Hypercholesterolemia affects a large portion of the population, yet its effects on the lymphatic system have received comparatively little attention. Collecting lymphatic vessels (CLVs) depend on synchronized contractions of lymphatic muscle cells (LMCs) and one-way valves to propel lymph. The function of ion channels that underlie lymphatic pacemaking and contractility is influenced by the cholesterol content of the plasma membrane. This study evaluated how acute modulation of membrane cholesterol affects CLV contractile performance.
The authors used isolated inguinal-axillary CLVs from C57BL6/J (wild-type) mice, cannulated and maintained under controlled pressure to measure contractile function. Two cholesterol-depleting agents, the cyclodextrins MβCD and HPβCD, were applied acutely to test the effects of membrane cholesterol removal. Cholesterol supplementation was performed using BODIPY-cholesterol incubation, intended to increase membrane cholesterol. Contractile metrics included contraction amplitude and calculated fluid volume displacement per contraction. Calcium imaging was used to assess LMC calcium events. The study also evaluated CLVs from hypercholesterolemic ApoEKO mice to test effects in a disease-relevant model.
Acute treatment with either MβCD or HPβCD produced significant increases in CLV pumping capacity. Specifically, cyclodextrin exposure increased contraction amplitudes by approximately 50±12% and increased estimated fluid volume displaced per contraction by about 35±11% relative to control conditions. These changes indicate a rapid enhancement of the intrinsic contractile mechanics of CLVs after membrane cholesterol depletion.
To probe mechanisms, the investigators used calcium imaging of LMCs. Treatment with HPβCD increased both the amplitude and the duration of large, Cav1.2-mediated calcium events, referred to as calcium flashes. The observed augmentation of these Cav1.2-dependent calcium events provides mechanistic evidence that cholesterol depletion enhances contractility by recruiting or augmenting the function of voltage-gated Cav1.2 channels in LMCs.
Incubating CLVs with BODIPY-cholesterol, a membrane-incorporating cholesterol probe, impaired contractile activity compared with controls. Quantitatively, contraction amplitude decreased from control values (control: 42±2 µm) to substantially lower values with BODIPY-cholesterol (20±7 µm). Calculated fluid volume displacement per contraction fell from 9.2±3.9 nL (control) to 3.3±1.2 nL with BODIPY-cholesterol. Importantly, subsequent treatment with HPβCD partially restored contractile measures (amplitude 36±11 µm; volume displacement 5.5±2.4 nL), showing that the inhibitory effect of cholesterol enrichment on contractility is at least partially reversible in this acute ex vivo preparation.
The study extended findings to a pathological model by testing CLVs isolated from hypercholesterolemic ApoEKO mice. Treatment with HPβCD significantly improved the contractile capacity of these dysfunctional CLVs, indicating that cholesterol depletion can rescue impaired lymphatic pumping associated with hypercholesterolemia in this genetic model.
The data demonstrate that acute modifications of membrane cholesterol content can modulate CLV contractility in isolated vessels, with depletion improving pumping through enhanced Cav1.2-mediated calcium signaling. The authors note that HPβCD has been used safely as a drug excipient in humans, which supports translational consideration. However, this report is a preprint and the experiments are acute, ex vivo interventions; the study does not provide in vivo efficacy, safety, dosing, long-term effects, or detailed pharmacokinetics for cholesterol depletion in lymphatics. The authors indicate planned future studies to test whether pharmacological membrane cholesterol depletion can improve or restore lymphatic contractile function in secondary lymphedema, including obesity/hypercholesterolemia-driven and cancer-related cases.
Acute depletion of membrane cholesterol by cyclodextrins (MβCD and HPβCD) increased CLV contraction amplitude and per-contraction volume displacement in mouse inguinal-axillary collecting lymphatic vessels. The improvement in contractility was associated with recruitment or enhanced activity of voltage-gated Cav1.2 channels in lymphatic muscle cells. Cholesterol enrichment impaired contractility, and subsequent cholesterol depletion partially reversed these effects. HPβCD also improved contractility in CLVs from hypercholesterolemic ApoEKO mice. The authors propose that targeted membrane cholesterol depletion may represent a potential therapeutic approach to improve lymphatic pumping in conditions linked to hypercholesterolemia, but additional in vivo and translational studies are required to evaluate safety, efficacy, and clinical applicability. The manuscript is a preprint and has not undergone peer review.