Monocyte tissue factor-dependent activation of coagulation in hypercholesterolemic mice and monkeys is inhibited by simvastatin

Abstract

Hypercholesterolemia is a major risk factor for atherosclerosis. It also is associated with platelet hyperactivity, which increases morbidity and mortality from cardiovascular disease. However, the mechanisms by which hypercholesterolemia produces a procoagulant state remain undefined. Atherosclerosis is associated with accumulation of oxidized lipoproteins within atherosclerotic lesions. Small quantities of oxidized lipoproteins are also present in the circulation of patients with coronary artery disease. We therefore hypothesized that hypercholesterolemia leads to elevated levels of oxidized LDL (oxLDL) in plasma and that this induces expression of the procoagulant protein tissue factor (TF) in monocytes. In support of this hypothesis, we report here that oxLDL induced TF expression in human monocytic cells and monocytes. In addition, patients with familial hypercholesterolemia had elevated levels of plasma microparticle (MP) TF activity. Furthermore, a high-fat diet induced a time-dependent increase in plasma MP TF activity and activation of coagulation in both LDL receptor-deficient mice and African green monkeys. Genetic deficiency of TF in bone marrow cells reduced coagulation in hypercholesterolemic mice, consistent with a major role for monocyte-derived TF in the activation of coagulation. Similarly, a deficiency of either TLR4 or TLR6 reduced levels of MP TF activity. Simvastatin treatment of hypercholesterolemic mice and monkeys reduced oxLDL, monocyte TF expression, MP TF activity, activation of coagulation, and inflammation, without affecting total cholesterol levels. Our results suggest that the prothrombotic state associated with hypercholesterolemia is caused by oxLDL-mediated induction of TF expression in monocytes via engagement of a TLR4/TLR6 complex.

Figure 1. Measurement of coagulation in FH patients.

Blood was obtained from matched healthy controls (n = 17) and FH patients (n = 25). Preapheresis is defined as blood drawn from the body that has not yet passed over the lipid-absorbing column. Postapheresis blood was collected after lipid absorbing, but prior to reentering the body. (A) oxLDL, (B) MP TF activity, (C) TAT, and (D) hsCRP. Histobars represent mean ± SEM. *P < 0.025, FH patients versus healthy controls; ^#P < 0.01, FH patients before versus after apheresis. Data were analyzed with 1-way ANOVA on ranks with a Dunn’s post hoc.

Figure 2. oxLDL induction of monocytic TF expression and the release of TF⁺ MPs.

THP-1 cells were treated with LDL (50 μg/ml) or oxLDL (50 μg/ml) for different times, and (A) cellular TF activity and (B) MP TF activity in the culture supernatant were analyzed. THP-1 cells were treated for either 8 hours and analyzed for (C) cellular TF activity or 24 hours and analyzed for (D) MP TF activity in the culture supernatant after pretreatment with simvastatin (20 μM, sim) for 12 hours, the TLR4 inhibitor TAK-242 (1 μg/ml) for 2 hours, or the LPS-neutralizing antibiotic peptide polymyxin B (10 μg/ml) for 30 minutes. Human monocytes were also pretreated with the same agents; (E) cellular TF activity was analyzed after 8 hours, and (F) MP TF activity in the culture supernatant was analyzed after 24 hours. Data are represented as mean ± SEM. All experiments were performed 5 times in triplicate. *P < 0.01, oxLDL-treated versus untreated and LDL-treated cells; ^#P < 0.01, simvastatin-treated versus untreated cells; ^†P < 0.001, simvastatin-treated or TAK-242–treated versus cells without inhibitor. Data in A and B were analyzed with 1-way ANOVA with Holm-Sidak post hoc, while data in C–F were analyzed via 2-way ANOVA with Holm-Sidak post hoc.

Figure 3. Simvastatin attenuates hypercholesterolemic activation of coagulation in mice.

Ldlr^–/– mice were fed a HFD for up to 12 weeks and then fed a HFD with simvastatin (50 mg/kg/d) for an additional 4 weeks (16 weeks total). (A) Total plasma cholesterol (TPC), (B) oxLDL, (C) wbc TF activity, (D) MP TF activity, (E) TAT, (F) D-dimer, (G) IL-6, and (H) PS⁺ MPs. Histobars represent means ± SEM of 5 mice per group (HFD) or 10 mice per group (simvastatin). *P < 0.01, HFD versus 0-week control (chow). ^#P < 0.05, simvastatin treated versus week 12. Data were analyzed with 1-way ANOVA on ranks with Dunn’s post hoc (A and D–G) or 1-way ANOVA with Holm-Sidak post hoc (B, C, and H).

Figure 4. Activation of coagulation in hypercholesterolemic mice is TF dependent.

Ldlr^–/– mice were fed a HFD for 12 weeks and injected with either a rat anti-mouse TF monoclonal antibody or a rat IgG control. (A) MP TF activity, (B) TAT, (C) D-dimer, (D) IL-6. Histobars represent mean ± SEM of n = 7 mice per group. *P < 0.001, rat anti-mouse TF treatment versus rat IgG. Data were analyzed using either a 2-tailed Student’s t test (A–C) or a Mann-Whitney rank sum (D).

Figure 5. Hematopoietic cell TF deficiency attenuates activation of coagulation in hypercholesterolemic mice.

Irradiated Ldlr^–/– mice were repopulated with either TF^+/– or low TF bone marrow. Mice were fed either chow (n = 7 each group) or HFD (TF^+/– n = 10, low TF n = 9) for 12 weeks and (A) MP TF activity, (B) TAT, (C) D-dimer, and (D) IL-6 were measured. Histobars represent mean ± SEM. *P < 0.001, TF^+/– HFD versus other groups; ^#P < 0.001, low TF HFD versus TF^+/– HFD; ^†P = 0.08 low TF HFD versus TF^+/– HFD. Data were analyzed with 2-way ANOVA with a Holm-Sidak post hoc.

Figure 6. Hypercholesterolemia increases fibrin deposition and platelet accumulation in a laser-induced cremaster arteriole model of thrombosis.

Male Ldlr^–/– mice fed either chow or HFD for 12 weeks (n = 3 mice per group) underwent cremaster arteriole laser injury (chow, 24 thrombi; HFD, 20 thrombi). (A) Fibrin and (D) platelet deposition were measured by intravital microscopy in which median fluorescent units (MFU) represented as AU were plotted against time for 200 seconds. Representative combined binarized fluorescence and bright field microscopy images of peak fibrin deposition in chow-fed (B) and HFD-fed (C) mice labeled with a fibrin-specific antibody conjugated to Alexa Fluor 647 (red) or peak platelet accumulation in chow-fed (E) and HFD-fed (F) mice labeled with an anti-CD42b antibody conjugated to DyLight 488 (green). *P < 0.05 fibrin HFD versus chow and platelet HFD versus chow. Data analyzed as AUC with a Mann-Whitney rank sum. Original magnification, ×60. Scale bars: 10 μm.

Figure 7. TLR4 and TLR6 deficiency attenuate activation of coagulation in hypercholesterolemic mice.

Ldlr^–/–Tlr4^+/+ (chow, n = 6; HFD, n = 10), Ldlr^–/–Tlr4^–/– (chow, n = 8; HFD, n = 26), Ldlr^–/–Tlr6^+/+ (chow and HFD, n = 7), and Ldlr^–/–Tlr6^–/– (chow, n = 6; HFD, n = 13) mice were fed either a chow diet or HFD for 12 weeks. (A) MP TF activity, (B) TAT, (C) D-dimer, and (D) IL-6. Histobars represent mean ± SEM. *P < 0.001, Tlr4^+/+ or Tlr6^+/+ versus other groups; ^#P < 0.001, Tlr4^–/– or Tlr6^–/– HFD versus Tlr4^+/+ or Tlr6^+/+ HFD. Data were analyzed with 2-way ANOVA with a Holm-Sidak post hoc test.

Figure 8. Simvastatin attenuates activation of coagulation in hypercholesterolemic monkeys.

African green monkeys were switched from a chow diet (0 weeks) to a HFD for 16 weeks before being fed a HFD containing simvastatin (10 mg/kg/d) for an additional 4 weeks (20 weeks total). Blood samples were collected at 0–20 weeks. (A) oxLDL, (B) MP TF activity, (C) TAT, (D) D-dimer, (E) hsCRP, (F) IL-8, (G) PS⁺ MPs, and (H) number of particles were measured. Histobars represent mean ± SEM of 12 monkeys. *P < 0.05, HFD versus chow; ^#P < 0.001, simvastatin treated versus 16 weeks. Data were analyzed with either 1-way ANOVA with a Holm-Sidak post hoc (A–C, E, and F) or 1-way ANOVA on ranks (D, G, and H).

Figure 9. Proposed sites of action of simvastatin that reduce TF expression and activation of coagulation during hypercholesterolemia.

In humans, but not mice or monkeys, statins reduce plasma cholesterol levels. In addition, our results indicate that simvastatin reduces activation of coagulation by inhibiting the oxidation of LDL, inhibiting the expression of the CD36/TLR4/TLR6 complex, and inhibiting monocyte TF expression and the release of TF⁺ MPs.