Assessing the effect of prolonged use of desloratadine on adipose Brillouin shift and composition in rats.

Antihistamines, which are commonly used to treat allergic reactions, are known for their side effects, which contribute to weight gain. It is hypothesized that simultaneous Brillouin elastography and Raman spectroscopy can be used to detect changes in adipose tissue associated with a prolonged intake of desloratadine, a commonly used second generation antihistamine. White and brown adipose tissue samples were excised from adult rats following 16 weeks of daily administration of desloratadine. It was found that the prolonged intake of desloratadine leads to an increase in Brillouin shift in both adipose tissue types. Raman spectra indicate that antihistamine use reduces protein-to-lipid ratio in brown adipose tissue but not white adipose tissue, indicating the effect on adipose tissue is location-dependent.

The Role of Lymphatics in Cholestasis: A Comprehensive Review.

Cholestatic liver disease affects millions of people worldwide and stems from a plethora of causes such as immune dysfunction, genetics, cancerous growths, and lifestyle choices. While not considered a classical lymphatic organ, the liver plays a vital role in the lymph system producing up to half of the body's lymph per day. The lymphatic system is critical to the health of an organism with its networks of vessels that provide drainage for lymphatic fluid and routes for surveilling immune cells. Cholestasis results in an increase of inflammatory cytokines, growth factors, and inflammatory infiltrate. Left unchecked, further disease progression will include collagen deposition which impedes both the hepatic and lymphatic ducts, eventually resulting in an increase in hepatic decompensation, increasing portal pressures, and accumulation of fluid within the abdominal cavity (ascites). Despite the documented interplay between these vital systems, little is known about the effect of liver disease on the lymph system and its biological response. This review looks at the current cholestatic literature from the perspective of the lymphatic system and summarizes what is known about the role of the lymph system in liver pathogenesis during hepatic injury and remodeling, immune-modulating events, or variations in interstitial pressures.

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.

Temporal Dynamics of the Rat Thoracic Duct Contractility in the Presence of Imposed Flow.

BACKGROUND: The initial periods of increased flow inside lymphatic vessels demonstrate specific temporary patterns of self-tuning of lymphatic vessel contractility that are heterogeneous across regional lymphatic networks. The current literature primarily refers to the immediate and fast reactions of the lymphangions to increases in basal flow. Until now, there were no available data on how the lymphatic vessels react to comparatively longer periods of imposed flow. METHODS AND RESULTS: In this study, we measured and analyzed the contractility of the rat thoracic duct segments, isolated, cannulated, and pressurized at 3 cm H2O at no imposed flow conditions and during 4 hours of imposed flow (constant transaxial pressure gradient of 2 cm H2O). We found the development of a progressing lymphatic tonic relaxation and inhibition of the lymphatic contraction frequency over 4 hours of imposed flow. After a short initial decrease, lymphatic phasic contraction amplitude rose significantly during the first hour of imposed flow, and it demonstrated a trend to return toward control levels after 3 hours of imposed flow. As a result, the fractional pump flow (active lymph pumping per minute) of isolated thoracic duct segments reached and maintained a statistically significant decrease (from control no-flow conditions) at the end of the third hour of imposed flow. CONCLUSIONS: Our new findings provide a better understanding of how lymphatic contractility changes during the development of prolonged periods of steady lymph flow. The latter may occur during the initial phases of development of an inflammatory-related tissue edema.

Depletion of MHC class II invariant chain peptide or γ-δ T-cells ameliorates experimental preeclampsia.

Excessive innate immune system activation and inflammation during pregnancy can lead to organ injury and dysfunction and preeclampsia (PE); however, the molecular mechanisms involved are unknown. We tested the hypothesis that Toll-like receptor (TLR) activation induces major histocompatibility complex (MHC) class II invariant chain peptide (CLIP) expression on immune cells, makes them pro-inflammatory, and are necessary to cause PE-like features in mice. Treatment with VG1177, a competitive antagonist peptide for CLIP in the groove of MHC class II, was able to both prevent and treat PE-like features in mice. We then determined that γ-δ T cells are critical for the development of PE-like features in mice since γ-δ T-cell knockout mice, like CLIP deficient mice, are resistant to developing PE-like features. Placentas from women with PE exhibit significantly increased levels of γ-δ T cells. These preclinical data demonstrate that CLIP expression and activated γ-δ T cells are responsible for the development of immunologic PE-like features and that temporarily antagonizing CLIP and/or γ-δ T cells may be a therapeutic strategy for PE.

Mast cells and histamine are triggering the NF-κB-mediated reactions of adult and aged perilymphatic mesenteric tissues to acute inflammation.

This study aimed to establish mechanistic links between the aging-associated changes in the functional status of mast cells and the altered responses of mesenteric tissue and mesenteric lymphatic vessels (MLVs) to acute inflammation. We used an in vivo model of acute peritoneal inflammation induced by lipopolysaccharide treatment of adult (9-month) and aged (24-month) F-344 rats. We analyzed contractility of isolated MLVs, mast cell activation, activation of nuclear factor-κB (NF-κB) without and with stabilization of mast cells by cromolyn or blockade of all types of histamine receptors and production of 27 major pro-inflammatory cytokines in adult and aged perilymphatic mesenteric tissues and blood. We found that the reactivity of aged contracting lymphatic vessels to LPS-induced acute inflammation was abolished and that activated mast cells trigger NF-κB signaling in the mesentery through release of histamine. The aging-associated basal activation of mesenteric mast cells limits acute inflammatory NF-κB activation in aged mesentery. We conclude that proper functioning of the mast cell/histamine/NF-κB axis is necessary for reactions of the lymphatic vessels to acute inflammatory stimuli as well as for interaction and trafficking of immune cells near and within the collecting lymphatics.

Blunted flow-mediated responses and diminished nitric oxide synthase expression in lymphatic thoracic ducts of a rat model of metabolic syndrome.

Shear-dependent inhibition of lymphatic thoracic duct (TD) contractility is principally mediated by nitric oxide (NO). Endothelial dysfunction and poor NO bioavailability are hallmarks of vasculature dysfunction in states of insulin resistance and metabolic syndrome (MetSyn). We tested the hypothesis that flow-dependent regulation of lymphatic contractility is impaired under conditions of MetSyn. We utilized a 7-wk high-fructose-fed male Sprague-Dawley rat model of MetSyn and determined the stretch- and flow-dependent contractile responses in an isobaric ex vivo TD preparation. TD diameters were tracked and contractile parameters were determined in response to different transmural pressures, imposed flow, exogenous NO stimulation by S-nitro-N-acetylpenicillamine (SNAP), and inhibition of NO synthase (NOS) by l-nitro-arginine methyl ester (l-NAME) and the reactive oxygen species (ROS) scavenging molecule 4-hydroxy-tempo (tempol). Expression of endothelial NO synthase (eNOS) in TD was determined using Western blot. Approximately 25% of the normal flow-mediated inhibition of contraction frequency was lost in TDs isolated from MetSyn rats despite a comparable SNAP response. Inhibition of NOS with l-NAME abolished the differences in the shear-dependent contraction frequency regulation between control and MetSyn TDs, whereas tempol did not restore the flow responses in MetSyn TDs. We found a significant reduction in eNOS expression in MetSyn TDs suggesting that diminished NO production is partially responsible for impaired flow response. Thus our data provide the first evidence that MetSyn conditions diminish eNOS expression in TD endothelium, thereby affecting the flow-mediated changes in TD lymphatic function.

Effects of dynamic shear and transmural pressure on wall shear stress sensitivity in collecting lymphatic vessels.

Given the known mechanosensitivity of the lymphatic vasculature, we sought to investigate the effects of dynamic wall shear stress (WSS) on collecting lymphatic vessels while controlling for transmural pressure. Using a previously developed ex vivo lymphatic perfusion system (ELPS) capable of independently controlling both transaxial pressure gradient and average transmural pressure on an isolated lymphatic vessel, we imposed a multitude of flow conditions on rat thoracic ducts, while controlling for transmural pressure and measuring diameter changes. By gradually increasing the imposed flow through a vessel, we determined the WSS at which the vessel first shows sign of contraction inhibition, defining this point as the shear stress sensitivity of the vessel. The shear stress threshold that triggered a contractile response was significantly greater at a transmural pressure of 5 cmH2O (0.97 dyne/cm(2)) than at 3 cmH2O (0.64 dyne/cm(2)). While contraction frequency was reduced when a steady WSS was applied, this inhibition was reversed when the applied WSS oscillated, even though the mean wall shear stresses between the conditions were not significantly different. When the applied oscillatory WSS was large enough, flow itself synchronized the lymphatic contractions to the exact frequency of the applied waveform. Both transmural pressure and the rate of change of WSS have significant impacts on the contractile response of lymphatic vessels to flow. Specifically, time-varying shear stress can alter the inhibition of phasic contraction frequency and even coordinate contractions, providing evidence that dynamic shear could play an important role in the contractile function of collecting lymphatic vessels.

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.

Nitric oxide formation by lymphatic bulb and valves is a major regulatory component of lymphatic pumping.

Microscopic lymphatics produce nitric oxide (NO) during contraction as flow shear activates the endothelial cells. The valve leaflets and bulbous valve housing contain a large amount of endothelial nitric oxide synthase (eNOS) due both to many endothelial cells and increased expression of eNOS. Direct NO measurements indicate the valve area has a 30-50% higher NO concentration ([NO]) than tubular regions although both regions generate equivalent relative increases in [NO] with each contraction. We hypothesize that 1) the greater eNOS and [NO] of the bulb region would have greater effects to lower pumping activity of the overall lymphatic than occurs in tubular regions and 2), the elevated [NO] in the bulb region may be because of high NO production in the valve leaflets that diffuses to the wall of the bulb. Measurement of [NO] with a micropipette inside the lymphatic bulb revealed the valve leaflets generate ~50% larger [NO] than the bulb wall in the in vivo rat mesenteric lymphatics. The valves add NO to the lymph that quickly diffuses to the bulb wall. Bradykinin locally released iontophoretically from a micropipette on both bulbs and tubes increased the [NO] in a dose-dependent manner up to ~50%, demonstrating agonist activation of the NO pathway. However, pumping output determined by contraction frequency and stroke volume decreased much more for the bulb than tubular areas in response to the bradykinin. In effect, NO generation by the bulb area and its valves limits the pumped flow of the total lymphatic by lowering frequency and stroke volume of individual contractions.

Phasic contractions of rat mesenteric lymphatics increase basal and phasic nitric oxide generation in vivo.

Multiple investigators have shown interdependence of lymphatic contractions on nitric oxide (NO) activity by pharmacological and traumatic suppression of endothelial NO synthase (eNOS). We demonstrated that lymphatic diastolic relaxation is particularly sensitive to NO from the lymphatic endothelium. The predicted mechanism is shear forces produced by the lymph flow during phasic pumping, activating eNOS in the lymphatic endothelium to produce NO. We measured [NO] during phasic contractions using microelectrodes on in situ mesenteric lymphatics in anesthetized rats under basal conditions and with an intravenous saline bolus (0.5 ml/100 g) or infusion (0.5 ml x 100 g(-1) x h(-1)). Under basal conditions, [NO] measured on the tubular portions of the lymphatics was approximately 200-250 nM, slightly higher than in the adjacent adipocyte microvasculature, whereas [NO] measured on the lymphatic bulb surface was approximately 400 nM. Immunohistochemistry of eNOS in isolated lympathics indicated a much greater expression in the lymph valves and surrounding bulb area than in the tubular regions. During phasic lymphatic contractions, the valve and tubular [NO] increased with each contraction, and during intravenous saline infusion, [NO] increased in proportion to the contraction frequency and, presumably, lymph flow. The partial blockade of eNOS over approximately 1 cm length with N(omega)-nitro-L-arginine methyl ester lowered the [NO]. These in vivo data document for the first time that both valvular and tubular lymphatic segments increase NO generation during each phasic contraction and that [NO] summated with increased contraction frequency. The combined data predict regional variations in eNOS and [NO] in the tubular and valve areas, plus the summated NO responses dependent on contraction frequency provide for a complex relaxation mechanism involving NO.

Methods for lymphatic vessel culture and gene transfection.

OBJECTIVE: To develop the techniques needed for the specific gene/protein targeting transfection experiments in isolated lymphatic vessels, we completed two major tasks: 1) optimize the experimental conditions to maintain the viability of isolated rat lymphatic vessels in culture for sufficiently long periods of time to permit knockdown or overexpression of selected proteins/genes and 2) develop effective transfection protocols for lymphatic muscle and endothelial cells in intact lymphatic vessels without nonspecific impairment of lymphatic contractile function due to the transfection protocol itself. METHODS: Experimental protocols were developed for the maintenance of isolated lymphatic vessels under nonpressurized and pressurized conditions for 3-12 days in culture and for adenoviral gene transfection of the lymphatic muscle and endothelial cells. RESULTS: The data demonstrate the effectiveness of the newly developed experimental protocols for the maintenance of isolated rat mesenteric lymphatic vessels and thoracic duct in culture up to 3-12 days without significant impairment of the parameters of their pumping and effective adenoviral/GFP transfection of lymphatic endothelial and muscle cells in isolated rat mesenteric lymphatic vessels. CONCLUSIONS: These experimental techniques will extend the set of the modern experimental tools available to researchers investigating the physiology of lymphatic function.

Age-related alterations of active pumping mechanisms in rat thoracic duct.

OBJECTIVE: To evaluate the age-related changes in active pumping in thoracic duct (TD) from 24-month-old Fisher-344 rats comparing with TD pumping in 9-month rats. METHODS: Lymphatic diameters, contraction amplitude and frequency, ejection fraction, and fractional pump flow were determined in isolated TD preparations. Western blot analyses were performed to evaluate relative levels of eNOS and iNOS in 9- and 24-month-old TD. RESULTS: Stretch-dependent regulation was altered in aged TD especially at higher levels of pressure: the negative inotropy, negative chronotropy and diminished minute pumping (2- to 3-fold decrease) were observed. Physiological NO/imposed-flow-dependent inhibition was completely abolished in aged TD, yet NO-synthase blockade by L-NAME (10(-4) M) increased pumping in a flow-independent manner. Western blot analyses indicated that the relative levels of eNOS were decreased approximately 7-fold in the 24-month-old TD when compared with 9-month-old TD; whereas iNOS levels were increased approximately 10-fold in 24-month-old TD. CONCLUSIONS: These data provide the first evidence that stretch- and imposed-flow-dependent regulatory mechanisms are greatly altered in aged TD. These alterations of active pumping mechanisms in TD appear to be related with age-related disturbances in NO-dependent regulatory pathways, and may reflect diminished lymphatic muscle contractility as well as altered lymphatic endothelium function.

Contraction-initiated NO-dependent lymphatic relaxation: a self-regulatory mechanism in rat thoracic duct.

The objectives of this study were to evaluate the physiological importance of the flow and shear generated by phasic contractions of lymphatic vessels and the mechanisms responsible for the influences of such shear on lymphatic pumping. Lymphatic segments of the rat thoracic duct were isolated, cannulated and pressurized. The diastolic diameters were measured in phasically non-active segments. The diastolic and systolic diameters, half-relaxation time (HRT), contraction frequency, ejection fraction and fractional pump flow were determined in phasically active segments. Since imposed flow was excluded, flow and shear occurred only as a result of the intrinsic contractions in phasically active segments whereas in phasically non-active segments contraction-generated flow and shear were absent. The influences of incrementally increased transmural pressure (from 1 to 5 cmH(2)O) were examined in control conditions and after NO synthase blockade (l-NAME 10(-4) m) or cyclooxygenase blockade (indomethacin 10(-5) m). The spontaneous phasic contractions produced a flow-dependent diastolic relaxation. This reduction of the lymphatic tone is a regulatory mechanism that maintains pumping in thoracic duct in an energy-saving/efficient mode: it improves diastolic filling (enhanced lusitropy - lowering HRT), makes lymphatic contractions stronger (enhanced inotropy - higher contraction amplitude) and propels more fluid forward during each contraction (elevated ejection fraction) while decreasing contraction frequency (reduced chronotropy). The findings also demonstrated that the NO pathway, not the cyclooxygenase pathway is responsible for this reduction of lymphatic tone and is the prevailing pathway responsible for the self-regulatory adjustment of thoracic duct pumping to changes in lymph flow pattern.