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Two major functions of the lymphatic system are the reabsorption of excess interstitial fluid/protein and the coordination of immune cell interactions and trafficking. Specialized junctions between lymphatic endothelial cells optimize reabsorption. The spontaneous contractions of collecting vessels provide active lymph propulsion. One-way valves prevent backflow, and chemokine gradients direct the migration of immune cells. Specialized compartments within the lymph node facilitate antigen-immune cell interactions to produce innate and acquired immunity. Lymphatic injury and/or mutations in genes controlling vessel/valve development result in contractile/valve dysfunction, reduced immune cell trafficking and, ultimately, lymphedema. Activated CD4+ T cells produce inflammatory mediators that exacerbate these processes, potentially leading to interstitial and lymphatic vessel remodeling and negatively impacting overall function. Mouse models have advanced our knowledge of lymphatic disease, but clinical trials to reduce the impact of inflammatory mediators have yielded mixed success, implying that additional factors underlying human lymphedema are not yet understood.

Collecting lymphatic vessels (cLVs) exhibit spontaneous contractions with a pressure-dependent frequency, but the identity of the lymphatic pacemaker cell is still debated. By analogy to pacemakers in the GI and lower urinary tracts, proposed cLV pacemaker cells include interstitial cells of Cajal like cells (ICLC) or the lymphatic muscle (LMCs) cells themselves. Here we combined immunofluorescence and scRNAseq analyses with electrophysiological methods to examine the cellular constituents of the mouse cLV wall and assess whether any cell type exhibited morphological and functional processes characteristic of pacemaker cells: a continuous if not contiguous network integrated into the electrical syncytium; spontaneous Ca 2+ transients; and depolarization-induced propagated contractions. We employed inducible Cre (iCre) mouse models routinely used to target these specific cell populations including: c-kitCreER T2 to target ICLC; PdgfrβCreER T2 to target pericyte-like cells; PdgfrαCreER ™ to target CD34 + adventitial cells and ICLC; and Myh11CreER T2 to target LMCs directly. These specific inducible Cre lines were crossed to the fluorescent reporter ROSA26mT/mG, the genetically encoded Ca 2+ sensor GCaMP6f, and the light-activated cation channel rhodopsin2 (ChR2). c-KitCreER T2 labeled both a sparse population of LECs and round adventitial cells that responded to the mast cell activator compound 48-80. PdgfrβCreER T2 drove recombination in both adventitial cells and LMCs, limiting its power to discriminate a pericyte-specific population. PdgfrαCreER ™ labeled a large population of interconnected, oak leaf-shaped cells primarily along the adventitial surface of the vessel. Of these cells, only LMCs consistently, but heterogeneously, displayed spontaneous Ca 2+ events during the diastolic period of the contraction cycle, and whose frequency was modulated in a pressure-dependent manner. Optogenetic depolarization through the expression of ChR2 under control of Myh11CreER T2 , but not PdgfrαCreER ™ or c-KitCreER T2 , resulted in propagated contractions upon photo-stimulation. Membrane potential recordings in LMCs demonstrated that the rate of diastolic depolarization significantly correlated with contraction frequency. These findings support the conclusion that LMCs, or a subset of LMCs, are responsible for mouse cLV pacemaking., Competing Interests: The authors declare no competing financial interests. This work was supported by NIH HL-122608 and HL-122578 to MJD, HL-143198 and HL-175083 SDZ, and HL-141143 and HL-168568 to JAC-G, and AHA CDA-931652 to CEN. A. Patro was supported by MizzouForward Undergraduate Research Fellow.

Lymphatic dysfunction is an underlying component of multiple metabolic diseases, including diabetes, obesity, and metabolic syndrome. We investigated the roles of KATP channels in lymphatic contractile dysfunction in response to acute metabolic stress induced by inhibition of the mitochondrial electron transport chain. Ex vivo popliteal lymphatic vessels from mice were exposed to the electron transport chain inhibitors antimycin A and rotenone, or the oxidative phosphorylation inhibitor/protonophore, CCCP. Each inhibitor led to a significant reduction in the frequency of spontaneous lymphatic contractions and calculated pump flow, without a significant change in contraction amplitude. Contraction frequency was restored by the KATP channel inhibitor, glibenclamide. Lymphatic vessels from mice with global Kir6.1 deficiency or expressing a smooth muscle-specific dominant negative Kir6.1 channel were resistant to inhibition. Antimycin A inhibited the spontaneous action potentials generated in lymphatic muscle and this effect was reversed by glibenclamide, confirming the role of KATP channels. Antimycin A, but not rotenone or CCCP, increased dihydrorhodamine fluorescence in lymphatic muscle, indicating ROS production. Pretreatment with tiron or catalase prevented the effect of antimycin A on wild-type lymphatic vessels, consistent with its action being mediated by ROS. Our results support the conclusion that KATP channels in lymphatic muscle can be directly activated by reduced mitochondrial ATP production or ROS generation, consequent to acute metabolic stress, leading to contractile dysfunction through inhibition of the ionic pacemaker controlling spontaneous lymphatic contractions. We propose that a similar activation of KATP channels contributes to lymphatic dysfunction in metabolic disease., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Physiological Society.)

The expression and functional relevance of the gap junction molecule connexin-45 (Cx45; GJC1) in lymphatic endothelium were not previously known. We found that Cx45 was expressed widely in the endothelium of murine lymphatics, in both valve and nonvalve regions. Cell-specific deletion of Cx45, driven by a constitutive Cre line (Lyve1-Cre) or an inducible Cre line (Prox1-CreERT2), compromised the function of lymphatic valves, as assessed by physiological tests (back leak and closure) of isolated, single-valve vessel segments. The defects were comparable to those previously reported for loss of Cx43, and as with Cx43, deletion of Cx45 resulted in shortening or increased asymmetry of lymphatic valve leaflets, providing an explanation for the compromised valve function. In contrast with Cx43, lymphatic endothelial cell-specific (LEC-specific) deletion of Cx45 did not alter the number of valves in mesenteric or dermal lymphatic networks or the expression patterns of the canonical valve-associated proteins PROX1, ITGA9, or CLAUDIN5. Constitutive deletion of Cx45 from LECs resulted in increased backflow of injected tracer in popliteal networks in vivo and compromised the integrity of the LEC permeability barrier in a subset of collecting vessels. These findings provide evidence for an unexpected role of Cx45 in the development and maintenance of lymphatic valves.

Ca 2+ signalling plays a crucial role in determining lymphatic muscle cell excitability and contractility through its interaction with the Ca 2+ -activated Cl - channel anoctamin 1 (ANO1). In contrast, the large-conductance (BK) Ca 2+ -activated K + channel (KCa) and other KCa channels have prominent vasodilatory actions by hyperpolarizing vascular smooth muscle cells. Here, we assessed the expression and contribution of the KCa family to mouse and rat lymphatic collecting vessel contractile function. The BK channel was the only KCa channel consistently expressed in fluorescence-activated cell sorting-purified mouse lymphatic muscle cell lymphatic muscle cells. We used a pharmacological inhibitor of BK channels, iberiotoxin, and small-conductance Ca 2+ -activated K + channels, apamin, to inhibit KCa channels acutely in ex vivo isobaric myography experiments and intracellular membrane potential recordings. In basal conditions, BK channel inhibition had little to no effect on either mouse inguinal-axillary lymphatic vessel (MIALV) or rat mesenteric lymphatic vessel contractions or action potentials (APs). We also tested BK channel inhibition under loss of ANO1 either by genetic ablation (Myh11CreERT 2 -Ano1 fl/fl, Ano1ismKO) or by pharmacological inhibition with Ani9. In both Ano1ismKO MIALVs and Ani9-pretreated MIALVs, inhibition of BK channels increased contraction amplitude, increased peak AP and broadened the peak of the AP spike. In rat mesenteric lymphatic vessels, BK channel inhibition also abolished the characteristic post-spike notch, which was exaggerated with ANO1 inhibition, and significantly increased the peak potential and broadened the AP spike. We conclude that BK channels are present and functional on mouse and rat lymphatic muscle cells but are otherwise masked by the dominance of ANO1. KEY POINTS: Mouse and rat lymphatic muscle cells express functional BK channels. BK channels make little contribution to either rat or mouse lymphatic collecting vessel contractile function in basal conditions across a physiological pressure range. ANO1 limits the peak membrane potential achieved in the action potential and sets a plateau potential limiting the voltage-dependent activation of BK. BK channels are activated when ANO1 is absent or blocked and slightly impair contractile strength by reducing the peak membrane potential achieved in the action potential spike and accelerating the post-spike repolarization., (© 2024 The Authors. The Journal of Physiology © 2024 The Physiological Society.)

Lymphatic collecting vessels exhibit spontaneous phasic contractions that are critical for lymph propulsion and tissue fluid homeostasis. This rhythmic activity is driven by action potentials conducted across the lymphatic muscle cell (LMC) layer to produce entrained contractions. The contraction frequency of a lymphatic collecting vessel displays exquisite mechanosensitivity, with a dynamic range from <1 to >20 contractions per minute. A myogenic pacemaker mechanism intrinsic to the LMCs was initially postulated to account for pressure-dependent chronotropy. Further interrogation into the cellular constituents of the lymphatic vessel wall identified non-muscle cell populations that shared some characteristics with interstitial cells of Cajal, which have pacemaker functions in the gastrointestinal and lower urinary tracts, thus raising the possibility of a non-muscle cell pacemaker. However, recent genetic knockout studies in mice support LMCs and a myogenic origin of the pacemaker activity. LMCs exhibit stochastic, but pressure-sensitive, sarcoplasmic reticulum calcium release (puffs and waves) from IP 3 R1 receptors, which couple to the calcium-activated chloride channel Anoctamin 1, causing depolarisation. The resulting electrical activity integrates across the highly coupled lymphatic muscle electrical syncytia through connexin 45 to modulate diastolic depolarisation. However, multiple other cation channels may also contribute to the ionic pacemaking cycle. Upon reaching threshold, a voltage-gated calcium channel-dependent action potential fires, resulting in a nearly synchronous calcium global calcium flash within the LMC layer to drive an entrained contraction. This review summarizes the key ion channels potentially responsible for the pressure-dependent chronotropy of lymphatic collecting vessels and various mechanisms of IP 3 R1 regulation that could contribute to frequency tuning., (© 2024 The Authors. The Journal of Physiology © 2024 The Physiological Society.)

Introduction: The bileaflet valves found in collecting lymphatic vessels and some veins are essential for maintaining a unidirectional flow, which is important for lymphatic and venous function. Under an adverse pressure gradient, the two leaflets tightly overlap to prevent backflow. Valves are proposed to share four main stages of development, based on images obtained from randomly oriented valves in fixed mouse embryos, with the best structural views obtained from larger venous valves. It is not known at what stage lymphatic valves (LVs) become functional (e.g., able to oppose backflow), although a requirement for stage 4 is presumed. Methods: To gain an insight into this sequence of events for LVs, we used Prox1CreER T2 :Foxo1 fl/fl mice and Foxc2CreER T2 :Foxo1 fl/fl mouse models, in which deletion of the valve repressor factor Foxo1 promotes the development of new LVs in adult lymphatic vessels. Both strains also contained a Prox1eGFP reporter to image the lymphatic endothelium. Mesenteric collecting lymphatic vessels were dissected, cannulated, and pressurized for ex vivo tests of valve function. LVs at various stages (1-4 and intermediate) were identified in multi-valve segments, which were subsequently shortened to perform the backleak test on single valves. The GFP signal was then imaged at high magnification using a confocal microscope. Z-stack reconstructions enabled 1:1 comparisons of LV morphology with a quantitative measurement of back leak. Results: As expected, LVs of stages 1-3 were completely leaky in response to outflow pressure elevation. Stage 4 valves were generally not leaky, but valve integrity depended on the Cre line used to induce new valve formation. A high percentage of valves at leaflet an intermediate stage (3.5), in which there was an insertion of a second commissure, but without proper luminal alignment, effectively resisted back leak when the outflow pressure was increased. Discussion: Our findings represent the first 3D images of developing lymphatic valves and indicate that valves become competent between stages 3 and 4 of development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Davis, Zawieja and Yang.)

Lymphatic system defects are involved in a wide range of diseases, including obesity, cardiovascular disease, and neurological disorders, such as Alzheimer's disease. Fluid return through the lymphatic vascular system is primarily provided by contractions of muscle cells in the walls of lymphatic vessels, which are in turn driven by electrochemical oscillations that cause rhythmic action potentials and associated surges in intracellular calcium ion concentration. There is an incomplete understanding of the mechanisms involved in these repeated events, restricting the development of pharmacological treatments for dysfunction. Previously, we proposed a model where autonomous oscillations in the membrane potential (M-clock) drove passive oscillations in the calcium concentration (C-clock). In this paper, to model more accurately what is known about the underlying physiology, we extend this model to the case where the M-clock and the C-clock oscillators are both active but coupled together, thus both driving the action potentials. This extension results from modifications to the model's description of the IP3 receptor, a key C-clock mechanism. The synchronised dual-driving clock behaviour enables the model to match IP3 receptor knock-out data, thus resolving an issue with previous models. We also use phase-plane analysis to explain the mechanisms of coupling of the dual clocks. The model has the potential to help determine mechanisms and find targets for pharmacological treatment of some causes of lymphoedema., (© 2023 Hancock et al.)

Pressure-dependent chronotropy of murine lymphatic collecting vessels relies on the activation of the Ca2+-activated chloride channel encoded by Anoctamin 1 (Ano1) in lymphatic muscle cells. Genetic ablation or pharmacological inhibition of ANO1 results in a significant reduction in basal contraction frequency and essentially complete loss of pressure-dependent frequency modulation by decreasing the rate of the diastolic depolarization phase of the ionic pacemaker in lymphatic muscle cells (LMCs). Oscillating Ca2+ release from sarcoendoplasmic reticulum Ca2+ channels has been hypothesized to drive ANO1 activity during diastole, but the source of Ca2+ for ANO1 activation in smooth muscle remains unclear. Here, we investigated the role of the inositol triphosphate receptor 1 (Itpr1; Ip3r1) in this process using pressure myography, Ca2+ imaging, and membrane potential recordings in LMCs of ex vivo pressurized inguinal-axillary lymphatic vessels from control or Myh11CreERT2;Ip3r1fl/fl (Ip3r1ismKO) mice. Ip3r1ismKO vessels had significant reductions in contraction frequency and tone but an increased contraction amplitude. Membrane potential recordings from LMCs of Ip3r1ismKO vessels revealed a depressed diastolic depolarization rate and an elongation of the plateau phase of the action potential (AP). Ca2+ imaging of LMCs using the genetically encoded Ca2+ sensor GCaMP6f demonstrated an elongation of the Ca2+ flash associated with an AP-driven contraction. Critically, diastolic subcellular Ca2+ transients were absent in LMCs of Ip3r1ismKO mice, demonstrating the necessity of IP3R1 activity in controlling ANO1-mediated diastolic depolarization. These findings indicate a critical role for IP3R1 in lymphatic vessel pressure-dependent chronotropy and contractile regulation., (© 2023 Zawieja et al.)

We previously identified two isoforms of T-type, voltage-gated calcium (Ca v 3) channels (Ca v 3.1, Ca v 3.2) that are functionally expressed in murine lymphatic muscle cells; however, contractile tests of lymphatic vessels from single and double Ca v 3 knock-out (DKO) mice, exhibited nearly identical parameters of spontaneous twitch contractions as wild-type (WT) vessels, suggesting that Ca v 3 channels play no significant role. Here, we considered the possibility that the contribution of Ca v 3 channels might be too subtle to detect in standard contraction analyses. We compared the sensitivity of lymphatic vessels from WT and Ca v 3 DKO mice to the L-type calcium channel (Ca v 1.2) inhibitor nifedipine and found that the latter vessels were significantly more sensitive to inhibition, suggesting that the contribution of Ca v 3 channels might normally be masked by Ca v 1.2 channel activity. We hypothesized that shifting the resting membrane potential (Vm) of lymphatic muscle to a more negative voltage might enhance the contribution of Ca v 3 channels. Because even slight hyperpolarization is known to completely silence spontaneous contractions, we devised a method to evoke nerve-independent, twitch contractions from mouse lymphatic vessels using single, short pulses of electric field stimulation (EFS). TTX was present throughout to block the potential contributions of voltage-gated Na + channels in perivascular nerves and lymphatic muscle. In WT vessels, EFS evoked single contractions that were comparable in amplitude and degree of entrainment to those occurring spontaneously. When Ca v 1.2 channels were blocked or deleted, only small residual EFS-evoked contractions (~ 5% of normal amplitude) were present. These residual, EFS-evoked contractions were enhanced (to 10-15%) by the K ATP channel activator pinacidil (PIN) but were absent in Ca v 3 DKO vessels. Our results point to a subtle contribution of Ca v 3 channels to lymphatic contractions that can be unmasked in the absence of Ca v 1.2 channel activity and when the resting Vm is more hyperpolarized than normal., (© 2023. Springer Nature Limited.)

Introduction: The widely-used, tamoxifen-inducible, smooth muscle (SM)-specific Cre, Myh11-CreER T2 , suffers from two disadvantages: 1) it is carried on the Y-chromosome and thus only effective for gene deletion in male mice, and 2) it recombines in both vascular and non-vascular SM, potentially leading to unwanted or confounding gastrointestinal phenotypes. Here, we tested the effectiveness of a new, SM-specific Cre, based on the integrin α8 promoter ( Itga8-CreER T2 ), that has been recently developed and characterized, to assess the effects of Cav1.2 deletion on mouse lymphatic SM function. Methods: Cav1.2 (the L-type voltage-gated calcium channel) is essential for lymphatic pacemaking and contraction and its deletion using either Myh11-CreER T2 or Itga8-CreER T2 abolished spontaneous lymphatic contractions. Mouse lymphatic contractile function was assessed using two ex vivo methods. Results: Myh11-CreER T2 ; Cav1.2 f/f mice died of gastrointestinal obstruction within 20 days of the first tamoxifen injection, preceded by several days of progressively poor health, with symptoms including weight loss, poor grooming, hunched posture, and reduced overall activity. In contrast, Itga8-CreER T2 ; Cav1.2 f/f mice survived for >80 days after induction and were in normal health until the time of sacrifice for experimental studies. Cav1.2 deletion was equally effective in male and female mice. Discussion: Our results demonstrate that Itga8-CreER T2 can be used to effectively delete genes in lymphatic smooth muscle while avoiding potentially lethal visceral myopathy and allowing comparative studies of lymphatic contractile function in both male and female mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Davis, Kim, Li and Zawieja.)

All current smooth muscle cell (SMC) Cre mice similarly recombine floxed alleles in vascular and visceral SMCs. Here, we present an Itga8-CreER T2 knock-in mouse and compare its activity with a Myh11-CreER T2 mouse. Both Cre drivers demonstrate equivalent recombination in vascular SMCs. However, Myh11-CreER T2 mice, but not Itga8-CreER T2 mice, display high activity in visceral SMC-containing tissues such as intestine, show early tamoxifen-independent activity, and produce high levels of CreER T2 protein. Whereas Myh11-CreER T2 -mediated knockout of serum response factor ( Srf ) causes a lethal intestinal phenotype precluding analysis of the vasculature, loss of Srf with Itga8-CreER T2 ( Srf Itga8 ) yields viable mice with no evidence of intestinal pathology. Male and female Srf Itga8 mice exhibit vascular contractile incompetence, and angiotensin II causes elevated blood pressure in wild type, but not Srf Itga8 , male mice. These findings establish the Itga8-CreER T2 mouse as an alternative to existing SMC Cre mice for unfettered phenotyping of vascular SMCs following selective gene loss.

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., Competing Interests: YL is currently working at GlaxoSmithKline. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lee, Zawieja and Muthuchamy.)

Chronic dysfunction of the lymphatic vascular system results in fluid accumulation between cells: lymphoedema. The condition is commonly acquired secondary to diseases such as cancer or the associated therapies. The primary driving force for fluid return through the lymphatic vasculature is provided by contractions of the muscularized lymphatic collecting vessels, driven by electrochemical oscillations. However, there is an incomplete understanding of the molecular and bioelectric mechanisms involved in lymphatic muscle cell excitation, hampering the development and use of pharmacological therapies. Modelling in silico has contributed greatly to understanding the contributions of specific ion channels to the cardiac action potential, but modelling of these processes in lymphatic muscle remains limited. Here, we propose a model of oscillations in the membrane voltage (M-clock) and intracellular calcium concentrations (C-clock) of lymphatic muscle cells. We modify a model by Imtiaz and colleagues to enable the M-clock to drive the C-clock oscillations. This approach differs from typical models of calcium oscillators in lymphatic and related cell types, but is required to fit recent experimental data. We include an additional voltage dependence in the gating variable control for the L-type calcium channel, enabling the M-clock to oscillate independently of the C-clock. We use phase-plane analysis to show that these M-clock oscillations are qualitatively similar to those of a generalised FitzHugh-Nagumo model. We also provide phase plane analysis to understand the interaction of the M-clock and C-clock oscillations. The model and methods have the potential to help determine mechanisms and find targets for pharmacological treatment of lymphoedema., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Key Points: Spontaneous contractions are essential for normal lymph transport and these contractions are exquisitely sensitive to the K ATP channel activator pinacidil. K ATP channel Kir6.1 and SUR2B subunits are expressed in mouse lymphatic smooth muscle (LSM) and form functional K ATP channels as verified by electrophysiological techniques. Global deletion of Kir6.1 or SUR2 subunits results in severely impaired lymphatic contractile responses to pinacidil. Smooth muscle-specific expression of Kir6.1 gain-of-function mutant (GoF) subunits results in profound lymphatic contractile dysfunction and LSM hyperpolarization that is partially rescued by the K ATP inhibitor glibenclamide. In contrast, lymphatic endothelial-specific expression of Kir6.1 GoF has essentially no effect on lymphatic contractile function. The high sensitivity of LSM to K ATP channel GoF offers an explanation for the lymphoedema observed in patients with Cantú syndrome, a disorder caused by gain-of-function mutations in genes encoding Kir6.1 or SUR2, and suggests that glibenclamide may be an appropriate therapeutic agent., Abstract: This study aimed to understand the functional expression of K ATP channel subunits in distinct lymphatic cell types, and assess the consequences of altered K ATP channel activity on lymphatic pump function. K ATP channel subunits Kir6.1 and SUR2B were expressed in mouse lymphatic muscle by PCR, but only Kir6.1 was expressed in lymphatic endothelium. Spontaneous contractions of popliteal lymphatics from wild-type (WT) (C57BL/6J) mice, assessed by pressure myography, were very sensitive to inhibition by the SUR2-specific K ATP channel activator pinacidil, which hyperpolarized both mouse and human lymphatic smooth muscle (LSM). In vessels from mice with deletion of Kir6.1 (Kir6.1 -/- ) or SUR2 (SUR2[STOP]) subunits, contractile parameters were not significantly different from those of WT vessels, suggesting that basal K ATP channel activity in LSM is not an essential component of the lymphatic pacemaker, and does not exert a strong influence over contractile strength. However, these vessels were >100-fold less sensitive than WT vessels to pinacidil. Smooth muscle-specific expression of a Kir6.1 gain-of-function (GoF) subunit resulted in severely impaired lymphatic contractions and hyperpolarized LSM. Membrane potential and contractile activity was partially restored by the K ATP channel inhibitor glibenclamide. In contrast, lymphatic endothelium-specific expression of Kir6.1 GoF subunits had negligible effects on lymphatic contraction frequency or amplitude. Our results demonstrate a high sensitivity of lymphatic contractility to K ATP channel activators through activation of Kir6.1/SUR2-dependent channels in LSM. In addition, they offer an explanation for the lymphoedema observed in patients with Cantú syndrome, a disorder caused by gain-of-function mutations in genes encoding Kir6.1/SUR2., (© 2020 The Authors. The Journal of Physiology © 2020 The Physiological Society.)

The spontaneous contractions of collecting lymphatic vessels provide an essential propulsive force to return lymph centrally. These contractions are driven by an intrinsic electrical pacemaker, working through an unknown underlying ionic mechanism that becomes compromised in some forms of lymphedema. In previous studies, T-type voltage-gated Ca 2+ channels (VGCCs) were implicated in this pacemaking mechanism, based on the effects of the reputedly selective T-type VGCC inhibitors mibefradil and Ni 2+ . Our goal was to test this idea in a more definitive way using genetic knock out mice. First, we demonstrated through both PCR and immunostaining that mouse lymphatic muscle cells expressed Ca v 3.1 and Ca v 3.2 and produced functional T-type VGCC currents when patch clamped. We then employed genetic deletion strategies to selectively test the roles of each T-type VGCC isoform in the regulation of lymphatic pacemaking. Surprisingly, global deletion of either, or both, isoform(s) was without significant effect on either the frequency, amplitude, or fractional pump flow of lymphatic collectors from two different regions of the mouse, studied ex vivo. Further, both WT and Ca v 3.1 -/- ; 3.2 -/- double knock-out lymphatic vessels responded similarly to mibefradil and Ni 2+ , which substantially reduced contraction amplitudes and slightly increased frequencies at almost all pressures in both strains: a pattern consistent with inhibition of L-type rather than T-type VGCCs. Neither T-type VGCC isoform was required for ACh-induced inhibition of contraction, a mechanism by which those channels in smooth muscle are thought to be targets of endothelium-derived nitric oxide. Sharp intracellular electrode measurements in lymphatic smooth muscle revealed only subtle, but not significant, differences in the resting membrane potential and action potential characteristics between vessels from wild-type and Ca v 3.1 -/- ; 3.2 -/- double knock-out mice. In contrast, smooth-muscle specific deletion of the L-type VGCC, Ca v 1.2, completely abolished all lymphatic spontaneous contractions. Collectively our results suggest that, although T-type VGCCs are expressed in mouse lymphatic smooth muscle, they do not play a significant role in modulating the frequency of the ionic pacemaker or the amplitude of spontaneous contractions. We conclude that the effects of mibefradil and Ni 2+ in other lymphatic preparations are largely or completely explained by off-target effects on L-type VGCCs, which are essential for controlling both the frequency and strength of spontaneous contractions.

Lymphatic collecting vessels exhibit spontaneous contractions with a pressure-dependent contraction frequency. The initiation of contraction has been proposed to be mediated by the activity of a Ca 2+ -activated Cl - channel (CaCC). Here, we show that the canonical CaCC Anoctamin 1 (Ano1, TMEM16a) plays an important role in lymphatic smooth muscle pacemaking. We find that isolated murine lymphatic muscle cells express Ano1, and demonstrate functional CaCC currents that can be inhibited by the Ano1 inhibitor benzbromarone. These currents are absent in lymphatic muscle cells from Cre transgenic mouse lines targeted for Ano1 genetic deletion in smooth muscle. We additionally show that loss of functional Ano1 in murine inguinal-axillary lymphatic vessels, whether through genetic manipulation or pharmacological inhibition, results in an impairment of the pressure-frequency relationship that is attributable to a hyperpolarized resting membrane potential and a significantly depressed diastolic depolarization rate preceding each action potential. These changes are accompanied by alterations in action potential shape and duration, and a reduced duration but increased amplitude of the action potential-induced global "Ca 2+ flashes" that precede lymphatic contractions. These findings suggest that an excitatory Cl - current provided by Ano1 is critical for mediating the pressure-sensitive contractile response and is a major component of the murine lymphatic action potential., (© 2019 Zawieja et al.)

Rationale: Mutations in GJC2 and GJA1, encoding Cxs (connexins) 47 and 43, respectively, are linked to lymphedema, but the underlying mechanisms are unknown. Because efficient lymph transport relies on the coordinated contractions of lymphatic muscle cells (LMCs) and their electrical coupling through Cxs, Cx-related lymphedema is proposed to result from dyssynchronous contractions of lymphatic vessels., Objective: To determine which Cx isoforms in LMCs and lymphatic endothelial cells are required for the entrainment of lymphatic contraction waves and efficient lymph transport., Methods and Results: We developed novel methods to quantify the spatiotemporal entrainment of lymphatic contraction waves and used optogenetic techniques to analyze calcium signaling within and between the LMC and the lymphatic endothelial cell layers. Genetic deletion of the major lymphatic endothelial cell Cxs (Cx43, Cx47, or Cx37) revealed that none were necessary for the synchronization of the global calcium events that triggered propagating contraction waves. We identified Cx45 in human and mouse LMCs as the critical Cx mediating the conduction of pacemaking signals and entrained contractions. Smooth muscle-specific Cx45 deficiency resulted in 10- to 18-fold reduction in conduction speed, partial-to-severe loss of contractile coordination, and impaired lymph pump function ex vivo and in vivo. Cx45 deficiency resulted in profound inhibition of lymph transport in vivo, but only under an imposed gravitational load., Conclusions: Our results (1) identify Cx45 as the Cx isoform mediating the entrainment of the contraction waves in LMCs; (2) show that major endothelial Cxs are dispensable for the entrainment of contractions; (3) reveal a lack of coupling between lymphatic endothelial cells and LMCs, in contrast to arterioles; (4) point to lymphatic valve defects, rather than contraction dyssynchrony, as the mechanism underlying GJC2- or GJA1-related lymphedema; and (5) show that a gravitational load exacerbates lymphatic contractile defects in the intact mouse hindlimb, which is likely critical for the development of lymphedema in the adult mouse.

Contractions of lymphangions, i.e., the segment between two one-way lymphatic valves, generate the pressure gradients that propel lymph back to the circulation. Each lymphangion is comprised of an inner sheet of lymphatic endothelial cells circumscribed by one or more layers of lymphatic muscle cells (LMCs). Each contraction is produced by an LMC action potential (AP) that propagates via gap junctions along the lymphangion. Yet, electrical coupling within and between cell layers and the impact on AP waves is poorly understood. Here, we combine studies in rat and mouse lymphatic vessels with mathematical modeling to show that initiation of AP waves depends on high input resistance (low current drain), whereas propagation depends on morphology and sufficient LMC:LMC coupling. Simulations show that 1) myoendothelial coupling is insignificant to facilitate AP generation and sustain an experimentally measured cross-junctional potential difference of 25 mV, i.e., AP waves propagate along the LMC layer only; 2) LMC:LMC resistance is estimated around 2-10 MΩ but depends on vessel structure and cell-cell coupling, e.g., some degree of LMC overlap protects AP waves against LMC decoupling; 3) the propensity of AP wave initiation is highest around the valves, where the density of LMCs is low; and 4) a single pacemaker cell embedded in the LMC layer must be able to generate very large currents to overcome the current drain from the layer. However, the required current generation to initiate an AP wave is reduced upon stimulation of multiple adjacent LMCs. With stimulation of all LMCs, AP waves can also arise from heterogeneity in the electrical activity of LMCs. The findings advance our understanding of the electrical constraints that underlie initiation of APs in the LMC layer and make testable predictions about how morphology, LMC excitability, and LMC:LMC electrical coupling interact to determine the ability to initiate and propagate AP waves in small lymphatic vessels., (Copyright © 2018 Biophysical Society. All rights reserved.)

We identified a regional dichotomy in murine lymphatic contractile function with regard to vessel location within the periphery or visceral cavity. All vessels isolated from peripheral regions [cervical, popliteal, inguinal, axillary, and internodal inguinal axillary (Ing-Ax)] developed robust contractions with maximal ejection fractions (EFs) of 50-80% in our ex vivo isobaric myograph experiments. Conversely, vessels isolated from the visceral cavity (mesenteric, thoracic duct, and iliac) demonstrated maximal EFs of ≤10%. Using pressure myography, sharp electrode membrane potential recordings, and Ca 2+ imaging, we assessed the role of L-type Ca 2+ channels in this contractile dichotomy. Ing-Ax membrane potential revealed a ~2-s action potential (AP) cycle (resting -35 mV, spike -5 mV, and plateau -11 mV) with a plateau phase that was significantly lengthened by the L-type Ca 2+ channel agonist Bay K8644 (BayK; 100 nM). APs recorded from mesenteric vessels, however, displayed a slower upstroke and an elongated time over threshold. BayK (100 nM) increased the mesenteric AP upstroke velocity and plateau duration but also significantly hyperpolarized the vessel. Contractions of vessels from both regions were preceded by Ca 2+ flashes, detected with a smooth muscle-specific endogenous Ca 2+ reporter, that typically were coordinated over the length of the vessel. Similar to the membrane potential recordings, Ca 2+ flashes in mesenteric vessels were weaker and had a slower rise time but were longer lasting than those in Ing-Ax vessels. BayK (100 nM) significantly increased the Ca 2+ transient amplitude and duration in both vessels and decreased time to peak Ca 2+ in mesenteric vessels. However, a higher concentration (1 μM) of BayK was required to produce even a modest increase in EF in visceral lymphatics, which remained at <20%. NEW & NOTEWORTHY Lymphatic collecting vessels isolated from murine peripheral tissues, but not from the visceral cavities, display robust contractile behavior similar to lymphatic vessels from other animal models and humans. These differences are partially explained by L-type Ca 2+ channel activity as revealed by the first measurements of murine lymphatic action potentials and contraction-associated Ca 2+ transients.

It is theorized that toxic agents are transported from the hyperpermeable gut of burn victims through the lymph, to the systemic circulation, causing global injury. We believe that immune cells respond to leakage of "toxic lymph" following trauma causing the attraction of these cells to the perilymphatic space. To test this, we utilized a model of burn on rats to examine changes in a single immune cell population associated with mesenteric lymphatic dysfunction. We examined the ability of serum from these animals to increase permeability in lymphatic endothelial monolayers and disrupt cellular junctions. We also treated burn animals with doxycycline, an inhibitor of microvascular permeability, and observed the effects on immune cell populations, morphometry, and lymphatic endothelial permeability. Burn injury increased the number of MHCII + immune cells along the vessel (>50%). The size and shape of these cells also changed significantly following burn injury. Serum from burn animals increased lymphatic endothelial permeability (∼1.5-fold) and induced breaks in VE-cadherin staining. Doxycycline treatment blocked the accumulation of immune cells along the vessel, whereas serum from doxycycline-treated animals failed to increase lymphatic endothelial permeability. The size of cells along the vessel in doxycycline-treated burn animals was not affected, suggesting that the cells already present on the lymphatic vessels still respond to substances in the lymph. These findings suggest that factors produced during burn can induce lymphatic endothelial barrier disruption and lymph produced during traumatic injury can influence the attraction and morphology of immune cell populations along the vessel.

Key Points: Endothelial cell function in resistance arteries integrates Ca 2+ signalling with hyperpolarization to promote relaxation of smooth muscle cells and increase tissue blood flow. Whether complementary signalling occurs in lymphatic endothelium is unknown. Intracellular calcium and membrane potential were evaluated in endothelial cell tubes freshly isolated from mouse collecting lymphatic vessels of the popliteal fossa. Resting membrane potential measured using intracellular microelectrodes averaged ∼-70 mV. Stimulation of lymphatic endothelium by acetylcholine or a TRPV4 channel agonist increased intracellular Ca 2+ with robust depolarization. Findings from Trpv4 -/- mice and with computational modelling suggest that the initial mobilization of intracellular Ca 2+ leads to influx of Ca 2+ and Na + through TRPV4 channels to evoke depolarization. Lymphatic endothelial cells lack the Ca 2+ -activated K + channels present in arterial endothelium to generate endothelium-derived hyperpolarization. Absence of this signalling pathway with effective depolarization may promote rapid conduction of contraction along lymphatic muscle during lymph propulsion., Abstract: Subsequent to a rise in intracellular Ca 2+ ([Ca 2+ ] i ), hyperpolarization of the endothelium coordinates vascular smooth muscle relaxation along resistance arteries during blood flow control. In the lymphatic vasculature, collecting vessels generate rapid contractions coordinated along lymphangions to propel lymph, but the underlying signalling pathways are unknown. We tested the hypothesis that lymphatic endothelial cells (LECs) exhibit Ca 2+ and electrical signalling properties that facilitate lymph propulsion. To study electrical and intracellular Ca 2+ signalling dynamics in lymphatic endothelium, we excised collecting lymphatic vessels from the popliteal fossa of mice and removed their muscle cells to isolate intact LEC tubes (LECTs). Intracellular recording revealed a resting membrane potential of ∼-70 mV. Acetylcholine (ACh) increased [Ca 2+ ] i with a time course similar to that observed in endothelium of resistance arteries (i.e. rapid initial peak with a sustained 'plateau'). In striking contrast to the endothelium-derived hyperpolarization (EDH) characteristic of arteries, LECs depolarized (>15 mV) to either ACh or TRPV4 channel activation. This depolarization was facilitated by the absence of Ca 2+ -activated K + (K Ca ) channels as confirmed with PCR, persisted in the absence of extracellular Ca 2+ , was abolished by LaCl 3 and was attenuated ∼70% in LECTs from Trpv4 -/- mice. Computational modelling of ion fluxes in LECs indicated that omitting K + channels supports our experimental results. These findings reveal novel signalling events in LECs, which are devoid of the K Ca activity abundant in arterial endothelium. Absence of EDH with effective depolarization of LECs may promote the rapid conduction of contraction waves along lymphatic muscle during lymph propulsion., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Recent years have seen a renewed interest in studies of the lymphatic system. This review addresses the differences between in vivo and ex vivo methods for visualization and functional studies of lymphatic networks, with an emphasis on studies of collecting lymphatic vessels. We begin with a brief summary of the historical uses of both approaches. For the purpose of detailed comparisons, we subdivide in vivo methods into those visualizing lymphatic networks through the intact skin and those using surgically opened skin. We subdivide ex vivo methods into isobaric studies (using a pressure myograph) or isometric studies (using a wire myograph). For all four categories, we compile a comprehensive list of the advantages, disadvantages, and limitations of each preparation, with the goal of informing the research community as to the appropriate kinds of experiments best suited, and ill suited, for each.

Many tissues exhibit subatmospheric interstitial pressures under normal physiologic conditions. The mechanisms by which the lymphatic system extracts fluid from these tissues against the overall pressure gradient are unknown. We address this important physiologic issue by combining experimental measurements of contractile function and pressure generation with a previously validated mathematical model. We provide definitive evidence for the existence of 'suction pressure' in collecting lymphatic vessels, which manifests as a transient drop in pressure downstream of the inlet valve following contraction. This suction opens the inlet valve and is required for filling in the presence of low upstream pressure. Positive transmural pressure is required for this suction, providing the energy required to reopen the vessel. Alternatively, external vessel tethering can serve the same purpose when the transmural pressure is negative. Suction is transmitted upstream, allowing fluid to be drawn in through initial lymphatics. Because suction plays a major role in fluid entry to the lymphatics and is affected by interstitial pressure, our results introduce the phenomenon as another important factor to consider in the study of lymphoedema and its treatment.

Capillary malformation-arteriovenous malformation (CM-AVM) is a blood and lymphatic vessel (LV) disorder that is caused by inherited inactivating mutations of the RASA1 gene, which encodes p120 RasGAP (RASA1), a negative regulator of the Ras small GTP-binding protein. How RASA1 mutations lead to the LV leakage defects that occur in CM-AVM is not understood. Here, we report that disruption of the Rasa1 gene in adult mice resulted in loss of LV endothelial cells (LECs) specifically from the leaflets of intraluminal valves in collecting LVs. As a result, valves were unable to prevent fluid backflow and the vessels were ineffective pumps. Furthermore, disruption of Rasa1 in midgestation resulted in LEC apoptosis in developing LV valves and consequently failed LV valvulogenesis. Similar phenotypes were observed in induced RASA1-deficient adult mice and embryos expressing a catalytically inactive RASA1R780Q mutation. Thus, RASA1 catalytic activity is essential for the function and development of LV valves. These data provide a partial explanation for LV leakage defects and potentially other LV abnormalities observed in CM-AVM.

A combination of extrinsic (passive) and intrinsic (active) forces move lymph against a hydrostatic pressure gradient in most regions of the body. The effectiveness of the lymph pump system impacts not only interstitial fluid balance but other aspects of overall homeostasis. This review focuses on the mechanisms that regulate the intrinsic, active contractions of collecting lymphatic vessels in relation to their ability to actively transport lymph. Lymph propulsion requires not only robust contractions of lymphatic muscle cells, but contraction waves that are synchronized over the length of a lymphangion as well as properly functioning intraluminal valves. Normal lymphatic pump function is determined by the intrinsic properties of lymphatic muscle and the regulation of pumping by lymphatic preload, afterload, spontaneous contraction rate, contractility and neural influences. Lymphatic contractile dysfunction, barrier dysfunction and valve defects are common themes among pathologies that directly involve the lymphatic system, such as inherited and acquired forms of lymphoedema, and pathologies that indirectly involve the lymphatic system, such as inflammation, obesity and metabolic syndrome, and inflammatory bowel disease., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Objective: The intrinsic lymphatic pump is critical to proper lymph transport and is impaired in models of the MetSyn. Lymphatic contractile inhibition under inflammatory conditions has been linked with elevated NO production by activated myeloid-derived cells. Hence we hypothesized that inhibition of the MLV pump function in MetSyn animals was dependent on NO and was associated with altered macrophage recruitment and polarization within the MLV., Methods: We used a high fructose-fed rat model of MetSyn. Macrophage polarization was determined by whole mount immunofluorescence in mesenteric neurovascular bundles based on expression of CD163, CD206, and MHCII. We also utilized isolated vessel isobaric preparations to determine the role for elevated NO production in the inhibition of MLV contractility. Both LECs and LMCs were used to assess the cytokines and chemokines to test how the lymphatic cells response to inflammatory conditions., Results: Data demonstrated a greater accumulation of M1-skewed (CD163 + MHCII + ) macrophages that were observed both within the perivascular adipose tissue and invested along the lymphatic vessels in MetSyn rats when compared to control rats. LECs and LMCs basally express the macrophage maturation polarization cytokines monocyte colony-stimulating factor and dramatically up regulate the M1 promoting cytokine granulocyte/monocyte colony-stimulating factor in response to lipopolysaccharide stimulation. MetSyn MLVs exhibited altered phasic contraction frequency. Incubation of MetSyn MLVs with LNAME or Glib had a partial restoration of lymphatic contraction frequency., Conclusion: The data presented here provide the first evidence for a correlation between alterations in macrophage status and lymphatic dysfunction that is partially mediated by NO and K ATP channel in MetSyn rats., (© 2016 John Wiley & Sons Ltd.)

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., (Copyright © 2016 the American Physiological Society.)

Impairment of the lymphatic system is apparent in multiple inflammatory pathologies connected to elevated endotoxins such as LPS. However, the direct mechanisms by which LPS influences the lymphatic contractility are not well understood. We hypothesized that a dynamic modulation of innate immune cell populations in mesentery under inflammatory conditions perturbs tissue cytokine/chemokine homeostasis and subsequently influences lymphatic function. We used rats that were intraperitoneally injected with LPS (10 mg/kg) to determine the changes in the profiles of innate immune cells in the mesentery and in the stretch-mediated contractile responses of isolated lymphatic preparations. Results demonstrated a reduction in the phasic contractile activity of mesenteric lymphatic vessels from LPS-injected rats and a severe impairment of lymphatic pump function and flow. There was a significant reduction in the number of neutrophils and an increase in monocytes/macrophages present on the lymphatic vessels and in the clear mesentery of the LPS group. This population of monocytes and macrophages established a robust M2 phenotype, with the majority showing high expression of CD163 and CD206. Several cytokines and chemoattractants for neutrophils and macrophages were significantly changed in the mesentery of LPS-injected rats. Treatment of lymphatic muscle cells (LMCs) with LPS showed significant changes in the expression of adhesion molecules, VCAM1, ICAM1, CXCR2, and galectin-9. LPS-TLR4-mediated regulation of pAKT, pERK pI-κB, and pMLC20 in LMCs promoted both contractile and inflammatory pathways. Thus, our data provide the first evidence connecting the dynamic changes in innate immune cells on or near the lymphatics and complex cytokine milieu during inflammation with lymphatic dysfunction., (Copyright © 2015 the American Physiological Society.)

Background: Lymphatic dysfunction has been linked to inflammation since the 1930s. Lymphatic function in the gut and mesentery is grossly underexplored in models of inflammatory bowel disease despite the use of lymphatic occlusion in early models of inflammatory bowel disease. Activation of the innate and adaptive immune system is a hallmark of TNBS-induced inflammation and is linked to disruption of the intrinsic lymph pump. Recent identification of crosstalk between lymphatic vessel resident immune cells and regulation of lymphatic vessel contractility underscore the importance of the timing of lymphatic dysfunction during tissue inflammation in response to TNBS., Methods: To evaluate lymphatic function in TNBS induced inflammation, lymph was collected and flow measured from mesenteric lymphatics. Cellularity and cytokine profile of the lymph was also measured. Histopathology was performed to determine severity of injury and immunofluorescent staining of the mesentery was done to evaluate changes in the population of immune cells that reside near and on gastro-intestinal collecting lymphatics., Results: Lymph transport fell 24 hours after TNBS administration and began recovering at 72 hours. Significant reduction of lymph flow preceded significant increase in histopathological score and occurred simultaneously with increased myeloperoxidase activity. These changes were preceded by increased MHCII cells surrounding mesenteric lymphatics leading to an altered lymphatic environment that would favor dysfunction., Conclusions: Alterations in environmental factors that effect lymphatic function occur before the development of gross GI inflammation. Reduced lymphatic function in TNBS-mediated inflammation is likely an early factor in the development of injury and that recovery of function is associated with resolution of inflammation.

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.

The principal function of the lymphatic system is to transport lymph from the interstitium to the nodes and then from the nodes to the blood. In doing so lymphatics play important roles in fluid homeostasis, macromolecular/antigen transport and immune cell trafficking. To better understand the genes that contribute to their unique physiology, we compared the transcriptional profile of muscular lymphatics (prenodal mesenteric microlymphatics and large, postnodal thoracic duct) to axillary and mesenteric arteries and veins isolated from rats. Clustering of the differentially expressed genes demonstrated that the lymph versus blood vessel differences were more profound than between blood vessels, particularly the microvessels. Gene ontology functional category analysis indicated that microlymphatics were enriched in antigen processing/presentation, IgE receptor signaling, catabolic processes, translation and ribosome; while they were diminished in oxygen transport, regulation of cell proliferation, glycolysis and inhibition of adenylate cyclase activity by G-proteins. We evaluated the differentially expressed microarray genes/products by qPCR and/or immunofluorescence. Immunofluorescence documented that multiple MHC class II antigen presentation proteins were highly expressed by an antigen-presenting cell (APC) type found resident within the lymphatic wall. These APCs also expressed CD86, a co-stimulatory protein necessary for T-cell activation. We evaluated the distribution and phenotype of APCs within the pre and postnodal lymphatic network. This study documents a novel population of APCs resident within the walls of muscular, prenodal lymphatics that indicates novel roles in antigen sampling and immune responses. In conclusion, these prenodal lymphatics exhibit a unique profile that distinguishes them from blood vessels and highlights the role of the lymphatic system as an immunovascular system linking the parenchymal interstitium, lymph nodes and the blood.

Numerous studies on metabolic syndrome (MetSyn), a cluster of metabolic abnormalities, have demonstrated its profound impact on cardiovascular and blood microvascular health; however, the effects of MetSyn on lymphatic function are not well understood. We hypothesized that MetSyn would modulate lymphatic muscle activity and alter muscularized lymphatic function similar to the impairment of blood vessel function associated with MetSyn, particularly given the direct proximity of the lymphatics to the chronically inflamed adipose depots. To test this hypothesis, rats were placed on a high-fructose diet (60%) for 7 wk, and their progression to MetSyn was assessed through serum insulin and triglyceride levels in addition to the expression of metabolic and inflammatory genes in the liver. Mesenteric lymphatic vessels were isolated and subjected to different transmural pressures while lymphatic pumping and contractile parameters were evaluated. Lymphatics from MetSyn rats had significant negative chronotropic effects at all pressures that effectively reduced the intrinsic flow-generating capacity of these vessels by ∼50%. Furthermore, lymphatics were remodeled to a significantly smaller diameter in the animals with MetSyn. Wire myograph experiments demonstrated that permeabilized lymphatics from the MetSyn group exhibited a significant decrease in force generation and were less sensitive to Ca(2+), although there were no significant changes in lymphatic muscle cell coverage or morphology. Thus, our data provide the first evidence that MetSyn induces a remodeling of collecting lymphatics, thereby effectively reducing their potential load capabilities and impairing the intrinsic contractility required for proper lymph flow.

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.