Evan Weitman

Th2 differentiation is necessary for soft tissue fibrosis and lymphatic dysfunction resulting from lymphedema

Lymphedema is a dreaded complication of cancer treatment. However, despite the fact that > 5 million Americans are affected by this disorder, the development of effective treatments is limited by the fact that the pathology of lymphedema remains unknown. The purpose of these studies was to determine the role of inflammatory responses in lymphedema pathology. Using mouse models of lymphedema, as well as clinical lymphedema specimens, we show that lymphatic stasis results in a CD4+ T‐cell inflammation and T‐helper 2 (Th2) differentiation. Using mice deficient in T cells or CD4+ cells, we show that this inflammatory response is necessary for the pathological changes of lymphedema, including fibrosis, adipose deposition, and lymphatic dysfunction. Further, we show that inhibition of Th2 differentiation using interleukin‐4 (IL‐4) or IL‐13 blockade prevents initiation and progression of lymphedema by decreasing tissue fibrosis and significantly improving lymphatic function, independent of lymphangiogenic growth factors. We show that CD4+ inflammation is a critical regulator of tissue fibrosis and lymphatic dysfunction in lymphedema and that inhibition of Th2 differentiation markedly improves lymphatic function independent of lymphangiogenic cytokine expression. Notably, preventing and/or reversing the development of pathological tissue changes that occur in lymphedema may be a viable treatment strategy for this disorder.—Avraham, T., Zampell, J. C., Yan, A., Elhadad, S., Weitman, E. S., Rockson, S. G., Bromberg, J., Mehrara, B. J. Th2 differentiation is necessary for soft tissue fibrosis and lymphatic dysfunction resulting from lymphedema. FASEB J. 27, 1114–1126 (2013). www.fasebj.org

CD4+ Cells Regulate Fibrosis and Lymphangiogenesis in Response to Lymphatic Fluid Stasis

Introduction Lymphedema is a chronic disorder that occurs commonly after lymph node removal for cancer treatment and is characterized by swelling, fibrosis, inflammation, and adipose deposition. Although previous histological studies have investigated inflammatory changes that occur in lymphedema, the precise cellular make up of the inflammatory infiltrate remains unknown. It is also unclear if this inflammatory response plays a causal role in the pathology of lymphedema. The purpose of this study was therefore to characterize the inflammatory response to lymphatic stasis and determine if these responses are necessary for the pathological changes that occur in lymphedema. Methods We used mouse-tail lymphedema and axillary lymph node dissection (ANLD) models in order to study tissue inflammatory changes. Single cell suspensions were created and analyzed using multi-color flow cytometry to identify individual cell types. We utilized antibody depletion techniques to analyze the causal role of CD4+, CD8+, and CD25+ cells in the regulation of inflammation, fibrosis, adipose deposition, and lymphangiogenesis. Results Lymphedema in the mouse-tail resulted in a mixed inflammatory cell response with significant increases in T-helper, T-regulatory, neutrophils, macrophages, and dendritic cell populations. Interestingly, we found that ALND resulted in significant increases in T-helper cells suggesting that these adaptive immune responses precede changes in macrophage and dendritic cell infiltration. In support of this we found that depletion of CD4+, but not CD8 or CD25+ cells, significantly decreased tail lymphedema, inflammation, fibrosis, and adipose deposition. In addition, depletion of CD4+ cells significantly increased lymphangiogenesis both in our tail model and also in an inflammatory lymphangiogenesis model. Conclusions Lymphedema and lymphatic stasis result in CD4+ cell inflammation and infiltration of mature T-helper cells. Loss of CD4+ but not CD8+ or CD25+ cell inflammation markedly decreases the pathological changes associated with lymphedema. In addition, CD4+ cells regulate lymphangiogenesis during wound repair and inflammatory lymphangiogenesis.

Obesity impairs lymphatic fluid transport and dendritic cell migration to lymph nodes.

Introduction Obesity is a major cause of morbidity and mortality resulting in pathologic changes in virtually every organ system. Although the cardiovascular system has been a focus of intense study, the effects of obesity on the lymphatic system remain essentially unknown. The purpose of this study was to identify the pathologic consequences of diet induced obesity (DIO) on the lymphatic system. Methods Adult male wild-type or RAG C57B6-6J mice were fed a high fat (60%) or normal chow diet for 8–10 weeks followed by analysis of lymphatic transport capacity. In addition, we assessed migration of dendritic cells (DCs) to local lymph nodes, lymph node architecture, and lymph node cellular make up. Results High fat diet resulted in obesity in both wild-type and RAG mice and significantly impaired lymphatic fluid transport and lymph node uptake; interestingly, obese wild-type but not obese RAG mice had significantly impaired migration of DCs to the peripheral lymph nodes. Obesity also resulted in significant changes in the macro and microscopic anatomy of lymph nodes as reflected by a marked decrease in size of inguinal lymph nodes (3.4-fold), decreased number of lymph node lymphatics (1.6-fold), loss of follicular pattern of B cells, and dysregulation of CCL21 expression gradients. Finally, obesity resulted in a significant decrease in the number of lymph node T cells and increased number of B cells and macrophages. Conclusions Obesity has significant negative effects on lymphatic transport, DC cell migration, and lymph node architecture. Loss of T and B cell inflammatory reactions does not protect from impaired lymphatic fluid transport but preserves DC migration capacity. Future studies are needed to determine how the interplay between diet, obesity, and the lymphatic system modulate systemic complications of obesity.

Lymphatic function is regulated by a coordinated expression of lymphangiogenic and anti-lymphangiogenic cytokines

Lymphangiogenic cytokines such as vascular endothelial growth factor-C (VEGF-C) are critically required for lymphatic regeneration; however, in some circumstances, lymphatic function is impaired despite normal or elevated levels of these cytokines. The recent identification of anti-lymphangiogenic molecules such as interferon-γ (IFN-γ), transforming growth factor-β1, and endostatin has led us to hypothesize that impaired lymphatic function may represent a dysregulated balance in the expression of pro/anti-lymphangiogenic stimuli. We observed that nude mice have significantly improved lymphatic function compared with wild-type mice in a tail model of lymphedema. We show that gradients of lymphatic fluid stasis regulate the expression of lymphangiogenic cytokines (VEGF-A, VEGF-C, and hepatocyte growth factor) and that paradoxically the expression of these molecules is increased in wild-type mice. More importantly, we show that as a consequence of T-cell-mediated inflammation, these same gradients also regulate expression patterns of anti-lymphangiogenic molecules corresponding temporally and spatially with impaired lymphatic function in wild-type mice. We show that neutralization of IFN-γ significantly increases inflammatory lymph node lymphangiogenesis independently of changes in VEGF-A or VEGF-C expression, suggesting that alterations in the balance of pro- and anti-lymphangiogenic cytokine expression can regulate lymphatic vessel formation. In conclusion, we show that gradients of lymphatic fluid stasis regulate not only the expression of pro-lymphangiogenic cytokines but also potent suppressors of lymphangiogenesis as a consequence of T-cell inflammation and that modulation of the balance between these stimuli can regulate lymphatic function.

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

Lymph Node Transplantation Results in Spontaneous Lymphatic Reconnection and Restoration of Lymphatic Flow

Background: Although lymph node transplantation has been shown to improve lymphatic function, the mechanisms regulating lymphatic vessel reconnection and functional status of lymph nodes remains poorly understood. Methods: The authors developed and used LacZ lymphatic reporter mice to examine the lineage of lymphatic vessels infiltrating transferred lymph nodes. In addition, the authors analyzed lymphatic function, expression of vascular endothelial growth factor (VEGF)-C, maintenance of T- and B-cell zone, and anatomical localization of lymphatics and high endothelial venules. Results: Reporter mice were specific and highly sensitive in identifying lymphatic vessels. Lymph node transfer was associated with rapid return of lymphatic function and clearance of technetium-99 secondary to a massive infiltration of recipient mouse lymphatics and putative connections to donor lymphatics. T- and B-cell populations in the lymph node were maintained. These changes correlated with marked increases in the expression of VEGF-C in the perinodal fat and infiltrating lymphatics. Newly formed lymphatic channels in transferred lymph nodes were in close anatomical proximity to high endothelial venules. Conclusions: Transferred lymph nodes have rapid infiltration of functional host lymphatic vessels and maintain T- and B-cell populations. This process correlates with increased endogenous expression of VEGF-C in the perinodal fat and infiltrating lymphatics. Anatomical proximity of newly formed lymphatics and high endothelial venules supports the hypothesis that lymph node transfer can improve lymphedema by exchanges with the systemic circulation.

Adipose-derived stem cells promote lymphangiogenesis in response to VEGF-C stimulation or TGF-β1 inhibition

AIMS Recent studies have demonstrated that augmentation of lymphangiogenesis and tissue engineering hold promise as a treatment for lymphedema. The purpose of this study was to determine whether adipose-derived stem cells (ASCs) can be used in lymphatic tissue-engineering by altering the balance between pro- and anti-lymphangiogenic cytokines. MATERIALS & METHODS ASCs were harvested and cultured in media with or without recombinant VEGF-C for 48 h. ASCs were then implanted in mice using Matrigel plugs. Additional groups of animals were implanted with ASCs transfected with a dominant-negative TGF-β1 receptor-II adenovirus with or without VEGF-C stimulation, since TGF-β1 has been shown to have potent antilymphangiogenic effects. Lymphangiogenesis, lymphatic differentiation and cellular proliferation were assessed. RESULTS Stimulation of ASCs with VEGF-C in vitro significantly increased expression of VEGF-A, VEGF-C and Prox-1. ASCs stimulated with VEGF-C prior to implantation induced a significant (threefold increase) lymphangiogenic response as compared with control groups (unstimulated ASCs or empty Matrigel plugs; p < 0.01). This effect was significantly potentiated when TGF-β1 signaling was inhibited using the dominant-negative TGF-β1 receptor-II virus (4.5-fold increase; p < 0.01). Stimulation of ASCs with VEGF-C resulted in a marked increase in the number of donor ASCs (twofold; p < 0.01) and increased the number of proliferating cells (sevenfold; p < 0.01) surrounding the Matrigel. ASCs stimulated with VEGF-C expressed podoplanin, a lymphangiogenic cell marker, whereas unstimulated cells did not. CONCLUSION Short-term stimulation of ASCs with VEGF-C results in increased expression of VEGF-A, VEGF-C and Prox-1 in vitro and is associated with a marked increase lymphangiogenic response after in vivo implantation. This lymphangiogenic response is significantly potentiated by blocking TGF-β1 function. Furthermore, stimulation of ASCs with VEGF-C markedly increases cellular proliferation and cellular survival after in vivo implantation and stimulated cells express podoplanin, a lymphangiogenic cell marker.

Toll-like receptor deficiency worsens inflammation and lymphedema after lymphatic injury

Mechanisms regulating lymphedema pathogenesis remain unknown. Recently, we have shown that lymphatic fluid stasis increases endogenous danger signal expression, and these molecules influence lymphatic repair (Zampbell JC, et al. Am J Physiol Cell Physiol 300: C1107-C1121, 2011). Endogenous danger signals activate Toll-like receptors (TLR) 2, 4, and 9 and induce homeostatic or harmful responses, depending on physiological context. The purpose of this study was to determine the role of TLRs in regulating tissue responses to lymphatic fluid stasis. A surgical model of lymphedema was used in which wild-type or TLR2, 4, or 9 knockout (KO) mice underwent tail lymphatic excision. Six weeks postoperatively, TLR KOs demonstrated markedly increased tail edema compared with wild-type animals (50-200% increase; P < 0.01), and this effect was most pronounced in TLR4 KOs (P < 0.01). TLR deficiency resulted in decreased interstitial and lymphatic transport, abnormal lymphatic architecture, and fewer capillary lymphatics (40-50% decrease; P < 0.001). Lymphedematous tissues of TLR KOs demonstrated increased leukocyte infiltration (P < 0.001 for TLR4 KOs), including higher numbers of infiltrating CD3+ cells (P < 0.05, TLR4 and TLR9 KO), yet decreased infiltrating F4/80+ macrophages (P < 0.05, all groups). Furthermore, analysis of isolated macrophages revealed twofold reductions in VEGF-C (P < 0.01) and LYVE-1 (P < 0.05) mRNA from TLR2-deficient animals. Finally, TLR deficiency was associated with increased collagen type I deposition and increased transforming growth factor-β1 expression (P < 0.01, TLR4 and TLR9 KO), contributing to dermal fibrosis. In conclusion, TLR deficiency worsens tissue responses to lymphatic fluid stasis and is associated with decreased lymphangiogenesis, increased fibrosis, and reduced macrophage infiltration. These findings suggest a role for innate immune responses, including TLR signaling, in lymphatic repair and lymphedema pathogenesis.

Tissue engineering and regeneration of lymphatic structures

Tissue engineering is the process by which biological structures are recreated using a combination of molecular signals, cellular components and scaffolds. Although the perceived potential of this approach to reconstruct damaged or missing tissues is seemingly limitless, application of these ideas in vivo has been more difficult than expected. However, despite these obstacles, important advancements have been reported for a number of organ systems, including recent reports on the lymphatic system. These advancements are important since the lymphatic system plays a central role in immune responses, regulation of inflammation, lipid absorption and interstitial fluid homeostasis. Insights obtained over the past two decades have advanced our understanding of the molecular and cellular mechanisms that govern lymphatic development and function. Utilizing this knowledge has led to important advancements in lymphatic tissue engineering, which is the topic of this review.

IL-6 regulates adipose deposition and homeostasis in lymphedema

Lymphedema (LE) is a morbid disease characterized by chronic limb swelling and adipose deposition. Although it is clear that lymphatic injury is necessary for this pathology, the mechanisms that underlie lymphedema remain unknown. IL-6 is a known regulator of adipose homeostasis in obesity and has been shown to be increased in primary and secondary models of lymphedema. Therefore, the purpose of this study was to determine the role of IL-6 in adipose deposition in lymphedema. The expression of IL-6 was analyzed in clinical tissue specimens and serum from patients with or without LE, as well as in two mouse models of lymphatic injury. In addition, we analyzed IL-6 expression/adipose deposition in mice deficient in CD4(+) cells (CD4KO) or IL-6 expression (IL-6KO) or mice treated with a small molecule inhibitor of IL-6 or CD4 depleting antibodies to determine how IL-6 expression is regulated and the effect of changes in IL-6 expression on adipose deposition after lymphatic injury. Patients with LE and mice treated with lymphatic excision of the tail had significantly elevated tissue and serum expression of IL-6 and its downstream mediator. The expression of IL-6 was associated with adipose deposition and CD4(+) inflammation and was markedly decreased in CD4KO mice. Loss of IL-6 function resulted in significantly increased adipose deposition after tail lymphatic injury. Our findings suggest that IL-6 is increased as a result of adipose deposition and CD4(+) cell inflammation in lymphedema. In addition, our study suggests that IL-6 expression in lymphedema acts to limit adipose accumulation.