Multiple aspects of lymphatic dysfunction in an ApoE -/- mouse model of hypercholesterolemia

Abstract

Introduction: Rodent models of cardiovascular disease have uncovered various types of lymphatic vessel dysfunction that occur in association with atherosclerosis, type II diabetes and obesity. Previously, we presented in vivo evidence for impaired lymphatic drainage in apolipoprotein E null (ApoE ^-/- ) mice fed a high fat diet (HFD). Whether this impairment relates to the dysfunction of collecting lymphatics remains an open question. The ApoE ^-/- mouse is a well-established model of cardiovascular disease, in which a diet rich in fat and cholesterol on an ApoE deficient background accelerates the development of hypercholesteremia, atherosclerotic plaques and inflammation of the skin and other tissues. Here, we investigated various aspects of lymphatic function using ex vivo tests of collecting lymphatic vessels from ApoE ^+/+ or ApoE ^-/- mice fed a HFD. Methods: Popliteal collectors were excised from either strain and studied under defined conditions in which we could quantify changes in lymphatic contractile strength, lymph pump output, secondary valve function, and collecting vessel permeability. Results: Our results show that all these aspects of lymphatic vessel function are altered in deleterious ways in this model of hypercholesterolemia. Discussion: These findings extend previous in vivo observations suggesting significant dysfunction of lymphatic endothelial cells and smooth muscle cells from collecting vessels in association with a HFD on an ApoE-deficient background. An implication of our study is that collecting vessel dysfunction in this context may negatively impact the removal of cholesterol by the lymphatic system from the skin and the arterial wall and thereby exacerbate the progression and/or severity of atherosclerosis and associated inflammation.

Keywords: back-leak; contractile dysfunction; high fat diet; lymphatic endothelium; lymphatic muscle; lymphatic valve; permeability; pump limit.

FIGURE 1

Contractile parameters of ApoE ^+/+ and ApoE ^−/− popliteal lymphatic vessels. Contractile strength was consistently reduced at all pressures between .5 and 10 cmH₂0 in ApoE ^−/− vessels. (A) Recording of spontaneous contractions from an ApoE ^+/+ popliteal lymphatic vessel at different levels of luminal pressure. Each downward deflection in diameter represents a single, twitch contraction. (B) Recording of spontaneous contractions from an ApoE ^−/− lymphatic in response to the same protocol. Note the relatively reduced contraction amplitude at all pressures and reduced contraction frequency at the lowest pressure. (C) Summary data for normalized amplitude; changes in ejection fraction (not shown) were similar. (D) Summary data for tone, which was significantly different at only the lowest pressure (the least reliable measurement of tone). (E) Summary data for frequency, which was significantly different at the two lowest pressures. (F) Fractional pump flow was significantly reduced in ApoE ^−/− vessels at about half the pressures. Statistical differences were assessed via mixed-effects model ANOVAs with Geisser-Greenhouse correction (allowing for missing values or unequal groups) with Fisher’s LSD multiple comparisons post hoc tests.

FIGURE 2

ApoE ^−/− valves exhibit increased back-leak. (A) Experimental configuration used for valve tests. Calibration bar = 40 μm. (B–C) Examples of back-leak tests over the pressure range 0–10 cmH₂O for isolated valves from ApoE ^+/+ and ApoE ^−/− popliteal lymphatics. When closed, the ApoE ^+/+ valve showed only very slight back leak (B), comparable to that of normal valves in previous studies; (C) in contrast, the ApoE ^−/− valve began to leak when Pout exceeded ∼3 cmH₂O. (D) Summary data show that ApoE ^+/+ valves, when closed, effectively prevent back-leak whereas ApoE ^−/− valves consistently showed back-leak when Pout exceeded ∼3 cmH₂O; back-leak was significant at all Pout levels ≥4 cmH₂O. Statistical differences were assessed via mixed-effects model ANOVAs with Geisser-Greenhouse correction with Sidak’s multiple comparisons tests.

FIGURE 3

ApoE ^−/− valves require a higher adverse pressure gradient for closure. (A–B) Examples of valve closure tests for isolated ApoE ^+/+ (A) and ApoE ^−/− (B) valves. Note the difference in the y-axis scales between the two panels. (C) Summary data showing that normal valves require a slightly higher adverse (Pout-Pin) pressure gradient (ΔP) to close a valve as baseline pressure (Pin) rises. (D) When ΔP for closure is plotted as a function of normalized diameter, the relationship is curvilinear. A gradient of .1 cmH₂O is sufficient to close a typical valve at low diameter (∼.6 D/Dmax), whereas a higher pressure gradient (2–15 cmH₂O) is needed near maximal diameter. 7 of 7 ApoE ^+/+ valves closed within the normal range of adverse pressure gradients. In contrast, 5 of 11 ApoE ^−/− valves showed normal closure curves, 4 of 11 closed at low diameters but would not close at higher diameters, and 2 of 11 ApoE ^−/− valves would not close at any adverse pressure gradient tested. Statistical differences were assessed via mixed-effects model ANOVAs with Geisser-Greenhouse correction with Sidak’s multiple comparisons tests.

FIGURE 4

ApoE ^−/− vessels have impaired pump strength. (A) Experimental configuration used for pump test of a 2-valve lymphangion. Calibration bar = 35 μm. Pump test examples for an ApoE ^+/+ vessel (B) and a ApoE ^−/− vessel (C). (D) Pump test results for isolated, 2-valve popliteal lymphatic segments from ApoE ^+/+ and ApoE ^−/− mice. On average ApoE ^−/− vessels were only able to pump against an adverse pressure gradient of 1.2 cmH₂O, compared to 4.8 cmH₂O for ApoE ^+/+ vessels. Statistical differences were assessed via Mann-Whitney test. Valve positions: 1 = open; 0 = closed.

FIGURE 5

ApoE ^−/− vessels exhibit hyperpermeability. (A–B) Increased leak of Evan’s Blue dye is evident in vivo in ApoE ^−/− vessels (B) compared to ApoE ^+/+ vessels (A). Arrows mark sites of leakage. (C) Ex vivo quantification of albumin permeability (P_s) for ApoE ^+/+ vessels (n = 15) and ApoE ^−/− vessels (n = 20). The averages were 21 × 10^–7 cm/s vs. 109 × 10^–7 cm/s for the controls vs. knockouts, respectively, which was significantly different based on a Mann-Whitney test. There was high variability in the ApoE ^−/− group, with 8 of 20 ApoE ^−/− vessels having severely elevated permeability (statistically significant by Mann-Whitney test) compared to ApoE ^+/+ vessels when the data were split into subpopulations, and 12 of 20 vessels were within the range of ApoE ^+/+ vessels (not significantly different by Mann-Whitney test).

FIGURE 6

Low Investiture of lymphatic muscle cells at valve areas. Three examples showing that, compared to ApoE ^+/+ vessels, ApoE ^−/− vessels have reduced LMC coverage in valve sinus regions. LMCs were stained with anti-SMA. Valve regions were identified by CD31 staining (not shown for clarity).