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BACKGROUND: Breast cancer-related lymphedema affects 700,000 breast cancer survivors in the United States. Although taxane-based chemotherapy regimens are commonly used in the treatment of breast cancer, the impact of taxanes on the lymphatic system remains poorly understood. This study aims to examine the influence of taxane-based chemotherapy on lymphatic function in breast cancer patients. METHODS: A retrospective review of a prospectively-maintained database was performed. Consecutive patients with node positive breast cancer who underwent preoperative indocyanine green (ICG) lymphangiograms were identified. Information including patient demographics, baseline measurements, cancer characteristics, and treatment information were retrieved. Preoperative ICG lymphangiography videos were analyzed and lymphatic contractility was quantified for each subject. Multiple regions of interest were selected on each lymphatic channel and signal intensity was recorded for 3 minutes to generate contractility curves. Each lymphatic contraction was identified using a novel, systematic, and algorithmic approach. RESULTS: Twenty-nine consecutive patients with unilateral node-positive breast cancer were included for analysis. Average patient age was 54.5 (13) years and mean BMI was 26.8 kg/m (4). The mean lymphatic contractility of patients who received taxane-based neoadjuvant chemotherapy was 0.7 contractions/minute (c/m) (n = 19) compared to 1.1 c/m in those who received no neoadjuvant therapy (n = 10), (P = 0.11). In subgroup analysis, patients who reported taxane induced neuropathy demonstrated significantly lower lymphatic contractility values than those who were asymptomatic or did not receive any chemotherapy (P = 0.018). CONCLUSIONS: In this study, we used a novel method for quantifying and evaluating lymphatic contractility rates in routine ICG lymphangiograms. Diminished lymphatic contractility was noted in patients who received taxane-based neoadjuvant chemotherapy compared with those who did not. Taxane-based neoadjuvant chemotherapy may adversely affect the lymphatic system in the breast cancer population. A larger patient cohort with longer follow-up time is needed to validate this finding and evaluate any potential association with breast cancer-related lymphedema development.
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Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Sistema Linfático/efeitos dos fármacos , Taxoides/farmacologia , Taxoides/uso terapêutico , Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Rheumatoid arthritis (RA) is a prevalent inflammatory joint disease with enigmatic flares, which causes swelling, pain, and irreversible connective tissue damage. Recently, it has been demonstrated in murine models of RA that the popliteal lymph node (PLN) is a biomarker of arthritic flare, as it "expands" in size and contrast enhancement during a prolonged asymptomatic phase, prior to when it "collapses" with accelerated synovitis and joint erosion. This PLN collapse is associated with adjacent knee flare, decreases in PLN volume and contrast enhancement, lymphatic pulse and pumping pressure, and an increase in PLN pressure. Currently, it is known that PLN collapse is accompanied by a translocation of B cells from the follicles to the sinuses, effectively clogging the lymphatic sinuses of the PLN, and that B cell depletion therapy ameliorates arthritic flare by eliminating these B cells and restoring passive lymphatic flow from inflamed joints. Here we review the technological advances that have launched this area of research, describe future directions to help elucidate the potential mechanism of PLN collapse, and speculate on clinical translation towards new diagnostics and therapies for RA.
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Artrite Reumatoide/patologia , Linfonodos/patologia , Sistema Linfático/fisiologia , Animais , Artrite Reumatoide/imunologia , Linfócitos B/imunologia , Humanos , Linfonodos/imunologia , Sistema Linfático/imunologia , Exacerbação dos SintomasRESUMO
Tumor necrosis factor (TNF) is a key cytokine in rheumatoid arthritis (RA) pathogenesis, as underscored by the clinical effectiveness of TNF antagonists. While several of TNF's key targets in RA are well understood, its many pleiotropic effects remain to be elucidated. TNF-transgenic mice develop inflammatory-erosive arthritis associated with disruption of draining lymph node histology and function, and accumulation of B cells with unique phenotypic and functional features consistent with contribution to pathogenesis (B cells in inflamed nodes, Bin). Bin cell induction depends on the inflamed microenvironment, but the specific signals are unknown. Using anti-TNF treatment and TNF-receptor-deficient mice, here we show that Bin cells are induced and maintained independently of B cell-intrinsic TNF signals.
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Anticorpos/farmacologia , Artrite Experimental/imunologia , Linfócitos B/imunologia , Linfonodos/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Transferência Adotiva , Animais , Antígenos CD1d/genética , Antígenos CD1d/imunologia , Artrite Experimental/tratamento farmacológico , Artrite Experimental/genética , Artrite Experimental/patologia , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Linfócitos B/transplante , Microambiente Celular/imunologia , Expressão Gênica , Humanos , Imunofenotipagem , Linfonodos/efeitos dos fármacos , Linfonodos/patologia , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Complemento 3b/genética , Receptores de Complemento 3b/imunologia , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/imunologia , Receptores de IgE/genética , Receptores de IgE/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/deficiência , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Rheumatoid arthritis (RA) is a chronic inflammatory joint disease with episodic flares. In TNF-Tg mice, a model of inflammatory-erosive arthritis, the popliteal lymph node (PLN) enlarges during the pre-arthritic 'expanding' phase, and then 'collapses' with adjacent knee flare associated with the loss of the intrinsic lymphatic pulse. As the mechanisms responsible are unknown, we developed in vivo methods to quantify lymph viscosity and pressure in mice with wild-type (WT), expanding and collapsed PLN. While no differences in viscosity were detected via multiphoton fluorescence recovery after photobleaching (MP-FRAP) of injected FITC-BSA, a 32.6% decrease in lymph speed was observed in vessels afferent to collapsed PLN (P < 0.05). Direct measurement of intra-lymph node pressure (LNP) demonstrated a decrease in expanding PLN versus WT pressure (3.41 ± 0.43 vs. 6.86 ± 0.56 cmH2O; P < 0.01), which dramatically increased to 9.92 ± 1.79 cmH2O in collapsed PLN. Lymphatic pumping pressure (LPP), measured indirectly by slowly releasing a pressurized cuff occluding indocyanine green (ICG), demonstrated an increase in vessels afferent to expanding PLN versus WT (18.76 ± 2.34 vs. 11.04 ± 1.47 cmH2O; P < 0.01), which dropped to 2.61 ± 0.72 cmH2O (P < 0.001) after PLN collapse. Herein, we document the first in vivo measurements of murine lymph viscosity and lymphatic pressure, and provide evidence to support the hypothesis that lymphangiogenesis and lymphatic transport are compensatory mechanisms to prevent synovitis via increased drainage of inflamed joints. Furthermore, the decrease in lymphatic flow and loss of LPP during PLN collapse are consistent with decreased drainage from the joint during arthritic flare, and validate these biomarkers of RA progression and possibly other chronic inflammatory conditions.
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Artrite Experimental/fisiopatologia , Artrite Reumatoide/fisiopatologia , Linfonodos/fisiologia , Linfa/fisiologia , Vasos Linfáticos/fisiopatologia , Animais , Artrite Experimental/etiologia , Artrite Reumatoide/etiologia , Progressão da Doença , Recuperação de Fluorescência Após Fotodegradação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pressão , Fator de Necrose Tumoral alfa/genética , ViscosidadeRESUMO
OBJECTIVE: B cell depletion therapy ameliorates rheumatoid arthritis by mechanisms that are incompletely understood. Arthritis flare in tumor necrosis factor (TNF)-transgenic mice is associated with efferent lymph node (LN) "collapse," triggered by B cell translocation into lymphatic spaces and decreased lymphatic drainage. The aim of this study was to examine whether the efficacy of B cell depletion therapy is associated with restoration of lymphatic drainage due to removal of obstructing nodal B cells. METHODS: We used contrast-enhanced magnetic resonance imaging, indocyanine green near-infrared imaging, and intravital immunofluorescence imaging to longitudinally assess synovitis, lymphatic flow, and cell migration in lymphatic vessels in TNF-transgenic mice. We conducted tests to determine whether the efficacy of B cell depletion therapy is associated with restoration of lymphatic draining and cell egress from arthritic joints. RESULTS: Unlike active lymphatics to normal and prearthritic knees, afferent lymphatic vessels to collapsed LNs in inflamed knees do not pulse. Intravital immunofluorescence imaging demonstrated that CD11b+ monocyte/macrophages in lymphatic vessels afferent to expanding LNs travel at high velocity (mean±SD 186±37 µm/second), while these cells are stationary in lymphatic vessels afferent to collapsed popliteal LNs. B cell depletion therapy for arthritis flares in TNF-transgenic mice significantly decreased knee synovium volume (by 50% from the baseline level) and significantly increased lymphatic clearance compared with placebo (P<0.05). This increased lymphatic drainage restored macrophage egress from inflamed joints without recovery of the lymphatic pulse. CONCLUSION: These results support a novel mechanism in which B cell depletion therapy for joint arthritis flares lessens inflammation by increasing lymphatic drainage and subsequent migration of cells and cytokines from the synovial space.
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Artrite Reumatoide/terapia , Articulação do Joelho/patologia , Vasos Linfáticos/patologia , Depleção Linfocítica/métodos , Sinovite/patologia , Animais , Artrite Reumatoide/patologia , Linfócitos B , Antígeno CD11b , Citometria de Fluxo , Imuno-Histoquímica , Linfonodos , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Resultado do TratamentoRESUMO
Lymphatic muscle cells (LMCs) within the wall of collecting lymphatic vessels exhibit tonic and autonomous phasic contractions, which drive active lymph transport to maintain tissue-fluid homeostasis and support immune surveillance. Damage to LMCs disrupts lymphatic function and is related to various diseases. Despite their importance, knowledge of the transcriptional signatures in LMCs and how they relate to lymphatic function in normal and disease contexts is largely missing. We have generated a comprehensive transcriptional single-cell atlas-including LMCs-of collecting lymphatic vessels in mouse dermis at various ages. We identified genes that distinguish LMCs from other types of muscle cells, characterized the phenotypical and transcriptomic changes in LMCs in aged vessels, and uncovered a pro-inflammatory microenvironment that suppresses the contractile apparatus in advanced-aged LMCs. Our findings provide a valuable resource to accelerate future research for the identification of potential drug targets on LMCs to preserve lymphatic vessel function as well as supporting studies to identify genetic causes of primary lymphedema currently with unknown molecular explanation.
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Background: Recent studies demonstrated lymphangiogenesis and expansion of draining lymph nodes during chronic inflammatory arthritis, and lymphatic dysfunction associated with collapse of draining lymph nodes in rheumatoid arthritis (RA) patients and TNF-transgenic (TNF-Tg) mice experiencing arthritic flare. As the intrinsic differences between lymphatic vessels afferent to healthy, expanding, and collapsed draining lymph nodes are unknown, we characterized the ex vivo behavior of popliteal lymphatic vessels (PLVs) from WT and TNF-Tg mice. We also interrogated the mechanisms of lymphatic dysfunction through inhibition of nitric oxide synthase (NOS). Methods: Popliteal lymph nodes (PLNs) in TNF-Tg mice were phenotyped as Expanding or Collapsed by in vivo ultrasound and age-matched to WT littermate controls. The PLVs were harvested and cannulated for ex vivo functional analysis over a relatively wide range of hydrostatic pressures (0.5-10 cmH2O) to quantify the end diastolic diameter (EDD), tone, amplitude (AMP), ejection fraction (EF), contraction frequency (FREQ), and fractional pump flow (FPF) with or without NOS inhibitors Data were analyzed using repeated measures two-way ANOVA with Bonferroni's post hoc test. Results: Real time videos of the cannulated PLVs demonstrated the predicted phenotypes of robust vs. weak contractions of the WT vs. TNF-Tg PLV, respectively. Quantitative analyses confirmed that TNF-Tg PLVs had significantly decreased AMP, EF, and FPF vs. WT (p < 0.05). EF and FPF were recovered by NOS inhibition, while the reduction in AMP was NOS independent. No differences in EDD, tone, or FREQ were observed between WT and TNF-Tg PLVs, nor between Expanding vs. Collapsed PLVs. Conclusion: These findings support the concept that chronic inflammatory arthritis leads to NOS dependent and independent draining lymphatic vessel dysfunction that exacerbates disease, and may trigger arthritic flare due to decreased egress of inflammatory cells and soluble factors from affected joints.
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Vascular endothelial growth factor (VEGF)-C is necessary for lymphangiogenesis, and excess VEGF-C has been shown to be ameliorative for edema produced by lymphatic obstruction in experimental models. However, it has recently been shown that edema can resolve in the mouse tail even in the complete absence of capillary lymphangiogenesis when distal lymph fluid crosses the regenerating wound site interstitially. This finding has raised questions about the action of VEGF-C/VEGF receptor (VEGFR) signaling during the resolution of experimental edema. Here, the roles of VEGFR-2 and VEGFR-3 signaling in edema resolution were explored. It was found that edema resolved following neutralization of either VEGFR-2 or VEGFR-3 in the mouse tail skin, which inhibited lymphangiogenesis. Neutralization of either VEGFR-2 or VEGFR-3 reduced angiogenesis at the site of obstruction at day 10 (9.2 +/- 1.2% and 11.5 +/- 1.0% blood capillary coverage, respectively) relative to controls (14.3 +/- 1.5% blood capillary coverage). Combined VEGFR-2/-3 neutralization more strongly inhibited angiogenesis (6.9 +/- 1.5% blood capillary coverage), leading to a reduced wound repair of the lymphatic obstruction and extended edema in the tail skin. In contrast, improved tissue repair of the obstruction site increased edema resolution. Macrophages in the swollen tissue were excluded as contributing factors in the VEGFR-dependent extended edema. These results support a role for VEGFR-2/-3-combined signaling in the resolution of experimental edema that is lymphangiogenesis independent.
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Linfangiogênese , Linfedema/fisiopatologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Cicatrização , Animais , Anticorpos Monoclonais/administração & dosagem , Capilares/metabolismo , Capilares/fisiopatologia , Modelos Animais de Doenças , Feminino , Linfa/metabolismo , Linfedema/metabolismo , Linfografia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica , Transdução de Sinais , Fatores de Tempo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
OBJECTIVES: Breast cancer related lymphedema (BCRL) is a common complication of current breast cancer treatment modalities, significantly lowering quality of life for these patients and often leading to recurrent infections. Here, based on pre-clinical literature, we aim to retrospectively evaluate the risks of prescribed medications on BCRL development. METHODS: All post-operative breast cancer patients who received radiotherapy from 2005-2013 at Massachusetts General Hospital and developed lymphedema(n=115) were included in the analysis. Comparable patients without lymphedema(n=230) were randomly selected as control. The following classes of medications were analyzed: NSAIDs, corticosteroids, angiotensin system inhibitors, calcium channel blockers and hormonal therapy. Known risk factors for lymphedema development were included as variables, including BMI, age at diagnosis, type of surgery, number of lymph nodes removed and radiation therapy. Outcomes were BCRL development and lymphedema severity. RESULTS: Similarly, to previous studies, we found that an increase in BMI increases the risk of BCRL(p=0.006) and axillary lymph node dissection has a higher risk of developing BCRL compared to sentinel lymph node biopsy(p=0.045). None of the drugs studied increased the risk of BCRL development or lymphedema severity. However, lymphedema severity was positively correlated with the number of lymph nodes removed(p=0.034). CONCLUSION: We found that anti-inflammatory drugs, anti-hypertensive drugs and hormonal therapy taken during the year postoperatively do not increase the risk of BCRL development or lymphedema severity in breast cancer patients. While others have demonstrated that the number of lymph nodes removed during surgery increases the risk of BCRL, we found it also correlates to lymphedema severity.
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Background: Metastatic tumor cells spread through lymphatic vessels and colonize draining lymph nodes (LNs). It is known that tumors induce lymphangiogenesis to enhance lymphatic metastasis and that metastatic cancer cells are carried by lymph flow to LNs. Methods and Results: Here, we investigated the molecular and cellular regulation of collecting lymphatic vessel contraction in vessels draining a metastatic tumor using intravital microscopy. In tumor-draining collecting lymphatic vessels, we found vessel contraction was suppressed. The infiltration of peritumor tissue by inducible nitric oxide synthase positive and CD11b+Gr1+ myeloid cells played a critical role in the suppression of lymphatic contraction. Depletion of Gr1+ cells with an anti-Gr1 antibody improved contraction of tumor-draining lymphatic vessels. In addition, inducing tumor cell death restored lymphatic contraction in nude mice. Conclusions: These findings indicate that tumors contribute to regulation of lymphatic transport in a reversible manner, warranting further investigation into the role of impaired lymphatic transport in cancer progression.
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Antígeno CD11b/metabolismo , Vasos Linfáticos/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores de Quimiocinas/metabolismo , Vasoconstrição , Animais , Biomarcadores , Antígeno CD11b/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Xenoenxertos , Linfonodos/patologia , Metástase Linfática , Vasos Linfáticos/fisiopatologia , Camundongos , Modelos Biológicos , Neoplasias/etiologia , Neoplasias/fisiopatologia , Óxido Nítrico Sintase Tipo II/genética , Receptores de Quimiocinas/genética , Vasoconstrição/genéticaRESUMO
Lymphedema arises from impaired lymphatic function. Quantification of lymphatic contractility has previously been shown using a custom-built near-infrared imaging system. However, to broaden the clinical use of functional lymphatic measurements, these measurements need to be performed using a standard-of-care, clinically available camera. The authors propose an objective, algorithmic, and clinically accessible approach to quantify lymphatic contractility using a 3-minute indocyanine green lymphangiograph recorded with a commercially available near-infrared camera. A retrospective review of the authors' indocyanine green lymphangiography video repository maintained in a Research Electronic Data Capture database was performed. All patients with a newly diagnosed unilateral breast cancer undergoing preoperative indocyanine green lymphangiography were included in the analysis. Patient medical records were then analyzed for patient demographics, and videos were analyzed for contractility. Seventeen consecutive patients with unilateral breast cancers underwent video processing to quantify lymphatic contractility of the ipsilateral extremity in contractions per minute. All patients were women, with an average age of 60.5 years (range, 38 to 84 years). The average lymphatic contractility rate was 1.13 contractions per minute (range, 0.67 to 2.5 contractions per minute). Using a clinically accessible standard-of-care device for indocyanine green lymphangiography, the authors were able to determine lymphatic contractility rates of a normal extremity. The authors' finding falls within the range of previously published data quantifying lymphatic contractility using a research device, suggesting that the authors' technique provides a clinically accessible, time-effective means of assessing lymphatic contractility. Potential future applications include both lymphedema surveillance and evaluation of nonsurgical and surgical interventions. CLINICAL QUESTION/LEVEL OF EVIDENCE:: Diagnostic, IV.
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Braço/fisiopatologia , Verde de Indocianina , Linfedema/diagnóstico por imagem , Linfografia/métodos , Mastectomia/efeitos adversos , Adulto , Idoso , Algoritmos , Neoplasias da Mama/cirurgia , Estudos de Coortes , Bases de Dados Factuais , Feminino , Humanos , Interpretação de Imagem Assistida por Computador , Vasos Linfáticos/diagnóstico por imagem , Linfedema/etiologia , Mastectomia/métodos , Pessoa de Meia-Idade , Contração Muscular/fisiologia , Prognóstico , Estudos Retrospectivos , Gravação em VídeoRESUMO
OBJECTIVE: To investigate the roles of the synovial lymphatic system in the severity and progression of joint tissue damage and functional responses of synovial lymphatic endothelial cells (LECs) to macrophage subsets, and to evaluate the therapeutic potential of the proteasome inhibitor bortezomib (BTZ) in a mouse model of experimental posttraumatic osteoarthritis (OA). METHODS: C57BL/6J wild-type mice received a meniscal ligamentous injury to induce posttraumatic knee OA. Lymphangiogenesis was blocked by a vascular endothelial growth factor receptor 3 (VEGFR-3) neutralizing antibody. Synovial lymphatic drainage was examined by near-infrared imaging. Joint damage was assessed by histology. RNA-sequencing and pathway analyses were applied to synovial LECs. Macrophage subsets in the mouse synovium were identified by flow cytometry and immunofluorescence staining. M1 and M2 macrophages were induced from mouse bone marrow cells, and their effects on LECs were examined in cocultures in the presence or absence of BTZ. The effects of BTZ on joint damage, LEC inflammation, and synovial lymphatic drainage were examined. RESULTS: Injection of a VEGFR-3 neutralizing antibody into the joints of mice with posttraumatic knee OA reduced synovial lymphatic drainage and accelerated joint tissue damage. Synovial LECs from the mouse OA joints had dysregulated inflammatory pathways and expressed high levels of inflammatory genes. The number of M1 macrophages was increased in the knee joints of mice with posttraumatic OA, thereby promoting the expression of inflammatory genes by LECs; this effect was blocked by BTZ. Treatment with BTZ decreased cartilage loss, reduced the expression of inflammatory genes by LECs, and improved lymphatic drainage in the knee joints of mice with posttraumatic OA. CONCLUSION: Experimental posttraumatic knee OA is associated with decreased synovial lymphatic drainage, increased numbers of M1 macrophages, and enhanced inflammatory gene expression by LECs, all of which was improved by treatment with BTZ. Intraarticular administration of BTZ may represent a new therapy for the restoration of synovial lymphatic function in subjects with posttraumatic knee OA.
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Bortezomib/farmacologia , Células Endoteliais/efeitos dos fármacos , Vasos Linfáticos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Osteoartrite do Joelho/imunologia , Inibidores de Proteassoma/farmacologia , Membrana Sinovial/efeitos dos fármacos , Animais , Anticorpos Neutralizantes/farmacologia , Bortezomib/uso terapêutico , Técnicas de Cocultura , Progressão da Doença , Inflamação , Traumatismos do Joelho/complicações , Linfangiogênese/efeitos dos fármacos , Vasos Linfáticos/imunologia , Macrófagos/imunologia , Camundongos , Osteoartrite do Joelho/tratamento farmacológico , Osteoartrite do Joelho/etiologia , Inibidores de Proteassoma/uso terapêutico , Espectroscopia de Luz Próxima ao Infravermelho , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/imunologiaRESUMO
Although clinical outcomes for patients with rheumatoid arthritis (RA) have greatly improved with the use of biologic and conventional DMARDs, approximately 40% of patients do not achieve primary clinical outcomes in randomized trials, and only a small proportion achieve lasting remission. Over the past decade, studies in murine models point to the critical role of the lymphatic system in the pathogenesis and therapy of inflammatory-erosive arthritis, presumably by the removal of catabolic factors, cytokines and inflammatory cells from the inflamed synovium. Murine studies demonstrate that lymphatic drainage increases at the onset of inflammatory-erosive arthritis but, as inflammation progresses to a more chronic phase, lymphatic clearance declines and both structural and cellular changes are observed in the draining lymph node. Specifically, chronic damage to the lymphatic vessel from persistent inflammation results in loss of lymphatic vessel contraction followed by lymph node collapse, reduced lymphatic drainage, and ultimately severe synovitis and joint erosion. Notably, clinical pilot studies in patients with RA report lymph node changes following treatment, and thus draining lymphatic vessels and nodes could represent a potential biomarker of arthritis activity and response to therapy. Most importantly, targeting lymphatics represents an innovative strategy for therapeutic intervention for RA.
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Artrite Reumatoide/terapia , Sistema Linfático/patologia , Fator C de Crescimento do Endotélio Vascular/genética , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Terapia Genética , Vetores Genéticos/administração & dosagem , Humanos , Sistema Linfático/efeitos dos fármacos , CamundongosRESUMO
Several imaging modalities have been used to assess lymphatic function, including fluorescence microscopy, near-infrared fluorescence (NIRF) imaging, and Doppler optical coherence tomography (DOCT). They vary in how the mouse is positioned, the invasiveness of the experimental setup, and the volume of contrast agent injected. Here, we present how each of these experimental parameters affects functional measurements of collecting lymphatic vessels. First, fluorescence microscopy showed that supine mice have a statistically lower contraction frequency compared with mice sitting upright. To assess the effect of different injection volumes on these endpoints, mice were injected with 4, 10, or 20 µl of dye. The lowest frequencies were observed after 20-µl injections. Interestingly, lymph-flow DOCT revealed that although there was lower contraction frequency in mice injected with 20 µl versus 4 µl, mice showed a higher volumetric flow with a 20-µl injection. This indicates that contraction frequency alone is not sufficient to understand lymphatic transport. Finally, NIRF revealed that removing the skin reduced contraction frequency. Therefore, this study reveals how sensitive these techniques are to mouse position, removal of skin, and dye volume. Care should be taken when comparing results obtained under different experimental conditions.
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Meios de Contraste/administração & dosagem , Vasos Linfáticos/diagnóstico por imagem , Posicionamento do Paciente , Animais , Efeito Doppler , Relação Dose-Resposta a Droga , Feminino , Camundongos , Microscopia de Fluorescência/métodos , Modelos Animais , Tomografia de Coerência Óptica/métodosRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of morbidity and mortality worldwide and is a frequent cause of skin and soft tissue infections (SSTIs). Lymphedema-fluid accumulation in tissue caused by impaired lymphatic vessel function-is a strong risk factor for SSTIs. SSTIs also frequently recur in patients and sometimes lead to acquired lymphedema. However, the mechanism of how SSTIs can be both the consequence and the cause of lymphatic vessel dysfunction is not known. Intravital imaging in mice revealed an acute reduction in both lymphatic vessel contractility and lymph flow after localized MRSA infection. Moreover, chronic lymphatic impairment is observed long after MRSA is cleared and inflammation is resolved. Associated with decreased collecting lymphatic vessel function was the loss and disorganization of lymphatic muscle cells (LMCs), which are critical for lymphatic contraction. In vitro, incubation with MRSA-conditioned supernatant led to LMC death. Proteomic analysis identified several accessory gene regulator (agr)-controlled MRSA exotoxins that contribute to LMC death. Infection with agr mutant MRSA resulted in sustained lymphatic function compared to animals infected with wild-type MRSA. Our findings suggest that agr is a promising target to preserve lymphatic vessel function and promote immunity during SSTIs.
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Vasos Linfáticos/imunologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Feminino , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Inflamação/imunologia , Masculino , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Células Musculares/imunologiaRESUMO
Lymph node metastases in cancer patients are associated with tumor aggressiveness, poorer prognoses, and the recommendation for systemic therapy. Whether cancer cells in lymph nodes can seed distant metastases has been a subject of considerable debate. We studied mice implanted with cancer cells (mammary carcinoma, squamous cell carcinoma, or melanoma) expressing the photoconvertible protein Dendra2. This technology allowed us to selectively photoconvert metastatic cells in the lymph node and trace their fate. We found that a fraction of these cells invaded lymph node blood vessels, entered the blood circulation, and colonized the lung. Thus, in mouse models, lymph node metastases can be a source of cancer cells for distant metastases. Whether this mode of dissemination occurs in cancer patients remains to be determined.
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Vasos Sanguíneos/patologia , Linfonodos/patologia , Metástase Linfática/patologia , Inoculação de Neoplasia , Animais , Carcinoma de Células Escamosas , Linhagem Celular Tumoral , Movimento Celular , Rastreamento de Células/métodos , Citosol/química , Feminino , Proteínas Luminescentes/análise , Pulmão/patologia , Melanoma Experimental , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Neoplásicas CirculantesRESUMO
To assess the utility of longitudinal ultrasound (US) to quantify volumetric changes in joint soft tissues during the progression of posttraumatic osteoarthritis (PTOA) in mice, and validate the US results with histological findings. A longitudinal cohort of 3-month-old wild-type C57BL/6 male mice received the Hulth-Telhag surgical procedure on right knee to induce PTOA, and sham surgery on their left knee as control. US scans were performed on both knees before, 2, 4, 6, and 8 weeks post-surgery. Joint space volume and Power-Doppler (PD) volume were obtained from US images via Amira software. A parallel cross-sectional cohort of mice was killed at each US time point, and knee joints were subjected to histological analysis to obtain synovial soft-tissue area and OARSI scores. The correlation between US joint space volume and histological synovial soft-tissue area or OARSI score was assessed via linear regression analysis. US images indicated increased joint space volume in PTOA joints over time, which was associated with synovial inflammation and cartilage damage by histology. These changes started from 2 weeks post-surgery and gradually became more severe. No change was detected in sham joints. Increased joint space volume was significantly correlated with increased synovial soft-tissue area and the OARSI score (P<0.001). PD signal was detected in the joint space of PTOA joints at 6 weeks post-surgery, which was consistent with the location of blood vessels that stained positively for CD31 and alpha-smooth muscle actin in the synovium. This study indicates that US is a cost-effective longitudinal outcome measure of volumetric and vascular changes in joint soft tissues during PTOA progression in mice, which positively correlates with synovial inflammation and cartilage damage.
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OBJECTIVE: Recent studies have demonstrated that there is an inverse relationship between lymphatic egress and inflammatory arthritis in affected joints. As a model, tumor necrosis factor (TNF)-transgenic mice develop advanced arthritis following draining lymph node (LN) collapse, and loss of lymphatic contractions downstream of inflamed joints. It is unknown if these lymphatic deficits are reversible. This study was undertaken to test the hypothesis that anti-TNF therapy reduces advanced erosive inflammatory arthritis, associated with restoration of lymphatic contractions, repair of damaged lymphatic vessels, and evidence of increased monocyte egress. METHODS: TNF-transgenic mice with advanced arthritis and collapsed popliteal LNs were treated with anti-TNF monoclonal antibody (10 mg/kg weekly) or placebo for 6 weeks, and effects on knee synovitis, lymphatic vessel ultrastructure and function, and popliteal LN cellularity were assessed by ultrasound, histology, transmission electron microscopy (TEM), near-infrared indocyanine green imaging, and flow cytometry. RESULTS: Anti-TNF therapy significantly decreased synovitis (â¼5-fold; P < 0.05 versus placebo), restored lymphatic contractions, and significantly increased the number of popliteal LN monocyte/macrophages (â¼2-fold; P < 0.05 versus placebo). TEM demonstrated large activated macrophages attached to damaged lymphatic endothelium in mice with early arthritis, extensively damaged lymphatic vessels in placebo-treated mice with advanced arthritis, and rolling leukocytes in repaired lymphatic vessels in mice responsive to anti-TNF therapy. CONCLUSION: These findings support the concept that anti-TNF therapy ameliorates erosive inflammatory arthritis, in part via restoration of lymphatic vessel contractions and potential enhancement of inflammatory cell egress.
Assuntos
Anticorpos Monoclonais/farmacologia , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Vasos Linfáticos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Artrite Experimental/etiologia , Artrite Experimental/fisiopatologia , Artrite Reumatoide/etiologia , Artrite Reumatoide/fisiopatologia , Citometria de Fluxo , Linfonodos/efeitos dos fármacos , Linfonodos/fisiopatologia , Vasos Linfáticos/fisiopatologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Monócitos/efeitos dos fármacos , Sinovite/tratamento farmacológico , Sinovite/etiologia , Resultado do TratamentoRESUMO
OBJECTIVES: The mechanisms that trigger flare in rheumatoid arthritis (RA) are unknown. In murine arthritis models, dysfunctional lymph node (LN) drainage is associated with joint flare. To examine if LN alterations are associated with RA flare, we analyzed the change in LN volume via contrast-enhanced magnetic resonance imaging (CE-MRI) in patients with active joint flare at baseline and 16 weeks after certolizumab pegol (CZP) therapy. We also assessed the changes in popliteal or epitrochlear LN volumes versus the Rheumatoid and Arthritis Outcome Score (RAOS) (knee), or the Michigan Hand Questionnaire (MHQ; wrist/hand), and Disease Activity Score 28 (DAS28), at baseline and 16 weeks. RESULTS: Total LN volume in 7 of 10 patients with measurable LN on CE-MRI significantly decreased 16 weeks after CZP therapy (mean decrease 37%; P = 0.0019). Improvement in knee pain measured by the RAOS (P = 0.03) inversely correlated with a decrease in total popliteal LN volume (R2 = 0.94). All patients demonstrated significant improvement in DAS28 (mean decrease 1.48; P = 0.0002). For flare in the hand, significant improvement in activities of daily living (ADL) as measured by the MHQ was observed (left hand mean improvement 20%; P = 0.02; right hand mean improvement 37%; P = 0.03). CONCLUSION: RA patients with the smallest change in LN volume during anti-tumor necrosis factor (anti-TNF) therapy experienced the greatest pain relief in symptomatic knee joints. Moreover, the remarkably linear inverse correlation between LN volume and joint pain observed in this small clinical pilot provides initial evidence to support the concept that dynamic changes in draining LN volume are a biomarker of clinical response to therapy in RA.
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UNLABELLED: Proliferation of draining lymphatic vessels coupled with dynamic changes in lymph node volume and flow are characteristic features in rheumatoid arthritis (RA). Furthermore, impaired lymph egress from inflamed synovium is associated with joint flare in murine models of inflammatory-erosive arthritis. Unfortunately, advances towards a greater understanding of lymphatic changes in RA pathogenesis have been slow due to the absence of outcome measures to quantify lymphatic function in vivo. While lymphoscintigraphy is the current standard to assess lymphedema and sentinel lymph nodes in cancer patients, its sensitivity and specificity are inadequate to study lymphatics in RA. The emergence of high-resolution MRI, power Doppler ultrasound, and near-infrared imaging that permits real-time quantification of lymphatic function in animal models has been a major advance, and these techniques have produced a new paradigm of altered lymphatic function that underlies both acute arthritic flare and chronic inflammation. In acute flare, lymphatic drainage increases several fold, whereas no lymphatic contractions are detected in lymph vessels draining chronic arthritic joints. Moreover, these outcomes are now being adapted to study lymphatics in RA towards the development of novel biomarkers of arthritic flare and the discovery of new therapeutic targets. In particular, interventions that directly increase lymphatic egress from diseased joints by opening collateral lymphatic vessels, and that restore lymphatic vessel contractions, provide novel therapeutic approaches with potential for minimal toxicity and immunosuppression. To summarize the origins of this field, recent advances, and future directions, we herein review: current knowledge of lymphatics in RA based on classic literature; new in-vivo imaging modalities that have elucidated how lymphatics modulate acute versus chronic joint inflammation in murine models; and how these preclinical outcome measures are being translated to study lymphatic function in RA inflammation and how effective RA therapies alter lymphatic flow and lymph nodes draining flaring joints. TRIAL REGISTRATION: ClinicalTrials.gov NCT02680067 . Registered 7 December 2015; ClinicalTrials.gov NCT01098201 . Registered 30 March 2010; and ClinicalTrials.gov NCT01083563 . Registered 8 March 2010.