Allison L. McDaniel

Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides

Cardiovascular disease remains the leading cause of mortality in westernized countries, despite optimum medical therapy to reduce the levels of low-density lipoprotein (LDL)-associated cholesterol. The pursuit of novel therapies to target the residual risk has focused on raising the levels of high-density lipoprotein (HDL)-associated cholesterol in order to exploit its atheroprotective effects. MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of lipid metabolism and are thus a new class of target for therapeutic intervention. MicroRNA-33a and microRNA-33b (miR-33a/b) are intronic miRNAs whose encoding regions are embedded in the sterol-response-element-binding protein genes SREBF2 and SREBF1 (refs 3–5), respectively. These miRNAs repress expression of the cholesterol transporter ABCA1, which is a key regulator of HDL biogenesis. Recent studies in mice suggest that antagonizing miR-33a may be an effective strategy for raising plasma HDL levels and providing protection against atherosclerosis; however, extrapolating these findings to humans is complicated by the fact that mice lack miR-33b, which is present only in the SREBF1 gene of medium and large mammals. Here we show in African green monkeys that systemic delivery of an anti-miRNA oligonucleotide that targets both miR-33a and miR-33b increased hepatic expression of ABCA1 and induced a sustained increase in plasma HDL levels over 12 weeks. Notably, miR-33 antagonism in this non-human primate model also increased the expression of miR-33 target genes involved in fatty acid oxidation (CROT, CPT1A, HADHB and PRKAA1) and reduced the expression of genes involved in fatty acid synthesis (SREBF1, FASN, ACLY and ACACA), resulting in a marked suppression of the plasma levels of very-low-density lipoprotein (VLDL)-associated triglycerides, a finding that has not previously been observed in mice. These data establish, in a model that is highly relevant to humans, that pharmacological inhibition of miR-33a and miR-33b is a promising therapeutic strategy to raise plasma HDL and lower VLDL triglyceride levels for the treatment of dyslipidaemias that increase cardiovascular disease risk.

The TMAO-Generating Enzyme Flavin Monooxygenase 3 Is a Central Regulator of Cholesterol Balance

Circulating levels of the gut microbe-derived metabolite trimethylamine-N-oxide (TMAO) have recently been linked to cardiovascular disease (CVD) risk. Here, we performed transcriptional profiling in mouse models of altered reverse cholesterol transport (RCT) and serendipitously identified the TMAO-generating enzyme flavin monooxygenase 3 (FMO3) as a powerful modifier of cholesterol metabolism and RCT. Knockdown of FMO3 in cholesterol-fed mice alters biliary lipid secretion, blunts intestinal cholesterol absorption, and limits the production of hepatic oxysterols and cholesteryl esters. Furthermore, FMO3 knockdown stimulates basal and liver X receptor (LXR)-stimulated macrophage RCT, thereby improving cholesterol balance. Conversely, FMO3 knockdown exacerbates hepatic endoplasmic reticulum (ER) stress and inflammation in part by decreasing hepatic oxysterol levels and subsequent LXR activation. FMO3 is thus identified as a central integrator of hepatic cholesterol and triacylglycerol metabolism, inflammation, and ER stress. These studies suggest that the gut microbiota-driven TMA/FMO3/TMAO pathway is a key regulator of lipid metabolism and inflammation.

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

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.

Biliary Sterol Secretion Is Not Required for Macrophage Reverse Cholesterol Transport

Recent evidence suggests that the intestine may play a direct facilitative role in reverse cholesterol transport (RCT), independent of hepatobiliary secretion. In order to understand the nonbiliary pathway for RCT, we created both genetic and surgical models of biliary cholesterol insufficiency. To genetically inhibit biliary cholesterol secretion, we generated mice in which Niemann-Pick C1-Like 1 (NPC1L1) was overexpressed in the liver. Compared to controls, NPC1L1(Liver-Tg) mice exhibit a >90% decrease in biliary cholesterol secretion, yet mass fecal sterol loss and macrophage RCT are normal. To surgically inhibit biliary emptying into the intestine, we have established an acute biliary diversion model. Strikingly, macrophage RCT persists in mice surgically lacking the ability to secrete bile into the intestine. Collectively, these studies demonstrate that mass fecal sterol loss and macrophage RCT can proceed in the absence of biliary sterol secretion, challenging the obligate role of bile in RCT.

Coordinately Regulated Alternative Splicing of Genes Involved in Cholesterol Biosynthesis and Uptake

Genes involved in cholesterol biosynthesis and uptake are transcriptionally regulated in response to cellular sterol content in a coordinated manner. A number of these genes, including 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and LDL receptor (LDLR), undergo alternative splicing, resulting in reductions of enzyme or protein activity. Here we demonstrate that cellular sterol depletion suppresses, and sterol loading induces, alternative splicing of multiple genes involved in the maintenance of cholesterol homeostasis including HMGCR and LDLR, the key regulators of cellular cholesterol biosynthesis and uptake, respectively. These changes were observed in both in vitro studies of the HepG2 human hepatoma derived cell line, as well as in vivo studies of St. Kitts vervets, also known as African green monkeys, a commonly used primate model for investigating cholesterol metabolism. These effects are mediated in part by sterol regulation of polypyrimidine tract binding protein 1 (PTBP1), since knock-down of PTBP1 eliminates sterol induced changes in alternative splicing of several of these genes. Single nucleotide polymorphisms (SNPs) that influence HMGCR and LDLR alternative splicing (rs3846662 and rs688, respectively), have been associated with variation in plasma LDL-cholesterol levels. Sterol-induced changes in alternative splicing are blunted in carriers of the minor alleles for each of these SNPs, indicating an interaction between genetic and non-genetic regulation of this process. Our results implicate alternative splicing as a novel mechanism of enhancing the robust transcriptional response to conditions of cellular cholesterol depletion or accumulation. Thus coordinated regulation of alternative splicing may contribute to cellular cholesterol homeostasis as well as plasma LDL levels.

Hypercholesterolemia increases supraspinatus tendon stiffness and elastic modulus across multiple species.

BACKGROUND More than one-quarter of Americans have hypercholesterolemia and/or are being treated with cholesterol-lowering medications. Given the systemic nature of hypercholesterolemia and remaining questions regarding its effect on tendons at a local level, we sought to assess the utility of small versus large animal model systems for translational studies by exploring the effect of hypercholesterolemia on supraspinatus tendon elastic mechanical properties in mice, rats, and monkeys. We hypothesized that stiffness and elastic modulus would be increased in tendons across species due to hypercholesterolemia. MATERIALS AND METHODS Supraspinatus tendons from normal (control) and high-cholesterol (HC) mice, rats, and monkeys were used in this study. After dissection, tendons were geometrically measured and tensile tested with tissue strain measured optically. RESULTS Overall, HC animals had significantly altered plasma lipid profiles. Biomechanical testing showed a significant increase in stiffness compared with control in HC mice and rats, as well as a nonsignificant trend for HC monkeys. Elastic modulus was also significantly increased in HC mice and monkeys, with HC rats showing a trend. CONCLUSIONS The consistency of our findings across species and between small and large animals, combined with the fact that the aged mice were exposed to lifelong hypercholesterolemia (compared with rats and nonhuman primates, which were fed HC diets), suggests that these increased properties may be inherent to the effect of hypercholesterolemia on supraspinatus tendon rather than due to an effect of cumulative exposure time to the effects of HC. Further investigation is needed to confirm this concept.

Dietary Cholesterol Promotes Adipocyte Hypertrophy and Adipose Tissue Inflammation in Visceral, but Not in Subcutaneous, Fat in Monkeys

Objective— Excessive caloric intake is associated with obesity and adipose tissue dysfunction. However, the role of dietary cholesterol in this process is unknown. The aim of this study was to determine whether increasing dietary cholesterol intake alters adipose tissue cholesterol content, adipocyte size, and endocrine function in nonhuman primates. Approach and Results— Age-matched, male African Green monkeys (n=5 per group) were assigned to 1 of 3 diets containing 0.002 (low [Lo]), 0.2 (medium [Med]), or 0.4 (high [Hi]) mg cholesterol/kcal. After 10 weeks of diet feeding, animals were euthanized for adipose tissue, liver, and plasma collection. With increasing dietary cholesterol, free cholesterol (FC) content and adipocyte size increased in a stepwise manner in visceral, but not in subcutaneous fat, with a significant association between visceral adipocyte size and FC content (r 2=0.298; n=15; P=0.035). In visceral fat, dietary cholesterol intake was associated with (1) increased proinflammatory gene expression and macrophage recruitment, (2) decreased expression of genes involved in cholesterol biosynthesis and lipoprotein uptake, and (3) increased expression of proteins involved in FC efflux. Conclusions— Increasing dietary cholesterol selectively increases visceral fat adipocyte size, FC and macrophage content, and proinflammatory gene expression in nonhuman primates. Visceral fat cells seem to compensate for increased dietary cholesterol by limiting cholesterol uptake/synthesis and increasing FC efflux pathways.

Phytosterol Feeding Causes Toxicity in ABCG5/G8 Knockout Mice

Plant sterols, or phytosterols, are very similar in structure to cholesterol and are abundant in typical diets. The reason for poor absorption of plant sterols by the body is still unknown. Mutations in the ABC transporters G5 and G8 are known to cause an accumulation of plant sterols in blood and tissues (sitosterolemia). To determine the significance of phytosterol exclusion from the body, we fed wild-type and ABCG5/G8 knockout mice a diet enriched with plant sterols. The high-phytosterol diet was extremely toxic to the ABCG5/G8 knockout mice but had no adverse effects on wild-type mice. ABCG5/G8 knockout mice died prematurely and developed a phenotype that included high levels of plant sterols in many tissues, liver abnormalities, and severe cardiac lesions. This study is the first to report such toxic effects of phytosterol accumulation in ABCG5/G8 knockout mice. We believe these new data support the conclusion that plant sterols are excluded from the body because they are toxic when present at high levels.

Intestinal SR-BI does not impact cholesterol absorption or transintestinal cholesterol efflux in mice

Reverse cholesterol transport (RCT) can proceed through the classic hepatobiliary route or through the nonbiliary transintestinal cholesterol efflux (TICE) pathway. Scavenger receptor class B type I (SR-BI) plays a critical role in the classic hepatobiliary route of RCT. However, the role of SR-BI in TICE has not been studied. To examine the role of intestinal SR-BI in TICE, sterol balance was measured in control mice and mice transgenically overexpressing SR-BI in the proximal small intestine (SR-BIhApoCIII-ApoAIV-Tg). SR-BIhApoCIII-ApoAIV-Tg mice had significantly lower plasma cholesterol levels compared with wild-type controls, yet SR-BIhApoCIII-ApoAIV-Tg mice had normal fractional cholesterol absorption and fecal neutral sterol excretion. Both in the absence or presence of ezetimibe, intestinal SR-BI overexpression had no impact on the amount of cholesterol excreted in the feces. To specifically study effects of intestinal SR-BI on TICE we crossed SR-BIhApoCIII-ApoAIV-Tg mice into a mouse model that preferentially utilized the TICE pathway for RCT (Niemann-Pick C1-like 1 liver transgenic), and likewise found no alterations in cholesterol absorption or fecal sterol excretion. Finally, mice lacking SR-BI in all tissues also exhibited normal cholesterol absorption and fecal cholesterol disposal. Collectively, these results suggest that SR-BI is not rate limiting for intestinal cholesterol absorption or for fecal neutral sterol loss through the TICE pathway.

Reduction of VLDL Secretion Decreases Cholesterol Excretion in Niemann-Pick C1-Like 1 Hepatic Transgenic Mice

An effective way to reduce LDL cholesterol, the primary risk factor of atherosclerotic cardiovascular disease, is to increase cholesterol excretion from the body. Our group and others have recently found that cholesterol excretion can be facilitated by both hepatobiliary and transintestinal pathways. However, the lipoprotein that moves cholesterol through the plasma to the small intestine for transintestinal cholesterol efflux (TICE) is unknown. To test the hypothesis that hepatic very low-density lipoproteins (VLDL) support TICE, antisense oligonucleotides (ASO) were used to knockdown hepatic expression of microsomal triglyceride transfer protein (MTP), which is necessary for VLDL assembly. While maintained on a high cholesterol diet, Niemann-Pick C1-like 1 hepatic transgenic (L1Tg) mice, which predominantly excrete cholesterol via TICE, and wild type (WT) littermates were treated with control ASO or MTP ASO. In both WT and L1Tg mice, MTP ASO decreased VLDL triglyceride (TG) and cholesterol secretion. Regardless of treatment, L1Tg mice had reduced biliary cholesterol compared to WT mice. However, only L1Tg mice treated with MTP ASO had reduced fecal cholesterol excretion. Based upon these findings, we conclude that VLDL or a byproduct such as LDL can move cholesterol from the liver to the small intestine for TICE.