Biology subjects

Zawieja, S. D.

Publications and source records attributed to Zawieja, S. D..

7 recordsLinked to original sources

bioRxiv2025

Roles of G-protein coupled receptors and mechanosensitive ion channels in pressure-induced chronotropy of lymphatic vessels

Active lymph pumping relies on the spontaneous contractions of collecting lymphatic vessels, whose contraction frequencies are exquisitely sensitive to changes in intraluminal pressure. This homeostatic and mechanosensitive mechanism, termed pressure-induced lymphatic chronotropy, enables lymph transport to be matched to the filling state of the lymphatic capillary network. The mechanistic basis of pressure-induced chronotropy was investigated using ex vivo contraction assays of mouse popliteal collecting vessels, in which contraction frequency increases >10-fold with pressure changed from 0.5 to 5 cmH2O. The contractile, electrophysiological and transcriptional similarities between lymphatic muscle cells (LMCs) and arterial smooth muscle led us to hypothesize that pressure-dependent chronotropy shares a parallel signaling process with pressure-induced arterial depolarization/constriction. Thus, we probed two major mechanisms: 1) pressure-induced activation of mechanosensitive cation channels, including TRPC6, TRPM4, PKD1/2, TRPV2 and ENaC, and 2) mechano-activation of GNAQ/GNA11-coupled G-protein receptors (GPCRs) that would generate second messengers to activate those channels. Contraction assays were combined with scRNAseq analysis of the respective targets, with maximum use made of transgenic mice to avoid non-specific effects of pharmacological inhibitors, particularly those used to block TRP channels. Our findings rule out significant roles for the above TRP channels and other putative mechanosensitive channels implicated in arterial myogenic constriction, as well as channels implicated in ionic pacemaking of other tissues. In contrast, smooth-muscle specific knock out or inhibition of ANO1 or IP3R1 significantly blunted the effect of pressure on frequency. Pressure-induced chronotropy was suppressed by [~]70-90% at all pressures in GNAQ/GNA11 double knockout vessels, but with responsiveness partially maintained at pressures above 5 cmH2O. Pressure-induced chronotropy was also suppressed after acute Gq/11 inhibition with YM254890, but was normal in vessels from GNA12/GNA13 double knock out mice. These results support a scheme whereby mechano-activation of one or more GNAQ/GNA11-coupled GPCRs generates IP3, which induces SR Ca2+ release through IP3R1 and drives depolarization through the activation of ANO1 Cl- channels. The major GPCRs expressed in LMCs were subsequently identified and ranked by scRNAseq analysis but knock out or pharmacological inhibition of each of the top 7 candidates failed to significantly affect pressure-induced chronotropy. Our results strongly implicate one or more GNAQ/GNA11-coupled GPCRs in mediating this homeostatic process; however, the specific mechanosensitive GPCRs remain to be identified.

physiology↗

bioRxiv2024

S1PR1 Regulates Lymphatic Valve Development And Prevents Ileitis-Independent Tertiary Lymphoid Organ Formation

Lymphatic vessels function throughout the body to drain interstitial fluids. Efficient lymph flow is ensured by lymphatic valves (LVs). However, the mechanisms that regulate LV development are incompletely understood. Here, we show that the deletion of the GPCR sphingosine 1-phosphate receptor-1 (S1PR1) from lymphatic endothelial cells (LECs) results in fewer LVs. Interestingly, LVs that remained in the terminal-ileum draining lymphatic vessels were specifically dysfunctional, and tertiary lymphoid organs (TLOs) formed in this location. TLOs in the terminal ileum are associated with ileitis in humans and mice. However, mice lacking S1PR1 did not develop obvious characteristics of ileitis. Sphingosine kinases 1 and 2 (SPHK1/2) are required for the synthesis of S1P, the ligand of S1PR1. Mice that lack Sphk1/2 in LECs recapitulate the LV and TLO phenotypes of mice that lack S1PR1. Mechanistically, S1PR1 regulates shear stress signaling and the expression of the valve-regulatory molecules FOXC2 and connexin-37. Importantly, Foxc2+/- mice, a model for lymphedema-distichiasis syndrome, also develop TLOs in the terminal ileum. Thus, we have discovered S1PR1 as a previously unknown regulator of LV and TLO development. We also suggest that TLOs are a sign of subclinical inflammation that can form due to lymphatic disorders in the absence of ileitis.

developmental biology↗

bioRxiv2024

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

RationaleTRPV4 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. ObjectiveTo characterize the expression of TRPV4 channels in collecting lymphatic vessels and to determine the extent to which these channels regulate the contractile function of lymphatics. Methods and ResultsPressure myography on intact, isolated, and cannulated lymphatic vessels showed that pharmacological activation of TRPV4 channels with GSK1016790A (GSK101) led to contractile dysregulation. The response to GSK101 was multiphasic and included, 1) initial robust constriction that was sustained for [≥]1 minute and in some instances remained for [≥]4 minutes; and 2) subsequent vasodilation and partial or complete inhibition of lymphatic contractions associated with release of nitric oxide. The functional response to activation of TRPV4 channels displayed differences across lymphatics from four anatomical regions, but these differences were consistent across different species (mouse, rat, and non-human primate). Importantly, similar responses were observed following activation of TRPV4 channels in arterioles. The initial and sustained constriction was prevented with the COX inhibitor, indomethacin. We generated a controlled and spatially defined single-cell RNA sequencing (scRNAseq) dataset from intact and microdissected collecting lymphatic vessels. Our data uncovered a subset of macrophages displaying the highest expression of Trpv4 compared to other cell types within and surrounding the lymphatic vessel wall. These macrophages displayed a transcriptomic profile consistent with that of tissue-resident macrophages (TRMs), including differential expression of Lyve1, Cd163, Folr2, Mrc1, Ccl8, Apoe, Cd209f, Cd209d, and Cd209g; and at least half of these macrophages also expressed Timd4. This subset of macrophages also highly expressed Txa2s, which encodes the thromboxane A2 (TXA2) synthase. Inhibition of TXA2 receptors (TXA2Rs) prevented TRPV4-mediated contractile dysregulation. TXA2R activation on LMCs caused an increase in mobilization of calcium from intracellular stores through Ip3 receptors which promoted store operated calcium entry and vasoconstriction. ConclusionsClinical studies have linked cancer-related lymphedema with an increased infiltration of macrophages. While these macrophages have known anti-inflammatory and pro-lymphangiogenic roles, as well as promote tissue repair, our results point to detrimental effects to the pumping capacity of collecting lymphatic vessels mediated by activation of TRPV4 channels in macrophages. Pharmacological targeting of TRPV4 channels in LYVE1-expressing macrophages or pharmacological targeting of TXA2Rs may offer novel therapeutic strategies to improve lymphatic pumping function and lymph transport in lymphedema. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/595189v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1ce67fforg.highwire.dtl.DTLVardef@dd1beorg.highwire.dtl.DTLVardef@1454c78org.highwire.dtl.DTLVardef@9f76a1_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2023

Lymphatic muscle cells are the innate pacemaker cells regulating mouse lymphatic collecting vessel contractions

Collecting lymphatic vessels (cLVs) exhibit spontaneous contractions with a pressure-dependent frequency, but the identity of the lymphatic pacemaker cell is still debated. 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. We employed inducible Cre (iCre) mouse models to target specific cell populations including c-kitCreERT2 to target interstitial cells of Cajal like cells; Pdgfr{beta}CreERT2to target pericyte-like cells; PdgfrCreERTMto target CD34+ adventitial cells; and Myh11CreERT2to target lymphatic muscle cells (LMCs) directly. These inducible Cre lines were crossed to the fluorescent reporter ROSA26mT/mG, the genetically encoded Ca2+ sensor GCaMP6f, and the light- activated cation channel rhodopsin2 (ChR2). Only LMCs consistently, but heterogeneously, displayed spontaneous Ca2+ events during the diastolic period of the contraction cycle, and whose frequency was modulated in a pressure-dependent manner. Further, optogenetic depolarization with ChR2 only induced propagated contractions in LMCs. Membrane potential recordings in LMCs demonstrated that the rate of diastolic depolarization significantly correlated with contraction frequency. These findings support the conclusion that LMCs, or a subset of LMCs, are responsible for mouse cLV pacemaking. ImpactLymphatic muscle cells, but not CD34+ adventitial cells, exhibited pacemaker behaviors such as pressure-dependent depolarization, pressure-dependent calcium mobilization during diastole, and propagated contraction waves induced by focal, optogenetic depolarization via enforced channel- rhodopsin2.

physiology↗

bioRxiv2022

A dual-clock-driven model emulating the effects of either Ano1 or IP3R knock-out on lymphatic muscle cell pace-making

Lymphoedema, a common dysfunction of the lymphatic system, results in fluid accumulating between cells. Fluid return through the lymphatic vascular system is primarily provided by contractions of muscle cells in the walls of lymphatic vessels, driven by electrochemical oscillations causing rhythmic action potentials and associated surges in intracellular calcium ion concentration. There is 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, and thus both driving the action potentials. This extension results from modifications to the model for the IP3 receptor, a key C-clock mechanism. The synchronized dual-driving clock behaviour enables the model to match IP3 receptor knock-out data, resolving an issue with previous models. We also use phase-plane analysis to explain the mechanisms for the dual-clock coupling. The model has the potential to help determine mechanisms and find targets for pharmacological treatment of lymphoedema.

biophysics↗

bioRxiv2022

An Inducible Cre Mouse with Preferential Activity in Vascular Smooth Muscle Evades a Previously Lethal Intestinal Phenotype

All smooth muscle cell (SMC) restricted Cre mice recombine floxed alleles in vascular and visceral SMCs. We generated a new tamoxifen-inducible CreERT2 mouse, Itga8-CreERT2, and compared its activity to the widely used Myh11-CreERT2 mouse. Both CreERT2 mice showed similar activity in vascular SMCs; however, Itga8-CreERT2 displayed limited activity in visceral SMC-containing tissues (e.g., intestine). Myh11-CreERT2 (but not Itga8-CreERT2) mice displayed high levels of CreERT2 protein, tamoxifen-independent activity, and an altered transcriptome. Whereas Myh11-CreERT2-mediated knockout of Srf resulted in a lethal intestinal phenotype, loss of Srf with Itga8-CreERT2 (SrfItga8) revealed viable mice with attenuated vascular SMC contractile gene expression, but no evidence of intestinal pathology. Male and female SrfItga8 mice presented with vascular contractile incompetence; however, only male SrfItga8 mice showed systemic changes in blood pressure. These results establish the Itga8-CreERT2 mouse as an alternative to existing SMC Cre strains, including Myh11-CreERT2, where SMC gene loss results in visceral myopathies that obfuscate accurate phenotyping in vascular SMCs.

genetics↗

bioRxiv2021

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

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

biophysics↗