Biology subjects

Castorena-Gonzalez, J. A.

Publications and source records attributed to Castorena-Gonzalez, J. A..

5 recordsLinked to original sources

bioRxiv2026

Cholesterol-Mediated Modulation of Collecting Lymphatic Vessel Contractility: Exploring Cholesterol Depletion as a Therapeutic Alternative to Improve Lymphatic Function in Hypercholesterolemia

Globally, hypercholesterolemia affects over 20% of the population; and while many studies have examined its impact on cardiovascular health, little is known about its effects on the lymphatic system. In mice, hypercholesterolemia has been linked to multiple aspects of lymphatic dysfunction; and a recent study demonstrated that cholesterol depletion by cyclodextrins promoted lymphatic vessel regeneration and restored lymphatic drainage in mouse models of lymphedema. Collecting lymphatic vessels rely on the spontaneous and highly entrained contractions of lymphatic muscle cells (LMCs) and competent unidirectional on-way valves to propel lymph forward. Critical to lymphatic pacemaking and contractility is the proper functioning of ion channels, which are known to be modulated by the cholesterol content in the plasma membrane. Therefore, we sought to understand the role cholesterol plays in regulating lymphatic contractility. The effects of cholesterol depletion by the cyclodextrins M{beta}CD and HP{beta}CD were assessed in cannulated and pressurized inguinal-axillary collecting lymphatic vessels (CLVs) from C57BL6/J (WT) mice. Noteworthy, studies have shown that HP{beta}CD is safe for human use, and in fact, it is commonly used as a drug excipient. Acute treatment with both cyclodextrins significantly increased the pumping capacity of CLVs, as demonstrated by the increased contraction amplitudes by ~50{+/-}12% and calculated fluid volume displacement by each contraction by ~35{+/-}11%. Calcium imaging demonstrated that HP{beta}CD increased the amplitude and duration of the large Cav1.2-mediated calcium events (termed calcium flashes. In contrast, cholesterol supplementation by incubation with BODIPY-cholesterol, which presumably incorporates cholesterol into the cell membrane, significantly impaired the contractile activity of CLVs compared to controls by decreasing contraction amplitude (control: 42{+/-}2 {micro}m versus BODIPY-cholesterol: 20{+/-}7 {micro}m) and calculated fluid volume displacement (control: 9.2{+/-}3.9 nL versus BODIPY cholesterol: 3.3{+/-}1.2 nL) which were significantly restored with subsequent cholesterol depletion using HP{beta}CD (amplitude: 36{+/-}11 {micro}m, volume displacement: 5.5{+/-}2.4 nL). Similarly, treatment with HP{beta}CD significantly improved the contractile capacity of dysfunctional CLVs isolated from hypercholesterolemic ApoEKO mice. In conclusion, changes to cell membrane cholesterol content acutely and significantly altered CLV contractility with depletion improving contractility associated with recruitment of voltage-gated Cav1.2 channels in lymphatic muscle cells (LMCs). Future studies from our lab will determine whether pharmacological depletion of membrane cholesterol can be therapeutic strategy to improve and/or restore lymphatic contractile function in secondary lymphedema, including obesity/hypercholesterolemia-induced and cancer-related lymphedemas.

physiology↗

bioRxiv2025

GLP-1R Agonism Directly Improves the Pumping Capacity of Murine Collecting Lymphatic Vessels

BackgroundGlucagon-like peptide-1 receptor (GLP-1R) agonists have recently been suggested as effective therapies to treat or reduce the risk of developing secondary lymphedema in patients with obesity; however, it is unknown whether the observed improvement in lymphatic function is solely due to weight loss-associated systemic benefits or in synergy with a lymphatic-specific effect of these pharmacological therapies. MethodsWe assessed the expression and localization GLP-1Rs in and around the lymphatic vasculature by single-cell RNA sequencing and fluorescence confocal microscopy. Using pressure myography we evaluated the direct effects of GLP-1R agonist, semaglutide, on modulating the contractile activity of lymphatic vessels from healthy wild-type (WT) mice, as well as lymphatics from diet-induced obese (DIO) WT mice, and hypercholesterolemic ApoE KO mice. ResultsExpression of Glp1r (encoding GLP-1Rs) was detected solely in LECs and was highly enriched in LECs from collecting lymphatics but absent in LECs from capillary regions. Pharmacological activation of GLP-1Rs using semaglutide led to robust vasodilation and an increase in the pumping capacity of isolated collecting lymphatics from WT, DIO, and ApoE KO mice. Compared to WT controls, lymphatics from ApoE KO mice displayed significant contractile dysfunction, which was restored with semaglutide. The GLP-1R-mediated response was in part facilitated by nitric oxide (NO), NADPH oxidase-mediated reactive oxygen species (ROS), and potentially vasodilatory prostanoids. ConclusionsOur results revealed a direct, beneficial effect of GLP-1R agonism on lymphatic pumping capacity mediated by robust vasodilation, allowing lymphatics to accommodate larger fluid volumes, while maintaining strong and highly efficient contractions. Our observations implicated NO, ROS, and potentially vasodilatory prostanoids in the underlying mechanism; however, additional signaling components remain to be elucidated. These findings support recent clinical reports and further suggest that GLP-1R agonism could be an effective therapy for improving lymphatic contractile function in secondary lymphedema.

physiology↗

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

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↗