Andrographolide promotes lymphangiogenesis and lymphatic vessel remodeling to alleviate secondary lymphedema.

Lymphedema, a prevalent, multifaceted, and chronic ailment, is mainly managed through physical manipulation and suffers from a lack of specific pharmacological treatments. Secondary lymphedema is mainly caused by impaired lymphatic drainage. Therapeutic lymphangiogenesis is a promising strategy in the treatment of lymphedema. Andrographolide, a natural product from Andrographis paniculata, is unknown whether andrographolide promotes lymphangiogenesis to improve secondary lymphedema. By using the murine tail lymphedema model, we demonstrated that andrographolide can reduce the thickness of subcutaneous tissue in the mice's tail and enhance lymphatic drainage. Moreover, immunofluorescence staining showed that the number of capillary lymphatic vessels in the ANDRO25 group was significantly more than that in the ANDRO50 and Model groups. Near-infrared lymphography images showed that highlighted sciatic lymph nodes could be seen in the ANDRO25 and ANDRO50 groups. In vitro, andrographolide could promote the proliferation and migration of LEC. In conclusion, andrographolide enhanced the recovery of lymphatic vessels, and promoted lymphatic drainage in the murine tail lymphedema model by promoting the proliferation of lymphatic endothelial cells, thereby reducing the symptoms of lymphedema. This suggested andrographolide may be used as a potential therapeutic drug or medical food ingredient to help patients with secondary lymphedema.

Lymphatics in bone arise from pre-existing lymphatics.

Bones do not normally have lymphatics. However, individuals with generalized lymphatic anomaly (GLA) or Gorham-Stout disease (GSD) develop ectopic lymphatics in bone. Despite growing interest in the development of tissue-specific lymphatics, the cellular origin of bone lymphatic endothelial cells (bLECs) is not known and the development of bone lymphatics has not been fully characterized. Here, we describe the development of bone lymphatics in mouse models of GLA and GSD. Through lineage-tracing experiments, we show that bLECs arise from pre-existing Prox1-positive LECs. We show that bone lymphatics develop in a stepwise manner where regional lymphatics grow, breach the periosteum and then invade bone. We also show that the development of bone lymphatics is impaired in mice that lack osteoclasts. Last, we show that rapamycin can suppress the growth of bone lymphatics in our models of GLA and GSD. In summary, we show that bLECs can arise from pre-existing LECs and that rapamycin can prevent the growth of bone lymphatics.

Live Imaging of Intracranial Lymphatics in the Zebrafish.

RATIONALE: The recent discovery of meningeal lymphatics in mammals is reshaping our understanding of fluid homeostasis and cellular waste management in the brain, but visualization and experimental analysis of these vessels is challenging in mammals. Although the optical clarity and experimental advantages of zebrafish have made this an essential model organism for studying lymphatic development, the existence of meningeal lymphatics has not yet been reported in this species. OBJECTIVE: Examine the intracranial space of larval, juvenile, and adult zebrafish to determine whether and where intracranial lymphatic vessels are present. METHODS AND RESULTS: Using high-resolution optical imaging of the meninges in living animals, we show that zebrafish possess a meningeal lymphatic network comparable to that found in mammals. We confirm that this network is separate from the blood vascular network and that it drains interstitial fluid from the brain. We document the developmental origins and growth of these vessels into a distinct network separated from the external lymphatics. Finally, we show that these vessels contain immune cells and perform live imaging of immune cell trafficking and transmigration in meningeal lymphatics. CONCLUSIONS: This discovery establishes the zebrafish as a important new model for experimental analysis of meningeal lymphatic development and opens up new avenues for probing meningeal lymphatic function in health and disease.

The role of fatty acid ß-oxidation in lymphangiogenesis.

Lymphatic vessels are lined by lymphatic endothelial cells (LECs), and are critical for health. However, the role of metabolism in lymphatic development has not yet been elucidated. Here we report that in transgenic mouse models, LEC-specific loss of CPT1A, a rate-controlling enzyme in fatty acid ß-oxidation, impairs lymphatic development. LECs use fatty acid ß-oxidation to proliferate and for epigenetic regulation of lymphatic marker expression during LEC differentiation. Mechanistically, the transcription factor PROX1 upregulates CPT1A expression, which increases acetyl coenzyme A production dependent on fatty acid ß-oxidation. Acetyl coenzyme A is used by the histone acetyltransferase p300 to acetylate histones at lymphangiogenic genes. PROX1-p300 interaction facilitates preferential histone acetylation at PROX1-target genes. Through this metabolism-dependent mechanism, PROX1 mediates epigenetic changes that promote lymphangiogenesis. Notably, blockade of CPT1 enzymes inhibits injury-induced lymphangiogenesis, and replenishing acetyl coenzyme A by supplementing acetate rescues this process in vivo.

Lymphotonic activity of Ruscus extract, hesperidin methyl chalcone and vitamin C in human lymphatic smooth muscle cells.

OBJECTIVE: Besides actions including their venotonic, anti-inflammatory, and anti-oxidant effects, venoactive drugs are expected to act on edema via their action on lymphatics. The objective of this study was to evaluate the effect of the combination of Ruscus, hesperidin methyl chalcone and Vitamin C (Ruscus/HMC/Vit C) on intracellular calcium mobilization and contraction of human lymphatic smooth muscle cells (LSMCs) to better characterize the mechanism of its lymphotonic activity. METHODS: Calcium mobilization was evidenced by videomicroscopy analysis of the fluorescence emitted by a specific Ca2+ sensitive dye and measured after injection of Ruscus/HMC/Vit C at 0.1, 0.3, 1.0, and 3.0 mg/mL into LSMCs. RESULTS: Ruscus/HMC/Vit C induced a strong and reproducible concentration-dependent calcium mobilization in LSMCs. On the contrary, another venoactive drug used as comparator, micronized purified flavonoid fraction (MPFF), did not induce calcium mobilization whatever the tested concentration. CONCLUSION: Although alternative mechanisms of action may result in potential lymphotonic effects, the efficacy of lymphotonic products is nonetheless related to their stimulating effect on the contractile activity of the smooth muscle cells surrounding lymphatic vessels. In the light of the results obtained in this study, the direct effect of Ruscus/HMC/Vit C on LSMC contraction may partially explain its clinical efficacy on lymphotonic activity, as has been observed in terms of objective signs of edema as reported in the recent guidelines on chronic venous disease.

Molecular mechanisms of cyclic phosphatidic acid-induced lymphangiogenic actions in vitro.

The lymphatic system plays important roles in various physiological and pathological phenomena. As a bioactive phospholipid, lysophosphatidic acid (LPA) has been reported to function as a lymphangiogenic factor as well as some growth factors, yet the involvement of phospholipids including LPA and its derivatives in lymphangiogenesis is not fully understood. In the present study, we have developed an in-vitro lymphangiogenesis model (termed a collagen sandwich model) by utilizing type-I collagen, which exists around the lymphatic endothelial cells of lymphatic capillaries in vivo. The collagen sandwich model has revealed that cyclic phosphatidic acid (cPA), and not LPA, augmented the tube formation of human dermal lymphatic endothelial cells (HDLECs). Both cPA and LPA increased the migration of HDLECs cultured on the collagen. As the gene expression of LPA receptor 6 (LPA6) was predominantly expressed in HDLECs, a siRNA experiment against LPA6 attenuated the cPA-mediated tube formation. A synthetic LPA1/3 inhibitor, Ki16425, suppressed the cPA-augmented tube formation and migration of the HDLECs, and the LPA-induced migration. The activity of Rho-associated protein kinase (ROCK) located at the downstream of the LPA receptors was augmented in both the cPA- and LPA-treated cells. A potent ROCK inhibitor, Y-27632, suppressed the cPA-dependent tube formation but not the migration of the HDLECs. Furthermore, cPA, but not LPA, augmented the gene expression of VE-cadherin and ß-catenin in the HDLECs. These results provide novel evidence that cPA facilitates the capillary-like morphogenesis and the migration of HDLECs through LPA6/ROCK and LPA1/3 signaling pathways in concomitance with the augmentation of VE-cadherin and ß-catenin expression. Thus, cPA is likely to be a potent lymphangiogenic factor for the initial lymphatics adjacent to type I collagen under physiological conditions.

GLP-1 (Glucagon-Like Peptide-1) Is Physiologically Relevant for Chylomicron Secretion Beyond Its Known Pharmacological Role.

[Figure: see text].

Roles of Thromboxane Receptor Signaling in Enhancement of Lipopolysaccharide-Induced Lymphangiogenesis and Lymphatic Drainage Function in Diaphragm.

[Figure: see text].

Pleiotropic neuroprotective effects of taxifolin in cerebral amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) results from amyloid-ß deposition in the cerebrovasculature. It is frequently accompanied by Alzheimer's disease and causes dementia. We recently demonstrated that in a mouse model of CAA, taxifolin improved cerebral blood flow, promoted amyloid-ß removal from the brain, and prevented cognitive dysfunction when administered orally. Here we showed that taxifolin inhibited the intracerebral production of amyloid-ß through suppressing the ApoE-ERK1/2-amyloid-ß precursor protein axis, despite the low permeability of the blood-brain barrier to taxifolin. Higher expression levels of triggering receptor expressed on myeloid cell 2 (TREM2) were associated with the exacerbation of inflammation in the brain. Taxifolin suppressed inflammation, alleviating the accumulation of TREM2-expressing cells in the brain. It also mitigated glutamate levels and oxidative tissue damage and reduced brain levels of active caspases, indicative of apoptotic cell death. Thus, the oral administration of taxifolin had intracerebral pleiotropic neuroprotective effects on CAA through suppressing amyloid-ß production and beneficially modulating proinflammatory microglial phenotypes.

VEGFR3 tyrosine kinase inhibition aggravates cisplatin nephrotoxicity.

Expansion of renal lymphatic networks, or lymphangiogenesis (LA), is well recognized during development and is now being implicated in kidney diseases. Although LA is associated with multiple pathological conditions, very little is known about its role in acute kidney injury. The purpose of this study was to evaluate the role of LA in a model of cisplatin-induced nephrotoxicity. LA is predominately regulated by vascular endothelial growth factor (VEGF)-C and VEGF-D, ligands that exert their function through their cognate receptor VEGF receptor 3 (VEGFR3). We demonstrated that use of MAZ51, a selective VEGFR3 inhibitor, caused significantly worse structural and functional kidney damage in cisplatin nephrotoxicity. Apoptotic cell death and inflammation were also increased in MAZ51-treated animals compared with vehicle-treated animals following cisplatin administration. Notably, MAZ51 caused significant upregulation of intrarenal phospho-NF-κB, phospho-JNK, and IL-6. Cisplatin nephrotoxicity is associated with vascular congestion due to endothelial dysfunction. Using three-dimensional tissue cytometry, a novel approach to explore lymphatics in the kidney, we detected significant vascular autofluorescence attributed to erythrocytes in cisplatin alone-treated animals. Interestingly, no such congestion was detected in MAZ51-treated animals. We found increased renal vascular damage in MAZ51-treated animals, whereby MAZ51 caused a modest decrease in the endothelial markers endomucin and von Willebrand factor, with a modest increase in VEGFR2. Our findings identify a protective role for de novo LA in cisplatin nephrotoxicity and provide a rationale for the development of therapeutic approaches targeting LA. Our study also suggests off-target effects of MAZ51 on the vasculature in the setting of cisplatin nephrotoxicity.NEW & NOTEWORTHY Little is known about injury-associated LA in the kidney and its role in the pathophysiology of acute kidney injury (AKI). Observed exacerbation of cisplatin-induced AKI after LA inhibition was accompanied by increased medullary damage and cell death in the kidney. LA inhibition also upregulated compensatory expression of LA regulatory proteins, including JNK and NF-κB. These data support the premise that LA is induced during AKI and lymphatic expansion is a protective mechanism in cisplatin nephrotoxicity.

KATP Channel Openers Inhibit Lymphatic Contractions and Lymph Flow as a Possible Mechanism of Peripheral Edema.

Pharmacological openers of ATP-sensitive potassium (KATP) channels are effective antihypertensive agents, but off-target effects, including severe peripheral edema, limit their clinical usefulness. It is presumed that the arterial dilation induced by KATP channel openers (KCOs) increases capillary pressure to promote filtration edema. However, KATP channels also are expressed by lymphatic muscle cells (LMCs), raising the possibility that KCOs also attenuate lymph flow to increase interstitial fluid. The present study explored the effect of KCOs on lymphatic contractile function and lymph flow. In isolated rat mesenteric lymph vessels (LVs), the prototypic KATP channel opener cromakalim (0.01-3 µmol/l) progressively inhibited rhythmic contractions and calculated intraluminal flow. Minoxidil sulfate and diazoxide (0.01-100 µmol/l) had similar effects at clinically relevant plasma concentrations. High-speed in vivo imaging of the rat mesenteric lymphatic circulation revealed that superfusion of LVs with cromakalim and minoxidil sulfate (0.01-10 µmol/l) maximally decreased lymph flow in vivo by 38.4% and 27.4%, respectively. Real-time polymerase chain reaction and flow cytometry identified the abundant KATP channel subunits in LMCs as the pore-forming Kir6.1/6.2 and regulatory sulfonylurea receptor 2 subunits. Patch-clamp studies detected cromakalim-elicited unitary K+ currents in cell-attached patches of LMCs with a single-channel conductance of 46.4 pS, which is a property consistent with Kir6.1/6.2 tetrameric channels. Addition of minoxidil sulfate and diazoxide elicited unitary currents of similar amplitude. Collectively, our findings indicate that KCOs attenuate lymph flow at clinically relevant plasma concentrations as a potential contributing mechanism to peripheral edema. SIGNIFICANCE STATEMENT: ATP-sensitive potassium (KATP) channel openers (KCOs) are potent antihypertensive medications, but off-target effects, including severe peripheral edema, limit their clinical use. Here, we demonstrate that KCOs impair the rhythmic contractions of lymph vessels and attenuate lymph flow, which may promote edema formation. Our finding that the KATP channels in lymphatic muscle cells may be unique from their counterparts in arterial muscle implies that designing arterial-selective KCOs may avoid activation of lymphatic KATP channels and peripheral edema.

The YAP signaling pathway promotes the progression of lymphatic malformations through the activation of lymphatic endothelial cells.

BACKGROUND: To investigate whether the YAP/TAZ (Yes-associated protein/transcriptional coactivator with PDZ binding motif) pathway contributes to the pathogenesis of lymphatic malformations (LMs). METHODS: YAP, TAZ, CTGF (connective tissue growth factor), and Ki-67 were detected in LMs by immunohistochemistry. The colocalization of YAP and Ki-67 was analyzed by double immunofluorescence. Pearson's correlation and cluster analyses were performed to analyze the relationships between these proteins. Human dermal lymphatic endothelial cells (HDLECs) were used for mechanistic investigation. Rat models of LMs were established to investigate the role of the YAP pathway in LM development. RESULTS: Compared with those in normal skin, the expression levels of YAP, TAZ, CTGF, and Ki-67 were significantly upregulated in lymphatic endothelial cells (LECs) of LMs. Interestingly, YAP and CTGF presented much higher expression levels in infected LMs. In experiments in vitro, lipopolysaccharide (LPS) enhanced the expression of YAP in a concentration- and time-dependent manner via the increased phosphorylation of Erk1/2 (extracellular signal-regulated kinase 1/2). Moreover, the proliferation, invasion, and tubule formation of HDLECs increased significantly in accordance with the activation of the YAP signaling pathway. Furthermore, LM rat models validated that LPS facilitated the development of LMs, which was dependent on the activation of YAP. CONCLUSIONS: The data reveal that activation of the YAP signaling pathway in LECs may play a crucial role in the progression of LMs. IMPACT: Compared with that in normal skin, the YAP signaling pathway was activated in LECs of LMs. Inhibiting the YAP signaling pathway attenuated the proliferation, invasion, and tubule formation of HDLECs. Additionally, the activation of the YAP signaling pathway could promote LM development in a rat model. Activation of the YAP signaling pathway in LECs may play a crucial role in the progression of LMs. The YAP signaling pathway was activated in LMs. Inhibition of the YAP signaling pathway could promote regression of the lesions.

Dieckol Decreases Caloric Intake and Attenuates Nonalcoholic Fatty Liver Disease and Hepatic Lymphatic Vessel Dysfunction in High-Fat-Diet-Fed Mice.

Increased inflammation is the main pathophysiology of nonalcoholic fatty liver disease (NAFLD). Inflammation affects lymphatic vessel function that contributes to the removal of immune cells or macromolecules. Dysfunctional lymphatic vessels with decreased permeability are present in NAFLD. High-fat diet (HFD) is known to increase body weight, food intake, and inflammation in the liver. Previously, it was reported that Ecklonia cava extracts (ECE) decreased food intake or weight gain, and low-calorie diet and weight loss is known as a treatment for NAFLD. In this study, the effects of ECE and dieckol (DK)-which is one component of ECE that decreases inflammation and increases lymphangiogenesis and lymphatic drainage by controlling lymphatic permeability in high-fat diet (HFD)-fed mice-on weight gain and food intake were investigated. ECE and DK decreased weight gain and food intake in the HFD-fed mice. NAFLD activities such as steatosis, lobular inflammation, and ballooning were increased by HFD and attenuated by ECE and DK. The expression of inflammatory cytokines such as IL-6 and TNF-α and infiltration of M1 macrophages were increased by HFD, and they were decreased by ECE or DK. The signaling pathways of lymphangiogenesis, VEGFR-3, PI3K/pAKT, and pERK were decreased by HFD, and they were restored by either ECE or DK. The expression of VE-cadherin (which represents lymphatic junctional function) was increased by HFD, although it was restored by either ECE or DK. In conclusion, ECE and DK attenuated NAFLD by decreasing weight gain and food intake, decreasing inflammation, and increasing lymphangiogenesis, as well as modulating lymphatic vessel permeability.

Clinical variability in multifocal lymphangioendotheliomatosis with thrombocytopenia: a review of the literature.

Multifocal lymphangioendotheliomatosis with thrombocytopenia (MLT) is a recently recognized disorder characterized by vascular lesions marked by distinct endothelial proliferation. Lesions affect multiple tissues, and MLT can be associated with refractory thrombocytopenia resulting in life-threatening bleeding. Diagnosing MLT may be challenging given its rarity and phenotypic variability. There is no consensus on the optimal management or treatment duration. We report a 4-month-old male who presented with multiple vascular malformations involving the gastrointestinal tract, lung, bones, choroid plexus, and spleen, with minimal cutaneous involvement and no thrombocytopenia. Wedge resection of a pulmonary nodule was strongly positive for lymphatic vessel endothelial hyaluronan receptor 1 favoring MLT despite the lack of thrombocytopenia. The patient's clinical symptoms and vascular lesions improved on sirolimus therapy. We review the literature to highlight the clinical variability of MLT and discuss the diagnostic and therapeutic options for MLT.

The Kinetics of Lymphatic Dysfunction and Leukocyte Expansion in the Draining Lymph Node during LTB4 Antagonism in a Mouse Model of Lymphedema.

The mechanisms of lymphedema development are not well understood, but emerging evidence highlights the crucial role the immune system plays in driving its progression. It is well known that lymphatic function deteriorates as lymphedema progresses; however, the connection between this progressive loss of function and the immune-driven changes that characterize the disease has not been well established. In this study, we assess changes in leukocyte populations in lymph nodes within the lymphatic drainage basin of the tissue injury site (draining lymph nodes, dLNs) using a mouse tail model of lymphedema in which a pair of draining collecting vessels are left intact. We additionally quantify lymphatic pump function using established near infrared (NIR) lymphatic imaging methods and lymph-draining nanoparticles (NPs) synthesized and employed by our team for lymphatic tissue drug delivery applications to measure lymphatic transport to and resulting NP accumulation within dLNs associated with swelling following surgery. When applied to assess the effects of the anti-inflammatory drug bestatin, which has been previously shown to be a possible treatment for lymphedema, we find lymph-draining NP accumulation within dLNs and lymphatic function to increase as lymphedema progresses, but no significant effect on leukocyte populations in dLNs or tail swelling. These results suggest that ameliorating this loss of lymphatic function is not sufficient to reverse swelling in this surgically induced disease model that better recapitulates the extent of lymphatic injury seen in human lymphedema. It also suggests that loss of lymphatic function during lymphedema may be driven by immune-mediated mechanisms coordinated in dLNs. Our work indicates that addressing both lymphatic vessel dysfunction and immune cell expansion within dLNs may be required to prevent or reverse lymphedema when partial lymphatic function is sustained.

Condition of Adrenergic Innervation Apparatus of the Thyroid Gland, Blood and Lymph Vessels, and Lymph Nodes during Correction of Hypothyrosis.

We used specific histochemical fluorescence-microscopic method of visualization of catecholamines to study adrenergic innervation of the thyroid gland tissue, blood vessels of the thyroid gland, cervical lymphatic vessel and lymph nodes in rats during correction of hypothyroidism with a bioactive formulation (Vozrozhdenie Plus balm with Potentilla alba L.). In experimental hypothyroidism, adrenergic innervation of the thyroid gland and the wall of the cervical lymph node, concentrated mainly along the arterial vessels and the cervical lymphatic vessel, retained its structural formations (plexuses and varicosities), but diffusion of catecholamines outside these formations was observed. Correction with the bioactive formulation restored of the contours of the nerve plexuses and varicosities and their brighter fluorescence in the thyroid gland and cervical lymphatic vessel and node. During correction of hypothyroidism with the bioactive formulation, reorganization of regional lymphatic vessels and nodes was more pronounced than reorganization of the thyroid gland.

Inhibition of receptor activity-modifying protein 1 suppresses the development of endometriosis and the formation of blood and lymphatic vessels.

Neuroimmune interactions are involved in the development of endometriosis. Here, we examined the role of a neuropeptide, calcitonin gene-related peptide (CGRP), and its receptor, receptor activity-modifying protein (RAMP) 1, in growth of endometrial tissues and the formation of blood and lymphatic vessels in a mouse ectopic endometrial transplantation model. Endometrial fragments from donor wild-type (WT) mice transplanted into the peritoneal wall of recipient WT mice grew with increased density of blood and lymphatic vessels. When tissues from RAMP1-deficient (RAMP1-/- ) mice were transplanted into RAMP1-/- mice, implant growth and angiogenesis/lymphangiogenesis were decreased. CGRP was up-regulated in dorsal root ganglia, and CGRP+ nerve fibres were distributed into the implants from the peritoneum. RAMP1 was co-expressed with CD11b (macrophages) and S100A4 (fibroblasts), but did not co-localize with blood vessel endothelial cell marker CD31 or lymphatic vessel endothelial hyaluronan receptor (LYVE)-1. Cultured with CGRP, macrophages up-regulated vascular endothelial growth factor (VEGF)-A, VEGF-C and VEGF-D, whereas fibroblasts up-regulated VEGF-C, but not VEGF-A or VEGF-D, in a RAMP1-dependent manner. CGRP receptor antagonist CGRP8-37 inhibited growth of and angiogenesis/lymphangiogenesis within endometrial tissue implants. These results suggest that RAMP1 signalling is crucial for growth and angiogenesis/lymphangiogenesis in endometrial tissue. Blockade of RAMP1 is a potential tool for the treatment of endometriosis.

Renal lymphatic vessel dynamics.

Similar to other organs, renal lymphatics remove excess fluid, solutes, and macromolecules from the renal interstitium. Given the kidney's unique role in maintaining body fluid homeostasis, renal lymphatics may be critical in this process. However, little is known regarding the pathways involved in renal lymphatic vessel function, and there are no studies on the effects of drugs targeting impaired interstitial clearance, such as diuretics. Using pressure myography, we showed that renal lymphatic collecting vessels are sensitive to changes in transmural pressure and have an optimal range of effective pumping. In addition, they are responsive to vasoactive factors known to regulate tone in other lymphatic vessels including prostaglandin E2 and nitric oxide, and their spontaneous contractility requires Ca2+ and Cl-. We also demonstrated that Na+-K+-2Cl- cotransporter Nkcc1, but not Nkcc2, is expressed in extrarenal lymphatic vessels. Furosemide, a loop diuretic that inhibits Na+-K+-2Cl- cotransporters, induced a dose-dependent dilation in lymphatic vessels and decreased the magnitude and frequency of spontaneous contractions, thereby reducing the ability of these vessels to propel lymph. Ethacrynic acid, another loop diuretic, had no effect on vessel tone. These data represent a significant step forward in our understanding of the mechanisms underlying renal lymphatic vessel function and highlight potential off-target effects of furosemide that may exacerbate fluid accumulation in edema-forming conditions.

TRPV4 channels' dominant role in the temperature modulation of intrinsic contractility and lymph flow of rat diaphragmatic lymphatics.

The lymphatic system drains and propels lymph by extrinsic and intrinsic mechanisms. Intrinsic propulsion depends upon spontaneous rhythmic contractions of lymphatic muscles in the vessel walls and is critically affected by changes in the surrounding tissue like osmolarity and temperature. Lymphatics of the diaphragm display a steep change in contraction frequency in response to changes in temperature, and this, in turn, affects lymph flow. In the present work, we demonstrated in an ex vivo diaphragmatic tissue rat model that diaphragmatic lymphatics express transient receptor potential channels of the vanilloid 4 subfamily (TRPV4) and that their blockade by both the nonselective antagonist Ruthenium Red and the selective antagonist HC-067047 abolished the response of lymphatics to temperature changes. Moreover, the selective activation of TRPV4 channels by means of GSK1016790A mirrored the behavior of vessels exposed to increasing temperatures, pointing out the critical role played by these channels in sensing the temperature of the lymphatic vessels' environment and thus inducing a change in contraction frequency and lymph flow.NEW & NOTEWORTHY The present work addresses the putative receptor system that enables diaphragmatic lymphatics to change intrinsic contraction frequency and thus lymph flow according to the changes in temperature of the surrounding environment, showing that this role can be sustained by TRPV4 channels alone.

Selected Factors Affecting Oral Bioavailability of Nanoparticles Surface-Conjugated with Glycocholic Acid via Intestinal Lymphatic Pathway.

Here, we describe the absorption pathways of nanoparticles whose surface is modified with bile acid and present environmental factors that influence oral bioavailability (BA) from the gastrointestinal tract (GIT). The approach utilized 100 nm sized fluorescence-labeled, carboxylated polystyrene nanoparticles (CPN) conjugated with glycocholic acid (G/CPN) to exclude potential artifacts, if existing, and instability issues in evaluating the transit of G/CPN in the GIT and measuring BA. The in vitro study using SK-BR-3 that expresses the apical sodium bile acid transporter showed that once G/CPN is internalized, it stayed 2.9 times longer in the cells than CPN, indirectly suggesting that G/CPN takes intracellular trafficking pathways different from CPN in SK-BR-3 cells. In a Caco-2 cell monolayer, G/CPN passed through the monolayer without damaging the tight junction. G/CPN, when administered orally in rodents, showed sustained transit time in the GIT for at least 4 h and was absorbed into the intestinal lymphatic system and circulated into the blood. Ingestion of food before and after oral administration delays G/CPN absorption and decreases BA. A decrease in gastrointestinal motility by anesthetic condition increased the relative BA of G/CPN by up to 74%. Thus, the oral BA of G/CPN can be optimized by taking food ingestion and gastrointestinal motility into account.