Apolipoprotein A1 deficiency in mice primes bone marrow stem cells for T cell lymphopoiesis.

The bone marrow has emerged as a potentially important target in cardiovascular disease as it generates all leukocytes involved in atherogenesis. In the current study, we evaluated whether a change in bone marrow functionality underlies the increased atherosclerosis susceptibility associated with high-density lipoprotein (HDL) deficiency. We found that HDL deficiency in mice due to the genetic lack of hepatocyte-derived apolipoprotein A1 (APOA1) was associated with an increase in the Lin-Sca-1+Kit+ (LSK) bone marrow stem cell population and lymphoid-primed multipotent progenitor numbers, which translated into a higher production and systemic flux of T cell subsets. In accordance with APOA1 deficiency-associated priming of stem cells to increase T lymphocyte production, atherogenic diet-fed low-density lipoprotein receptor knockout mice transplanted with bone marrow from APOA1-knockout mice displayed marked lymphocytosis as compared to wild-type bone marrow recipients. However, atherosclerotic lesion sizes and collagen contents were similar in the two groups of bone marrow recipients. In conclusion, systemic lack of APOA1 primes bone marrow stem cells for T cell lymphopoiesis. Our data provide novel evidence for a regulatory role of HDL in bone marrow functioning in normolipidemic mice.

Pcpe2, a Novel Extracellular Matrix Protein, Regulates Adipocyte SR-BI-Mediated High-Density Lipoprotein Uptake.

Objective: To investigate the role of adipocyte Pcpe2 (procollagen C-endopeptidase enhancer 2) in SR-BI (scavenger receptor class BI)-mediated HDL-C (high-density lipoprotein cholesterol) uptake and contributions to adipose lipid storage. Approach and Results: Pcpe2, a glycoprotein devoid of intrinsic proteolytic activity, is believed to participate in extracellular protein-protein interactions, supporting SR-BI- mediated HDL-C uptake. In published studies, Pcpe2 deficiency increased the development of atherosclerosis by reducing SR-BI-mediated HDL-C catabolism, but the biological impact of this deficiency on adipocyte SR-BI-mediated HDL-C uptake is unknown. Differentiated cells from Ldlr-/-/Pcpe2-/- (Pcpe2-/-) mouse adipose tissue showed elevated SR-BI protein levels, but significantly reduced HDL-C uptake compared to Ldlr-/- (control) adipose tissue. SR-BI-mediated HDL-C uptake was restored by preincubation of cells with exogenous Pcpe2. In diet-fed mice lacking Pcpe2, significant reductions in visceral, subcutaneous, and brown adipose tissue mass were observed, despite elevations in plasma triglyceride and cholesterol concentrations. Significant positive correlations exist between adipose mass and Pcpe2 expression in both mice and humans. Conclusions: Overall, these findings reveal a novel and unexpected function for Pcpe2 in modulating SR-BI expression and function as it relates to adipose tissue expansion and cholesterol balance in both mice and humans.

Impact of bone marrow ATP-binding cassette transporter A1 deficiency on atherogenesis is independent of the presence of the low-density lipoprotein receptor.

BACKGROUND AND AIMS: There is extensive evidence from bone marrow transplantation studies that hematopoietic ATP binding cassette A1 (Abca1) is atheroprotective in low-density lipoprotein receptor (Ldlr) deficient mice. In contrast, studies using lysosyme M promoter-driven deletion of Abca1 in Ldlr deficient mice failed to show similar effects. It was hypothesized that the discrepancy between these studies might be due to the presence of Ldlr in bone marrow-derived cells in the transplantation model. In this study, we aim to determine the contribution of Ldlr to the atheroprotective effect of hematopoietic Abca1 in the murine bone marrow transplantation model. METHODS: Wild-type, Ldlr-/-, Abca1-/-, and Abca1-/-Ldlr-/- bone marrow was transplanted into hypercholesterolemic Ldlr-/- mice. RESULTS: Bone marrow Lldr deficiency did not influence the effects of Abca1 on macrophage cholesterol efflux, foam cell formation, monocytosis or plasma cholesterol. Ldlr deficiency did reduce circulating and peritoneal lymphocyte counts, albeit only in animals lacking Abca1 in bone marrow-derived cells. Importantly, the effects of Abca1 deficiency on atherosclerosis susceptibility were unaltered by the presence or absence of Ldlr. Bone marrow Ldlr deficiency did lead to marginally but consistently decreased atherosclerosis, regardless of Abca1 deficiency. Thus, Ldlr expression on bone marrow-derived cells does, to a minimal extent, influence atherosclerotic lesion development, albeit independent of Abca1. CONCLUSIONS: This study provides novel insight into the relative impact of Ldlr and Abca1 in bone marrow-derived cells on macrophage foam cell formation and atherosclerosis development in vivo. We have shown that Ldlr and Abca1 differentially and independently influence atherosclerosis development in a murine bone marrow transplantation model of atherosclerosis.

SR-BI deficiency disassociates obesity from hepatic steatosis and glucose intolerance development in high fat diet-fed mice.

Scavenger receptor BI (SR-BI) has been suggested to modulate adipocyte function. To uncover the potential relevance of SR-BI for the development of obesity and associated metabolic complications, we compared the metabolic phenotype of wild-type and SR-BI deficient mice fed an obesogenic diet enriched in fat. Both male and female SR-BI knockout mice gained significantly more weight as compared to their wild-type counterparts in response to 12 weeks high fat diet feeding (1.5-fold; P < .01 for genotype). Plasma free cholesterol levels were ~2-fold higher (P < .001) in SR-BI knockout mice of both genders, whilst plasma cholesteryl ester and triglyceride concentrations were only significantly elevated in males. Strikingly, the exacerbated obesity in SR-BI knockout mice was paralleled by a better glucose handling. In contrast, only SR-BI knockout mice developed atherosclerotic lesions in the aortic root, with a higher predisposition in females. Biochemical and histological studies in male mice revealed that SR-BI deficiency was associated with a reduced hepatic steatosis degree as evident from the 29% lower (P < .05) liver triglyceride levels. Relative mRNA expression levels of the glucose uptake transporter GLUT4 were increased (+47%; P < .05), whilst expression levels of the metabolic PPARgamma target genes CD36, HSL, ADIPOQ and ATGL were reduced 39%-58% (P < .01) in the context of unchanged PPARgamma expression levels in SR-BI knockout gonadal white adipose tissue. In conclusion, we have shown that SR-BI deficiency is associated with a decrease in adipocyte PPARgamma activity and a concomitant uncoupling of obesity development from hepatic steatosis and glucose intolerance development in high fat diet-fed mice.

The ins and outs of lipid rafts: functions in intracellular cholesterol homeostasis, microparticles, and cell membranes: Thematic Review Series: Biology of Lipid Rafts.

Cellular membranes are not homogenous mixtures of proteins; rather, they are segregated into microdomains on the basis of preferential association between specific lipids and proteins. These microdomains, called lipid rafts, are well known for their role in receptor signaling on the plasma membrane (PM) and are essential to such cellular functions as signal transduction and spatial organization of the PM. A number of disease states, including atherosclerosis and other cardiovascular disorders, may be caused by dysfunctional maintenance of lipid rafts. Lipid rafts do not occur only in the PM but also have been found in intracellular membranes and extracellular vesicles (EVs). Here, we focus on discussing newly discovered functions of lipid rafts and microdomains in intracellular membranes, including lipid and protein trafficking from the ER, Golgi bodies, and endosomes to the PM, and we examine lipid raft involvement in the production and composition of EVs. Because lipid rafts are small and transient, visualization remains challenging. Future work with advanced techniques will continue to expand our knowledge about the roles of lipid rafts in cellular functioning.

Vulnerable plaque and vulnerable blood: Two critical factors for spontaneous atherothrombosis in mouse models.

Atherothrombotic events such as myocardial infarction and ischemic stroke are a major cause of morbidity and mortality worldwide. Understanding the molecular and cellular mechanisms of atherosclerotic plaque destabilization or erosion, and developing new therapeutics to prevent acute cardiovascular events is important for vascular biology research and clinical cardiovascular medicine. However, basic research on plaque destabilization, rupture and erosion is hampered by the lack of appropriate animal models of atherothrombosis. Unprovoked atherothrombosis is very scarce in commonly used mouse models for atherosclerosis, the low-density lipoprotein receptor knockout and apolipoprotein E knockout mice. Therefore, specific interventions are required to induce atherothrombosis in these models. Two strategies can be employed to induce atherothrombosis: 1) plaque destabilization and 2) induction of blood hypercoagulability. Although the individual strategies yield atherothrombosis at low incidence, it appears that the combination of both plaque destabilization and an increase in blood coagulability is the most promising strategy to induce atherothrombosis on a larger scale. In this review, we summarize the recent developments on mouse models for the investigation of atherothrombosis.

ATP-binding cassette transporter G1 deficiency is associated with mild glucocorticoid insufficiency in mice.

OBJECTIVE: Since cholesterol is the sole precursor for glucocorticoid synthesis, it is hypothesized that genetic defects in proteins that impact the cellular cholesterol pool may underlie glucocorticoid insufficiency in humans. In the current study, we specifically focused on the cholesterol efflux mediator ATP-binding cassette transporter G1 (ABCG1) as gene candidate. METHODS: The adrenal transcriptional response to fasting stress was measured in wild-type mice to identify putative novel gene candidates. Subsequently, the adrenal glucocorticoid function was compared between ABCG1 knockout mice and wild-type controls. RESULTS: Overnight food deprivation induced a change in relative mRNA expression levels of cholesterol metabolism-related proteins previously linked to steroidogenesis, i.e. scavenger receptor class B type I (+149%; P < 0.001), LDL receptor (-70%; P < 0.001) and apolipoprotein E (-41%; P < 0.01). Strikingly, ABCG1 transcript levels were also markedly decreased (-61%; P < 0.05). In contrast to our hypothesis that decreasing cholesterol efflux would increase the adrenal cholesterol pool and enhance glucocorticoid output, ABCG1 knockout mice as compared to wild-type mice exhibited a reduced ability to secrete corticosterone in response to an ACTH challenge (two-way ANOVA: P < 0.001 for genotype) or fasting stress. As a result, glucocorticoid target gene expression levels in liver and hypothalamus were reduced and blood lymphocyte concentrations and spleen weights increased in ABCG1 knockout mice under fasting stress conditions. This was paralleled by a 48% reduction in adrenal cholesteryl ester stores and stimulation of adrenal NPC intracellular cholesterol transporter 2 (+37%; P < 0.05) and apolipoprotein E (+59%; P < 0.01) mRNA expression. CONCLUSION: ABCG1 deficiency is associated with mild glucocorticoid insufficiency in mice.

Hypercholesterolemia impairs megakaryopoiesis and platelet production in scavenger receptor BI knockout mice.

BACKGROUND AND AIMS: Thrombocytopenia in scavenger receptor BI (SR-BI) knockout mice is suggested to result from augmented platelet clearance induced by elevated intracellular unesterified cholesterol (UC) levels. We hypothesize that SR-BI deficiency may also influence platelet production at the level of its precursor cell in the bone marrow, the megakaryocyte. METHODS: In this study, we compared megakaryopoiesis and platelet production in SR-BI knockout and wild-type mice. RESULTS: In line with our hypothesis, megakaryocytes from SR-BI knockout mice exhibited UC accumulation while no accumulation of UC was detectable in wild-type megakaryocytes. Bone marrow expression of transcription factors involved in megakaryocyte maturation was induced, but megakaryocyte counts were unchanged in bone marrow of SR-BI knockout mice. Interestingly, we did find a striking 62% decrease (p < 0.01) in proplatelet production by SR-BI knockout megakaryocytes. SR-BI knockout mice displayed an impaired increase in circulating platelet concentrations and bone marrow megakaryocyte numbers upon thrombopoietin challenge. Importantly, megakaryocytes from normolipidemic bone marrow-specific SR-BI knockout mice exhibited a normal ability to produce proplatelets. Moreover, bone marrow-specific deletion of SR-BI did not impair the thrombopoietin response or induce thrombocytopenia, confirming that absence of megakaryocyte SR-BI does not underlie the thrombocytopenic phenotype in total body SR-BI knockout mice. CONCLUSIONS: In conclusion, the elevation of plasma unesterified cholesterol levels impairs megakaryopoiesis and platelet production in SR-BI knockout mice. Our findings suggest that, in addition to an increased platelet clearance, a decrease in platelet production may also, in part, explain the thrombocytopenic phenotype associated with SR-BI deficiency in mice.

Predilection of Low Protein C-induced Spontaneous Atherothrombosis for the Right Coronary Sinus in Apolipoprotein E deficient mice.

Silencing of anticoagulant protein C using RNA interference (siProc) evokes low incident but spontaneous atherothrombosis in the aortic root of apolipoprotein E-deficient (Apoe-/-) mice. The aims of the current study were (1) to analyze if plaque characteristics or circulating factors could be linked to atherothrombosis susceptibility, (2) to increase the incidence of atherothrombosis by transiently increasing blood pressure, and (3) to direct atherothrombosis to an additional predefined vascular site by applying a semi-constrictive collar around the carotid artery. siProc-driven spontaneous atherothrombosis in the aortic root of Apoe-/- mice was reproduced and occurred at an incidence of 23% (9 out of 39 mice), while the incidence of collar-induced atherothrombosis in the carotid artery was 2.6% (1 out of 39 mice). Treatment with phenylephrine, to transiently increase blood pressure, did not increase atherothrombosis in the aortic root of the Apoe-/- mice nor in the carotid arteries with collars. Plaques in the aortic root with an associated thrombus were lower in collagen and macrophage content, and mice with atherothrombosis had significantly more circulating platelets. Plasma protein C, white blood cell counts, total cholesterol, fibrinogen, serum amyloid A, and IL-6 were not different amongst siProc treated mice with or without thrombosis. Remarkably, our data revealed that thrombus formation preferably occurred on plaques in the right coronary sinus of the aortic root. In conclusion, there is a predilection of low protein C-induced spontaneous atherothrombosis in Apoe-/- mice for the right coronary sinus, a process that is associated with an increase in platelets and plaques lower in collagen and macrophage content.

Simvastatin treatment aggravates the glucocorticoid insufficiency associated with hypocholesterolemia in mice.

BACKGROUND AND AIMS: Statin treatment disrupts HMG-CoA reductase-mediated endogenous cholesterol synthesis and lowers plasma LDL-cholesterol levels. Although statin treatment can theoretically impair adrenal steroid hormone synthesis, thus far, no effect on glucocorticoid output has been described, as LDL-cholesterol levels usually remain within the physiological range. However, novel statin-based treatment regimens that dramatically decrease LDL-cholesterol levels are currently employed. Here, we assessed whether inhibition of cholesterol synthesis under these relatively hypocholesterolemic conditions may alter adrenal glucocorticoid output. METHODS: Hypocholesterolemic apolipoprotein A1 (apoA1) knockout mice were administered high dose simvastatin twice daily for 3 days. RESULTS: Simvastatin treatment did not change plasma cholesterol levels or modify the adrenal expression levels of genes involved in cholesterol metabolism. However, simvastatin treatment lowered basal plasma levels of the primary glucocorticoid corticosterone (-62%; p < 0.05). Upon injection with adrenocorticotropic hormone, control-treated apoA1 knockout mice already showed only a mild increase in plasma corticosterone levels, indicative of relative glucocorticoid insufficiency. Importantly, simvastatin treatment further diminished the adrenal glucocorticoid response to adrenocorticotropic hormone exposure (two-way ANOVA p < 0.05 for treatment). Peak corticosterone levels were 49% lower (p < 0.01) upon simvastatin treatment. CONCLUSIONS: We have shown that simvastatin treatment aggravates the glucocorticoid insufficiency associated with hypocholesterolemia in mice. Our data suggest that (1) HMG-CoA reductase activity controls the adrenal steroidogenic capacity under hypocholesterolemic conditions and (2) imply that it might be important to monitor adrenal function in humans subjected to statin-based treatments aimed at achieving sub-physiological LDL-cholesterol levels, as these may potentially execute a negative impact on the glucocorticoid function in humans.

Silencing of Anticoagulant Protein C Evokes Low-Incident but Spontaneous Atherothrombosis in Apolipoprotein E-Deficient Mice-Brief Report.

OBJECTIVE: Murine atherosclerosis models do not spontaneously develop atherothrombotic complications. We investigated whether disruption of natural anticoagulation allows preexisting atherosclerotic plaques to progress toward an atherothrombotic phenotype. APPROACH AND RESULTS: On lowering of plasma protein C levels with small interfering RNA (siProc) in 8-week Western-type diet-fed atherosclerotic apolipoprotein E-deficient mice, 1 out of 4 mice displayed a large, organized, and fibrin- and leukocyte-rich thrombus on top of an advanced atherosclerotic plaque located in the aortic root. Although again at low incidence (3 in 25), comparable thrombi at the same location were observed during a second independent experiment in 9-week Western-type diet-fed apolipoprotein E-deficient mice. Mice with thrombi on their atherosclerotic plaques did not show other abnormalities and had equally lowered plasma protein C levels as siProc-treated apolipoprotein E-deficient mice without thrombi. Fibrinogen and thrombin-antithrombin concentrations and blood platelet numbers were also comparable, and plaques in siProc mice with thrombi had a similar composition and size as plaques in siProc mice without thrombi. Seven out of 25 siProc mice featured clots in the left atrium of the heart. CONCLUSIONS: Our findings indicate that small interfering RNA-mediated silencing of protein C in apolipoprotein E-deficient mice creates a condition that allows the occurrence of spontaneous atherothrombosis, albeit at a low incidence. Lowering natural anticoagulation in atherosclerosis models may help to discover factors that increase atherothrombotic complications.

Endogenous glucocorticoids exacerbate cholestasis-associated liver injury and hypercholesterolemia in mice.

Cholestatic liver disease is characterized by a disruption of bile flow, bile acid toxicity, liver injury, and hypercholesterolemia. Relatively high secretion of glucocorticoids by the adrenals has been observed under cholestatic conditions. Here we investigated a contribution of the rise in endogenous glucocorticoids to initial stage cholestasis pathology. Adrenalectomized or sham-operated control C57BL/6 mice were given an oral dose of alpha-naphthylisothiocyanate to induce cholestasis. Adrenalectomy effectively lowered plasma corticosterone levels (18±5ng/ml vs 472±58ng/ml; P<0.001) and disrupted the metabolic and anti-inflammatory glucocorticoid function. Adrenal removal did not exacerbate the cholestasis extent. In contrast, the cholestasis-associated liver injury was markedly lower in adrenalectomized mice as compared to controls as evidenced by a 84%-93% decrease in liver necrosis and plasma alanine aminotransferase and bile acid levels (P<0.001 for all). Gene expression analysis on livers from adrenalectomized mice suggested the absence of bile acid toxicity-associated farnesoid X receptor signaling in the context of a 44% (P<0.01) and 82% (P<0.001) reduction in sodium/bile acid cotransporter member 1 transcript level as compared to respectively control and non-diseased mice. Adrenalectomy reduced the expression of the cholesterol synthesis gene HMG-CoA reductase by 70% (P<0.05), which translated into a 73% lower plasma total cholesterol level (P<0.05). Treatment of C57BL/6 mice with the glucocorticoid receptor antagonist RU-486 recapitulated the protective effect of adrenalectomy on indices of liver injury and hypercholesterolemia. In conclusion, we have shown that endogenous glucocorticoids exacerbate the liver injury and hypercholesterolemia associated with acute cholestasis in mice.

Lipoproteins as modulators of atherothrombosis: From endothelial function to primary and secondary coagulation.

Atherothrombosis is a complication of atherosclerosis that causes acute cardiovascular events such as myocardial infarction and stroke. Circulating lipid levels are highly correlated with atherosclerotic plaque development. In addition, experimental evidence suggests that lipids also directly influence thrombosis and influence the risk and the outcome of acute cardiovascular events. Plasma lipoproteins influence three aspects important to atherothrombosis: endothelial function, platelet aggregation (primary coagulation) and secondary coagulation. Overall, VLDL, LDL and oxLDL promote thrombus formation, whereas HDL shows antithrombotic actions. In this review we will address the current knowledge about modulation of atherothrombosis by lipoproteins, summarizing findings from in vitro and in vivo animal studies, as well as from observational and interventional studies in humans. We will conclude with future perspectives for lipid modulation in the prevention of atherothrombosis.