Lymphatic Contractile Activity Research Articles

Midazolam nanocrystal facilitates submandibular lymphatic metabolism via cAMP signal transduction pathway

Midazolam is a short-acting benzodiazepine for sedation, but its application is limited by its poor solubility. This study explored the impact of midazolam nanocrystals on Cyclic adenosine monophosphate (cAMP0 signal transduction. A rat model of hypercalcemia was established and divided into model group, control group, midazolam nanocrystal group, midazolam group, inhibitor group and agonist group. After treatment, the contents of cAMP and cGMP were detected by radioimmunoassay, and transformation rate, proliferation and apoptosis of lymphocytes were analyzed. Additionally, we observed microcirculation of lymphocytes and calculated contraction activity, overall lymphatic contractile activity and lymph dynamic index. The structure of the prepared nanocrystals was uniform and stable. Higher cGMP concentration and lymph viscosity of rats in the midazolam nanocrystal, midazolam and inhibitor groups were lower, while cGMP was raised in the agonist group. Besides, modeled rats exhibited decreased cAMP concentration, T lymphocyte conversion rate, lymphatic vessel contraction frequency, contraction activity index, total contraction activity index, lymph dynamic index/flow and lymphocyte output (p < 0.05), which were all increased in the midazolam nanocrystal group, midazolam group and agonist group but decreased in the inhibitor group (p < 0.05). Moreover, Midazolam nanocrystals improved lymphocyte proliferation and decreased apoptosis (<0.05). Midazolam nanocrystals can increase the concentration of cAMP and conversion rate of T lymphocytes, thereby reducing the concentration of cGMP, promoting lymphocyte proliferation, inhibiting apoptosis, and effectively improving the contraction frequency and contraction activity of lymphatic vessels. The cGMP can elevate total contractile activity index and lymph dynamics index, and enhance lymphatic transport function, while reducing lymph viscosity.

Hypertension is linked to enhanced lymphatic contractile response via RGS16/RhoA/ROCK pathway.

Lymph capillary network can be expected to alter blood pressure via regulating interstitial electrolyte and volume balance. However, the pathophysiology of lymphatic vessel in hypertension is poorly understood. In this study, we examined lymph vessel function focusing on contractile response in hypertensive rats. It was found that thoracic ducts isolated from adult (10-14 wk old) spontaneously hypertensive rats (SHRs) exhibited increased agonist-mediated contraction compared with age-matched Wistar-Kyoto (WKY) rats, whereas lymphatic contractions in younger (4 wk old) SHRs, exhibiting normal blood pressure, were no different compared with age-matched control rats. Tight regulation of blood pressure with antihypertensive drugs (hydrochlorothiazide/hydralazine) did not prevent the augmented lymphatic contraction in adult SHRs; however, treatment of SHRs with angiotensin II (ANG II) type 1 receptor blocker (losartan) for 6 wk abolished the augmentation of lymphatic contractions. In addition, ANG II infusion in Wistar rat caused augmented lymphatic contractile responses in the thoracic duct. The augmented contractions in adult SHRs were diminished by a ROCK inhibitor (Y-27632). Consistently, the thoracic ducts in SHRs showed significantly higher phosphorylation of myosin phosphatase targeting protein-1 than WKY rats. Furthermore, gene expression profiling of adult SHR lymphatics showed marked loss of regulator of G-protein signaling 16 (RGS16) mRNA, which was confirmed by the real-time PCR. Treatment with the RGS inhibitor CCG-63808 enhanced contractions in thoracic ducts from Wistar rats, which were abolished by the ROCK inhibitor. It is concluded that lymphatic contractile function was enhanced in hypertensive model rats, which could be mediated by dysregulation of the ROCK pathway possibly through RGS16.NEW & NOTEWORTHY Lymph capillary controls interstitial electrolyte and volume balance, which may blunt increased blood pressure. However, the function of lymphatic vessel in hypertension is poorly understood. Our study showed that the lymphatic smooth muscle contractility is hyperreactive in two different hypertensive models. The lymphatic dysfunction could be mediated by dysregulation of ROCK pathway possibly through RGS16. The present finding supports a new concept showing the functional relationship between lymphatic contractile activity and hypertension.

Lymphatic Collecting Vessel: New Perspectives on Mechanisms of Contractile Regulation and Potential Lymphatic Contractile Pathways to Target in Obesity and Metabolic Diseases.

Obesity and metabolic syndrome pose a significant risk for developing cardiovascular disease and remain a critical healthcare challenge. Given the lymphatic system’s role as a nexus for lipid absorption, immune cell trafficking, interstitial fluid and macromolecule homeostasis maintenance, the impact of obesity and metabolic disease on lymphatic function is a burgeoning field in lymphatic research. Work over the past decade has progressed from the association of an obese phenotype with Prox1 haploinsufficiency and the identification of obesity as a risk factor for lymphedema to consistent findings of lymphatic collecting vessel dysfunction across multiple metabolic disease models and organisms and characterization of obesity-induced lymphedema in the morbidly obese. Critically, recent findings have suggested that restoration of lymphatic function can also ameliorate obesity and insulin resistance, positing lymphatic targeted therapies as relevant pharmacological interventions. There remain, however, significant gaps in our understanding of lymphatic collecting vessel function, particularly the mechanisms that regulate the spontaneous contractile activity required for active lymph propulsion and lymph return in humans. In this article, we will review the current findings on lymphatic architecture and collecting vessel function, including recent advances in the ionic basis of lymphatic muscle contractile activity. We will then discuss lymphatic dysfunction observed with metabolic disruption and potential pathways to target with pharmacological approaches to improve lymphatic collecting vessel function.

Effect of Human Synovial Fluid From Osteoarthritis Patients and Healthy Individuals on Lymphatic Contractile Activity.

The lymphatic system has been proposed to play a crucial role in preventing the development and progression of osteoarthritis (OA). As OA develops and progresses, inflammatory cytokines and degradation by-products of joint tissues build up in the synovial fluid (SF) providing a feedback system to exacerbate disease. The lymphatic system plays a critical role in resolving inflammation and maintaining overall joint homeostasis; however, there is some evidence that the lymphatics can become dysfunctional during OA. We hypothesized that the functional mechanics of lymphatic vessels (LVs) draining the joint could be directly compromised due to factors within SF derived from osteoarthritis patients (OASF). Here, we utilized OASF and SF derived from healthy (non-OA) individuals (healthy SF (HSF)) to investigate potential effects of SF entering the draining lymph on migration of lymphatic endothelial cells (LECs) in vitro, and lymphatic contractile activity of rat femoral LVs (RFLVs) ex vivo. Dilutions of both OASF and HSF containing serum resulted in a similar LEC migratory response to the physiologically endothelial basal medium-treated LECs (endothelial basal medium containing serum) in vitro. Ex vivo, OASF and HSF treatments were administered within the lumen of isolated LVs under controlled pressures. OASF treatment transiently enhanced the RFLVs tonic contractions while phasic contractions were significantly reduced after 1 h of treatment and complete ceased after overnight treatment. HSF treatment on the other hand displayed a gradual decrease in lymphatic contractile activity (both tonic and phasic contractions). The observed variations after SF treatments suggest that the pump function of lymphatic vessel draining the joint could be directly compromised in OA and thus might present a new therapeutic target.

Abstract P1-24-06: Risk factor modeled microenvironment effects lymphatics activity and IBC invasiveness and progression

Abstract Background. Lymphovascular invasion (LVI) and breast tumor emboli within dermal and breast lymphatic vessels are prognostic for metastatic spread and poor outcomes, and are abundant in Inflammatory breast cancer (IBC). IBC is an aggressive breast cancer that presents suddenly with breast swelling and redness due to tumor emboli in lymphatics. Lack of breast-feeding and obesity are IBC risk factors. We sought to demonstrate the combinatorial effects of a high-fat diet and nursing on lymphatic function and compare these to IBC tumor induced changes in lymphatic function. We hypothesize that risk factors for aggressive breast cancer may alter lymphatic function in the normal gland prior to tumor initiation. Methods. Following two rounds of pregnancy in 20 multiparous SCID Beige immunocompromised mice, half of the mice were force weaned while half nursed pups. Prior to forced weaning, half of each of these groups were fed a high fat diet (HFD: 60 Kcal %, N = 10) while the other half received a low-fat diet (LFD: 10 Kcal %, N = 10). Consecutive dynamic near-infrared fluorescence (NIRF) lymphatic imaging was performed at 6-7 months (covid interruption) and 14 months after initiating the diet by injecting a near-IR fluorophore into the mammary fatpad and recording lymphatic pulsing over 8 minutes using V++. Matlab and ImageJ were used to quantify pulsing rates on the ventral lymphatics in each animal. Fatpads were subsequently inoculated with SUM149 IBC cells and imaging was repeated 16 months post diet initiation. Lymphatic imaging over time by HFD vs LFD was further studied in nulliparous animals. Tissues were collected for further analyses. ResultsData analysis prior to tumor injection, demonstrated lymphatic pulsing (pulses/4 minutes) increased over time in HFD force weaned (HFFW) and HFD nursing (HFN) animals only (65.5 vs 72.6, P=0.059; 60.1 vs 76.6, P=0.0099, respectively). Comparing HFFW and HFN to matched LFD groups (LFFW and LFN), at 14 weeks HFD was associated with increased pumping after forced weaning (62.3 vs. 72.6, P=0.074), and nursing (62.5 vs 76.6, P=0.0023). There was an increase in pulsing after tumor initiation (16 months after initiation of diet) in all groups (80.1, 84.1, 83.2, 82.4, P > 0.05 all comparisons to initial timepoint). In a separate experiment examining HFD (N=5) vs LFD (N=5) in nulliparous mice, lymphatic contractile activity increased in all animals over. time, average ventral lymphatic contractile frequency for LFD and HFD at week 8,11 and 14 weeks after diet initiation were 5, 8.64, 15.9 pumps/4 mins vs 11.8, 18.5, 28.2 pumps/4 mins, (P = 0.01, 0.05, and 0.0005 respectively). ConclusionsHFD increased lymphatic pulsing rate over time to a significantly greater extent than LFD continuing over 14 months independent of reproductive and nursing status. Tumor initiation prompted further increased pulsing rates beyond that observed after HFD across all groups. The magnitude of the effect of HFD on lymphatic pulsing approached the rate after tumor initiation, while reproductive variables did not impact lymphatic pulsing. Further studies are warranted to demonstrate the relationship if any between lymphatic pumping pre-initiation and LVI after tumor initiation and examine the role of intervention on reducing LVI. Citation Format: Wintana Balema, Janelle Morton, Richard Larson, Fred C Velasquez, Eva Sevick-Muraca, Wendy A Woodward. Risk factor modeled microenvironment effects lymphatics activity and IBC invasiveness and progression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-24-06.

Roles of sarcoplasmic reticulum Ca2+ ATPase pump in the impairments of lymphatic contractile activity in a metabolic syndrome rat model

The intrinsic lymphatic contractile activity is necessary for proper lymph transport. Mesenteric lymphatic vessels from high-fructose diet-induced metabolic syndrome (MetSyn) rats exhibited impairments in its intrinsic phasic contractile activity; however, the molecular mechanisms responsible for the weaker lymphatic pumping activity in MetSyn conditions are unknown. Several metabolic disease models have shown that dysregulation of sarcoplasmic reticulum Ca2+ ATPase (SERCA) pump is one of the key determinants of the phenotypes seen in various muscle tissues. Hence, we hypothesized that a decrease in SERCA pump expression and/or activity in lymphatic muscle influences the diminished lymphatic vessel contractions in MetSyn animals. Results demonstrated that SERCA inhibitor, thapsigargin, significantly reduced lymphatic phasic contractile frequency and amplitude in control vessels, whereas, the reduced MetSyn lymphatic contractile activity was not further diminished by thapsigargin. While SERCA2a expression was significantly decreased in MetSyn lymphatic vessels, myosin light chain 20, MLC20 phosphorylation was increased in these vessels. Additionally, insulin resistant lymphatic muscle cells exhibited elevated intracellular calcium and decreased SERCA2a expression and activity. The SERCA activator, CDN 1163 partially restored lymphatic contractile activity in MetSyn lymphatic vessel by increasing phasic contractile frequency. Thus, our data provide the first evidence that SERCA2a modulates the lymphatic pumping activity by regulating phasic contractile amplitude and frequency, but not the lymphatic tone. Diminished lymphatic contractile activity in the vessels from the MetSyn animal is associated with the decreased SERCA2a expression and impaired SERCA2 activity in lymphatic muscle.

Sigma‐1 Receptors Promote Lymphatic Endothelial Barrier Function

The lymphatic endothelium plays important roles in lymphatic contractile activity, lymph transport and maintaining a barrier between the lymph and interstitial compartments and its dysfunction is a key factor in the pathogenesis of lymphatic diseases such as lymphedema. Sigma‐1 (σ1) receptors are chaperone proteins located in the endoplasmic reticulum were recently identified to promote collecting lymphatic dilation through endothelial production of nitric oxide. However, the role of σ1 in other functions of lymphatic endothelial cells are not clear. The aim of the current study was to test the hypothesis that σ1 promotes barrier function of lymphatic endothelium. We confirmed expression of σ1 in adult and juvenile human dermal lymphatic endothelial cells (HDLEC) using the ProteinSimple Western (WES) system and immunofluorescence microscopy. To test whether activation of σ1 promotes lymphatic endothelial barrier function, we applied the selective agonist PRE‐084 to cultured monolayers of HDLEC and determined the transendothelial electrical resistance (TER) as an index of barrier function using electrical cell‐substrate impedance sensing. The contribution of basal σ1 activity to HDLEC TER was also assessed either with the σ1 antagonist BD1047 (200 nM) or by silencing σ1 expression with siRNA. Because σ1 has been previously described to be located in the ER‐mitochondrial membrane interface, we also evaluated its role in endothelial bioenergetics using the Agilent Seahorse system glycolytic rate assay. Lastly, we tested the ability of PRE‐084 to counteract IL‐1β‐induced barrier dysfunction in HDLEC monolayers. The results show that the activation of σ1 receptor with PRE‐084 increases HDLEC TER in a concentration‐dependent manner, with 50, 100, 150, and 200 μM PRE‐084 eliciting significant elevations in TER (100 μM was chosen for subsequent experiments). Blockade of σ1 either with the antagonist BD‐1047 or reducing σ1 expression with siRNA resulted in decreased HDLEC TER. PRE‐084 also enhanced basal and compensatory glycolysis as well as proton efflux rate in HDLEC. Moreover, PRE‐084 partially counteracted the IL‐1β‐induced TER drop indicating its potential anti‐inflammatory impact on lymphatic endothelial cells. Collectively our results suggest that σ1 contributes to basal lymphatic endothelial barrier function, potentially through its ability to enhance glycolytic ATP production. Our work also highlights the therapeutic potential of σ1 agonists for preventing lymphatic barrier dysfunction caused by inflammatory mediators.Support or Funding InformationThis work was supported by NIH Grant R01GM120774.

Assessing lymphatic route of CSF outflow and peripheral lymphatic contractile activity during head-down tilt using near-infrared fluorescence imaging.

Evidence overwhelmingly suggests that the lymphatics play a critical role in the clearance of cerebrospinal fluid (CSF) from the cranial space. Impairment of CSF outflow into the lymphatics is associated with a number of pathological conditions including spaceflight‐associated neuro‐ocular syndrome (SANS), a problem that limits long‐duration spaceflight. We used near‐infrared fluorescence lymphatic imaging (NIRFLI) to dynamically visualize the deep lymphatic drainage pathways shared by CSF outflow and disrupted during head‐down tilt (HDT), a method used to mimic the cephalad fluid shift that occurs in microgravity. After validating CSF clearance into the lymph nodes of the neck in swine, a pilot study was conducted in human volunteers to evaluate the effect of gravity on the flow of lymph through these deep cervical lymphatics. Injected into the palatine tonsils, ICG was imaged draining into deep jugular lymphatic vessels and subsequent cervical lymph nodes. NIRFLI was performed under HDT, sitting, and supine positions. NIRFLI shows that lymphatic drainage through pathways shared by CSF outflow are dependent upon gravity and are impaired under short‐term HDT. In addition, lymphatic contractile rates were evaluated from NIRFLI following intradermal ICG injections of the lower extremities. Lymphatic contractile activity in the legs was slowed in the gravity neutral, supine position, but increased under the influence of gravity regardless of whether its force direction opposed (sitting) or favored (HDT) lymphatic flow toward the heart. These studies evidence the role of a lymphatic contribution in SANS.

Lymphatic remodelling in response to lymphatic injury in the hind limbs of sheep.

Contractile activity in the lymphatic vasculature is essential for maintaining fluid balance within organs and tissues. However, the mechanisms by which collecting lymphatics adapt to changes in fluid load and how these adaptations influence lymphatic contractile activity are unknown. Here we report a model of lymphatic injury based on the ligation of one of two parallel lymphatic vessels in the hind limb of sheep and the evaluation of structural and functional changes in the intact, remodelling lymphatic vessel over a 42-day period. We show that the remodelled lymphatic vessel displayed increasing intrinsic contractile frequency, force generation and vessel compliance, as well as decreasing flow-mediated contractile inhibition via the enzyme endothelial nitric oxide synthase. A computational model of a chain of lymphatic contractile segments incorporating these adaptations predicted increases in the flow-generation capacity of the remodelled vessel at the expense of normal mitochondrial function and elevated oxidative stress within the lymphatic muscle. Our findings may inform interventions for mitigating lymphatic muscle fatigue in patients with dysfunctional lymphatics.

The Regulation of Lymphatic Muscle Cell Contractile Activity by Intracellular Calcium Signals

Lymphedema is characterized by chronic edema due to lymphatic insufficiency. Normally, the spontaneous contractions of the collecting lymphatic vessels (cLVs) function to pump lymph against a pressure gradient back to the blood stream, however cLVs from lymphedema patients often display weak or irregular pumping activity. We have recently shown that that mouse lymphatic muscle cells (LMCs) exhibit a diastolic depolarization that sets contraction frequency and is the basis of cLV pacemaking and autorhythmicity. In murine cLVs, this diastolic depolarization is pressure‐dependent and is mediated by the Ca2+ activated chloride channel Anoctamin1 (Ano1). Ano1 is presumed to be activated by the spontaneous Ca2+ transients originating from the sarcoplasmic reticulum (SR), however the identity of the SR Ca2+ channels underlying these diastolic Ca2+ transients has not been rigorously tested using genetic models. We tested the hypothesis that intracellular Ca2+ signals released from the SR are critical in regulating lymphatic pacemaking and contractile activity. We sorted murine LMCs and observed expression of both Itpr1 and RyR2. We tested the role of Itpr1 and RyR2 in the electrical and contractile regulation of the murine inguinal‐axillary cLV (Ing‐Ax) through the use of inducible deletion models (MYH11CreERT2) or mice harboring knock‐in gain of function (GOF) of loss of function (LOF) mutations. These mouse models include Ano1fl/fl, Itpr1fl/fl, Itpr1DK (GOF mutant), Itpr1DA (LOF mutant), RyR2fl/fl, and RyR2VF (GOF mutant). We performed ex vivo isobaric vessel myography in response to a physiological pressure range to determine Ing‐Ax contraction frequency (Freq), contraction amplitude (Amp), and vessel tone (Tone). In some experiments, we also recorded membrane potential using “sharp electrodes” and imaged Ca2+ signals using the genetically encoded cytosolic Ca2+ sensor GCaMP6f or GCaMP8 from pressurized Ing‐Ax cLVs. Ing‐Ax cLVs isolated from the heterozygous Itpr1DA/WT, the homozygous Itpr1DA/DA, and Itpr1smKO mice had significantly reduced contraction Freq and Tone, but also observed significantly higher Amp. Strikingly, Itpr1smKO Ing‐Ax cLVs also lacked the pressure‐dependent chronotropy similar to cLVs from Ano1smKO mice, however Itpr1smKO vessels had unstable periodicity in the contraction rhythm. Ing‐Ax cLVs from RyR2smKO mice had normal contraction Freq, Amp and Tone. In contrast, Ing‐Ax cLVs from the GOF RyR2VF/KO mice had significantly higher Tone and Freq, but significantly reduced Amp. Ing‐Ax cLVs from GOF Iptr1DK mice contracted into a paralytic rigor during the vessel warming and equilibration period likely due to Ca2+overload. These findings suggest that the pressure‐dependent Freq in murine cLVs is mediated by the activation of Ano1 by SR Ca2+ release through Itpr1 and an acceleration of the diastolic depolarization.Support or Funding InformationR01‐H122578 (MD), R01‐HL133256 & R01‐HL137745 (JJ)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Imaging the Lymphatic Response to Manual Lymphatic Drainage and Pneumatic Compression Devices

Manual lymphatic drainage (MLD), as a component of complete decongestive therapy, has formed the basis of the initial treatment for lymphedema (LED), and pneumatic compression (PC) has been used in the maintenance phase (at-home treatment). The physiologic mechanism, however, of methods to promote lymph drainage for both MLD and PC has not been rigorously established. Recent developments in near-infrared fluorescence lymphatic imaging (NIRFLI) have enabled the visualization of the lymphatic response to these therapies nearly in real time and provide opportunities to assess the physiologic impact of these manual interventions. Herein we summarize the results of seven studies, published and unpublished, assessing the acute and longitudinal impact of MLD or PC therapy in patients with phlebolymphedema or standard arm, leg, or head and neck LED. After informed consent and under Food and Drug Administration-approved investigational new drugs for off-label administration of indocyanine green (ICG) as an imaging agent for NIRFLI, we visualized lymphatic anatomy and contractile pumping activity in study participants. Patients received intradermal injections of ICG distal to the area of vascular interest, typically in the hands and feet. Imaging was performed by illuminating the skin with near-infrared light and collecting the fluorescent light emanating from the ICG-laden lymph. Whereas imaging times varied between each study, imaging was conducted immediately before and after treatment and, when possible, during treatment. In one study, NIRFLI was also performed before and after 2 weeks of PC therapy in patients with head and neck LED. Images were assessed for changes in lymphatic uptake and contractile pumping. Overall 69 patients, 51 affected and 18 control, undergoing MLD or PC therapy were imaged. The first study of 10 LED and 12 control patients showed a 23% and 28% increase in lymph velocity and a 9% and 23% reduction in propulsion period in symptomatic and control limbs, respectively, in response to MLD. Other NIRFLI studies demonstrated enhanced lymphatic uptake after PC therapy in control patients and patients diagnosed with phlebolymphedema or standard LED. One study, using a clear PC device, demonstrated the ability to image the statistically significant improvement in lymphatic contractile propulsion during and after PC therapy. A recent unpublished study of 10 patients with head and neck LED demonstrated enhanced lymphatic uptake in all patients and a reduction of abnormal lymphatic anatomy in 75% of patients after 2 weeks of daily at-home PC therapy. NIRFLI enables the assessment of manual lymphatic interventions, demonstrating enhanced lymphatic uptake and improved lymphatic contractile pumping in response to MLD and PC therapy in patients with LED.

Effect of pneumatic compression therapy on lymph movement in lymphedema-affected extremities, as assessed by near-infrared fluorescence lymphatic imaging.

Previous studies have shown cost effectiveness and quality-of-life benefit of pneumatic compression therapy (PCT) for lymphedema. Insurers, such as the Centers for Medicare/Medicaid (CMS), however, desire visual proof that PCT moves lymph. Near-infrared fluorescence lymphatic imaging (NIRFLI) was used to visualize lymphatic anatomy and function in four subjects with primary and cancer treatment-related lymphedema (LE) of the lower extremities before, during, and after pneumatic compression therapy (PCT). Optically transparent and windowed PCT garments allowed visualization of lymph movement during single, one-hour PCT treatment sessions. Visualization revealed significant extravascular and lymphatic vascular movement of intradermally injected dye in all subjects. In one subject with sufficient patent lymphatic vessels to allow quantification of lymph pumping velocities and frequencies, these values were significantly increased during and after PCT as compared to pre-treatment values. Lymphatic contractile activity in patent lymphatic vessels occurred in concert with the sequential cycling of PCT. Direct visualization revealed increased lymphatic function, during and after PCT therapy, in all lymphedema-affected extremities. Further studies are warranted to assess the effects of PCT pressure and sequences on lymph uptake and movement.

Effect of lidocaine with and without epinephrine on lymphatic contractile activity in mice in vivo.

A local anesthetic, lidocaine, is known to affect cutaneous blood flow when injected into the skin. However, it is unknown if dermal lymphatic function can also be affected. Therefore, we characterized lymphatic function in response to administration of lidocaine with and without epinephrine. Non-invasive near-infrared fluorescence imaging (NIRFI) with intradermal injection of indocyanine green (ICG) was used to characterize the lymphatic "pumping" function in mice after subcutaneous injection of 2% lidocaine with and without 1:100,000 epinephrine or saline. NIRFI was performed for 10-20min immediately after and 1, 3, and 5h after these interventions. Lymphatic contraction frequencies significantly decreased 10min after subcutaneous injection of lidocaine and remained plateaued for another 5min, before returning to baseline. However, addition of 1:100,000 epinephrine to 2% lidocaine rapidly increased lymphatic contraction frequencies at 5min post-injection, which returned to baseline levels 15min later. Injection of saline also increased lymphatic contraction frequency 5min after injection, which returned to baseline 10min post-injection. Although lidocaine administration showed a decrease in lymphatic function, the combination of epinephrine with lidocaine resulted in a predominant net effect of increased contractile activity.

Dietary Sodium and Lymphatic Contractile Activity

Dietary Sodium and Lymphatic Contractile Activity

Dextran sulfate sodium-induced acute colitis impairs dermal lymphatic function in mice.

To investigate whether dermal lymphatic function and architecture are systemically altered in dextran sulfate sodium (DSS)-induced acute colitis. Balb/c mice were administered 4% DSS in lieu of drinking water ad libitum for 7 d and monitored to assess disease activity including body weight, diarrhea severity, and fecal bleeding. Control mice received standard drinking water with no DSS. Changes in mesenteric lymphatics were assessed following oral administration of a fluorescently-labelled fatty acid analogue, while dermal lymphatic function and architecture was longitudinally characterized using dynamic near-infrared fluorescence (NIRF) imaging following intradermal injection of indocyanine green (ICG) at the base of the tail or to the dorsal aspect of the left paw prior to, 4, and 7 d after DSS administration. We also measured dye clearance rate after injection of Alexa680-bovine serum albumin (BSA). NIRF imaging data was analyzed to reveal lymphatic contractile activity after selecting fixed regions of interest (ROIs) of the same size in fluorescent lymphatic vessels on fluorescence images. The averaged fluorescence intensity within the ROI of each fluorescence image was plotted as a function of imaging time and the lymphatic contraction frequency was computed by assessing the number of fluorescent pulses arriving at a ROI. Mice treated with DSS developed acute inflammation with clinical symptoms of loss of body weight, loose feces/watery diarrhea, and fecal blood, all of which were aggravated as disease progressed to 7 d. Histological examination of colons of DSS-treated mice confirmed acute inflammation, characterized by segmental to complete loss of colonic mucosa with an associated chronic inflammatory cell infiltrate that extended into the deeper layers of the wall of the colon, compared to control mice. In situ intravital imaging revealed that mice with acute colitis showed significantly fewer fluorescent mesenteric lymphatic vessels, indicating impaired uptake of a lipid tracer within mesenteric lymphatics. Our in vivo NIRF imaging data demonstrated dilated dermal lymphatic vessels, which were confirmed by immunohistochemical staining of lymphatic vessels, and significantly reduced lymphatic contractile function in the skin of mice with DSS-induced acute colitis. Quantification of the fluorescent intensity remaining in the depot as a function of time showed that there was significantly higher Alexa680-BSA fluorescence in mice with DSS-induced acute colitis compared to pre-treatment with DSS, indicative of impaired lymphatic drainage. The lymphatics are locally and systemically altered in acute colitis, and functional NIRF imaging is useful for noninvasively monitoring systemic lymphatic changes during inflammation.

Distinct roles of L- and T-type voltage-dependent Ca2+ channels in regulation of lymphatic vessel contractile activity.

Lymph drainage maintains tissue fluid homeostasis and facilitates immune response. It is promoted by phasic contractions of collecting lymphatic vessels through which lymph is propelled back into the blood circulation. This rhythmic contractile activity (i.e. lymphatic pumping) increases in rate with increase in luminal pressure and relies on activation of nifedipine-sensitive voltage-dependent Ca(2+) channels (VDCCs). Despite their importance, these channels have not been characterized in lymphatic vessels. We used pressure- and wire-myography as well as intracellular microelectrode electrophysiology to characterize the pharmacological and electrophysiological properties of L-type and T-type VDCCs in rat mesenteric lymphatic vessels and evaluated their particular role in the regulation of lymphatic pumping by stretch. We complemented our study with PCR and confocal immunofluorescence imaging to investigate the expression and localization of these channels in lymphatic vessels. Our data suggest a delineating role of VDCCs in stretch-induced lymphatic vessel contractions, as the stretch-induced increase in force of lymphatic vessel contractions was significantly attenuated in the presence of L-type VDCC blockers nifedipine and diltiazem, while the stretch-induced increase in contraction frequency was significantly decreased by the T-type VDCC blockers mibefradil and nickel. The latter effect was correlated with a hyperpolarization. We propose that activation of T-type VDCCs depolarizes membrane potential, regulating the frequency of lymphatic contractions via opening of L-type VDCCs, which drive the strength of contractions.

Human lymphatic vessel contractile activity is inhibited in vitro but not in vivo by the calcium channel blocker nifedipine.

Calcium channel blockers (CCB) are widely prescribed anti-hypertensive agents. The commonest side-effect, peripheral oedema, is attributed to a larger arterial than venous dilatation causing increased fluid filtration. Whether CCB treatment is detrimental to human lymphatic vessel function and thereby exacerbates oedema formation is unknown. We observed that spontaneous lymphatic contractions in isolated human vessels (thoracic duct and mesenteric lymphatics) maintained under isometric conditions were inhibited by therapeutic concentrations (nanomolar) of the CCB nifedipine while higher than therapeutic concentrations of verapamil (micromolar) were necessary to inhibit activity. Nifedipine also inhibited spontaneous action potentials measured by sharp microelectrodes. Furthermore, noradrenaline did not elicit normal increases in lymphatic vessel tone when maximal constriction was reduced to 29.4±4.9% of control in the presence of 20nmoll(-1) nifedipine. Transcripts for the L-type calcium channel gene CACNA1C were consistently detected from human thoracic duct samples examined and the CaV1.2 protein was localized by immunoreactivity to lymphatic smooth muscle cells. While human lymphatics ex vivo were highly sensitive to nifedipine, this was not apparent in vivo when nifedipine was compared to placebo in a randomized, double-blinded clinical trial: conversely, lymphatic vessel contraction frequency was increased and refill time was faster despite all subjects achieving target nifedipine plasma concentrations. We conclude that human lymphatic vessels are highly sensitive to nifedipine in vitro but that care must be taken when extrapolating in vitro observations of lymphatic vessel function to the clinical situation, as similar changes in lymphatic function were not evident in our clinical trial comparing nifedipine treatment to placebo.

Rho Kinase Enhances Contractions of Rat Mesenteric Collecting Lymphatics

The mechanisms that control phasic and tonic contractions of lymphatic vessels are poorly understood. We hypothesized that rho kinase ROCK, previously shown to increase calcium (Ca2+) sensitivity in vascular smooth muscle, enhances lymphatic contractile activity in a similar fashion. Contractions of isolated rat mesenteric lymphatic vessels were observed at a luminal pressure of 2 cm H2O in a 37°C bath. The expression of ROCK in isolated rat mesenteric lymphatic vessels was assessed by Western blotting and confocal microscopy. The role of ROCK in contractile function was tested using two specific yet structurally distinct inhibitors: H1152 (0.1–10 μM) and Y-27632 (0.5–50 μM). In addition, lymphatics were transfected with constitutively active (ca)-ROCK protein (2 μg/ml) to assess gain of contractile function. Vessel diameter and the concentration of intracellular free Ca2+ ([Ca2+]i) were simultaneously measured in a subset of isolated lymphatics loaded with the Ca2+-sensing dye fura-2. The results show expression of both the ROCK1 and ROCK2 isoforms in lymphatic vessels. Inhibition of ROCK increased lymphatic end diastolic diameter and end systolic diameter in a concentration-dependent manner. Significant reductions in lymphatic tone and contraction amplitude were observed after treatment 1–10 μM H1152 or 25–50 μM Y-27632. H1152 (10 μM) also significantly reduced contraction frequency. Transient increases in [Ca2+]i preceded each phasic contraction, however this pattern was disrupted by either 10 μM H1152 or 50 μM Y-27632 in the majority of lymphatics studied. The significant decrease in tone caused by H1152 or Y-27632 was not associated with a significant change in the basal [Ca2+]i between transients. Transfection with ca-ROCK protein enhanced lymphatic tone, but was not associated with a significant change in basal [Ca2+]i. Our data suggest that ROCK mediates normal tonic constriction and influences phasic contractions in lymphatics. We propose that ROCK modulates Ca2+ sensitivity of contractile proteins in lymphatics.

Potential for intranasal drug delivery to alter cerebrospinal fluid outflow via the nasal turbinate lymphatics

BackgroundCerebrospinal fluid absorption (CSF) at the cribriform plate is mediated by direct extracranial connections to the lymphatic system. Given the accessibility of these pharmacologically responsive vessels we hypothesized that the rate of CSF outflow can be modulated via the intranasal delivery of drugs known to affect lymphatic contractile activity.FindingsFluid was infused into the lateral ventricle of anesthetized sheep and inflow rate and CSF pressure measured during intranasal administration of pharmacological agents. CSF absorption was calculated at steady-state CSF pressures. The ability of a pharmacological agent to alter CSF absorption was related to the steady-state intracranial pressure (ICP), the concentration and the class of pharmacological agent delivered. An increase in drug concentration correlated with an increase in CSF absorption at high ICP (45 cm H2O, r = 0.42, p = 0.0058). Specifically, the delivery of NG-monomethyl L-arginine (L-NMMA) significantly increased CSF absorption by 2.29 fold over no treatment (2.29 ± 0.34 mL/min), while the thromboxane A2 analogue U46619 resulted in a 2.44 fold increase in CSF absorption over no treatment (2.44 ± 0.55 mL/min). Saline delivery did not significantly increase CSF absorption (0.88 ± 0.097 mL/min). A trend of increased CSF absorption upon noradrenaline delivery was observed: however, this did not reach statistical significance. Increasing drug concentrations inversely correlated with CSF outflow resistance across all drug classes (r = -0.26, p = 0.046).ConclusionsThe administration of nebulized pharmacological agents intranasally has the potential to provide an alternate method to non-invasively modulate CSF absorption and outflow resistance.

The effects of inflammatory cytokines on lymphatic endothelial barrier function

Proper lymphatic function is necessary for the transport of fluids, macromolecules, antigens and immune cells out of the interstitium. The lymphatic endothelium plays important roles in the modulation of lymphatic contractile activity and lymph transport, but it's role as a barrier between the lymph and interstitial compartments is less well understood. Alterations in lymphatic function have long been associated with edema and inflammation although the integrity of the lymphatic endothelial barrier during inflammation is not well-defined. In this paper we evaluated the integrity of the lymphatic barrier in response to inflammatory stimuli commonly associated with increased blood endothelial permeability. We utilized in vitro assays of lymphatic endothelial cell (LEC) monolayer barrier function after treatment with different inflammatory cytokines and signaling molecules including TNF-α, IL-6, IL-1β, IFN-γ and LPS. Moderate increases in an index of monolayer barrier dysfunction were noted with all treatments (20-60% increase) except IFN-γ which caused a greater than 2.5-fold increase. Cytokine-induced barrier dysfunction was blocked or reduced by the addition of LNAME, except for IL-1β and LPS treatments, suggesting a regulatory role for nitric oxide. The decreased LEC barrier was associated with modulation of both intercellular adhesion and intracellular cytoskeletal activation. Cytokine treatments reduced the expression of VE-cadherin and increased scavenging of β-catenin in the LECs and this was partially reversed by LNAME. Likewise the phosphorylation of myosin light chain 20 at the regulatory serine 19 site, which accompanied the elevated monolayer barrier dysfunction in response to cytokine treatment, was also blunted by LNAME application. This suggests that the lymphatic barrier is regulated during inflammation and that certain inflammatory signals may induce large increases in permeability.