Seth Z. Aschen

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.

Mechanisms of Lymphatic Regeneration after Tissue Transfer

Introduction Lymphedema is the chronic swelling of an extremity that occurs commonly after lymph node resection for cancer treatment. Recent studies have demonstrated that transfer of healthy tissues can be used as a means of bypassing damaged lymphatics and ameliorating lymphedema. The purpose of these studies was to investigate the mechanisms that regulate lymphatic regeneration after tissue transfer. Methods Nude mice (recipients) underwent 2-mm tail skin excisions that were either left open or repaired with full-thickness skin grafts harvested from donor transgenic mice that expressed green fluorescent protein in all tissues or from LYVE-1 knockout mice. Lymphatic regeneration, expression of VEGF-C, macrophage infiltration, and potential for skin grafting to bypass damaged lymphatics were assessed. Results Skin grafts healed rapidly and restored lymphatic flow. Lymphatic regeneration occurred beginning at the peripheral edges of the graft, primarily from ingrowth of new lymphatic vessels originating from the recipient mouse. In addition, donor lymphatic vessels appeared to spontaneously re-anastomose with recipient vessels. Patterns of VEGF-C expression and macrophage infiltration were temporally and spatially associated with lymphatic regeneration. When compared to mice treated with excision only, there was a 4-fold decrease in tail volumes, 2.5-fold increase in lymphatic transport by lymphoscintigraphy, 40% decrease in dermal thickness, and 54% decrease in scar index in skin-grafted animals, indicating that tissue transfer could bypass damaged lymphatics and promote rapid lymphatic regeneration. Conclusions Our studies suggest that lymphatic regeneration after tissue transfer occurs by ingrowth of lymphatic vessels and spontaneous re-connection of existing lymphatics. This process is temporally and spatially associated with VEGF-C expression and macrophage infiltration. Finally, tissue transfer can be used to bypass damaged lymphatics and promote rapid lymphatic regeneration.

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.

Temporal and spatial patterns of endogenous danger signal expression after wound healing and in response to lymphedema

While acute tissue injury potently induces endogenous danger signal expression, the role of these molecules in chronic wound healing and lymphedema is undefined. The purpose of this study was to determine the spatial and temporal expression patterns of the endogenous danger signals high-mobility group box 1 (HMGB1) and heat shock protein (HSP)70 during wound healing and chronic lymphatic fluid stasis. In a surgical mouse tail model of tissue injury and lymphedema, HMGB1 and HSP70 expression occurred along a spatial gradient relative to the site of injury, with peak expression at the wound and greater than twofold reduced expression within 5 mm (P < 0.05). Expression primarily occurred in cells native to injured tissue. In particular, HMGB1 was highly expressed by lymphatic endothelial cells (>40% positivity; twofold increase in chronic inflammation, P < 0.001). We found similar findings using a peritoneal inflammation model. Interestingly, upregulation of HMGB1 (2.2-fold), HSP70 (1.4-fold), and nuclear factor (NF)-κβ activation persisted at least 6 wk postoperatively only in lymphedematous tissues. Similarly, we found upregulation of endogenous danger signals in soft tissue of the arm after axillary lymphadenectomy in a mouse model and in matched biopsy samples obtained from patients with secondary lymphedema comparing normal to lymphedematous arms (2.4-fold increased HMGB1, 1.9-fold increased HSP70; P < 0.01). Finally, HMGB1 blockade significantly reduced inflammatory lymphangiogenesis within inflamed draining lymph nodes (35% reduction, P < 0.01). In conclusion, HMGB1 and HSP70 are expressed along spatial gradients and upregulated in chronic lymphatic fluid stasis. Furthermore, acute expression of endogenous danger signals may play a role in inflammatory lymphangiogenesis.

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.

Sterile inflammation after lymph node transfer improves lymphatic function and regeneration.

Background: The aim of this study was to determine whether sterile inflammatory reactions can serve as a physiologic means of augmenting lymphangiogenesis in transplanted lymph nodes using a murine model. Methods: The authors used their previously reported model of lymph node transfer to study the effect of sterile inflammation on lymphatic regeneration. Mice were divided into three groups: group 1 (controls) underwent lymphadenectomy followed by immediate lymph node transplantation without inflammation; group 2 (inflammation before transfer) underwent transplantation with lymph nodes harvested from donor animals in which a sterile inflammatory reaction was induced in the ipsilateral donor limb; and group 3 (inflammation after transfer) underwent transplantation with lymph nodes and then inflammation was induced in the ipsilateral limb. Lymphatic function, lymphangiogenesis, and lymph node histology were examined 28 days after transplantation and compared with those of normal lymph nodes. Results: Animals that had sterile inflammation after transplantation (group 3) had significantly improved lymphatic function (>2-fold increase) on lympho scintigraphy, increased perinodal lymphangiogenesis, and functional lymphatics compared with the groups with no inflammation and inflammation before transplantation (p < 0.01). Inflammation after transplantation was associated with a more normal lymph node architecture, expansion of B-cell zones, and decreased percentage of T cells compared with the other experimental groups. Conclusions: Sterile inflammation is a potent method of augmenting lymphatic function and lymphangiogenesis after lymph node transplantation and is associated with maintenance of lymph node architecture. Induction of inflammation after transplantation is the most effective method and promotes maintenance of normal lymph node B- and T-cell architecture.

Abstract 146: diet-induced obesity results in lymphatic dysfunction and impaired T cell function.

Suday, M arch 9, 2014 producing two dividing cells and 10% one differentiated and one proliferating cell. Here too, division outcomes were independent of neighbours or close relations. These stem-like cells generated colonies containing hundreds of cells, mostly proliferating. Combinatorial statistics using the timelapse data predicted the distribution of clone sizes in a large sample of cells (n=1462) cultured for 7days, indicating the probabilities of each type of division were balanced as in vivo. Hypersupplementation (20ng/mL) or withdrawal (0ng/mL) of EGF, or addition of R-Spondin to the culture imbalanced the generation of proliferating or differentiating cells indicating that the fate of the progenitor-like cells was subject to external regulation. Transcriptional analysis revealed a distinct pattern of gene expression in 8-cell colonies derived from stem and progenitor-like cells. Validation of these results by immunostaining revealed the proportion of cells expressing differentiation associated genes in progenitor-derived colonies in accordance with the timelapse data.

Abstract 86: Lymph Node Transplantation Generates Spontaneous Lymphatic Reconnection and Restoration of Lymphatic Flow

PurPose: Although lymph node transplantation has been shown to improve lymphatic function in patients with lymphedema, the mechanisms regulating lymphatic vessel reconnection and the functional status of lymph nodes (LNs) remains poorly understood. In this study we developed a novel reporter mouse that allowed us to determine the origin of lymphatic endothelial cells (LECs) and vessels forming functional connections with transferred LNs.

Abstract 145: Lymph Node Transfer Restores Immune Responses in a Mouse Model of Lymphedema

RESULTS: Growth in hypoxia at 5% O2 increased cyclinD2, a marker of cell cycle progression (1.7-fold, p<0.05) and showed no change in p21, a marker of replicative senescence, indicating that hypoxia promotes proliferation. MSCs grown in hypoxia demonstrated preservation of stemness, as measured by flow cytometry (97.9% CD90+, CD29+ MSCs in hypoxia versus 63.9% in normoxia). There was no significant change in markers of differentiation or immunosuppression in hypoxia versus normoxia. Growth at 0.5% O2 resulted in a decrease in all markers assessed, including those for immunomodulation, replication, and differentiation.

Abstract 144: cd4+ cells are key regulators of pathologic changes in lymphedema.

our oBjeCtives are: 1. Demonstrate that grafting human skin onto TCR (T-cell receptor) αβ-/-γδ-/-, RAG (recombination activating gene)1-/and RAG-2-/-γc-/mice results in scars morphologically and histologically consistent with human HSc. 2. Characterize histologic and cellular changes that occur in scars with removal of specific immune cell subsets. 3. Compare scar response over time nude and knockout mice.