Modelling pacemaker oscillations in lymphatic muscle cells: lengthened action potentials by two distinct system effects.

Lymphatic system failures contribute to cardiovascular and various other diseases. A critical function of the lymphatic vascular system is the active pumping of fluid from the interstitium back into the blood circulation by periodic contractions of lymphatic muscle cells (LMCs) in the vessel walls. As in cardiac pacemaking, these periodic contractions can be interpreted as occurring due to linked pacemaker oscillations in the LMC membrane potential (M-clock) and calcium concentration (C-clock).

Acute Metabolic Stress Induces Lymphatic Dysfunction Through KATP Channel Activation.

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.

Connexin-45 is expressed in mouse lymphatic endothelium and required for lymphatic valve function.

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.

TRPV4-Expressing Tissue-Resident Macrophages Regulate the Function of Collecting Lymphatic Vessels via Thromboxane A2 Receptors in Lymphatic Muscle Cells.

Rationale: TRPV4 channels are critical regulators of blood vascular function and have been shown to be dysregulated in many disease conditions in association with inflammation and tissue fibrosis. These are key features in the pathophysiology of lymphatic system diseases, including lymphedema and lipedema; however, the role of TRPV4 channels in the lymphatic system remains largely unexplored. TRPV4 channels are calcium permeable, non-selective cation channels that are activated by diverse stimuli, including shear stress, stretch, temperature, and cell metabolites, which may regulate lymphatic contractile function.

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

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

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.

Developmental progression of lymphatic valve morphology and function.

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.

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.

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.

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 (Ca3) channels (Ca3.1, Ca3.2) that are functionally expressed in murine lymphatic muscle cells; however, contractile tests of lymphatic vessels from single and double Ca3 knock-out (DKO) mice, exhibited nearly identical parameters of spontaneous twitch contractions as wild-type (WT) vessels, suggesting that Ca3 channels play no significant role.

Characterization of the cellular components of mouse collecting lymphatic vessels reveals that lymphatic muscle cells are the innate pacemaker cells regulating lymphatic contractions.

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 transients; and depolarization-induced propagated contractions.

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

The widely-used, tamoxifen-inducible, smooth muscle (SM)-specific Cre, , 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 ( ), that has been recently developed and characterized, to assess the effects of deletion on mouse lymphatic SM function. (the L-type voltage-gated calcium channel) is essential for lymphatic pacemaking and contraction and its deletion using either or abolished spontaneous lymphatic contractions.

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

All current smooth muscle cell (SMC) mice similarly recombine floxed alleles in vascular and visceral SMCs. Here, we present an knock-in mouse and compare its activity with a mouse. Both drivers demonstrate equivalent recombination in vascular SMCs.

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.

Modelling the coupling of the M-clock and C-clock in lymphatic muscle cells.

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.

Kir6.1-dependent K 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 K channel activator pinacidil. K channel Kir6.1 and SUR2B subunits are expressed in mouse lymphatic smooth muscle (LSM) and form functional K channels as verified by electrophysiological techniques.

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 Ca channels (VGCCs) were implicated in this pacemaking mechanism, based on the effects of the reputedly selective T-type VGCC inhibitors mibefradil and Ni.

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.

Electrical Communication in Lymphangions.

Contractions of lymphangions, i.e., the segment between two one-way lymphatic valves, generate the pressure gradients that propel lymph back to the circulation.