I Have Been Studying Cholesterol and Lipoprotein Metabolism, Atherosclerosis, and Cardiovascular

1. Personal Statement

I have been studying cholesterol and lipoprotein metabolism, atherosclerosis, and cardiovascular disease for 20 years, and on these research subjects I have authors 30 peer-reviewed research articles and 8 reviews or editorials ( Much of my research uses nonhuman primates (NHPs) to bridge the translational gap between rodents and humans. There are many biological factors and pathways in humans and NHPs that are not present in mice or other lower mammalian species. For instance, we have a funded R01 to determine whether antagonism of miR-33a/b in NHPs will beneficially alter plasma lipids, increase reverse cholesterol transport, and regress atherosclerosis. NHPs are being used for this study because humans and NHPs express both miR-33a/b while mice have only miR-33a. In addition,we recently completed a NHP project with Drs. Cindy Hong and Peter Tontonoz in which we studied the species-specific regulation of plasma LDL levels by the LXR-Idol pathway.

We are also researching how hepatobiliary and transintestinal cholesterol excretion impact reverse cholesterol transport and atherosclerosis development in NHPs and mice. We are specifically interested in the contributions of hepatic Niemann-Pick C1-like 1 and ABCG5/G8 to these two pathways.

1. Positions and Honors

• ATVB Leadership Committee

• ATVB Early Career Committee

• Chair

• Immediate Past Chair

• ATVB Scientific Sessions Programming Committee

• AHA Scientific Sessions Programming Committee

1. Contribution to Science

Impact of SR-BI and apoAI on selective uptake of HDL cholesterol by adrenocortical cells

The ovaries, testes, and adrenal glands produce steroid hormones from cholesterol, much of which is derived from high density lipoproteins (HDL). Prior to me joining Dr. David Williams’ lab in 1995, it was known that steroidogenic tissuesinternalized HDL cholesteryl ester (CE) in the absence of whole particle uptake and degradation. However, the receptor that mediated selective uptake of HDL CE had not been characterized. In addition, the Williams and Breslow labs had shown that mice deficient in apoAI, themain HDL apolipoprotein, had reduced corticosteroid levels presumably due to the depletion of CE in the adrenal glands. During my Ph.D. studies in the Williams lab, we were the first to report that in adrenocortical cells, scavenger receptor B-I (SR-BI) mediated the selective uptake of HDL CE and droveHDL cholesterol into the steroidogenic pathway (1). We also analyzed the composition and function of HDL from apoAI deficient mice and found that HDL lacking apoAI had a blunted capacity to delivery CE to adrenocortical cells via selective uptake (3,4). Our in vitro studies established SR-BI and apoAI as critical factors in HDL CE selective uptake and steroidogenesis.

1. Scavenger receptor class B, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells. Temel RE, Trigatti B, DeMattos RB, Azhar S, Krieger M, Williams DL. Proc Natl AcadSci U S A. 1997;94:13600-5.
2. Apolipoprotein A-I is necessary for the in vivo formation of high density lipoprotein competent for scavenger receptor BI-mediated cholesteryl ester-selective uptake. Temel RE, Walzem RL, Banka CL, Williams DL. J Biol Chem. 2002;277:26565-72.
3. Enhancement of scavenger receptor class B type I-mediated selective cholesteryl ester uptake from apoA-I(-/-) high density lipoprotein (HDL) by apolipoprotein A-I requires HDL reorganization by lecithin cholesterol acyltransferase. Temel RE, Parks JS, Williams DL. J Biol Chem. 2003;278:4792-9.

Determination of the molecular mechanisms of transintestinal cholesterol efflux

A way to reduce LDL cholesterol, the primary risk factor for coronary heart disease (CHD), is to increase cholesterol excretion from the body. The majority of fecal cholesterol is derived from bile. However, there is mounting evidence that the liver may create lipoproteins that can traffic excess cholesterol to the intestine. Through a process known as transintestinal cholesterol efflux (TICE), lipoprotein-associated cholesterol is internalized by the enterocytes and is secreted into the lumen of the small intestine. Our lab studies TICE using theNiemann-Pick C1-like 1 hepatic transgenic (L1Tg) mouse. While working as a post-doctoral scholar in Dr. Larry Rudel’s lab and collaborating with Dr. Liqing Yu, we found that L1Tg mice that had an 80-90% decrease in biliary cholesterol but had normal cholesterol absorption and fecal cholesterol excretion (1). Dr. Mark Brown and I subsequently reported that L1Tg mice had normal macrophage-to-feces reverse cholesterol transport (2) thus indicating that TICE was stimulated in this mouse model. We have also shown that apoB-containing lipoproteins may feed cholesterol into the TICE pathway since reducing hepatic VLDL secretion decreased fecal cholesterol excretion in L1Tg mice (3). Although most of the molecular pathways contributing to TICE are undefined, we recently reported that flavin monooxygenase 3 (FMO3), a central regulator of cholesterol balance, was dramatically reduced in L1Tg mice (4). We believe our ongoing studies on TICE could lead to TICE-stimulating therapies that could be used to reduce the risk of CHD.

1. Hepatic Niemann-Pick C1-like 1 regulates biliary cholesterol concentration and is a target of ezetimibe. Temel RE, Tang W, Ma Y, Rudel LL, Willingham MC, Ioannou YA, Davies JP, Nilsson LM, Yu L. J Clin Invest. 2007;117:1968-78
2. Biliary sterol secretion is not required for macrophage reverse cholesterol transport. Temel RE, Sawyer JK, Yu L, Lord C, Degirolamo C, McDaniel A, Marshall S, Wang N, Shah R, Rudel LL, Brown JM. Cell Metab. 2010;12:96-102
3. Reduction of VLDL secretion decreases cholesterol excretion in Niemann-Pick C1-like 1 hepatic transgenic mice. Marshall SM, Kelley KL, Davis MA, Wilson MD, McDaniel AL, Lee RG, Crooke RM, Graham MJ, Rudel LL, Brown JM, Temel RE. PLoS One. 2014;9:e84418
4. The TMAO-Generating Enzyme Flavin Monooxygenase 3 Is a Central Regulator of Cholesterol Balance. Warrier M, Shih DM, Burrows AC, Ferguson D, Gromovsky AD, Brown AL, Marshall S, McDaniel A, Schugar RC, Wang Z, Sacks J, Rong X, Vallim TA, Chou J, Ivanova PT, Myers DS, Brown HA, Lee RG, Crooke RM, Graham MJ, Liu X, Parini P, Tontonoz P, Lusis AJ, Hazen SL, Temel RE, Brown JM. Cell Rep. 2015;10:326-338.

Effects of miR-33 antagonism on HDL metabolism and atherosclerosis

HDL has the ability to blunt inflammation, reduce oxidation, and drive reverse cholesterol transport (RCT). Therapies that increase HDL concentration and function could reduce atherosclerosis and the risk for CHD. A promising target for increasing HDL function is microRNA-33 (miR-33). In humans, two isoforms of this microRNA, miR-33a and miR-33b, are encoded in introns of the sterol response element binding factor (SREBF) 2 and SREBF1 genes, and co-regulate cellular lipid homeostasis with their host genes. Notably, miR-33a/b induce mRNA degradation and/or translational repression of genes involved in cholesterol efflux and fatty acid oxidation. A major target of miR-33a/b is the ATP binding cassette transporter A1 (ABCA1), a protein essential for cholesterol efflux from foam cells and the formation of HDL. In mice, which encode only miR-33a, we showed that an antisense oligonucleotide targeting miR-33 (anti-miR-33) increased hepatic and macrophage ABCA1, HDL cholesterol, RCT, and atherosclerosis regression (1). However the translational value of the studies in mice was limited by the lack of miR-33b, which is expressed in humans and NHPs. To test the effects of inhibiting both miR-33a and b, we treated NHPs with anti-miR-33 and found that hepatic ABCA1 and HDL were elevated VLDL triglyceride was decreased (2). We are currently working on a R01-funded project to determine the effects of anti-miR-33 on atherosclerosis regression in NHPs. The results from our project will greatly aid in assessing anti-miR-33 as a potential clinical treatment for CHD.

1. Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis. Rayner KJ, Sheedy FJ, Esau CC, Hussain FN, Temel RE, Parathath S, van Gils JM, Rayner AJ, Chang AN, Suarez Y, Fernandez-Hernando C, Fisher EA, Moore KJ. J Clin Invest. 2011;121:2921-31
2. Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides. Rayner KJ, Esau CC, Hussain FN, McDaniel AL, Marshall SM, van Gils JM, Ray TD, Sheedy FJ, Goedeke L, Liu X, Khatsenko OG, Kaimal V, Lees CJ, Fernandez-Hernando C, Fisher EA, Temel RE*, Moore KJ. Nature. 2011;478:404-7 (* equal contribution to study)

Species-specific regulation of plasma LDL levels by the LXR–Idol pathway

The lab of our collaborator Dr. Peter Tontonoz discovered that activation of liver X receptors (LXR) increases expression of the inducible degrader of the LDL receptor (Idol), an E3 ubiquitin ligase that tags the LDL receptor (LDLR) protein for degradation. The Tontonoz lab found that treatment of mice with the synthetic LXR agonist GW3965 significantly increased Idol expression in most tissues and consequently reduced LDLR protein. However, mice treated with LXR agonist did not have an increase in LDL cholesterol (LDLc) since hepatic Idol and LDLR levels were minimally changed. In contrast, human and NHP hepatocytes treated with LXR agonist displayed significantly increased Idol and low levels of LDLR protein (1). Based upon this data, we hypothesized that in primates LXR agonist treatment would increaseLDLc by inducing Idol-dependent degradation of hepatic LDLR. After treating NHPs with GW3965, we found that hepatic Idol was increased, hepatic LDLR was decreased, and LDLc was dramatically elevated. To determine whether the increase in hepatic Idol expression was responsible for the increase in LDLc, NHPs were pre-treated with an antisense oligonucleotide (ASO) targeting Idol and then given GW3965. ASO-mediated knockdown of Idol expression blunted the effect of LXR agonist on LDLc in the NHPs (1). These studies implicate the LXR–Idol axis in the control of plasma lipid levels in primates and supports Idol inhibition as a strategy for LDL lowering in humans.

1. The LXR-Idol Axis Differentially Regulates Plasma LDL Levels in Primates and Mice. Hong C, Marshall SM, McDaniel AL, Graham M, Layne JD, Cai L, Scotti E, Boyadjian R, Kim J, Chamberlain BT, Tangirala RK, Jung ME, Fong L, Lee R, Young SG, Temel RE*, Tontonoz. Cell Metab. 2010;20:910-918 (* co-corresponding author)

Complete List of Published Work in MyBibliography:

1. Research Support

Ongoing Research Support

Title: Effects of Anti-miR-33 on Atherosclerosis Regression and RCT in Nonhuman Primates

Principal Investigator: Ryan E. Temel

Agency: NIH, NHLBI

Grant No.: R01HL111932

Period: 02/01/13 – 11/30/17

Major Goal of Research: Determine the impact of miR-33 antagonism on atherosclerosis regression and reverse cholesterol transport using a nonhuman primate model

Title: A novel mechanism for ART-associated dyslipidemia and atherosclerosis

Principal Investigator: Changcheng Zhou

Co-Investigator: Ryan Temel

Agency: NIH, NHLBI

Grant No.: R01HL123358

Period:08/01/15 - 05/31/19

Major Goal of Research: Determine whether antiviral therapy for HIVincreases dyslipidemia and atherosclerosis by stimulating PXR

Completed Research Projects within the Last Three Years

Title: Center of Research on Obesity and Cardiovascular Diseases

Principal Investigator: Lisa Cassis

Co-Investigator: Ryan E. Temel

Agency: NIH, NIGMS

Grant No.: P20GM103527

Period:05/13/15 – 07-31/15

Major Goal of Research: Pilot project to determine how SR-BI imapcts adipocyte function and the development of obesity

Title: Mechanisms for PPAR∂ Agonist-Induced Elevation of HDL in Non-Human Primates

Principal Investigator:Ryan E. Temel

Agency: NIH, NHLBI

Grant No.: R00HL088528

Period: 02/15/09 – 01/31/13

Major Goal of Research: Determine the mechanisms by which PPAR∂ agonists increase HDL by employing an nonhuman primate model