECs) and lymphatic function. The tails of wild-type or acid sphingomyelinase (ASM)-deficient mice were treated with 0, 15, or 30 Gy of radiation and then analyzed for LEC apoptosis and lymphatic function at various time points. To analyze the effects of radiation fibrosis on lymphatic function, we determined the effects of transforming growth factor (TGF)-beta1 blockade after radiation in vivo. Finally, we determined the effects of radiation and exogenous TGF-beta1 on LECs in vitro. Radiation caused mild edema that resolved after 12-24 wk. Interestingly, despite resolution of tail edema, irradiated animals displayed persistent lymphatic dysfunction. Radiation caused loss of capillary lymphatics and was associated with a dose-dependent increase in LEC apoptosis. ASM-/- mice had significantly less LEC apoptosis; however, this finding did not translate to improved lymphatic function at later time points. Short-term blockade of TGF-beta1 function after radiation markedly decreased tissue fibrosis and significantly improved lymphatic function but did not alter LEC apoptosis. Radiation therapy decreases lymphatic reserve by causing depletion of lymphatic vessels and LECs as well as promoting soft tissue fibrosis. Short-term inhibition of TGF-beta1 activity following radiation improves lymphatic function and is associated with decreased soft tissue fibrosis. ASM deficiency confers LEC protection from radiation-induced apoptosis but does not prevent lymphatic dysfunction.

HIF-1α coordinates lymphangiogenesis during wound healing and in response to inflammation

This study aimed to investigate the mechanisms that coordinate lymphangiogenesis. Using mouse models of lymphatic regeneration and inflammatory lymphangiogenesis, we explored the hypothesis that hypoxia inducible factor‐α (HIF‐1α) is a central regulator of lymphangiogenesis. We show that HIF‐1α inhibition by small molecule inhibitors (YC‐1 and 2‐methyoxyestradiol) results in delayed lymphatic repair, decreased local vascular endothelial growth factor‐C (VEGF‐C) expression, reduced numbers of VEGF‐C+ cells, and reductions in inflammatory lymphangiogenesis. Using transgenic HIF‐1α/luciferase mice to image HIF‐1α expression in real time in addition to Western blot analysis and pimonidazole staining for cellular hypoxia, we demonstrate that hypoxia stabilizes HIF‐1α during initial stages of wound repair (1‐2 wk); whereas inflammation secondary to gradients of lymphatic fluid stasis stabilizes HIF‐1α thereafter (3‐6 wk). In addition, we show that CD4+ cell‐mediated inflammation is necessary for this response and regulates HIF‐1α expression by macrophages, as CD4‐deficient or CD4‐depleted mice demonstrate 2‐fold reductions in HIF‐1α expression as compared to wild‐types. In summary, we show that HIF‐1α is a critical coordinator of lymphangiogenesis by regulating the expression of lymphangiogenic cytokines as part of an early response mechanism to hypoxia, inflammation, and lymphatic fluid stasis.—Zampell, J. C., Yan, A., Avraham, T., Daluvoy, S., Weitman, E. S., Mehrara, B. J. HIF‐1α coordinates lymphangiogenesis during wound healing and in response to inflammation. FASEB J. 26, 1027‐1039 (2012). www.fasebj.org

Anatomical and Surgical Concepts in Lymphatic Regeneration

Abstract:  Chronic post‐surgical lymphedema is common condition that afflicts nearly 2 million Americans. In the USA, it is most commonly encountered in the upper extremities of patients who have undergone axillary lymph node dissection for breast cancer. Lymphedema has a significant negative effect on cosmesis, limb function, and overall quality of life. Despite the impact of this condition, very little is known about how to effectively prevent or treat lymphedema. While therapeutic options for chronic extremity lymphedema remain limited, several surgical approaches have been suggested. These include techniques aimed at reducing limb volume, as well as techniques that aim to reconstitute disrupted lymphatic channels. Operations proposed to re‐establish lymphatic continuity include lymphatico‐venous anastomoses, lymphatico‐lymphatico anastomoses, and tissue transfer.

9: LYMPHEDEMA IS A FIBROPROLIFERATIVE CONDITION ASSOCIATED WITH A PRO-FIBROTIC CHRONIC INFLAMMATORY RESPONSE

Results: Similar to clinical lymphedema, sustained lymphedema resulted in a chronic inflammatory reaction consisting predominantly CD4+ t-cells. Furthermore, sustained stasis resulted in a mixed Th1-Th2 response whereas transient stasis was associated with only a Th1 response. Application of our surgical model to athymic nude mice, demonstrated that pathological fibrosis resulting from sustained lymphedema is T-cell dependent. Inhibition of Th2 differentiation starting at the time of surgery prevented the establishment of fibrosis and lymphedema, with restoration of near baseline lymphatic function. Inhibition of Th2 differentiation in animals with established lymphedema using a monoclonal antibody against IL-4 resulted in a marked reduction in tail volume, improved lymphatic function, and resolution of inflammatory reaction. These improvements persisted after withdrawal of therapy. In contrast, isotype control treated animals demonstrated no improvement.

Abstract 3830: Inhibition of IL-4 function decreases fibrosis and lymphatic dysfunction in a mouse model of lymphedema

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Lymphedema, commonly encountered in cancer survivors, is characterized by chronic swelling and fibrosis. Previous studies have shown that fibrotic disorders involving other organ systems are dependent on T-helper 2 (Th-2) chronic inflammatory changes. The purpose of these experiments was therefore to evaluate the role of Th-2 cells in the pathological changes associated with lymphedema. Methods: We developed a surgical mouse model that results in tail lymphedema for prolonged periods (>9 weeks) and is histologically is similar to clinical lymphedema. Using this model in wild-type and athymic nude mice we evaluated the role of T-cells in general on the development of lymphedema and fibrosis. Furthermore, using a specific monoclonal neutralizing antibody against IL-4, a molecule necessary for Th-2 differentiation, we evaluated the role of Th2 cells in the initiation and maintenance of chronic lymphedema. Results: Lymphedema in wild-type mice resulted in a chronic inflammation consisting predominantly of CD-4+cells with a mixed Th1-Th2+ differentiation. Interestingly, application of this model to athymic nude mice resulted in markedly decreased tail edema and fibrosis, demonstrating that these outcomes are T-cell dependent. While inhibition of Th2 differentiation immediately after surgery did not prevent initiation of lymphedema, it did result in early resolution, with a marked reduction in fibrosis and improved lymphatic function 6 weeks post-operatively. Consistent with this, inhibition of Th2 cell function in mice with established lymphedema resulted in resolution of lymphedema and fibrosis with just 3 weekly treatments. In contrast, lymphedema was sustained in animals treated with isotype control. Conclusions: We have shown that Th2 differentiation is necessary for maintenance of the pathologic changes associated with lymphedema. Inhibition of Th2 differentiation completely reversed the fibrosis and lymphatic dysfunction in animals with established lymphedema, with resultant resolution of swelling. These results indicate that similar approaches may be clinically useful in patients with chronic lymphedema. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3830.

Cellular and Molecular Mechanisms of Lymphangiogenesis and Lymphedema

Abstract: Chronic secondary lymphedema is a potentially devastating condition affecting 90-150 million people world-wide. In the US, lymphedema is most commonly encountered in the upper extremity of women who have undergone axil-lary lymph node dissection for staging and treatment of breast cancer, though it may also occur following lymph node dis-section for melanoma as well as certain gynecology oncologic operations. While a great deal has been elucidated about the biology of lymphatics, lymphatic development, and the lymphatic system in the past 20 years, considerably less is known about impaired lymphatic regeneration and the mechanisms that lead to secondary lymphedema. This deficit in knowledge presents a barrier to the development of effective treatments or prophylactic measures for chronic secondary lymphedema. Recent advances in this arena have showed that pathogenesis of lymphedema is complex, and that effective treatments for this often devastating condition will likely require the use of multiple modalities. In this review, we will discuss the development, anatomy, and physiology of lymphatics as means of introducing this system to the reader. Fur-ther, the latest advances in the scientific exploration of lymphedema and lymphatic regeneration will be presented.