Preventing periprosthetic osteolysis in aging populations through lymphatic activation and stem cell-associated secretory phenotype inhibition.

With increases in life expectancy, the number of patients requiring joint replacement therapy and experiencing periprosthetic osteolysis, the most common complication leading to implant failure, is growing or underestimated. In this study, we found that osteolysis progression and osteoclast differentiation in the surface of the skull bone of adult mice were accompanied by significant expansion of lymphatic vessels within bones. Using recombinant VEGF-C protein to activate VEGFR3 and promote proliferation of lymphatic vessels in bone, we counteracted excessive differentiation of osteoclasts and osteolysis caused by titanium alloy particles or inflammatory cytokines LPS/TNF-α. However, this effect was not observed in aged mice because adipogenically differentiated mesenchymal stem cells (MSCs) inhibited the response of lymphatic endothelial cells to agonist proteins. The addition of the JAK inhibitor ruxolitinib restored the response of lymphatic vessels to external stimuli in aged mice to protect against osteolysis progression. These findings suggest that inhibiting SASP secretion by adipogenically differentiated MSCs while activating lymphatic vessels in bone offers a new method to prevent periprosthetic osteolysis during joint replacement follow-up.

Platelet extracellular vesicles preserve lymphatic endothelial cell integrity and enhance lymphatic vessel function.

Lymphatic vessels are essential for preventing the accumulation of harmful components within peripheral tissues, including the artery wall. Various endogenous mechanisms maintain adequate lymphatic function throughout life, with platelets being essential for preserving lymphatic vessel integrity. However, since lymph lacks platelets, their impact on the lymphatic system has long been viewed as restricted to areas where lymphatics intersect with blood vessels. Nevertheless, platelets can also exert long range effects through the release of extracellular vesicles (EVs) upon activation. We observed that platelet EVs (PEVs) are present in lymph, a compartment to which they could transfer regulatory effects of platelets. Here, we report that PEVs in lymph exhibit a distinct signature enabling them to interact with lymphatic endothelial cells (LECs). In vitro experiments show that the internalization of PEVs by LECs maintains their functional integrity. Treatment with PEVs improves lymphatic contraction capacity in atherosclerosis-prone mice. We suggest that boosting lymphatic pumping with exogenous PEVs offers a novel therapeutic approach for chronic inflammatory diseases characterized by defective lymphatics.

Meningeal lymphatic vessel dysfunction driven by CGRP signaling causes migraine-like pain in mice.

Migraines are a type of headache that occur with other neurological symptoms, but the pathophysiology remains unclear. In this issue of the JCI, Nelson-Maney and authors used constitutive and inducible knockouts of the CGRP receptor components, elegantly demonstrating an essential function of CGRP in modulating meningeal lymphatic vessels (MLVs) in migraine. CGRP was shown to induce rearrangement of membrane-bound gap junction proteins in MLVs, resulting in a reduced CSF flux into cervical lymph nodes. The authors also provided evidence of a primary role for CGRP in modulating neuro-immune function. Finally, by showing that blocking CGRP signaling in MLVs attenuated pain behavior associated with acute migraine in rodents, the authors provided a target for pharmacological blockade of CGRP in relation to primary headache disorders.

Lymphangiogenesis in the liver of biliary atresia.

BACKGROUND: Lymphatic vessels (LVs) play a crucial role in immune reactions by serving as the principal conduits for immune cells. However, to date, no study has analyzed the morphological changes in the LVs of patients with biliary atresia (BA). In this study, we aimed to determine the morphological changes in the LVs irrigating the liver in patients with BA, elucidate their correlations with the morphology of the portal vein (PV) branches, and discuss their etiopathogenetic significance. METHODS: Morphometric analyses of liver biopsy specimens from patients treated between 1986 and 2016 were performed. The parameters measured were as follows: the whole liver area of the specimen, fibrotic area, number of LVs, LVs without patent lumen (designated as Ly0) and PV branches, and diameters of the LVs with patent lumen and the PVs. RESULTS: The numbers of LVs, Ly0, and PV branches per unit area of the whole liver specimen were significantly higher in patients with BA than in control participants with liver disease and those with normal livers. However, no correlation was observed between the fibrotic area and the average diameter of LVs or PVs, and between the fibrotic area and the number of LVs or PV branches. Furthermore, no correlation was observed between the total number of LVs and the number of PV branches. CONCLUSIONS: The present study showed a significant increase in the number of total LVs and Ly0, characterized by a high Ly0 to total LVs ratio, suggesting that lymphangiogenesis occurs in the liver of patients with BA.

The anatomy of brainwashing.

Glymphatic-lymphatic brain cleansing may reveal new therapeutic strategies.

Contribution of initial lymphatics to oral wound healing after tooth extraction.

Lymphatics are involved in the resolution of inflammation and wound healing, but their role in the oral wound healing process after tooth extraction has never been investigated. We therefore sought to evaluate the healing process following the extraction of maxillary molars in two transgenic mouse models: K14-VEGFR3-Ig mice, which lack initial mucosal lymphatic vessels, and K14-VEGFC mice, which have hyperplastic mucosal lymphatics. Maxillary molars were extracted from both transgenic mouse types and their corresponding wild-type (WT) controls. Mucosal and alveolar bone healing were evaluated. A delayed epithelialization and bone regeneration were observed in K14-VEGFR3-Ig mice compared with their WT littermates. The hampered wound closure was accompanied by decreased levels of epidermal growth factor (EGF) and persistent inflammation, characterized by infiltrates of immune cells and elevated levels of pro-inflammatory markers in the wounds. Hyperplastic mucosal lymphatics did not enhance the healing process after tooth extraction in K14-VEGFC mice. The findings indicate that initial mucosal lymphatics play a major role in the initial phase of the oral wound healing process.

Photobiomodulation under Electroencephalographic Controls of Sleep for Stimulation of Lymphatic Removal of Toxins from Mouse Brain.

The meningeal lymphatic vessels (MLVs) play an important role in the removal of toxins from the brain. The development of innovative technologies for the stimulation of MLV functions is a promising direction in the progress of the treatment of various brain diseases associated with MLV abnormalities, including Alzheimer's and Parkinson's diseases, brain tumors, traumatic brain injuries, and intracranial hemorrhages. Sleep is a natural state when the brain's drainage processes are most active. Therefore, stimulation of the brain's drainage and MLVs during sleep may have the most pronounced therapeutic effects. However, such commercial technologies do not currently exist. This study presents a new portable technology of transcranial photobiomodulation (tPBM) under electroencephalographic (EEG) control of sleep designed to photo-stimulate removal of toxins (e.g., soluble amyloid beta (Aß)) from the brain of aged BALB/c mice with the ability to compare the therapeutic effectiveness of different optical resources. The technology can be used in the natural condition of a home cage without anesthesia, maintaining the motor activity of mice. These data open up new prospects for developing non-invasive and clinically promising photo-technologies for the correction of age-related changes in the MLV functions and brain's drainage processes and for effectively cleansing brain tissues from metabolites and toxins. This technology is intended both for preclinical studies of the functions of the sleeping brain and for developing clinically relevant treatments for sleep-related brain diseases.

Prognostic significance of lymphovascular space invasion in early-stage low-grade endometrioid endometrial cancer: a fifteen-year retrospective Chinese cohort study.

OBJECTIVE: In 2016, the ESMO-ESGO-ESTRO consensus included LVSI (Lymph-vascular space invasion, LVSI) status as a risk stratification factor for stage I endometrioid endometrial cancer (EEC) patients and as one of the indications for adjuvant therapy. Furthermore, LVSI is included in the new FIGO staging of endometrial cancer (EC) in 2023. However, the data contribution of the Chinese population in this regard is limited. The present study aimed to further comfirm the influence of LVSI on the prognosis of early-stage low-grade EEC in a fifteen-year retrospective Chinese cohort study. METHODS: This retrospective analysis cohort included 702 EEC patients who underwent TAH/BSO surgery, total abdominal hysterectomy, bilateral salpingooophorectomy in Peking University People's Hospital from 2006 to 2020. Patients were stratified based on LVSI expression status as: LVSI negative group and LVSI positive group. Clinical outcome measures related to LVSI, assessed with a univariate and multivariate Cox proportional hazards regression model. RESULTS: 702 EEC patients with stage I and grade 1-2 were analyzed. 58 patients (8.3%) were LVSI-positive and 14 patients (2.0%) was relapse. Recurrence rates in LVSI-negative and LVSI-positive were 1.6% and 6.9%, respectively. 5-year disease-free survival (DFS) rate in LVSI-negative and LVSI-positive were 98.4% and 93.1%, respectively. These rates for 5-year overall (OS) survival in LVSI-negative were 98.9% while it was 94.8% in LVSI-positive. Multivariate analysis showed that LVSI is an independent risk factor for 5-year DFS (HR = 4.60, p = 0.010). LVSI has a similar result for 5-year OS(HR = 4.39, p = 0.028). CONCLUSIONS: LVSI is an independent predictor of relapse and poor prognosis in early-stage low-grade endometrioid endometrial cancer in the Chinese cohort.

Lymphatic-localized Treg-mregDC crosstalk limits antigen trafficking and restrains anti-tumor immunity.

The tumor microenvironment (TME) has a significant impact on tumor growth and immunotherapy efficacies. However, the precise cellular interactions and spatial organizations within the TME that drive these effects remain elusive. Using advanced multiplex imaging techniques, we have discovered that regulatory T cells (Tregs) accumulate around lymphatic vessels in the peripheral tumor stroma. This localized accumulation is facilitated by mature dendritic cells enriched in immunoregulatory molecules (mregDCs), which promote chemotaxis of Tregs, establishing a peri-lymphatic Treg-mregDC niche. Within this niche, mregDCs facilitate Treg activation, which in turn restrains the trafficking of tumor antigens to the draining mesenteric lymph nodes, thereby impeding the initiation of anti-tumor adaptive immune responses. Disrupting Treg recruitment to mregDCs inhibits tumor progression. Our study provides valuable insights into the organization of TME and how local crosstalk between lymphoid and myeloid cells suppresses anti-tumor immune responses.

[Imaging for displaying lymphatic vessels in whole-mount aorta adventitia of ApoE-/- mice by immunofluorescent histochemical staining].

Objective To explore a simple and feasible method for whole-mount immunofluorescence staining of lymphatic vessels in the ApoE-/- mouse model of atherosclerosis. Methods Aortic specimens were carefully excised from the ApoE-/- mouse model. Following immunostaining with specific antibodies against smooth muscle actin (SMA) and lymphatic vessel endothelial receptor 1 (LYVE1), the aortas, including the aortic root, were subjected to a 30-minute treatment with 5 g/L Sudan Black B solution. This step was instrumental in minimizing the autofluorescent background of the tissue. Thereafter, the aortas were processed through a clearing protocol and imaged within a purpose-built chamber under a fluorescence microscope. Results The pretreatment with 5 g/L Sudan Black B effectively suppressed the autofluorescent signals emanating from the vascular structures, thereby enhancing the contrast and clarity of the specific fluorescence signals associated with the lymphatic vessels. This enhancement in signal quality did not compromise the integrity or specificity of the immunofluorescent markers. Conclusion A facile, highly specific, and effective approach for the visualization of lymphatic vessels in whole-mount aortic preparations from ApoE-/- mice is established.

Pannexin1 deletion in lymphatic endothelium affects lymphatic function in a sex-dependent manner.

The lymphatic network of capillaries and collecting vessels ensures tissue fluid homeostasis, absorption of dietary fats and trafficking of immune cells. Pannexin1 (Panx1) channels allow for the passage of ions and small metabolites between the cytosol and extracellular environment. Panx1 channels regulate the pathophysiological function of several tissues in a sex-dependent manner. Here, we studied the role of Panx1 in lymphatic function, and potential sex-dependent differences therein, in Prox1-CreERT2Panx1fl/fl and Panx1fl/fl control mice. Panx1 expression was higher in lymphatic endothelial cells (LECs) of male mice. Lymphatic vessel morphology was not affected in Prox1-CreERT2Panx1fl/fl male and female mice. Lymphatic drainage was decreased by 25% in male Prox1-CreERT2Panx1fl/fl mice, but was similar in females of both genotypes. Accordingly, only male Prox1-CreERT2Panx1fl/fl mice exhibited tail swelling, pointing to interstitial fluid accumulation in males upon Panx1 deletion in LECs. Moreover, serum triglyceride and free fatty acid levels raised less in Prox1-CreERT2Panx1fl/fl mice of both sexes in an oral lipid tolerance test. Finally, the percentage of migratory dendritic cells arriving in draining lymph nodes was increased in Prox1-CreERT2Panx1fl/fl female mice, but was comparable between male mice of both genotypes. Our results point to a LEC-specific role for Panx1 in the functions of the lymphatic system.

Meningeal lymphatic supporting cells govern the formation and maintenance of zebrafish mural lymphatic endothelial cells.

The meninges are critical for the brain functions, but the diversity of meningeal cell types and intercellular interactions have yet to be thoroughly examined. Here we identify a population of meningeal lymphatic supporting cells (mLSCs) in the zebrafish leptomeninges, which are specifically labeled by ependymin. Morphologically, mLSCs form membranous structures that enwrap the majority of leptomeningeal blood vessels and all the mural lymphatic endothelial cells (muLECs). Based on its unique cellular morphologies and transcriptional profile, mLSC is characterized as a unique cell type different from all the currently known meningeal cell types. Because of the formation of supportive structures and production of pro-lymphangiogenic factors, mLSCs not only promote muLEC development and maintain the dispersed distributions of muLECs in the leptomeninges, but also are required for muLEC regeneration after ablation. This study characterizes a newly identified cell type in leptomeninges, mLSC, which is required for muLEC development, maintenance, and regeneration.

Fluid-Dynamic Modeling of Flow in Embryonic Tissue Indicates That Lymphatic Valve Location Is Not Consistently Determined by the Local Fluid Shear or Its Gradient.

OBJECTIVE: Intravascular lymphatic valves often occur in proximity to vessel junctions. It is commonly held that disturbed flow at junctions is responsible for accumulation of valve-forming cells (VFCs) at these locations as the initial step in valve creation, and the one which explains the association with these sites. However, evidence in favor is largely limited to cell culture experiments. METHODS: We acquired images of embryonic lymphatic vascular networks from day E16.5, when VFC accumulation has started but the developing valve has not yet altered the local vessel geometry, stained for Prox1, which co-localizes with Foxc2. Using finite-element computational fluid mechanics, we simulated the flow through the networks, under conditions appropriate to this early development stage. Then we correlated the Prox1 distributions with the distributions of simulated fluid shear and shear stress gradient. RESULTS: Across a total of 16 image sets, no consistent correlation was found between Prox1 distribution and the local magnitude of fluid shear, or its positive or negative gradient. CONCLUSIONS: This, the first direct semi-empirical test of the localization hypothesis to interrogate the tissue from in vivo at the critical moment of development, does not support the idea that a feature of the local flow determines valve localization.

Combination of tumor antigen drainage and immune activation to promote a cancer-immunity cycle against glioblastoma.

While conventional cancer modalities, such as chemotherapy and radiotherapy, act through direct killing of tumor cells, cancer immunotherapy elicits potent anti-tumor immune responses thereby eliminating tumors. Nevertheless, promising outcomes have not been reported in patients with glioblastoma (GBM) likely due to the immune privileged status of the central nervous system and immunosuppressive micro-environment within GBM. In the past years, several exciting findings, such as the re-discovery of meningeal lymphatic vessels (MLVs), three-dimensional anatomical reconstruction of MLV networks, and the demonstration of the promotion of GBM immunosurveillance by lymphatic drainage enhancement, have revealed an intricate communication between the nervous and immune systems, and brought hope for the development of new GBM treatment. Based on conceptual framework of the updated cancer-immunity (CI) cycle, here we focus on GBM antigen drainage and immune activation, the early events in driving the CI cycle. We also discuss the implications of these findings for developing new therapeutic approaches in tackling fatal GBM in the future.

Periarterial or perivenous invasion is an independent indicator of lymph node metastasis in invasive breast carcinoma of no special type.

Pathological diagnosis of breast cancer often includes cases of lymph node metastases without lymphatic or lymphovascular invasion by the primary tumor. In this study, to resolve this discrepancy, we designed a sensitive method to detect lymphatic invasion and correlate it with lymph node metastasis. Elastica van Gieson (EVG) staining and D2-40 immunohistochemistry revealed the abundant distribution of lymphatic vessels around blood vessels in the mammary tissue in close proximity to the elastic fibers around the arteries and veins. Based on the histological location of the blood and lymphatic vessels, we hypothesized that, in breast cancer, perivascular invasion is similar to lymphatic invasion and correlates with the presence of lymph node metastasis. Using EVG staining, perivascular invasion was histologically classified into periarterial invasion (periA), perivenous invasion (periV), and periarterial or perivenous invasion (periA/V). We tested our method and compared it to other methods commonly used for identifying lymphatic invasion in 105 patients with invasive breast carcinoma of no special type (IBC-NST) who received minimal preoperative therapy. The correlation between perivascular invasion and lymph node metastasis in these patients was statistically analyzed, including findings related to lymphatic invasion, such as retractile artifacts and perineural invasion. PeriA, periV, and periA/V showed significant correlations with lymph node metastasis. PeriA/V had high sensitivity and negative predictive value. The odds ratio (OR) for periV was significantly high in the univariate analysis, while the ORs for periA/V, retraction artifacts, and perineural invasion were significantly high in both the univariate and multivariate analyses. In particular, periA/V revealed a strong correlation with lymph node metastasis (OR: 61.8). These findings indicate that the IBC-NST periA/V ratio is a sensitive pointer of lymphatic invasion and could be an independent and reliable indicator of lymph node metastasis.

Preliminary report of a thoracic duct-to-pulmonary vein lymphovenous anastomosis in swine: A novel technique and potential treatment for lymphatic failure.

OBJECTIVE: The thoracic duct is the largest lymphatic vessel in the body, and carries fluid and nutrients absorbed in abdominal organs to the central venous circulation. Thoracic duct obstruction can cause significant failure of the lymphatic circulation (i.e., protein-losing enteropathy, plastic bronchitis, etc.). Surgical anastomosis between the thoracic duct and central venous circulation has been used to treat thoracic duct obstruction but cannot provide lymphatic decompression in patients with superior vena cava obstruction or chronically elevated central venous pressures (e.g., right heart failure, single ventricle physiology, etc.). Therefore, this preclinical feasibility study sought to develop a novel and optimal surgical technique for creating a thoracic duct-to-pulmonary vein lymphovenous anastomosis (LVA) in swine that could remain patent and preserve unidirectional lymphatic fluid flow into the systemic venous circulation to provide therapeutic decompression of the lymphatic circulation even at high central venous pressures. METHODS: A thoracic duct-to-pulmonary vein LVA was attempted in 10 piglets (median age 80 [IQR 80-83] days; weight 22.5 [IQR 21.4-26.8] kg). After a right thoracotomy, the thoracic duct was mobilized, transected, and anastomosed to the right inferior pulmonary vein. Animals were systemically anticoagulated on post-operative day 1. Lymphangiography was used to evaluate LVA patency up to post-operative day 7. RESULTS: A thoracic duct-to-pulmonary vein LVA was successfully completed in 8/10 (80.0%) piglets, of which 6/8 (75.0%) survived to the intended study endpoint without any complication (median 6 [IQR 4-7] days). Initially, 2/10 (20.0%) LVAs were aborted intraoperatively, and 2/10 (20.0%) animals were euthanized early due to post-operative complications. However, using an optimized surgical technique, the success rate for creating a thoracic duct-to-pulmonary vein LVA in six animals was 100%, all of which survived to their intended study endpoint without any complications (median 6 [IQR 4-7] days). LVAs remained patent for up to seven days. CONCLUSION: A thoracic duct-to-pulmonary vein LVA can be completed safely and remain patent for at least one week with systemic anticoagulation, which provides an important proof-of-concept that this novel intervention could effectively offload the lymphatic circulation in patients with lymphatic failure and elevated central venous pressures.

Contribution of the cadaveric recirculation system in the anatomical study of lymphatic drainage of the ovary: applications in the management of ovarian cancer.

PURPOSE: The present knowledge about lymphatic drainage of the ovary is based on carcinological studies, but it has only rarely been studied under physiological conditions. However, it is one of the preferential routes of dissemination in ovarian cancer, and understanding it is therefore vital for optimal carcinological management.Our purpose was to evaluate the feasibility of an innovative technique to study the lymphatic drainage territories of the ovary using a recirculation module on the cadaveric model. METHODS: We injected patent blue into the cortex of twenty "revascularised" cadaver ovaries with the Simlife recirculation model. We observed the migration of the dye live and described the drainage territories of each ovary. RESULTS: We observed a staining of the lymphatic vessels and migration of the dye in all the subjects, systematically ipsilateral to the injected ovary. We identified a staining of the lumbo-aortic territory in 65% of cases, with a preferential lateral-caval involvement (60%) for the right ovary and lateral-aortic territory (40%) for the left ovary. A common iliac involvement was observed in only 10% of cases. In 57% of cases, the staining of the lumbo-aortic territory was associated with a staining of the suspensory ligament. The pelvic territory was involved in 50% of cases, with an external iliac staining in 25% of cases and internal in 20%. CONCLUSION: Our study provides for a better understanding of lymphatic drainage of the ovary using a new detection method, and allows the possibility of improving the teaching for operators with a realistic model. Continuation of this work could lead to considering more targeted and thus less morbid lymph node sampling for lymph node staging in early-stage ovarian cancer.

Congenital Vascular and Lymphatic Diseases.

Over the past several centuries, the integration of contemporary medical techniques and innovative technologies, like genetic sequencing, have played a pivotal role in enhancing our comprehension of congenital vascular and lymphatic disorders. Nonetheless, the uncommon and complex characteristics of these disorders, especially considering their formation during the intrauterine stage, present significant obstacles in diagnosis and treatment. Here, we review the intricacies of these congenital abnormalities, offering an in-depth examination of key diagnostic approaches, genetic factors, and therapeutic methods.

Efficacy of Neonatal Porcine Bone Marrow-Derived Mesenchymal Stem Cell Xenotransplantation for the Therapy of Hind Limb Lymphedema in Mice.

Lymphedema is an intractable disease with few effective therapeutic options. Autologous mesenchymal stem cell (MSC) transplantation is a promising therapy for this disease. However, its use is limited by the cost and time for preparation. Recently, xenotransplantation of porcine MSCs has emerged as an alternative to autologous MSC transplantation. In this study, we aimed to clarify the usefulness of neonatal porcine bone marrow-derived MSC (NpBM-MSC) xenotransplantation for the treatment of lymphedema. One million NpBM-MSCs were xenotransplanted into the hind limbs of mice with severe lymphedema (MSC transplantation group). The therapeutic effects were assessed by measuring the femoral circumference, the volume of the hind limb, the number and diameter of lymphatic vessels in the hind limb, and lymphatic flow using a near-infrared fluorescence (NIRF) imaging system. We compared the effects using mice with lymphedema that did not undergo NpBM-MSC transplantation (negative control group). The condition of the transplanted NpBM-MSCs was also evaluated histologically. The femoral circumference and volume of the hind limb had been normalized by postoperative day (POD) 14 in the MSC transplantation group, but not in the negative control group (P = 0.041). NIRF imaging revealed that lymphatic flow had recovered in the MSC transplantation group by POD 14, as shown by an increase in luminance in the hind limb. Histological assessment also showed that the xenotransplantation of NpBM-MSC increased the proliferation of lymphatic vessels, but they had been rejected by POD 14. The xenotransplantation of NpBM-MSCs is an effective treatment for lymphedema, and this is mediated through the promotion of lymphangiogenesis.