Surgical Delay-Induced Hemodynamic Alterations of the Superficial Inferior Epigastric Artery Flap for Autologous Breast Reconstruction.

BACKGROUND: The superficial inferior epigastric artery (SIEA) flap allows transfer of tissue without violating the rectus fascia. Traditionally it is best used in single stage reconstruction when vessel caliber is 1.5 mm; 56% to 70% of SIEAs are less than 1.5 mm and, therefore, not reliable. We aim to demonstrate the increased reliability of SIEA through surgical delay by quantifying reconstructive outcomes and delay-induced hemodynamic alterations. METHODS: Patients presenting for autologous breast reconstruction between May 2019 and October 2020 were evaluated with preoperative imaging and received either delayed SIEA or delayed deep inferior epigastric (DIEP) reconstruction based on clinical considerations, such as prior surgery and perforator size/location. Prospective data were collected on operative time, length of stay, and complications. Arterial diameter and peak flow were quantified with Doppler ultrasound predelay and postdelay. RESULTS: Seventeen delayed SIEA flaps were included. The mean age (± SD) was 46.2 ± 10.55 years, and body mass index was 26.7 ± 4.26 kg/m2. Average hospital stay after delay was 0.85 ± 0.90 days, and duration before reconstruction was 6 days to 14.5 months. Delay complications included 1 abdominal seroma (n = 1, 7.7%). Superficial inferior epigastric artery diameter predelay (mean ± 95% confidence interval) was 1.37 ± 0.20 mm and increased to 2.26 ± 0.24 mm postdelay. A significant increase in diameter was noted 0.9 ± 0.22 mm (P < 0.0001). Mean peak flow predelay was 14.43 ± 13.38 cm/s and 44.61 ± 60.35 cm/s (n = 4, P = 0.1822) postdelay. CONCLUSIONS: Surgical delay of the SIEA flap augments SIEA diameter, increasing the reliability of this flap for breast reconstruction. Superficial inferior epigastric artery delay results in low rates of complications and no failures in our series. Although more patients are needed to assess increase in arterial flow, use of surgical delay can expand the use of SIEA flap reconstruction and reduce abdominal morbidity associated with abdominal flap breast reconstruction.

Stratification of Surgical Risk in DIEP Breast Reconstruction Based on Classification of Obesity.

BACKGROUND: From both a medical and surgical perspective, obese breast cancer patients are considered to possess higher risk when undergoing autologous breast reconstruction relative to nonobese patients. However, few studies have evaluated the continuum of risk across the full range of obesity. This study sought to compare surgical risk between the three World Health Organization (WHO) classes of obesity in patients undergoing deep inferior epigastric perforator (DIEP) flap breast reconstruction. METHODS: A retrospective review of 219 obese patients receiving 306 individual DIEP flaps was performed. Subjects were stratified into WHO obesity classes I (body mass index [BMI]: 30-34), II (BMI: 35-39), and III (BMI: ≥ 40) and assessed for risk factors and postoperative donor and recipient site complications. RESULTS: When examined together, the rate of any complication between the three groups only trended toward significance (p = 0.07), and there were no significant differences among rates of specific individual complications. However, logistic regression analysis showed that class III obesity was an independent risk factor for both flap (odds ratio [OR]: 1.71, 95% confidence interval [CI]: 0.91-3.20, p = 0.03) and donor site (OR: 2.34, 95% CI: 1.09-5.05, p = 0.03) complications. CONCLUSION: DIEP breast reconstruction in the obese patient is more complex for both the patient and the surgeon. Although not a contraindication to undergoing surgery, obese patients should be diligently counseled regarding potential complications and undergo preoperative optimization of health parameters. Morbidly obese (class III) patients should be approached with additional caution, and perhaps even delay major reconstruction until specific BMI goals are met.

Regulation of inflammation and fibrosis by macrophages in lymphedema.

Lymphedema, a common complication of cancer treatment, is characterized by inflammation, fibrosis, and adipose deposition. We have previously shown that macrophage infiltration is increased in mouse models of lymphedema. Because macrophages are regulators of lymphangiogenesis and fibrosis, this study aimed to determine the role of these cells in lymphedema using depletion experiments. Matched biopsy specimens of normal and lymphedema tissues were obtained from patients with unilateral upper extremity breast cancer-related lymphedema, and macrophage accumulation was assessed using immunohistochemistry. In addition, we used a mouse tail model of lymphedema to quantify macrophage accumulation and analyze outcomes of conditional macrophage depletion. Histological analysis of clinical lymphedema biopsies revealed significantly increased macrophage infiltration. Similarly, in the mouse tail model, lymphatic injury increased the number of macrophages and favored M2 differentiation. Chronic macrophage depletion using lethally irradiated wild-type mice reconstituted with CD11b-diphtheria toxin receptor mouse bone marrow did not decrease swelling, adipose deposition, or overall inflammation. Macrophage depletion after lymphedema had become established significantly increased fibrosis and accumulation of CD4(+) cells and promoted Th2 differentiation while decreasing lymphatic transport capacity and VEGF-C expression. Our findings suggest that macrophages home to lymphedematous tissues and differentiate into the M2 phenotype. In addition, our findings suggest that macrophages have an antifibrotic role in lymphedema and either directly or indirectly regulate CD4(+) cell accumulation and Th2 differentiation. Finally, our findings suggest that lymphedema-associated macrophages are a major source of VEGF-C and that impaired macrophage responses after lymphatic injury result in decreased lymphatic function.

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.

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.

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.

Regulation of adipogenesis by lymphatic fluid stasis: part I. Adipogenesis, fibrosis, and inflammation.

BACKGROUND: Although fat deposition is a defining clinical characteristic of lymphedema, the cellular mechanisms that regulate this response remain unknown. The goals of this two-part study were to determine the effect of lymphatic fluid stasis on adipogenesis and inflammation (part I) and how these changes regulate the temporal and spatial expression of fat differentiation genes (part II). METHODS: Adult female mice underwent tail lymphatic ablation and were euthanized 6 weeks after surgery (n = 20). Fat deposition, fibrosis, and inflammation were then analyzed in the regions of the tail exposed to lymphatic fluid stasis as compared with normal lymphatic flow. RESULTS: Lymphatic fluid stasis in the tail resulted in significant subcutaneous fat deposition, with a 2-fold increase in fat thickness (p < 0.01). In addition, lymphatic stasis was associated with subcutaneous fat fibrosis and collagen deposition. Adipogenesis in response to lymphatic fluid stasis was associated with a marked mononuclear cell inflammatory response (5-fold increase in CD45 cells; p < 0.001). In addition, the authors noted a significant increase in the number of monocytes/macrophages as identified by F4/80 immunohistochemistry (p < 0.001). CONCLUSIONS: The mouse-tail model has pathologic findings that are similar to clinical lymphedema, including fat deposition, fibrosis, and inflammation. Adipogenesis in response to lymphatic fluid stasis closely resembles this process in obesity. This model therefore provides an excellent means with which to study the molecular mechanisms that regulate the pathophysiology of lymphedema.

Regulation of adipogenesis by lymphatic fluid stasis: part II. Expression of adipose differentiation genes.

BACKGROUND: Although fat deposition is a defining clinical characteristic of lymphedema, the cellular mechanisms that regulate this response remain unknown. The goal of this study was to determine how lymphatic fluid stasis regulates adipogenic gene activation and fat deposition. METHODS: Adult female mice underwent tail lymphatic ablation and were euthanied at 1, 3, or 6 weeks postoperatively (n = 8 per group). Samples were analyzed by immunohistochemistry and Western blot analysis. An alternative group of mice underwent axillary dissections or sham incisions, and limb tissues were harvested 3 weeks postoperatively (n = 8 per group). RESULTS: Lymphatic fluid stasis resulted in significant subcutaneous fat deposition and fibrosis in lymphedematous tail regions (p < 0.001). Western blot analysis demonstrated that proteins regulating adipose differentiation including CCAAT/enhancer-binding protein-α and adiponectin were markedly up-regulated in response to lymphatic fluid stasis in the tail and axillary models. Expression of these markers increased in edematous tissues according to the gradient of lymphatic stasis distal to the wound. Immunohistochemical analysis further demonstrated that adiponectin and peroxisome proliferator-activated receptor-γ, another critical adipogenic transcription factor, followed similar expression gradients. Finally, adiponectin and peroxisome proliferator-activated receptor-γ expression localized to a variety of cell types in newly formed subcutaneous fat. CONCLUSIONS: The mouse-tail model of lymphedema demonstrates pathologic findings similar to clinical lymphedema, including fat deposition and fibrosis. The authors show that lymphatic fluid stasis potently up-regulates the expression of fat differentiation markers both spatially and temporally. These studies elucidate mechanisms regulating abnormal fat deposition in lymphedema pathogenesis and therefore provide a basis for developing targeted treatments.

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-ß1, 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.

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-ß1 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.

Adipose-derived stem cells promote lymphangiogenesis in response to VEGF-C stimulation or TGF-ß1 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-ß1 receptor-II adenovirus with or without VEGF-C stimulation, since TGF-ß1 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-ß1 signaling was inhibited using the dominant-negative TGF-ß1 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-ß1 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.

Surgical management of lymphedema: past, present, and future.

Recent advances in surgical management of lymphedema have provided options for patients who have failed conservative management with manual lymphatic massage and/or compression garments. The purpose of this review is to provide a historical background to the surgical treatment of lymphedema and how these options have evolved over time. In addition, we aim to delineate the various types of surgical approaches available, indications for surgery, and reported outcomes. Our goal is to increase awareness of these options and foster research to improve their outcomes.

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.

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)-κß 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.

p21cip/WAF is a key regulator of long-term radiation damage in mesenchyme-derived tissues.

This study aimed to determine the mechanisms responsible for long-term tissue damage following radiation injury. We irradiated p21-knockout (p21(-/-)) and wild-type (WT) mice and determined the long-term deleterious effects of this intervention on mesenchyme-derived tissues. In addition, we explored the mechanisms of radiation-induced mesenchymal stem cell (MSC) dysfunction in isolated bone marrow-derived cells. p21 expression was chronically elevated >200-fold in irradiated tissues. Loss of p21 function resulted in a >4-fold increase in the number of skin MSCs remaining after radiation. p21(-/-) mice had significantly less radiation damage, including 6-fold less scarring, 40% increased growth potential, and 4-fold more hypertrophic chondrocytes in the epiphyseal plate (P<0.01). Irradiated p21(-/-) MSCs had 4-fold increased potential for bone or fat differentiation, 4-fold greater proliferation rate, and nearly 7-fold lower senescence as compared to WT MSCs (P<0.01). Ectopic expression of p21 in knockout cells decreased proliferation and differentiation potential and recapitulated the WT phenotype. Loss of p21 function markedly decreases the deleterious effects of radiation injury in mesenchyme-derived tissues and preserves tissue-derived MSCs. In addition, p21 is a critical regulator of MSC proliferation, differentiation, and senescence both at baseline and in response to radiation.

Radiation therapy causes loss of dermal lymphatic vessels and interferes with lymphatic function by TGF-beta1-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.

Patterns of axillary surgical care for breast cancer in the era of sentinel lymph node biopsy.

BACKGROUND: Population-based overall patterns of surgical management of the axilla in women with operable breast cancer during the era of adoption of sentinel lymph node biopsy (SLNB) were studied. METHODS: Women with operable breast carcinoma residing in 14 geographic areas of the Surveillance, Epidemiology, and End Results (SEER) cancer registries (1998-2004, n=239,661) were assessed for axillary surgical patterns of care. RESULTS: Use of SLNB increased from 11 to 59%. Use of no axillary surgery decreased from 14 to 6.6%. In pathologic node-negative women, use of axillary lymph node dissection (ALND) decreased from 94 to 36%. Independent factors most associated with failure to receive SLNB included diagnosis year (2000: 62%; 2004: 29%), surgery (mastectomy: 64%; breast-conserving surgery: 36%), tumor size (T3: 71%; T2: 56%; T1: 40%), age (>or= 70 years: 50%; <70 years: 45%), grade (high: 42%; low: 38%), urbanity (non-large metropolitan area: 49%; large metropolitan area: 42%), and, by quartile, poverty (highest: 47%; lowest: 35%), and white-collar employment (lowest: 56%; highest: 47%). In pathologic node-positive women who had SLNB, failure to undergo completion ALND increased from 20% in 1998 to 32% in 2004. Patients with smaller, lower-grade tumors, and those with smaller size of nodal metastasis, lack of extracapsular extension, age >or= 70 years, increased linguistic isolation, African-American or Hispanic race/ethnicity, and white-collar employment were less likely to undergo completion ALND. CONCLUSIONS: Management of the axilla changed dramatically during the period of rapid adoption of SLNB. Patterns of care suggest both appropriate and inappropriate selection for SLNB and ALND.

Modified cell ELISA to determine the solubilization of cell surface proteins: Applications in GPI-anchored protein purification.

A major step in purifying membrane bound proteins involves the solubilization of the protein of interest from the cell membranes. Glycosylphosphatidyl inositol (GPI)-anchored proteins pose a singular problem in this solubilization step since they are found in detergent-resistant membrane complexes and accordingly are insoluble in cold Triton X-100. In this study we have developed a modified cell ELISA that determines the solubility of these cell surface proteins under various solubilization conditions. Using this non-radioactive method we show that the combination of saponin/Triton X-100 at 4 degrees C solubilized GPI-anchored proteins more efficiently than Triton X-100 at 4 degrees C. The combination of saponin/Triton X-100 at 4 degrees C avoids the potential of activating proteases that occurs when using Triton X-100 at 37 degrees C. Furthermore, our method also shows the saponin/Triton X-100 solubilized GPI-anchored proteins equivalent to the more expensive octyl beta-glucoside. This is a particularly important consideration in large-scale protein purification. This method obviates the need to use radioactivity, gel electrophoresis and immunoblotting procedures. The solubilization conditions determined by this modified ELISA are readily translated to the practical application of large-scale protein purification as demonstrated in the purification of two different recombinant GPI-anchored proteins, GPI-hB7-1 (CD80) and GPI-mICAM-1 (CD54).