Lymphatic Muscle Cells Research Articles

What Is the Mechanism Underlying Pressure‐induced Chronotropy in Collecting Lymphatic Vessels?

The rhythmic contractions of lymphatic smooth muscle (LSM) cells enable lymph propulsion against adverse pressure gradients. These contractions are initiated by LSM action potentials (APs), generated by an ionic pacemaker, whose rate is sensitive to the transmural pressure difference. We recently showed that SM‐specific deletion of the Ca2+‐activated Cl− channel, anoctamin1 (Ano1), reduced the basal AP firing rate of mouse inguinal‐axillary lymphatic (IALs) vessels to ~25% of normal and abrogated pressure‐induced chronotopy. However, IALs are efferent vessels that show only a modest (2‐fold) increase in contraction frequency over the presumed physiological pressure range 0.5 – 8 cmH2O. In contrast, popliteal afferent lymphatics (PLs) exhibit an 8‐fold increase in chronotropy from 0.5 – 5 cmH2O and SM‐specific deletion of Ano1 attenuates but does not abolish their pressure‐induced chronotropy. Here we asked: 1) what other ionic mechanisms participate in pressure‐induced chronotropy of PLs? 2) what accounts for the residual, pressure‐independent pacemaking in IALs and PLs after Ano1 deletion?To address these questions we measured the frequency‐pressure relationship (F‐P) of cannulated, pressurized PLs from control mice and mice deficient in key mechanosensitive and second‐messenger‐gated ion channels implicated in arterial pressure sensing. SM‐specific deletion of the mechanosensitive cation channel Piezo1 had no significant effect on F‐P. Global deletion of the osmosensitive cation channel TRPV4, or the DAG‐gated cation channels TRPC3 or TRPC6, or both, had no significant effect on F‐P. TRPM4−/− mice were not available, but the selective TRPM4 inhibitors, CBA and NBA, had minimal effects on F‐P. SM‐specific deletion of the cation channel TRPP1 (Pkd2) had no significant effect on F‐P. Deletion of T‐type Ca2+ channels Cav3.1 and/or Cav3.2 (previously implicated in lymphatic pacemaking), had no significant effect on F‐P. However, SM‐specific deletion of IP3R1, the only IP3R isoform expressed in mouse LSM, produced a phenotype similar to Ano1 deletion: a reduction in basal frequency and blunting of the F‐P relationship, suggesting a pressure‐induced IP3 generation downstream from mechanosensitive G‐protein(s). We then tested the prominent arterial SM mechanosensitive GPCR, AT1R. The inverse agonist losartan had no effect on F‐P nor did global knock out of AT1bR, the only isoform expressed in mouse LSM. In conclusion, our results point to a GPCR mechanosensing mechanism, independent of AT1R, that generates IP3 in a pressure‐sensitive manner to activate Ano1 and regulate the cycling rate of the ionic pacemaker in lymphatic smooth muscle.Support or Funding InformationNIH‐HL‐122578

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.

Doxorubicin Activates Ryanodine Receptors in Rat Lymphatic Muscle Cells to Attenuate Rhythmic Contractions and Lymph Flow.

Doxorubicin is a risk factor for secondary lymphedema in cancer patients exposed to surgery or radiation. The risk is presumed to relate to its cytotoxicity. However, the present study provides initial evidence that doxorubicin directly inhibits lymph flow and this action appears distinct from its cytotoxic activity. We used real-time edge detection to track diameter changes in isolated rat mesenteric lymph vessels. Doxorubicin (0.5-20 μmol/l) progressively constricted lymph vessels and inhibited rhythmic contractions, reducing flow to 24.2% ± 7.7% of baseline. The inhibition of rhythmic contractions by doxorubicin paralleled a tonic rise in cytosolic Ca2+ concentration in lymphatic muscle cells, which was prevented by pharmacological antagonism of ryanodine receptors. Washout of doxorubicin partially restored lymph vessel contractions, implying a pharmacological effect. Subsequently, high-speed optical imaging was used to assess the effect of doxorubicin on rat mesenteric lymph flow in vivo. Superfusion of doxorubicin (0.05-10 μmol/l) maximally reduced volumetric lymph flow to 34% ± 11.6% of baseline. Likewise, doxorubicin (10 mg/kg) administered intravenously to establish clinically achievable plasma concentrations also maximally reduced volumetric lymph flow to 40.3% ± 6.0% of initial values. Our findings reveal that doxorubicin at plasma concentrations achieved during chemotherapy opens ryanodine receptors to induce "calcium leak" from the sarcoplasmic reticulum in lymphatic muscle cells and reduces lymph flow, an event linked to lymph vessel damage and the development of lymphedema. These results infer that pharmacological block of ryanodine receptors in lymphatic smooth muscle cells may mitigate secondary lymphedema in cancer patients subjected to doxorubicin chemotherapy. SIGNIFICANCE STATEMENT: Doxorubicin directly inhibits the rhythmic contractions of collecting lymph vessels and reduces lymph flow as a possible mechanism of secondary lymphedema, which is associated with the administration of anthracycline-based chemotherapy. The inhibitory effects of doxorubicin on rhythmic contractions and flow in isolated lymph vessels were prevented by pharmacological block of ryanodine receptors, thereby identifying the ryanodine receptor family of proteins as potential therapeutic targets for the development of new antilymphedema medications.

The effects of valve leaflet mechanics on lymphatic pumping assessed using numerical simulations

The lymphatic system contains intraluminal leaflet valves that function to bias lymph flow back towards the heart. These valves are present in the collecting lymphatic vessels, which generally have lymphatic muscle cells and can spontaneously pump fluid. Recent studies have shown that the valves are open at rest, can allow some backflow, and are a source of nitric oxide (NO). To investigate how these valves function as a mechanical valve and source of vasoactive species to optimize throughput, we developed a mathematical model that explicitly includes Ca2+ -modulated contractions, NO production and valve structures. The 2D lattice Boltzmann model includes an initial lymphatic vessel and a collecting lymphangion embedded in a porous tissue. The lymphangion segment has mechanically-active vessel walls and is flanked by deformable valves. Vessel wall motion is passively affected by fluid pressure, while active contractions are driven by intracellular Ca2+ fluxes. The model reproduces NO and Ca2+ dynamics, valve motion and fluid drainage from tissue. We find that valve structural properties have dramatic effects on performance, and that valves with a stiffer base and flexible tips produce more stable cycling. In agreement with experimental observations, the valves are a major source of NO. Once initiated, the contractions are spontaneous and self-sustained, and the system exhibits interesting non-linear dynamics. For example, increased fluid pressure in the tissue or decreased lymph pressure at the outlet of the system produces high shear stress and high levels of NO, which inhibits contractions. On the other hand, a high outlet pressure opposes the flow, increasing the luminal pressure and the radius of the vessel, which results in strong contractions in response to mechanical stretch of the wall. We also find that the location of contraction initiation is affected by the extent of backflow through the valves.

A Distinct Role of the Autonomic Nervous System in Modulating the Function of Lymphatic Vessels under Physiological and Tumor-Draining Conditions

SummaryLymphatic vessels (LVs) are important in the regulation of tissue fluid homeostasis and the pathogenesis of tumor progression. We investigated the innervation of LVs and the response to agonists and antagonists of the autonomic nervous system in vivo. While skin-draining collecting LVs express muscarinic, α1- and β2-adrenergic receptors on lymphatic endothelial cells and smooth muscle cells, intestinal lacteals express only β-adrenergic receptors and muscarinic receptors on their smooth muscle cells. Quantitative in vivo near-infrared imaging of the exposed flank-collecting LV revealed that muscarinic and α1-adrenergic agonists increased LV contractility, whereas activation of β2-adrenergic receptors inhibited contractility and initiated nitric oxide (NO)-dependent vasodilation. Tumor-draining LVs were expanded and showed a higher innervation density and contractility that was reduced by treatment with atropine, phentolamine, and, most potently, isoproterenol. These findings likely have clinical implications given the impact of lymphatic fluid drainage on intratumoral fluid pressure and thus drug delivery.

Methicillin‐resistant Staphylococcus aureus causes sustained collecting lymphatic vessel dysfunction

Methicillin‐resistant Staphylococcus aureus (MRSA) is a major cause of morbidity and mortality worldwide and is a frequent cause of skin and soft tissue infections (SSTIs). Lymphedema—fluid accumulation in tissue caused by impaired lymphatic vessel function—is a strong risk factor for SSTIs. SSTIs also frequently recur in patients and sometimes lead to acquired lymphedema. However, the mechanism of how SSTIs can be both the consequence and the cause of lymphatic vessel dysfunction is not known.Here, we used the endemic USA300 strain of community‐associated MRSA (CA‐MRSA), a common cause of serious bacterial infections in the United States, to establish a model of localized MRSA infection. We used intravital imaging to determine whether MRSA infections inhibit lymphatic vessel contractility and lymph flow. In vivo studies used C57BL/6, iNOS−/− C57BL/6, MyD88−/− C57BL/6, LysMgfp/gfp C57BL/6, and alpha SMAP‐DsRed/C57BL/6 mice to assess the effects of host‐derived molecules to lymphatic contractility after infection. Alpha SMA‐DsRed transgenic mice were visualized and immunofluorescence staining of alpha SMA+ lymphatic muscle cells (LMCs) of WT mice was performed to analyze LMC coverage of lymphatic vessels on multiple days after infection. To define the molecular mechanisms related to this response, we performed cell viability and cell lysis assays and measured killing of LMCs by MRSA‐conditioned supernatant from wild type and mutant MRSA strain(s). Similar experiments were performed using recombinant MRSA toxins.Intravital imaging in mice revealed an acute reduction in both lymphatic vessel contractility and lymph flow after localized MRSA infection. Moreover, chronic lymphatic impairment is observed long after MRSA is cleared and inflammation is resolved. Associated with decreased collecting lymphatic vessel function was the loss and disorganization of lymphatic muscle cells (LMCs), which are critical for lymphatic contraction. In vitro, incubation with MRSA‐conditioned supernatant led to LMC death. Proteomic analysis identified several accessory gene regulator (agr)–controlled MRSA exotoxins that contribute to LMC death. Infection with agr mutant MRSA resulted in sustained lymphatic function compared to animals infected with wild‐type MRSA.Our findings suggest that agr is a promising target to preserve lymphatic vessel function and promote immunity during SSTIs.Support or Funding InformationThis work was supported by the National Institutes of Health under award numbers R21AI097745, DP2OD008780, R01CA214913, and R01HL128168 to Timothy Padera. Dennis Jones was supported in part by the UNCF‐Merck Science Initiative Postdoctoral Fellowship, Burroughs Wellcome Fund Postdoctoral Enrichment Program Award, and NIH NCI F32CA183465.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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

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

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.

Linfangioleiomiomatosis pulmonar, una rara enfermedad pulmonar: presentación de un caso clínico

Introduction: Pulmonary lymphangioleiomyomatosis (LAM) is a rare and progressive disease; characterized by airway, lymphatic and blood vessels-smooth muscle cells excessive proliferation. Added to the abnormal cell growth, parenchymal cystic degeneration is present, which can be reflected initially as a asymptomatic course and can progress to severe gaseous exchange deterioration and fulminating respiratory insufficiency. Case description: A 41-year-old female patient with a clinical course consisting of occasional dry cough, associated with mild pleuritic pain on the right side of thorax. As no improvement was achieved, thoracic imaging study was performed, where a right pneumothorax was found. Tomography images showed multiple lung cystic lesions. Anatomopathological study reports structural changes compatible with LAM. Conclusion: Given the simplicity of the symptoms that LAM can debut with, its diagnostic confirmation is generated in advanced stages of the disease, when the important pulmonary damage leads to the appearance of clinical factors with greater impact on the general state of patients so early thoracic imaging studies gain vital importance.

Abstract ES11-1: Lymphedema management in 2018: Are we making any progress?

Abstract Lymphedema is a common and morbid complication of cancer treatment. To date, lymphedema treatment has been palliative aiming to prevent disease progression and improve symptoms of swelling. Development of novel therapies for lymphedema has been limited by the fact that the pathophysiology of the disease remains unknown. While it is true that lymphatic injury during surgery is an important initiating event, the mechanisms that regulate the development of the disease in a subset of patients remain unknown. Recent studies in our lab have suggested that the pathophysiology of lymphedema is related to inflammatory changes that occur following surgery. We have shown that CD4+ T cells play a key role in the pathology of lymphedema by orchestrating a variety of changes including tissue fibrosis, lymphatic leakiness, impaired collecting vessel pumping, and changes in the lymphatic smooth muscle cells. More importantly, in preclinical models we have shown that inhibition of this response can prevent development of lymphedema following surgery or reverse it once it has been initiated. These findings have led to novel treatments for lymphedema that may be effective either as standalone therapies or used in conjunction with surgical treatments for lymphedema. Citation Format: Mehrara B. Lymphedema management in 2018: Are we making any progress? [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr ES11-1.

In vitro model reveals a role for mechanical stretch in the remodeling response of lymphatic muscle cells.

Using primary LMCs in vitro, we sought to characterize the impact of LMC remodeling on their functional and molecular response to mechanical loading and culture conditions. Primary "wounded leg" LMCs were derived from the hindlimb of three sheep who underwent lymphatic injury 6weeks prior, while "control leg" LMCs were derived from the contralateral, unwounded, limb. Function of the LMCs was characterized in response to media of variable levels of serum (10% vs 0.2%) and glucose (4.5 vs 1g/L). Functional and proteomic data were evaluated in LMCs exposed to cyclic stretch (0.1Hz, 7.5% elongation) for 1week. LMCs were sensitive to changes in serum levels, significantly reducing overall activity and collagen synthesis under low serum conditions. LMCs from the remodeled vessel had higher baseline levels of metabolic activity but not collagen synthesis. Cyclic loading induced cellular alignment perpendicular to the axis of stretch and alterations in signaling pathways associated with metabolism. Remodeled LMCs had consistently higher levels of metabolic activity and were more resistant to strain-induced apoptosis. LMCs exist on a functional spectrum, becoming more active in response to stretching and maintaining phenotypic remodeling in response to local lymphatic/tissue damage.

Mechanisms of Connexin-Related Lymphedema

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. To determine which Cx isoforms in LMCs and lymphatic endothelial cells are required for the entrainment of lymphatic contraction waves and efficient lymph transport. 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. 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.

In This Issue

HomeCirculation ResearchVol. 123, No. 8In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published27 Sep 2018https://doi.org/10.1161/RES.0000000000000229Circulation Research. 2018;123:925is related toIn Vivo Ryr2 Editing Corrects Catecholaminergic Polymorphic Ventricular TachycardiaSerum Bioavailable and Free 25-Hydroxyvitamin D Levels, but Not Its Total Level, Are Associated With the Risk of Mortality in Patients With Coronary Artery Diseaseis related toMechanisms of Connexin-Related LymphedemaGenome Editing Cures CPVT in Mice (p 953)Download figureDownload PowerPointGene editing cures deadly tachycardia in mutant mice, report Pan et al.Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited condition characterized by arrhythmias brought on by stress or exercise. The episodes of fast and irregular heartbeats tend to start in childhood and can cause dizziness, fainting, and even sudden death. Although β-blockers are often used to treat CPVT, many patients continue to exhibit symptoms. Nearly two thirds of CPVT cases are caused by mutations to the calcium channel protein ryanodine receptor 2 (RYR2). Now, Pan and colleagues show that editing of the Ryr2 gene in mice carrying a heterozygous Ryr2 mutation can eliminate their susceptibility to electrical stimulation-induced tachycardia. They injected newborn mice with the editing vector, or a control, and then 5 to 6 weeks later they subjected the mice to cardiac electrical stimulation. They found that while 71% of the unedited mutant mice developed tachycardia in response to the stimulation, none of the treated animals did. They saw no off-target effects and their gene sequencing experiments confirmed specific editing of the mutant Ryr2 allele. These promising results pave the way for further, clinically translatable RYR2 gene-editing experiments.Mechanisms of Connexin-Related Lymphedema (p 964)Download figureDownload PowerPointCastorena-Gonzalez et al investigate the possible mechanisms underlying connexin-related primary lymphadema.Connexins are transmembrane proteins that form gap junctions for cell-cell communication. In humans, mutations in the connexin proteins Cx43 and Cx47 lead to the development of primary lymphedema. However, the underlying pathogenic mechanisms remain unknown. In mice, deletion of these connexins cause lymph valve defects, but such pathology has not been reported in humans. Moreover, evidence indicates connexin defects may cause aberrant electrical coupling and unsynchronized contractions in lymphatic smooth muscle cells (LMCs), which might impair proper fluid flow. Now, however, investigations by Castorena-Gonzalez and colleagues have largely ruled-out this alternative theory. Having identified the major connexin isoforms in mouse LMCs—Cx43, Cx47, Cx37, and Cx45—the team created animals in which these proteins were genetically deleted. Of these isoforms, only Cx45 was shown to be necessary for proper contractions of lymph vessels ex vivo. Hence, it seems unlikely that in humans Cx43 or Cx47 deficiency would cause contraction issues. Instead, perhaps hitherto unidentified valve problems are to blame.Bioavailable Vitamin D and Mortality (p 996)Download figureDownload PowerPointYu et al suggest a more meaningful method for measuring serum vitamin D.Due in part to indoor lifestyles and the need to protect skin from damaging ultraviolet rays, vitamin D deficiency has become a widespread health issue. Vitamin D deficiency has been linked to coronary artery disease; however, clinical trials with vitamin D supplementation have yielded uncertain results. Yu and colleagues now argue that standard measurements of vitamin D may have confounded such trial results. They point out that almost 90% of vitamin D in the body is attached to vitamin D–binding protein (DBP), with the remainder being either free or loosely attached to albumin and thus available to enter cells and perform functions. It is this bioavailable form, rather than total vitamin D, that should, therefore, be measured, the authors say. They drew this conclusion from their analysis of bioavailable and total vitamin D levels in 1387 coronary artery patients who were followed for an average of 6 years. The study revealed a significant inverse correlation between levels of bioavailable vitamin D and both all-cause and cardiovascular-linked mortality. No such correlation was observed with total vitamin D. These findings suggest that bioavailable vitamin D may be protective against adverse effects in coronary artery disease, and that measuring this form of the vitamin could help predict patient prognosis. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesIn Vivo Ryr2 Editing Corrects Catecholaminergic Polymorphic Ventricular TachycardiaXiaolu Pan, et al. Circulation Research. 2018;123:953-963Serum Bioavailable and Free 25-Hydroxyvitamin D Levels, but Not Its Total Level, Are Associated With the Risk of Mortality in Patients With Coronary Artery DiseaseChao Yu, et al. Circulation Research. 2018;123:996-1007Mechanisms of Connexin-Related LymphedemaJorge A. Castorena-Gonzalez, et al. Circulation Research. 2018;123:964-985 September 28, 2018Vol 123, Issue 8 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000229PMID: 30355044 Originally publishedSeptember 27, 2018 PDF download Advertisement

IL-1β reduces cardiac lymphatic muscle contraction via COX-2 and PGE2 induction: Potential role in myocarditis

The role of lymphatic vessels in myocarditis is largely unknown, while it has been shown to play a key role in other inflammatory diseases. We aimed to investigate the role of lymphatic vessels in myocarditis using in vivo model induced with Theiler’s murine encephalomyelitis virus (TMEV) and in vitro model with rat cardiac lymphatic muscle cells (RCLMC). In the TMEV model, we found that upregulation of a set of inflammatory mediator genes, including interleukin (IL)-1β, tumor necrosis factor (TNF)-αand COX-2 were associated with disease activity. Thus, using in vitro collagen gel contraction assays, we decided to clarify the role(s) of these mediators by testing contractility of RCLMC in response to IL-1β and TNF-α individually and in combination, in the presence or absence of: IL-1 receptor antagonist (Anakinra); cyclooxygenase (COX) inhibitors inhibitors (TFAP, diclofenac and DuP-697). IL-1β impaired RCLMC contractility dose-dependently, while co-incubation with both IL-1β and TNF-α exhibited synergistic effects in decreasing RCLMC contractility with increased COX-2 expression. Anakinra maintained RCLMC contractility; Anakinra blocked the mobilization of COX-2 induced by IL-1β with or without TNF-α. COX-2 inhibition blocked the IL-1β-mediated decrease in RCLMC contractility. Mechanistically, we found that IL-1β increased prostaglandin (PG) E2 release dose-dependently, while Anakinra blocked IL-1β mediated PGE2 release. Using prostaglandin E receptor 4 (EP4) receptor antagonist, we demonstrated that EP4 receptor blockade maintained RCLMC contractility following IL-1β exposure. Our results indicate that IL-1β reduces RCLMC contractility via COX-2/PGE2 signaling with synergistic cooperation by TNF-α. These pathways may help provoke inflammatory mediator accumulation within the heart, driving progression from acute myocarditis into dilated cardiomyopathy.

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. 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.

Spontaneous and α-adrenoceptor-induced contractility in human collecting lymphatic vessels require chloride.

Human lymphatic vessels are myogenically active and respond to sympathetic stimulation. The role of various cations in this behavior has recently been investigated, but whether the anion Cl- is essential is unclear. With ethical approval and informed consent, human thoracic duct and mesenteric lymphatic vessels were obtained from surgical patients. Spontaneous or norepinephrine-induced isometric force production from isolated vessels was measured by wire myography; the transmembrane Cl- gradient and Cl- channels were investigated by substitution of extracellular Cl- with the impermeant anion aspartate and inhibition of Cl- transport and channels with the clinical diuretics furosemide and bendroflumethiazide as well as DIDS and 5-nitro-2-(3-phenylpropylamino)benzoic acid. The molecular expression of Ca2+-activated Cl- channels was investigated by RT-PCR, and proteins were localized using immunoreactivity. Spontaneous and norepinephrine-induced contractility in human lymphatic vessels was highly abrogated after Cl- substitution with aspartate. About 100-300 µM DIDS or 5-nitro-2-(3-phenylpropylamino)benzoic acid inhibited spontaneous contractile behavior. Norepinephrine-stimulated tone was furthermore markedly abrogated by 200 µM DIDS. Furosemide lowered only spontaneous constrictions, whereas bendroflumethiazide had nonspecific inhibitory effects. Consistent expression of transmembrane member 16A [TMEM16A (anoctamin-1)] was found in both the thoracic duct and mesenteric lymphatic vessels, and immunoreactivity with different antibodies localized TMEM16A to lymphatic smooth muscle cells and interstitial cells. The significant change in contractile function observed with inhibitors and anion substitution suggests that Cl- movement over the plasma membrane of lymphatic myocytes is integral for spontaneous and α-adrenoceptor-evoked contractility in human collecting lymphatic vessels. Consistent detection and localization of TMEM16A to myocytes suggests that this channel could play a major functional role. NEW & NOTEWORTHY In this study, we report the first observations of Cl- being a critical ionic component of spontaneous and agonist-evoked contractility in human lymphatics. The most consistently expressed Ca2+-activated Cl- channel gene in the human thoracic duct and mesenteric lymphatic vessels appears to be transmembrane member 16A, suggesting that this channel plays a major role.

SUBSTANCE P REGULATES INFLAMMATORY PATHWAYS IN LYMPHATIC MUSCLE

Substance P (SP), a neuropeptide that acts through the neurokinin receptors is associated with the sensory innervation of lymphoid tissue and modulates lymphatic contractile function and lymph flow. SP signaling is associated with several inflammatory pathologies. The role of lymphatics in body fluid homeostasis, lipid absorption and inflammation and immunity rely on the contractility of the lymphatic muscle cells (LMCs). We have shown that lymphatic contractile dysfunction is associated with perturbations in inflammatory mechanisms and that SP activates both inflammatory and contractile pathways in lymphatics. However, the specific inflammatory pathways and the resultant cytokines activated by SP in LMCs is unknown. We hypothesized that SP stimulates the pro‐survival AKT and MAPK pathways, and increases pro‐inflammatory cytokines. Cultured rat mesenteric LMCs were treated with SP (1 mM) and total RNA and protein were isolated at different time points (6 hr and 24 hr). Western analysis was carried out to detect the total and phosphorylated forms of AKT, JNK, GSK3b, b‐catenin, FOXO1 and PDCD4. b‐actin was used as control. The relative levels of inflammatory cytokines and chemokines, and specific inflammatory microRNAs (miRNAs) were analyzed by real time PCR. Our results indicate that SP activates the AKT and JNK pathways to significantly increase inflammatory cytokine and chemokine expression, and induces expression of miR 17–5p, miR19b and miR186 in the LMCs. Additionally, SP stimulates expression of genes, involved in lymphatic remodeling. Taken together our data demonstrate that SP activates multiple inflammatory signaling pathways in the LMCs that contribute to an inflammatory response in lymphatics during pathological conditions.Support or Funding InformationAmerican Heart AssociationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Improved methods for characterizing ion channel expression in smooth muscle, endothelial and adventitial cells from collecting lymphatic vessels

Lymph is propelled centrally by collecting lymphatic vessels though active, twitch‐like contractions, which are initiated by a pacemaker that generates action potentials to drive calcium influx through voltage‐gated calcium channels in lymphatic smooth muscle cells (LMCs). Whether the pacemaker is intrinsic to LMCs or to another cell type in the vessel wall is not yet established. Both the frequency and amplitude of lymphatic contractions are regulated in turn by a number of additional factors, including the transmural pressure difference, wall shear stress, neurotransmitters, locally‐ and remotely‐generated vasoactive factors, and endothelial‐derived products. Difficulties in identifying the complex interactions of the various cell types within the vessel wall limit discernment of the mechanisms underlying lymphatic pacemaking and the factors that modulate it. Cells within the wall include LMCs, lymphatic endothelial cells (LECs), dendritic cells, macrophages, mast cells, fibroblasts, nerve endings and possibly glia. We developed methods for purifying specific cell populations and determining the expression and function of important ion channels with a focus on identifying the ion channels that control and modulate lymphatic function. Our strategy was to utilize fluorescent reporter mice (SMMHCGFP, Prox1GFP and PDGFRaGFP, respectively) to 1) identify the distribution of LMCs, LECs and a population of adventitial fibroblast‐like cells in mouse inguinal‐axillary lymphatic (IALs) vessels; 2) enzymatically dissociate IALs and utilize fluorescent activated cell sorting of GFP+ cells to obtain pure cell populations for transcriptional analysis of key ion channels; 3) confirm the presence and functionality of identified ion channels through whole cell patch clamp of GFP+ cells. LMCs expressed message for SMA, TMEM16A, SK3, BK and Cav1.2 but were negative for eNOS, Prox1 and VE‐caderin. LECs were positive for VE‐cadherin and eNOS but negative for SMA, SMMHC, PDGFRa, Cav1.2, TMEM16A and all SK‐IK‐BK channels. PDGFRa+ cells were positive for VE‐cadherin, SMA, SMMHC, TMEM16A, Cav1.2 and SK1, but negative for eNOS and all other SK‐IK‐BK channels. Single GFP+ cells used for patch clamping revealed spindle‐shaped LMCs expressing functional TMEM16A, Cav1.2, Cav3 and variable BK currents, LECs (also spindle‐shaped) that did not exhibit inward cation current or SK‐IK current, and rounded PDGFRa+ cells with SK currents. Our techniques improve on whole‐vessel and explant analyses, align with the results of recent functional studies, and show the feasibility of using these methods to more accurately determine the functional roles of the various cell populations in the lymphatic wall.Support or Funding InformationNIH HL122578This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lymphatics limp along after MRSA

Vascular Biology Lymphedema is associated with skin and soft tissue infections, and both can be recurring, causing continual suffering in affected patients. To better understand the relationship between bacterial infections and lymphedema, Jones et al. used intravital imaging. They examined the lymphatics of mice infected with MRSA (methicillin-resistant Staphylococcus aureus ) and observed lymphatic muscle cell death, which led to prolonged dysfunction months after the bacteria had been cleared. In vitro experiments with human cells indicated bacterial toxins were responsible for damaging the lymphatic muscle cells. The findings suggest that these bacterial toxins could be targeted in patients to interrupt this brutal cycle. Sci. Transl. Med. 10 , eaam7964 (2018).

Methicillin-resistant Staphylococcus aureus causes sustained collecting lymphatic vessel dysfunction.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of morbidity and mortality worldwide and is a frequent cause of skin and soft tissue infections (SSTIs). Lymphedema-fluid accumulation in tissue caused by impaired lymphatic vessel function-is a strong risk factor for SSTIs. SSTIs also frequently recur in patients and sometimes lead to acquired lymphedema. However, the mechanism of how SSTIs can be both the consequence and the cause of lymphatic vessel dysfunction is not known. Intravital imaging in mice revealed an acute reduction in both lymphatic vessel contractility and lymph flow after localized MRSA infection. Moreover, chronic lymphatic impairment is observed long after MRSA is cleared and inflammation is resolved. Associated with decreased collecting lymphatic vessel function was the loss and disorganization of lymphatic muscle cells (LMCs), which are critical for lymphatic contraction. In vitro, incubation with MRSA-conditioned supernatant led to LMC death. Proteomic analysis identified several accessory gene regulator (agr)-controlled MRSA exotoxins that contribute to LMC death. Infection with agr mutant MRSA resulted in sustained lymphatic function compared to animals infected with wild-type MRSA. Our findings suggest that agr is a promising target to preserve lymphatic vessel function and promote immunity during SSTIs.