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.

BACKGROUND: Lymphoedema is a chronic and progressive disease characterised by excessive accumulation of lymph in the interstitial compartment, leading to tissue swelling and fibroadipose deposition. Lymphangiogenesis is partly regulated by ketone body oxidation, and a ketogenic diet (KD) has shown therapeutic efficacy in a preclinical mouse tail lymphoedema model. Therefore, we aimed to investigate the potential therapeutic effect of a KD in patients with secondary lymphoedema.
METHODS: Nine patients with unilateral stage 2 lymphoedema secondary to lymphadenectomy were included in this quasi-experimental exploratory study consisting of a short run-in phase to gradually induce ketosis, followed by a classic KD (CKD) and modified Atkins diet (MAD) phase during which patients consumed a CKD and MAD, respectively. Lymphatic function and oedema volume, the primary outcomes, were assessed at baseline and at the end of both the CKD and MAD phase. Secondary outcomes included health-related and lymphedema-specific quality of life (QoL).
RESULTS: Seven out of nine patients completed the study protocol. Lymphatic function was improved upon consumption of both a CKD (dermal backflow score [mean ± SD]: 7.29 ± 2.98 vs. 10.86 ± 2.19 at baseline; p = 0.03) and MAD (6.71 ± 2.06; p = 0.02), whereas oedema volume did not decrease during the course of the study (excess limb volume [mean ± SD]: 20.13 ± 10.25% at end of CKD and 24.07 ± 17.77% at end of MAD vs. 20.79 ± 12.96% at baseline; p > 0.99 and p > 0.30, respectively). No changes were observed in health-related, nor lymphoedema-specific QoL at the end of CKD and MAD.
CONCLUSIONS: The consumption of a KD improved lymphatic function and was associated with a clinically meaningful reduction in oedema volume in some patients (3/7 at end of CKD, 2/7 at end of MAD) with unilateral stage 2 secondary lymphoedema. These results highlight the potential of a KD to improve lymphatic function in patients with lymphoedema. However, further studies are required to substantiate our findings.

Lymphatic dysfunction is a pivotal pathological mechanism underlying the development of early atherosclerotic plaques. Potential targets of lymphatic function must be identified to realize the early prevention and treatment of atherosclerosis (AS). The immunity-related GTPase Irgm1 is involved in orchestrating cellular autophagy and apoptosis. However, the effect of Irgm1 on early AS progression, particularly through alterations in lymphatic function, remains unclear. In this study, we confirmed the protective effect of lymphangiogenesis on early-AS in vivo. Subsequently, an in vivo model of early AS mice with Irgm1 knockdown shows that Irgm1 reduces early atherosclerotic plaque burden by promoting lymphangiogenesis. Given that lymphatic endothelial cell (LEC) autophagy significantly contributes to lymphangiogenesis, Irgm1 may enhance lymphatic circulation by promoting LEC autophagy. Moreover, Irgm1 orchestrates autophagy in LECs by inhibiting mTOR and facilitating nuclear translocation of Tfeb. Collectively, these processes lead to lymphangiogenesis. Thus, this study establishes a link between Irgm1 and early AS, thus revealing a novel mechanism by which Irgm1 exerts an early protective influence on AS within the context of lymphatic circulation. The insights gained from this study have the potential to revolutionize the approach and management of AS onset.

Lymphatics participate in reverse cholesterol transport, and their presence in the arterial wall of the great vessels and prior experimental results suggest their possible role in the development of atherosclerosis. The aim of this study was to characterize the lymphatic vasculature of the arterial wall in atherosclerosis. Tissue sections and tissue-cleared aortas of wild-type mice unveiled significant differences in the density of the arterial lymphatic network throughout the arterial tree. Male and female Ldlr-/- and ApoE-/- mice on a Western diet showed sex-dependent differences in plaque formation and calcification. Female mice on a Western diet developed more calcification of atherosclerotic plaques than males. The lymphatic vessels within the aortic wall of these mice showed no major changes regarding the number of lymphatic junctions and end points or the lymphatic area. However, female mice on a Western diet showed moderate dilation of lymphatic vessels in the abdominal aorta and exhibited indications of increased peripheral lymphatic function, findings that require further studies to understand the role of lymphatics in the arterial wall during the development of atherosclerosis.

Physiological properties and function of the lymphatic system is still somewhat of a mystery. We report the current knowledge about human lymphatic vessel contractility and capability of adaptation. A literature search in PubMed identified studies published January 2000-September 2022. Inclusion criteria were studies investigating parameters related to contraction frequency, fluid velocity, and lymphatic pressure in vivo and ex vivo in human lymphatic vessels. The search returned 2885 papers of which 28 met the inclusion criteria. In vivo vessels revealed baseline contraction frequencies between 0.2 ± 0.2 and 1.8 ± 0.1 min1 , velocities between 0.008 ± 0.002 and 2.3 ± 0.3 cm/s, and pressures between 4.5 (range 0.5-9.2) and 60.3 ± 2.8 mm Hg. Gravitational forces, hyperthermia, and treatment with nifedipine caused increases in contraction frequency. Ex vivo lymphatic vessels displayed contraction frequencies between 1.2 ± 0.1 and 5.5 ± 1.2 min-1 . Exposure to agents affecting cation and anion channels, adrenoceptors, HCN channels, and changes in diameter-tension properties all resulted in changes in functional parameters as known from the blood vascular system. We find that the lymphatic system is dynamic and adaptable. Different investigative methods yields alternating results. Systematic approaches, consensus on investigative methods, and larger studies are needed to fully understand lymphatic transport and apply this in a clinical context.

Inflammation causes bronchopulmonary dysplasia (BPD), a common lung disease of preterm infants. One reason this disease lacks specific therapies is the paucity of information on the mechanisms regulating inflammation in developing lungs. We address this gap by characterizing the lymphatic phenotype in an experimental BPD model because lymphatics are major regulators of immune homeostasis. We hypothesized that hyperoxia (HO), a major risk factor for experimental and human BPD, disrupts lymphatic endothelial homeostasis using neonatal mice and human dermal lymphatic endothelial cells (HDLECs). Exposure to 70% O2 for 24-72 h decreased the expression of prospero homeobox 1 (Prox1) and vascular endothelial growth factor c (Vegf-c) and increased the expression of heme oxygenase 1 and NAD(P)H dehydrogenase [quinone]1 in HDLECs, and reduced their tubule formation ability. Next, we determined Prox1 and Vegf-c mRNA levels on postnatal days (P) 7 and 14 in neonatal murine lungs. The mRNA levels of these genes increased from P7 to P14, and 70% O2 exposure for 14 d (HO) attenuated this physiological increase in pro-lymphatic factors. Further, HO exposure decreased VEGFR3+ and podoplanin+ lymphatic vessel density and lymphatic function in neonatal murine lungs. Collectively, our results validate the hypothesis that HO disrupts lymphatic endothelial homeostasis.

Advancements in lymphography technology are essential for comprehensive investigation of the lymphatic system and its function. Here, a shortwave infrared (SWIR) luminescence imaging of lymphatic vessels is proposed in both normal and lymphatic dysfunction in rat models with PbS quantum dots (PbS Qdots). The lymphography with PbS Qdots can clearly and rapidly demonstrate the normal lymphatic morphology in both the tail and hind limb. More importantly, compared to ICG, SWIR luminescence imaging with PbS Qdots can easily identify the dominant lymphatic vessel and node with higher luminescence signal in rats. Moreover, lymphatic pump is identified as segment contracting sections with a size of ≈1 cm in rat by in vivo SWIR lymphograhy, which propose a direct feature for precise evaluation of lymphatic function. Notably, in vivo SWIR luminescence imaging with PbS Qdots also clearly deciphers the in vivo pattern of morphological and function recovery from lymphatic system in rat model. In summary, SWIR luminescence imaging with PbS Qdots can improve the lymphography and thus deepen the understanding of the morphology and structure of the lymphatic system as well as lymphatic function such as lymphatic pump, which will facilitate the diagnosis of lymphatic dysfunction in the future.

Rheumatoid arthritis (RA), as an autoimmune inflammatory disease, is featured by enhanced vascular permeability, irreversible cartilage destroys and bone erosion. Although the pathogenesis of RA is still unclear, the immune environment, particularly the lymphatic system, which is instrumental to immune cell surveillance and interstitial fluid balance, plays vital roles in the process of RA. Herein, an inflammation specific environment activated methotrexate-encapsulated nanomedicine (MTX@NPs) was constructed for RA treatment, which accumulated in inflamed joints, and released MTX in the specific RA microenvironment. Notably, MTX@NPs could regulate the immune environment including reducing the expressions of inflammatory cytokines of macrophages and the inflammatory level of lymphatic epithelial cells (LECs), and ameliorating the lymphatic vessel contraction and drainage. In vitro and In vivo studies illustrated that MTX@NPs exhibited a high RA therapeutic efficacy and insignificant systemic toxicity owing to the suppression of the inflammation response and the improved lymphatic functions of RA joints. It suggests that the nanomedicine paves a potential way to the clinical practice of autoimmune diseases treatments via the regulation of immune environment and lymphatic functions. STATEMENT OF SIGNIFICANCE: Although 1.0% of the population in the world suffers from rheumatoid arthritis (RA), the pathogenesis of RA is still unclear and the therapeutic effect of the first-line clinical drugs is relatively low. Herein, we propose a specific RA-microenvironment triggered nanomedicine (MTX@NPs), which enhances RA treatment of a first-line antirheumatic drug (methotrexate, MTX) by immune environment reconstruction. The nanomedicine exhibits RA joints accumulation by EPR effect, and releases MTX under the specific RA environment, leading to the dramatical drop of M1-type macrophages and acceleration of lymphatic vessel contraction and drainage. Finally, the inflammatory cytokines in RA immune environment are reduced sharply, indicating the outstanding therapeutic efficacy of MTX@NPs to RA.

The term \"subpleural sparing\" refers to computed tomography (CT) images that indicate that there is limited disease/infiltrate in the immediate subpleural location. This observation is often associated with nonspecific interstitial pneumonitis and is a characteristic that distinguishes this pathology from usual interstitial pneumonitis (idiopathic pulmonary fibrosis). Subpleural sparing can also occur in acute respiratory disorders, including pulmonary contusion in children, acute lung disease associated with electronic cigarettes (vaping), and aspiration of exogenous lipids. Potential explanations for this observation include nonuniform distribution of lung injury/inflammation, nonuniform clearing/resolution of injury, and variations in CT image acquisition and presentation. The subpleural region contains lymphatic structures on the interior surface of the visceral pleura and in interlobular septa. The density of subpleural lymphatics decreases in more interior zones of the lung that largely contain alveolar-capillary units. These lymphatics transfer fluid and other inflammatory mediators from the peripheral lung into central lymphatics and veins. Consequently, the density and distribution of lymphatics could explain preferential clearing of the subpleural regions during acute injury. The acquisition of CT images also depends on the configuration of detectors, slice thickness, and the energy of the electron beam. Clinicians should carefully consider the disease process, lymphatic function and other clearance mechanisms, and the vagaries in CT image acquisition when they evaluate patients with subpleural sparing.

Dysfunction of the lymphatic system following injury, disease, or cancer treatment can lead to lymphedema, a debilitating condition with no cure. Despite the various physical therapy and surgical options available, most treatments are palliative and fail to address the underlying lymphatic vascular insufficiency driving lymphedema progression. Stem cell therapy provides a promising alternative in the treatment of various chronic diseases with a wide range of therapeutic effects that reduce inflammation, fibrosis, and oxidative stress, while promoting lymphatic vessel (LV) regeneration. Specifically, stem cell transplantation is suggested to promote LV restoration, rebuild lymphatic circulation, and thus potentially be utilized towards an effective lymphedema treatment. In addition to stem cells, studies have proposed the administration of vascular endothelial growth factor C (VEGFC) to promote lymphangiogenesis and decrease swelling in lymphedema.
Here, we seek to combine the benefits of stem cell therapy, which provides a cellular therapeutic approach that can respond to the tissue environment, and VEGFC administration to restore lymphatic drainage.
Specifically, we engineered mesenchymal stem cells (MSCs) to overexpress VEGFC using a lentiviral vector (hVEGFC MSC) and investigated their therapeutic efficacy in improving LV function and tissue swelling using near infrared (NIR) imaging, and lymphatic regeneration in a single LV ligation mouse tail lymphedema model.
First, we showed that overexpression of VEGFC using lentiviral transduction led to an increase in VEGFC protein synthesis in vitro. Then, we demonstrated hVEGFC MSC administration post-injury significantly increased the lymphatic contraction frequency 14-, 21-, and 28-days post-surgery compared to the control animals (MSC administration) in vivo, while also reducing tail swelling 28-days post-surgery compared to controls.
Our results suggest a therapeutic potential of hVEGFC MSC in alleviating the lymphatic dysfunction observed during lymphedema progression after secondary injury and could provide a promising approach to enhancing autologous cell therapy for treating lymphedema.