Loss of anoctamin 1 reveals a subtle role for BK channels in lymphatic muscle action potentials.

Ca2+ signalling plays a crucial role in determining lymphatic muscle cell excitability and contractility through its interaction with the Ca2+-activated Cl- channel anoctamin 1 (ANO1). In contrast, the large-conductance (BK) Ca2+-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 Ca2+-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 (Myh11CreERT2-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.

Pacemaking in the lymphatic system.

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 IP3R1 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 IP3R1 regulation that could contribute to frequency tuning.

Developmental progression of lymphatic valve morphology and function.

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.

A dual-clock-driven model of lymphatic muscle cell pacemaking to emulate knock-out of Ano1 or IP3R.

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.

IP3R1 underlies diastolic ANO1 activation and pressure-dependent chronotropy in lymphatic collecting vessels.

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.

Electric field stimulation unmasks a subtle role for T-type calcium channels in regulating lymphatic contraction.

We previously identified two isoforms of T-type, voltage-gated calcium (Cav3) channels (Cav3.1, Cav3.2) that are functionally expressed in murine lymphatic muscle cells; however, contractile tests of lymphatic vessels from single and double Cav3 knock-out (DKO) mice, exhibited nearly identical parameters of spontaneous twitch contractions as wild-type (WT) vessels, suggesting that Cav3 channels play no significant role. Here, we considered the possibility that the contribution of Cav3 channels might be too subtle to detect in standard contraction analyses. We compared the sensitivity of lymphatic vessels from WT and Cav3 DKO mice to the L-type calcium channel (Cav1.2) inhibitor nifedipine and found that the latter vessels were significantly more sensitive to inhibition, suggesting that the contribution of Cav3 channels might normally be masked by Cav1.2 channel activity. We hypothesized that shifting the resting membrane potential (Vm) of lymphatic muscle to a more negative voltage might enhance the contribution of Cav3 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 Cav1.2 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 KATP channel activator pinacidil (PIN) but were absent in Cav3 DKO vessels. Our results point to a subtle contribution of Cav3 channels to lymphatic contractions that can be unmasked in the absence of Cav1.2 channel activity and when the resting Vm is more hyperpolarized than normal.

Electric field stimulation unmasks a subtle role for T-type calcium channels in regulating lymphatic contraction.

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.

Generation and Comparative Analysis of an Itga8-CreER T2 Mouse with Preferential Activity in Vascular Smooth Muscle Cells.

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 CreERT2 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.

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.

A vascular smooth muscle-specific integrin-α8 Cre mouse for lymphatic contraction studies that allows male-female comparisons and avoids visceral myopathy.

Introduction: The widely-used, tamoxifen-inducible, smooth muscle (SM)-specific Cre, Myh11-CreERT2 , 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-CreERT2 ), 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-CreERT2 or Itga8-CreERT2 abolished spontaneous lymphatic contractions. Mouse lymphatic contractile function was assessed using two ex vivo methods. Results: Myh11-CreERT2 ; 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-CreERT2 ; 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.

Kir6.1-dependent KATP channels in lymphatic smooth muscle and vessel dysfunction in mice with Kir6.1 gain-of-function.

KEY POINTS: Spontaneous contractions are essential for normal lymph transport and these contractions are exquisitely sensitive to the KATP channel activator pinacidil. KATP channel Kir6.1 and SUR2B subunits are expressed in mouse lymphatic smooth muscle (LSM) and form functional KATP 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 KATP 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 KATP 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 KATP channel subunits in distinct lymphatic cell types, and assess the consequences of altered KATP channel activity on lymphatic pump function. KATP 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 KATP 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 KATP 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 KATP 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 KATP 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.

T-type, but not L-type, voltage-gated calcium channels are dispensable for lymphatic pacemaking and spontaneous contractions.

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 Ca2+ channels (VGCCs) were implicated in this pacemaking mechanism, based on the effects of the reputedly selective T-type VGCC inhibitors mibefradil and Ni2+. 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 Cav3.1 and Cav3.2 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 Cav3.1-/-; 3.2-/- double knock-out lymphatic vessels responded similarly to mibefradil and Ni2+, 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 Cav3.1-/-; 3.2-/- double knock-out mice. In contrast, smooth-muscle specific deletion of the L-type VGCC, Cav1.2, 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 Ni2+ 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.

Ano1 mediates pressure-sensitive contraction frequency changes in mouse lymphatic collecting vessels.

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 Ca2+-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 "Ca2+ 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.

Mechanisms of Connexin-Related Lymphedema.

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.

Burn Injury-Associated MHCII+ Immune Cell Accumulation Around Lymphatic Vessels of the Mesentery and Increased Lymphatic Endothelial Permeability Are Blocked by Doxycycline Treatment.

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.

Differences in L-type Ca2+ channel activity partially underlie the regional dichotomy in pumping behavior by murine peripheral and visceral lymphatic vessels.

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 Ca2+ imaging, we assessed the role of L-type Ca2+ 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 Ca2+ 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 Ca2+ flashes, detected with a smooth muscle-specific endogenous Ca2+ reporter, that typically were coordinated over the length of the vessel. Similar to the membrane potential recordings, Ca2+ 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 Ca2+ transient amplitude and duration in both vessels and decreased time to peak Ca2+ 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 Ca2+ channel activity as revealed by the first measurements of murine lymphatic action potentials and contraction-associated Ca2+ transients.

Experimental Models Used to Assess Lymphatic Contractile Function.

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.

Calcium and electrical dynamics in lymphatic endothelium.

KEY POINTS: Endothelial cell function in resistance arteries integrates Ca2+ 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 Ca2+ with robust depolarization. Findings from Trpv4-/- mice and with computational modelling suggest that the initial mobilization of intracellular Ca2+ leads to influx of Ca2+ and Na+ through TRPV4 channels to evoke depolarization. Lymphatic endothelial cells lack the Ca2+ -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 Ca2+ ([Ca2+ ]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 Ca2+ and electrical signalling properties that facilitate lymph propulsion. To study electrical and intracellular Ca2+ 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 [Ca2+ ]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 Ca2+ -activated K+ (KCa ) channels as confirmed with PCR, persisted in the absence of extracellular Ca2+ , was abolished by LaCl3 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 KCa 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.

Demonstration and Analysis of the Suction Effect for Pumping Lymph from Tissue Beds at Subatmospheric Pressure.

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.