Cholesterol efflux in megakaryocyte progenitors suppresses platelet production and thrombocytosis

Abstract

Platelets have a key role in atherogenesis and its complications. Both hypercholesterolemia and increased platelet production promote atherothrombosis; however, a potential link between altered cholesterol homeostasis and platelet production has not been explored. Here we show that transplantation of bone marrow deficient in ABCG4, a transporter of unknown function, into Ldlr(-/-) mice resulted in thrombocytosis, accelerated thrombosis and atherosclerosis. Although not detected in atherosclerotic lesions, Abcg4 was highly expressed in bone marrow megakaryocyte progenitors (MkPs). Abcg4(-/-) MkPs had defective cholesterol efflux to high-density lipoprotein (HDL), increased cell surface expression of the thrombopoietin (TPO) receptor (c-MPL) and enhanced proliferation. These consequences of ABCG4 deficiency seemed to reflect disruption of negative feedback regulation of c-MPL signaling by the E3 ligase c-CBL and the cholesterol-sensing LYN kinase. HDL infusion reduced platelet counts in Ldlr(-/-) mice and in a mouse model of myeloproliferative neoplasm in an ABCG4-dependent fashion. HDL infusions may offer a new approach to reducing atherothrombotic events associated with increased platelet production.

Figure 1

BM ABCG4 deficiency increases platelet count and accelerates atherosclerosis and thrombosis. Ldlr^−/− mice were transplanted with donor BM cells from WT, Abcg4^−/−, Abcg1^−/− or Abca1^−/−Abcg1^−/− mice and fed a WTD diet for 12 weeks. (a) Quantification of proximal aortic root lesion area (individual and mean) by morphometric analysis of H&E-stained sections. Scale Bar=50μm. (b) Representative of LacZ stained proximal aortas from mice receiving Abca1^−/−Abcg1^−/− or Abcg4^−/− BM. Original magnification 40x. (c) Platelet counts from mice receiving WT or Abcg4^−/− BM. Data are means ± SEM (n=12), representative result of 4 independent studies. (d) Cell surface CD11b levels of platelet-associated Ly6C^hi monocytes or neutrophils in WTD-fed Ldlr^−/− mice transplanted with WT or Abcg4^−/− BM cells. (e) Plasma platelet-derived microparticle and (f) percentage reticulated platelets levels in WTD-fed Ldlr^−/− recipient mice. (g) Microthrombi formation on collagen under shear flow with blood from WTD-fed Ldlr^−/− recipient mice. (h) FeCl₃ induced carotid artery thrombosis in vivo in WTD-fed Ldlr^−/− recipient mice. Data are means ± SEM, * P<0.05 between genotypes. ^{^}P<0.05 between the basal and treatment.

Figure 2

Abcg4 is highly expressed in MkPs, regulating megakaryopoiesis and c-MPL levels. (a) Abcg4 mRNA expression in various types of BM and peripheral white blood cells in WT mice determined by q-PCR. (n=5). (b) Flow cytometry analysis of CD41^lo/CD71⁺ (ErP), CD41⁺/CD71^lo (MkP) and CD41^lo/CD71^lo (MEP) cells from the parent MEP population of Fig. S4. (c) Abcg4 expression in MEPs and MkPs as assessed by quantitative RT-PCR. (d) ABCG4 protein expression in MkPs assessed by immunofluorescence confocal microscopy. The cells were stained with isotype control or anti-ABCG4 (green) and DAPI (nuclei, blue). Scale bar=5μm. (e) Confocal microscopy of WT MkPs immunostained with anti-ABCG4 (green), anti-58K Golgi or anti-TGN38 (red) and DAPI (blue). Scale bar=5μm. (f) Quantification of BM cell populations and (g) cell surface c-MPL levels of Ldlr^−/− recipient mice fed WTD for 12 weeks (n=5). (h) MK-CFU assay using HPCs harvested by FACS from WT or Abcg4^−/− mice. Scale bar=50μm. (i) Platelet count in the WT and Abcg4^−/− mice (n=5) receiving a single dose of TPO (50μg/kg BW) or the vehicle control. Data are means ± SEM, *P<0.05 WT versus Abcg4^−/− groups and ^{^}P<0.05 for treatment effect.

Figure 3

ABCG4 deficiency decreases cholesterol efflux, increases membrane cholesterol content and proliferation of MkPs. (a) Bodipy-cholesterol efflux from WT or Abcg4^−/− MkPs to CD (2 mM) or rHDL (20 μg /ml) for 2 hours (n=4). (b) Bodipy-cholesterol levels in WT or Abcg4^−/− MkPs following CD/Bodipy-cholesterol loading (n=4). (c) Confocal fluorescence microscopy of MkPs from WT and Abcg4^−/− mice incubated with CD/Bodipy-cholesterol (green) and TO-PRO-3 (nuclei, blue). Scale bar=5μm. (d) Confocal microscopy of WT and Abcg4^−/− MkPs stained with fillipin (red) and DAPI (blue) and quantification. Scale bar=10 μm. (e) EdU incorporation into and (f) cell surface c-MPL of WT or Abcg4^−/− MkPs were determined by flow cytometry (n=4). Data are means ± SEM, *P<0.05 WT versus Abcg4^−/− groups and ^{^}P<0.05 for treatment effect.

Figure 4

Increased MkP c-MPL and proliferation in ABCG4 deficiency involves altered activity of c-CBL and LYN. Shown are results all from WT, Abcg4^−/− or Lyn^−/− MkPs (n=4). (a) c-CBL phosphorylation in response to TPO was quantified by phosphor-flow cytometry. Representative histograms before and after 10 mins of TPO. (b) Cell surface c-MPL levels with or without MG132 treatment (10 μM) for 2 h in the presence of TPO. (c) c-CBL phosphorylation 5 min after TPO treatment with or without pretreatment with CD (3 mM), CD-chol (3 mM CD) for 30 min or rHDL (20 μg apoA-I/ml) for 2 hours. (d) c-CBL phosphorylation with or without 5 min TPO treatment or SU6656 pretreatment (10 μg/ml for 2 h). (e) Cell surface c-MPL levels on MkPs with or without TPO or SU6656 treatment for 2 h. (f) Tyrosine-phosphorylated c-CBL (5 min in response to TPO) or (g) cell surface c-MPL (2 h TPO treatment) with or without pretreatment with CD-chol (3 mM CD) for 30 min. (h) 16 h EdU incorporation in the presence of TPO and with or without CD (3 mM), CD-Chol (3 mM CD) pretreatment for 30 min or rHDL (20 μg/ml) for 16 h. (i) BM was isolated from WTD-fed BMT Ldlr^−/− recipients and cell surface c-MPL levels quantified after treatment with or without the LYN activator Tolimidone (10 μM) in the presence of TPO for 2 hours. (j) p-ERK1/2, p-AKT and p-STAT-5 levels with or without TPO for 10 minutes. TPO was 30 ng/ml for all the assays shown. Data are means ± SEM, *P<0.05 WT vs Abcg4^−/− TPO and ^{^}P<0.05 for treatment effect.

Figure 5

rHDL suppresses platelet production in an ABCG4-dependent fashion in vivo. WTD-fed Ldlr^−/− recipient mice (n=5) received a single infusion of vehicle or rHDL (100 mg apoA-I/kg BW). 5 days post infusion (a) platelet counts were determined using a hematology analyzer. Flow cytometry was used to determine (b) abundance of BM MkPs and (c) BM MkP c-MPL experssion. (d) WT mice were transplanted with donor BM cells from WT (n=10) or Abcg4^−/−mice (n=10), both transduced with Mpl^W515L. Platelet counts were monitored weekly and at 9 weeks post-transplant mice received two weekly infusions of rHDL (100mg apoA-I/kg BW) or vehicle as indicated (n=5 per subgroup). Data are means ± SEM, *P<0.05 WT versus Abcg4^−/− and ^P<0.05 for treatment effect. (e, f) Patients with peripheral vascular disease received a single infusion of rHDL (80mg/kg BW) or placebo. (e) Platelet counts were measured pre- and 5 days post-infusion. (f) Data is presented as mean decrease in total platelets post-infusion (n=7/group). (g) Schematic model depicting the involvement of ABCG4 in the regulation of c-MPL levels.