Modulation of the Tryptophan Hydroxylase 1/Monoamine Oxidase-A/5-Hydroxytryptamine/5-Hydroxytryptamine Receptor 2A/2B/2C Axis Regulates Biliary Proliferation and Liver Fibrosis During Cholestasis.

BACKGROUND AND AIMS: Serotonin (5HT) is a neuroendocrine hormone synthetized in the central nervous system (CNS) as well as enterochromaffin cells of the gastrointestinal tract. Tryptophan hydroxylase (TPH1) and monoamine oxidase (MAO-A) are the key enzymes for the synthesis and catabolism of 5HT, respectively. Previous studies demonstrated that 5-hydroxytryptamine receptor (5HTR)1A/1B receptor agonists inhibit biliary hyperplasia in bile-duct ligated (BDL) rats, whereas 5HTR2B receptor antagonists attenuate liver fibrosis (LF) in mice. Our aim was to evaluate the role of 5HTR2A/2B/2C agonists/antagonists in cholestatic models. APPROACH AND RESULTS: While in vivo studies were performed in BDL rats and the multidrug resistance gene 2 knockout (Mdr2-/- ) mouse model of PSC, in vitro studies were performed in cell lines of cholangiocytes and hepatic stellate cells (HSCs). 5HTR2A/2B/2C and MAO-A/TPH1 are expressed in cholangiocytes and HSCs from BDL rats and Mdr2-/- - mice. Ductular reaction, LF, as well as the mRNA expression of proinflammatory genes increased in normal, BDL rats, and Mdr2-/- - mice following treatment 5HTR2A/2B/2C agonists, but decreased when BDL rats and Mdr2-/- mice were treated with 5HTR2A/2B/2C antagonists compared to BDL rats and Mdr2-/- mice, respectively. 5HT levels increase in Mdr2-/- mice and in PSC human patients compared to their controls and decrease in serum of Mdr2-/- mice treated with 5HTR2A/2B/2C antagonists compared to untreated Mdr2-/- mice. In vitro, cell lines of murine cholangiocytes and human HSCs express 5HTR2A/2B/2C and MAO-A/TPH1; treatment of these cell lines with 5HTR2A/2B/2C antagonists or TPH1 inhibitor decreased 5HT levels as well as expression of fibrosis and inflammation genes compared to controls. CONCLUSIONS: Modulation of the TPH1/MAO-A/5HT/5HTR2A/2B/2C axis may represent a therapeutic approach for management of cholangiopathies, including PSC.

Lymphatic Cannulation for Lymph Sampling and Molecular Delivery.

Unlike the blood, the interstitial fluid and the deriving lymph are directly bathing the cellular layer of each organ. As such, composition analysis of the lymphatic fluid can provide more precise biochemical and cellular information on an organ's health and be a valuable resource for biomarker discovery. In this study, we describe a protocol for cannulation of mouse and rat lymphatic collectors that is suitable for the following: the "omic" sampling of pre- and postnodal lymph, collected from different anatomical districts; the phenotyping of immune cells circulating between parenchymal organs and draining lymph nodes; injection of known amounts of molecules for quantitative immunological studies of nodal trafficking and/or clearance; and monitoring an organ's biochemical omic changes in pathological conditions. Our data indicate that probing the lymphatic fluid can provide an accurate snapshot of an organ's physiology/pathology, making it an ideal target for liquid biopsy.

Histamine-mediated autocrine signaling in mesenteric perilymphatic mast cells.

Lymphatic vessels play a critical role in mounting a proper immune response by trafficking peripheral immune cells to draining lymph nodes. Mast cells (MCs) are well known for their roles in type I hypersensitivity reactions, but little is known about their secretory regulation in the lymphatic niche. MCs, as innate sensor and effector cells, reside close to mesenteric lymphatic vessels (MLVs), and their activation and ability to release histamine influences the lymphatic microenvironment in a histamine-NF-κB-dependent manner. Using an established experimental protocol involving surgical isolation of rat mesenteric tissue segments, including MLVs and surrounding perilymphatic tissues, we tested the hypothesis that perilymphatic mesenteric MCs possess histamine receptors (HRs) that bind and respond to the histamine released from these same MCs. Under various experimental conditions, including inflammatory stimulation by LPS, we measured histamine in mesenteric perilymphatic tissues, evaluated expression of histidine decarboxylase in MCs along with the degree of MC degranulation, assessed the functional status of HRs in MCs, and evaluated the ability of histamine itself to induce MC activation. Finally, we evaluated the importance of MCs and HR1 and -2 for MLV-directed trafficking of CD11b/c-positive cells during acute tissue inflammation. Our data indicate the existence of a functionally potent MC-histamine autocrine regulatory loop, the elements of which are crucially important for acute inflammation-induced trafficking of the CD11b/c-positive cells toward MLVs. This MC-histamine loop serves as a first-line cellular servo control system, playing a key role in the innate and adaptive immune response as well as NF-κB-mediated maintenance of body homeostasis.

Prolonged intake of desloratadine: mesenteric lymphatic vessel dysfunction and development of obesity/metabolic syndrome.

This study aimed to establish mechanistic links between the prolonged intake of desloratadine, a common H1 receptor blocker (i.e., antihistamine), and development of obesity and metabolic syndrome. Male Sprague-Dawley rats were treated for 16 wk with desloratadine. We analyzed the dynamics of body weight gain, tissue fat accumulation/density, contractility of isolated mesenteric lymphatic vessels, and levels of blood lipids, glucose, and insulin, together with parameters of liver function. Prolonged intake of desloratadine induced development of an obesity-like phenotype and signs of metabolic syndrome. These alterations in the body included excessive weight gain, increased density of abdominal subcutaneous fat and intracapsular brown fat, high blood triglycerides with an indication of their rerouting toward portal blood, high HDL, high fasting blood glucose with normal fasting and nonfasting insulin levels (insulin resistance), high liver/body weight ratio, and liver steatosis (fatty liver). These changes were associated with dysfunction of mesenteric lymphatic vessels, specifically high lymphatic tone and resistance to flow together with diminished tonic and abolished phasic responses to increases in flow, (i.e., greatly diminished adaptive reserves to respond to postprandial increases in lymph flow). The role of nitric oxide in this flow-dependent adaptation was abolished, with remnants of these responses controlled by lymphatic vessel-derived histamine. Our current data, considered together with reports in the literature, support the notion that millions of the United States population are highly likely affected by underevaluated, lymphatic-related side effects of antihistamines and may develop obesity and metabolic syndrome due to the prolonged intake of this medication. NEW & NOTEWORTHY Prolonged intake of desloratadine induced development of obesity and metabolic syndrome associated with dysfunction of mesenteric lymphatic vessels, high lymphatic tone, and resistance to flow together with greatly diminished adaptive reserves to respond to postprandial increases in lymph flow. Data support the notion that millions of the USA population are highly likely affected by underevaluated, lymphatic-related side effects of antihistamines and may develop obesity and metabolic syndrome due to the prolonged intake of this medication.

Collecting lymphatic vessel permeability facilitates adipose tissue inflammation and distribution of antigen to lymph node-homing adipose tissue dendritic cells.

Collecting lymphatic vessels (CLVs), surrounded by fat and endowed with contractile muscle and valves, transport lymph from tissues after it is absorbed into lymphatic capillaries. CLVs are not known to participate in immune responses. In this study, we observed that the inherent permeability of CLVs allowed broad distribution of lymph components within surrounding fat for uptake by adjacent macrophages and dendritic cells (DCs) that actively interacted with CLVs. Endocytosis of lymph-derived Ags by these cells supported recall T cell responses in the fat and also generated Ag-bearing DCs for emigration into adjacent lymph nodes (LNs). Enhanced recruitment of DCs to inflammation-reactive LNs significantly relied on adipose tissue DCs to maintain sufficient numbers of Ag-bearing DCs as the LN expanded. Thus, CLVs coordinate inflammation and immunity within adipose depots and foster the generation of an unexpected pool of APCs for Ag transport into the adjacent LN.

Aged Lymphatic Vessels and Mast Cells in Perilymphatic Tissues.

This review provides a comprehensive summary of research on aging-associated alterations in lymphatic vessels and mast cells in perilymphatic tissues. Aging alters structure (by increasing the size of zones with low muscle cell investiture), ultrastructure (through loss of the glycocalyx), and proteome composition with a concomitant increase in permeability of aged lymphatic vessels. The contractile function of aged lymphatic vessels is depleted with the abolished role of nitric oxide and an increased role of lymphatic-born histamine in flow-dependent regulation of lymphatic phasic contractions and tone. In addition, aging induces oxidative stress in lymphatic vessels and facilitates the spread of pathogens from these vessels into perilymphatic tissues. Aging causes the basal activation of perilymphatic mast cells, which, in turn, restricts recruitment/activation of immune cells in perilymphatic tissues. This aging-associated basal activation of mast cells limits proper functioning of the mast cell/histamine/NF-κB axis that is essential for the regulation of lymphatic vessel transport and barrier functions as well as for both the interaction and trafficking of immune cells near and within lymphatic collecting vessels. Cumulatively, these changes play important roles in the pathogenesis of alterations in inflammation and immunity associated with aging.

Involvement of histamine in endothelium-dependent relaxation of mesenteric lymphatic vessels.

OBJECTIVES: The knowledge of the basic principles of lymphatic function, still remains, to a large degree, rudimentary and will require significant research efforts. Recent studies of the physiology of the MLVs suggested the presence of an EDRF other than NO. In this study, we tested the hypothesis that lymphatic endothelium-derived histamine relaxes MLVs. METHODS: We measured and analyzed parameters of lymphatic contractility in isolated and pressurized rat MLVs under control conditions and after pharmacological blockade of NO by L-NAME (100 µM) or/and histamine production by α-MHD (10 µM). Effectiveness of α-MHD was confirmed immunohistochemically. We also used immunohistochemical labeling and Western blot analysis of the histamine-producing enzyme, HDC. In addition, we blocked HDC protein expression in MLVs by transient transfection with vivo-morpholino oligos. RESULTS: We found that only combined pharmacological blockade of NO and histamine production completely eliminates flow-dependent relaxation of lymphatic vessels, thus confirming a role for histamine as an EDRF in MLVs. We also confirmed the presence of HDC and histamine inside lymphatic endothelial cells. CONCLUSIONS: This study supports a role for histamine as an EDRF in MLVs.

Functional adaptation of bovine mesenteric lymphatic vessels to mesenteric venous hypertension.

Lymph flow is the primary mechanism for returning interstitial fluid to the blood circulation. Currently, the adaptive response of lymphatic vessels to mesenteric venous hypertension is not known. This study sought to determine the functional responses of postnodal mesenteric lymphatic vessels. We surgically occluded bovine mesenteric veins to create mesenteric venous hypertension to elevate mesenteric lymph flow. Three days after surgery, postnodal mesenteric lymphatic vessels from mesenteric venous hypertension (MVH; n = 7) and sham surgery (Sham; n = 6) group animals were evaluated and compared. Contraction frequency (MVH: 2.98 ± 0.75 min(-1); Sham: 5.42 ± 0.81 min(-1)) and fractional pump flow (MVH: 1.14 ± 0.30 min(-1); Sham: 2.39 ± 0.32 min(-1)) were significantly lower in the venous occlusion group. These results indicate that postnodal mesenteric lymphatic vessels adapt to mesenteric venous hypertension by reducing intrinsic contractile activity.

Cyclic guanosine monophosphate and the dependent protein kinase regulate lymphatic contractility in rat thoracic duct.

We have previously demonstrated a principal role for nitric oxide (NO) in the endothelium/shear-dependent regulation of contractility in rat thoracic duct (TD). In this study we tested the hypothesis that cyclic guanosine monophosphate (cGMP) and the dependent protein kinase (PKG) are central to the intrinsic and extrinsic flow-dependent modulation of lymphatic contractility. Lymphatic diameters and indices of pumping in isolated, cannulated and pressurized segments of rat TD were measured. The influences of increased transmural pressure (1-5 cmH2O) and imposed flow (1-5 cm H2O transaxial pressure gradients) on lymphatic function were studied before and after: (1) inhibition of guanylate cyclase (GC) with and without a NO donor; (2) application of stable cGMP analogue; and (3) inhibition of the cGMP activation of PKG. Additionally, Western blotting and immunofluorescent tissue staining were used to analyse the PKG isoforms expressed in TD. We found that the GC inhibitor ODQ induced changes in TD contractility similar to NO synthase blockade and prevented the relaxation induced by the NO donor S-nitroso-N-acetylpenicillamine. The cGMP analogue, 8-(4-Chlorophenylthio)-guanosine 3,5-cyclic monophosphate sodium salt (8pCPTcGMP), mimicked the extrinsic flow-induced relaxation in a dose-dependent manner, whereas treatment with the cGMP/PKG inhibitor, guanosine 3,5-cyclic monophosphorothioate, 8-(4-chlorophenylthio)-, Rp-isomer, triethylammonium salt (Rp-8-Br-PETcGMPS), eliminated intrinsic flow-dependent relaxation, and largely inhibited extrinsic flow-dependent relaxation. Western blotting demonstrated that both PKG-Iα and -Iß isoforms are found in TD, with ∼10 times greater expression of the PKG-Iα protein in TD compared with the aorta and vena cava. The PKG-Iß isoform expressed equally in TD and vena cava, both being ∼2 times higher than that in the aorta. Immunofluorescent labelling of PKG-Iα protein in the wall of rat thoracic duct confirmed its localization inside TD muscle cells. These findings demonstrate that cGMP is critical to the flow-dependent regulation of TD contractility; they also indicate an important involvement of PKG, especially PKG-Iα in these processes and identifies PKG protein as a potential therapeutic target.

Maximum shortening velocity of lymphatic muscle approaches that of striated muscle.

Lymphatic muscle (LM) is widely considered to be a type of vascular smooth muscle, even though LM cells uniquely express contractile proteins from both smooth muscle and cardiac muscle. We tested the hypothesis that LM exhibits an unloaded maximum shortening velocity (Vmax) intermediate between that of smooth muscle and cardiac muscle. Single lymphatic vessels were dissected from the rat mesentery, mounted in a servo-controlled wire myograph, and subjected to isotonic quick release protocols during spontaneous or agonist-evoked contractions. After maximal activation, isotonic quick releases were performed at both the peak and plateau phases of contraction. Vmax was 0.48 ± 0.04 lengths (L)/s at the peak: 2.3 times higher than that of mesenteric arteries and 11.4 times higher than mesenteric veins. In cannulated, pressurized lymphatic vessels, shortening velocity was determined from the maximal rate of constriction [rate of change in internal diameter (-dD/dt)] during spontaneous contractions at optimal preload and minimal afterload; peak -dD/dt exceeded that obtained during any of the isotonic quick release protocols (2.14 ± 0.30 L/s). Peak -dD/dt declined with pressure elevation or activation using substance P. Thus, isotonic methods yielded Vmax values for LM in the mid to high end (0.48 L/s) of those the recorded for phasic smooth muscle (0.05-0.5 L/s), whereas isobaric measurements yielded values (>2.0 L/s) that overlapped the midrange of values for cardiac muscle (0.6-3.3 L/s). Our results challenge the dogma that LM is classical vascular smooth muscle, and its unusually high Vmax is consistent with the expression of cardiac muscle contractile proteins in the lymphatic vessel wall.

Aging-associated shifts in functional status of mast cells located by adult and aged mesenteric lymphatic vessels.

We had previously proposed the presence of permanent stimulatory influences in the tissue microenvironment surrounding the aged mesenteric lymphatic vessels (MLV), which influence aged lymphatic function. In this study, we performed immunohistochemical labeling of proteins known to be present in mast cells (mast cell tryptase, c-kit, prostaglandin D(2) synthase, histidine decarboxylase, histamine, transmembrane protein 16A, and TNF-α) with double verification of mast cells in the same segment of rat mesentery containing MLV by labeling with Alexa Fluor 488-conjugated avidin followed by toluidine blue staining. Additionally, we evaluated the aging-associated changes in the number of mast cells located by MLV and in their functional status by inducing mast cell activation by various activators (substance P; anti-rat DNP Immunoglobulin E; peptidoglycan from Staphyloccus aureus and compound 48/80) in the presence of ruthenium red followed by subsequent staining by toluidine blue. We found that there was a 27% aging-associated increase in the total number of mast cells, with an ∼400% increase in the number of activated mast cells in aged mesenteric tissue in resting conditions with diminished ability of mast cells to be newly activated in the presence of inflammatory or chemical stimuli. We conclude that higher degree of preactivation of mast cells in aged mesenteric tissue is important for development of aging-associated impairment of function of mesenteric lymphatic vessels. The limited number of intact aged mast cells located close to the mesenteric lymphatic compartments to react to the presence of acute stimuli may be considered contributory to the aging-associated deteriorations in immune response.

Independent and interactive effects of preload and afterload on the pump function of the isolated lymphangion.

We tested the responses of single, isolated lymphangions to selective changes in preload and the effects of changing preload on the response to an imposed afterload. The methods used were similar to those described in our companion paper. Step-wise increases in input pressure (P(in); preload) over a pressure range between 0.5 and 3 cmH(2)O, at constant output pressure (P(out)), led to increases in end-diastolic diameter, decreases in end-systolic diameter, and increases in stroke volume. From a baseline of 1 cmH(2)O, P(in) elevation by 2-7 cmH(2)O consistently produced an immediate fall in stroke volume that subsequently recovered over a time course of 2-3 min. Surprisingly, this adaptation was associated with an increase in the slope of the end-systolic pressure-volume relationship, indicative of an increase in contractility. Lymphangions subjected to P(out) levels exceeding their initial ejection limit would often accommodate by increasing diastolic filling to strengthen contraction sufficiently to match P(out). The lymphangion adaptation to various pressure combinations (P(in) ramps with low or high levels of P(out), P(out) ramps at low or intermediate levels of P(in), and combined P(in) + P(out) ramps) were analyzed using pressure-volume data to calculate stroke work. Under relatively low imposed loads, stroke work was maximal at low preloads (P(in) ∼2 cmH(2)O), whereas at more elevated afterloads, the optimal preload for maximal work displayed a broad plateau over a P(in) range of 5-11 cmH(2)O. These results provide new insights into the normal operation of the lymphatic pump, its comparison with the cardiac pump, and its potential capacity to adapt to increased loads during edemagenic and/or gravitational stress.

Intrinsic increase in lymphangion muscle contractility in response to elevated afterload.

Collecting lymphatic vessels share functional and biochemical characteristics with cardiac muscle; thus, we hypothesized that the lymphatic vessel pump would exhibit behavior analogous to homeometric regulation of the cardiac pump in its adaptation to elevated afterload, i.e., an increase in contractility. Single lymphangions containing two valves were isolated from the rat mesenteric microcirculation, cannulated, and pressurized for in vitro study. Pressures at either end of the lymphangion [input pressure (P(in)), preload; output pressure (P(out)), afterload] were set by a servo controller. Intralymphangion pressure (P(L)) was measured using a servo-null micropipette while internal diameter and valve positions were monitored using video methods. The responses to step- and ramp-wise increases in P(out) (at low, constant P(in)) were determined. P(L )and diameter data recorded during single contraction cycles were used to generate pressure-volume (P-V) relationships for the subsequent analysis of lymphangion pump behavior. Ramp-wise P(out) elevation led to progressive vessel constriction, a rise in end-systolic diameter, and an increase in contraction frequency. Step-wise P(out) elevation produced initial vessel distention followed by time-dependent declines in end-systolic and end-diastolic diameters. Significantly, a 30% leftward shift in the end-systolic P-V relationship accompanied an 84% increase in dP/dt after a step increase in P(out), consistent with an increase in contractility. Calculations of stroke work from the P-V loop area revealed that robust pumps produced net positive work to expel fluid throughout the entire afterload range, whereas weaker pumps exhibited progressively more negative work as gradual afterload elevation led to pump failure. We conclude that lymphatic muscle adapts to output pressure elevation with an intrinsic increase in contractility and that this compensatory mechanism facilitates the maintenance of lymph pump output in the face of edemagenic and/or gravitational loads.

Mesenteric lymph flow in adult and aged rats.

The objective of study was to evaluate the aging-associated changes, contractile characteristics of mesenteric lymphatic vessels (MLV), and lymph flow in vivo in male 9- and 24-mo-old Fischer-344 rats. Lymphatic diameter, contraction amplitude, contraction frequency, and fractional pump flow, lymph flow velocity, wall shear stress, and minute active wall shear stress load were determined in MLV in vivo before and after N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME) application at 100 µM. The active pumping of the aged rat MLV in vivo was found to be severely depleted, predominantly through the aging-associated decrease in lymphatic contractile frequency. Such changes correlate with enlargement of aged MLV, which experienced much lower minute active shear stress load than adult vessels. At the same time, pumping in aged MLV in vivo may be rapidly increased back to levels of adult vessels predominantly through the increase in contraction frequency induced by nitric oxide (NO) elimination. Findings support the idea that in aged tissues surrounding the aged MLV, the additional source of some yet unlinked lymphatic contraction-stimulatory metabolites is counterbalanced or blocked by NO release. The comparative analysis of the control data obtained from experiments with both adult and aged MLV in vivo and from isolated vessel-based studies clearly demonstrated that ex vivo isolated lymphatic vessels exhibit identical contractile characteristics to lymphatic vessels in vivo.

Determinants of valve gating in collecting lymphatic vessels from rat mesentery.

Secondary lymphatic valves are essential for minimizing backflow of lymph and are presumed to gate passively according to the instantaneous trans-valve pressure gradient. We hypothesized that valve gating is also modulated by vessel distention, which could alter leaflet stiffness and coaptation. To test this hypothesis, we devised protocols to measure the small pressure gradients required to open or close lymphatic valves and determine if the gradients varied as a function of vessel diameter. Lymphatic vessels were isolated from rat mesentery, cannulated, and pressurized using a servo-control system. Detection of valve leaflet position simultaneously with diameter and intraluminal pressure changes in two-valve segments revealed the detailed temporal relationships between these parameters during the lymphatic contraction cycle. The timing of valve movements was similar to that of cardiac valves, but only when lymphatic vessel afterload was elevated. The pressure gradients required to open or close a valve were determined in one-valve segments during slow, ramp-wise pressure elevation, either from the input or output side of the valve. Tests were conducted over a wide range of baseline pressures (and thus diameters) in passive vessels as well as in vessels with two levels of imposed tone. Surprisingly, the pressure gradient required for valve closure varied >20-fold (0.1-2.2 cmH(2)O) as a passive vessel progressively distended. Similarly, the pressure gradient required for valve opening varied sixfold with vessel distention. Finally, our functional evidence supports the concept that lymphatic muscle tone exerts an indirect effect on valve gating.

Aging-associated alterations in contractility of rat mesenteric lymphatic vessels.

OBJECTIVE: To evaluate the age-related changes in pumping of mesenteric lymphatic vessels in 9- and 24-month-old male Fisher-344 rats. METHODS: Lymphatic diameters, contraction amplitude, contraction frequency, and fractional pump flow were determined in isolated MLV before and after l-NAME application. RESULTS: The data demonstrate a severe weakening of the lymphatic pump in aged MLV including diminished lymphatic contraction amplitude, contraction frequency, and as a result, lymphatic pump activity. The data also suggest that the imposed flow gradient-generated shear-dependent relaxation does not exist in aged rat MLV, and the sensitivity of both adult and aged MLV to such shear cannot be eliminated by nitric oxide (NO) synthases blockade. CONCLUSIONS: These data provide new evidence of lymphatic regional heterogeneity for both adult and aged MLV. In MLV, a constant interplay between the tonic and phasic components of the myogenic response and the shear-dependent release of NO predominantly determine the level of contractile activity; the existence of another shear-dependent, but NO-independent regulatory mechanism is probably present. Aging remarkably weakens MLV contractility, which would predispose this lymphatic network to lower total lymph flow in resting conditions and limit the ability to respond to an edemagenic challenge in the elderly.

Emerging Roles of Mast Cells in the Regulation of Lymphatic Immuno-Physiology.

Mast cells (MCs) are abundant in almost all vascularized tissues. Furthermore, their anatomical proximity to lymphatic vessels and their ability to synthesize, store and release a large array of inflammatory and vasoactive mediators emphasize their significance in the regulation of the lymphatic vascular functions. As a major secretory cell of the innate immune system, MCs maintain their steady-state granule release under normal physiological conditions; however, the inflammatory response potentiates their ability to synthesize and secrete these mediators. Activation of MCs in response to inflammatory signals can trigger adaptive immune responses by dendritic cell-directed T cell activation. In addition, through the secretion of various mediators, cytokines and growth factors, MCs not only facilitate interaction and migration of immune cells, but also influence lymphatic permeability, contractility, and vascular remodeling as well as immune cell trafficking through the lymphatic vessels. In summary, the consequences of these events directly affect the lymphatic niche, influencing inflammation at multiple levels. In this review, we have summarized the recent advancements in our understanding of the MC biology in the context of the lymphatic vascular system. We have further highlighted the MC-lymphatic interaction axis from the standpoint of the tumor microenvironment.

Antihistamines Increase Body Mass Index Percentiles and Z-Scores in Hispanic Children.

The prevalence of childhood obesity has increased over the years in the United States and contributed to a rise in metabolic syndrome and non-alcoholic fatty liver disease (NAFLD). Animal studies suggested the role of histamine blockade on mesenteric lymphatics tone, contributing to weight gain and hepatic steatosis. This study aimed to investigate an association between antihistamines (AH) use in children and obesity. A single-center retrospective cohort study on children with a diagnosis of NAFLD, followed in the gastroenterology clinic, was performed between January 2018 and April 2019. The demographics, medications, and body mass index (BMI) were assessed. Participants were divided into an AH group with documented use and comparison group, antihistamine naïve. Of the 32 participants in the study, 13 used AH, and 19 did not. Antihistamine users had a mean increase in BMI percentile per year of 1.17 compared to a decrease of 0.06 in comparison group (p = 0.0008). AH usage correlated with a mean increase in BMI z-score of 0.23 per year, as opposed to a decrease by 0.012 in comparison group (p = 0.0016). No difference was found in triglycerides (TG), glucose, and liver enzymes. AH use increases BMI percentiles and z-scores over time and is associated with obesity in children.

Rate-sensitive contractile responses of lymphatic vessels to circumferential stretch.

Phasic contractile activity in rat portal vein is more sensitive to the rate of change in length than to absolute length and this response is widely assumed to be a general characteristic of myogenic behaviour for vascular smooth muscle. Previously, we found that rat lymphatic vessels exhibit phasic contractile behaviour similar to that of portal vein. In the present study, we hypothesized that lymphatic muscle would exhibit rate-sensitive contractile responses to stretch. The hypothesis was tested on rat mesenteric lymphatics (90-220 microm, i.d.) using servo-controlled wire- and pressure-myograph systems to enable ramp increases in force or pressure at different rates. Under isometric conditions in wire-myograph preparations, both the amplitude and the frequency of phasic activity were enhanced at more optimal preloads, but superimposed upon this effect were bursts of contractions that occurred only during fast preload ramps. In such cases, the ratio of contraction frequency during the ramp to that at the subsequent plateau (at optimal preload) was > 1. Further, the frequency ratio increased as a function of the preload ramp speed, consistent with a rate-sensitive mechanism. In contrast, the amplitude ratio was < 1 and declined further with higher ramp speeds. Downward preload ramps produced corresponding rate-sensitive inhibition of contraction frequency but not amplitude. Similar findings were obtained in pressurized lymphatics in response to pressure ramps and steps. Our results suggest that lymphatics are sensitive to the rate of change in preload/pressure in a way that is different from portal vein, possibly because the pacemaker for generating electrical activity is rate sensitive but lymphatic muscle is not. The behaviour may be widely present in collecting lymphatic vessels and is probably an important mechanism for rapid adaptation of the lymphatic pump to local vascular occlusion.

Inhibition of myosin light chain phosphorylation decreases rat mesenteric lymphatic contractile activity.

Muscular lymphatics use both phasic and tonic contractions to transport lymph for conducting their vital functions. The molecular mechanisms regulating lymphatic muscle contractions are not well understood. Based on the well-established finding that the phosphorylation of myosin light chain 20 (MLC(20)) plays an essential role in blood vessel smooth muscle contraction, we investigated if phosphorylated MLC(20) (pMLC(20)) would modulate the tonic and/or phasic contractions of lymphatic muscle. The effects of ML-7, a MLC kinase inhibitor (1-10 microM), were tested on the contractile parameters of isolated and cannulated rat mesenteric lymphatics during their responses to the known modulators, pressure (1-5 cm H(2)O) and substance P (SP; 10(-7) M). Immunohistochemical and Western blot analyses of pMLC(20) were also performed on isolated lymphatics. The results showed that 1) increasing pressure decreased both the lymphatic tonic contraction strength and pMLC(20)-to-MLC(20) ratio; 2) SP increased both the tonic contraction strength and phosphorylation of MLC(20); 3) ML-7 decreased both the lymphatic tonic contraction strength and pMLC(20)-to-MLC(20) ratio; and 4) the increase in lymphatic phasic contraction frequency in response to increasing pressure was diminished by ML-7; however, the phasic contraction amplitude was not significantly altered by ML-7 either in the absence or presence of SP. These data provide the first evidence that tonic contraction strength and phasic contraction amplitude of the lymphatics can be differentially regulated, whereby the increase in MLC(20) phosphorylation produces an activation in the tonic contraction without significant changes in the phasic contraction amplitude. Thus, tonic contraction of rat mesenteric lymphatics appears to be MLC kinase dependent.