Apolipoprotein E 2 deficiency alters smooth muscle cell and macrophage characteristics to promote atherosclerotic lesion necrosis

Meaghan D. Waltmann University of South Carolina B.S. Biology

Doctor of Philosophy Pathobiology and Molecular Medicine Graduate Program University of Cincinnati College of Medicine

Defense Date: October 14th, 2013 Dissertation Submission Date: November 13th, 2013

Committee Chair: David Y. Hui, Ph.D.

Abstract

Cardiovascular disease is the leading cause of death in the U.S.

Atherosclerosis, a progressive, multi-factorial disease that is characterized by the

accumulation of particles, cells, and fibrous tissue in the vascular wall, is the underlying cause of the majority of deaths related to cardiovascular disease. In the late stages of atherosclerosis, the rupturing of the atherosclerotic plaque results in the

exposure of the inner constituents of the plaque to the blood and subsequent

thrombosis which is the leading cause of myocardial infarction (MI) and stroke. As the

atherosclerosis disease progresses, atherosclerotic plaques can develop into complex

lesions that are more likely to rupture. These unstable plaques are associated with

distinct morphological and histological features including increased plaque macrophages, reduced plaque smooth muscle cells (SMCs), and the formation of a necrotic core. Previous studies have shown that the low-density lipoprotein receptor

(Ldlr) family of receptors plays an integral role in atherosclerosis. Genetic analyses

have associated one member of this receptor family, the E receptor 2

(apoER2; Name: LRP8), with familial and premature coronary artery disease and

MI in humans. Although this receptor is expressed by endothelial cells, SMCs,

monocytes/macrophages, and platelets, the contribution of this receptor to

atherosclerosis is currently unknown.

The goal of this study was to determine the effect apoER2 has on

atherosclerosis. Since previous studies have suggested a role for apoER2 in

maintaining cell viability, we hypothesized that apoER2 deficiency would enhance

atherosclerotic lesion severity through a mechanism involving reduced macrophage and

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smooth muscle cell viability. This study demonstrated that inactivation of the Lrp8 gene in Ldlr deficient mice (Lrp8-/- Ldlr-/-) resulted in the formation of highly necrotic complex

lesions rich in macrophages and apoptotic cells, but relatively devoid of SMCs after 24

weeks on a high fat, high Western-type diet compared to Lrp8+/+ Ldlr-/-.

Consistent with the in vivo phenotype, apoER2 deficient macrophages in vitro accumulated more neutral , were under increased oxidative stress, and were more susceptible to stress-induced cell death through a mechanism involving defective activation of Akt and enhanced activation of the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ). Additionally, apoER2 deficient SMCs had reduced proliferative capacity, reduced viability, and were under increased oxidative stress in vitro through a mechanism involving defective activation of Akt. Taken together, these data suggest that apoER2 protects against the formation of highly necrotic, complex lesions through maintenance of macrophage and SMC viability and function.

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Acknowledgements

This work was partially supported by a pre-doctoral fellowship from the U.S.

National Institutes of Health training grant T32 HL007382. I would like to thank my advisor, Dr. David Hui, for giving me the opportunity to work on this project and for providing me with outstanding mentorship and training. I would also like to thank

Joshua Basford, James Cash, David Kuhel, Eddy Konaniah, Robyn Pilcher-Roberts,

Colleen Goodin, Patrick Roe, and Michelle Adams for their assistance and contributions

to this project. Additionally, I would like to thank my thesis committee members, Dr.

David Hui, Dr. Philip Howles, Dr. Neal Weintraub, Dr. Sean Davidson, and Dr. Keith

Jones, for their guidance and support with my thesis project. Finally, I would like to

thank my family and friends for their support throughout the duration of my graduate

work.

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Table of Contents

INTRODUCTION...... 1

SIGNIFICANCE...... 1 PATHOGENESIS OF ATHEROSCLEROSIS...... 1 LOW-DENSITY LIPOPROTEIN RECEPTORS AND ATHEROSCLEROSIS...... 3 RATIONALE...... 7 METHODS ...... 9

MICE ...... 9 EN FACE STAINING OF AORTAS ...... 9 PLASMA CHOLESTEROL MEASUREMENTS...... 10 PLASMA LIPOPROTEIN PROFILE ...... 10 ATHEROSCLEROTIC LESION ANALYSIS ...... 10 ASSESSMENT OF CIRCULATING MONOCYTES AND NEUTROPHILS...... 11 MACROPHAGE CELL CULTURE...... 12 INACTIVATION OF LRP8 IN RAW 264.7 CELLS...... 13 QUANTITATIVE REAL-TIME PCR ...... 13 IMMUNOBLOT ANALYSIS ...... 14 COPPER-OXIDATION OF LDL ...... 15 IN VITRO NEUTRAL ACCUMULATION...... 16 FLOW CYTOMETRY ANALYSIS OF APOPTOTIC CELLS...... 16 MACROPHAGE DICHLORODIHYDROFLUORESCEIN DIACETATE FLOW CYTOMETRY ...... 17 MICROARRAY...... 17 SMOOTH MUSCLE CELL ISOLATION AND CULTURE ...... 17 POPULATION DOUBLING MEASUREMENT ...... 18 SMOOTH MUSCLE CELL DICHLORODIHYDROFLUORESCEIN DIACETATE MICROSCOPY ...... 18 F2α-ISOPROSTANE STAINING...... 19 NADPH OXIDASE ACTIVITY ASSAY...... 19 TISSUE SELENIUM MEASUREMENT...... 20 BLOOD PRESSURE MEASUREMENT ...... 20 STATISTICAL ANALYSIS ...... 21 RESULTS...... 22

INACTIVATION OF THE LRP8 GENE ENHANCES ATHEROSCLEROTIC PLAQUE COMPLEXITY AND NECROSIS INDEPENDENT OF PLASMA LIPIDS ...... 22 LRP8 DEFICIENCY DOES NOT ALTER CIRCULATING MONOCYTES AND NEUTROPHILS ...... 24 APOER2 IS EXPRESSED IN MOUSE MACROPHAGES...... 25 REDUCED EXPRESSION OF APOER2 RESULTS IN INCREASED LIPID ACCUMULATION IN MACROPHAGES ...... 25 APOER2 DEFICIENT MACROPHAGES ARE MORE SUSCEPTIBLE TO STRESS-INDUCED DEATH...... 26 MACROPHAGE OXIDATIVE STRESS IS ENHANCED IN THE ABSENCE OF APOER2...... 27 APOER2 REGULATES THE EXPRESSION AND ACTIVATION OF PRO-SURVIVAL AND PRO-APOPTOTIC ...... 28 PPARγ EXPRESSION AND ACTIVATION ARE INCREASED IN THE ABSENCE OF APOER2 ...... 29 APOER2 REGULATES SMC PROLIFERATION AND VIABILITY IN VITRO ...... 30 DEFECTIVE ACTIVATION OF AKT OCCURS IN SMCS IN THE ABSENCE OF APOER2 ...... 31 APOER2 PROTECTS AGAINST OXIDATIVE STRESS IN SMCS AND THE WHOLE AORTA INDEPENDENT OF SELENIUM UPTAKE ...... 32 INACTIVATION OF THE LRP8 GENE DOES NOT AFFECT BLOOD PRESSURE IN MICE...... 33 DISCUSSION...... 35 REFERENCES...... 57 TABLES...... 90

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TABLE 1 ...... 90 TABLE 2 ...... 91 FIGURE LEGENDS ...... 92 FIGURES ...... 103

FIGURE 1...... 103 FIGURE 2...... 104 FIGURE 3...... 105 FIGURE 4...... 106 FIGURE 5...... 107 FIGURE 6...... 108 FIGURE 7...... 109 FIGURE 8...... 110 FIGURE 9...... 111 FIGURE 10...... 112 FIGURE 11...... 113 FIGURE 12...... 114 FIGURE 13...... 115 FIGURE 14...... 116 FIGURE 15...... 117 FIGURE 16...... 118 FIGURE 17...... 119 FIGURE 18...... 120 FIGURE 19...... 121 FIGURE 20...... 122 FIGURE 21...... 123 FIGURE 22...... 124 FIGURE 23...... 125 FIGURE 24...... 126 FIGURE 25...... 127 FIGURE 26...... 128

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Introduction

Significance

Cardiovascular disease (CVD) is the leading cause of death in the U.S. despite

annual reductions in CVD-related mortality over the past ten years. It was estimated

that in 2009, 787,931 deaths (32.3% of all U.S. deaths) occurred as a result of CVD.

Forty-nine percent of these CVD-related deaths were caused by coronary heart disease

(CHD). In addition to causing nearly half of all CVD-related deaths, over 15 million

Americans (6.4% of the U.S. population) were estimated to have CHD in 2010 (1).

Atherosclerosis, the underlying cause of CHD, is a progressive, multi-factorial disease

that is defined by the accumulation of lipids, cells, and fibrous tissue in the vascular wall

in the form of atherosclerotic plaques.

Pathogenesis of Atherosclerosis

The structure of a healthy large artery consists of the endothelium made up

exclusively of endothelial cells which serve as a barrier between the vessel wall and the lumen. The subendothelial space, or tunica intima, is a very small layer rich in extracellular matrix components and devoid of cells. The tunica intima is separated from the tunica media, which is composed primarily of smooth muscle cells (SMCs), by the internal elastic lamina. The outermost layer of the vessel wall is the adventitia which contains connective tissue, SMCs, and fibroblasts (2). Atherosclerosis is a disease which occurs in the large arteries and is characterized by the accumulation of lipids, cells, and fibrous tissue in the intima (3). Differences in blood flow typically dictate where atherogenesis will occur. Areas of low-shear stress and turbulent blood flow,

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such as branch points in arteries, are the primary locations for atherosclerosis to occur

(4-7). Perturbed blood flow results in the activation of endothelial cells and the

accumulation of subendothelial lipoprotein particles (8, 9). Additional factors, such as smoking, high blood pressure, and high fat diet, also promote atherogenesis (10, 11).

Activated endothelial cells recruit monocytes and other leukocytes to the area of lesion formation. Recruited monocytes migrate across the endothelium and accumulate in the intima where they proliferate and differentiate into macrophages. Intimal macrophages function to “clean-up” the intima by scavenging extracellular lipoprotein particles and facilitating cholesterol efflux into the blood. As the macrophages accumulate more lipids, they also begin secreting pro-inflammatory cytokines and growth factors, such as platelet-derived growth factor (PDGF), which stimulate the migration of medial SMCs into the intima and promote the proliferation of these SMCs (12, 13). This process is characteristic of the early stages of atherosclerosis otherwise known as fatty streak formation.

As the atherosclerotic disease process continues, macrophages accumulate large amounts of lipids to form “foam cells” (14-16). The accumulation of SMCs and extracellular matrix proteins produced by these SMCs in the intima, along with the intimal accumulation of macrophage foam cells, results in atherosclerotic plaque formation. As the plaque becomes more advanced, a fibrous cap rich is SMCs and connective tissue proteins forms over the luminal side of the plaque (17). This plaque cap serves as a barrier that contains the highly thrombotic contents of the plaque separated from the blood. As macrophage foam cells and SMCs undergo death within

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the plaque, insufficient clearance of these cells results in the formation of a necrotic

core which is primarily composed of dead cells, extracellular lipids, and cellular debris.

A number of events occurring in the late stages of atherosclerosis can result in

plaques more likely to induce fatal complications. Plaque rupture occurs when the

plaque cap is compromised resulting in the exposure of the inner constituents of the

plaque to the blood. This interaction results in thrombosis which can completely

occlude the flow of blood in the vessel resulting in myocardial infarction (MI) (3, 18).

The thrombus may also break off resulting in stroke or embolism. Plaque stability is a determinant of the risk of plaque rupture, where unstable plaques are more likely to rupture. Thinning of the fibrous cap is believed to be the leading cause of plaque rupture. Alterations in SMC viability and proliferation within the cap as well as enhanced infiltration of macrophages into the cap can cause it to thin. Macrophages produce highly proteolytic matrix metalloproteinases which break down the extracellular matrix composition of the cap (19). In the absence of adequate SMC production of more extracellular matrix proteins, as wells as reduced SMC structural support, the plaque cap can weaken significantly resulting in rupture of the plaque (20-24). The growth of the necrotic core within the plaque also puts large amounts of pressure of the plaque cap further promoting plaque rupture.

Low-Density Lipoprotein Receptors and Atherosclerosis

The low-density lipoprotein receptor (Ldlr) family is a group of cell surface receptors that participate in various activities including the endocytosis of

and the facilitation of outside-in signaling (25). The members of this receptor family

3 have structural similarities to the founding member of the family the Ldlr. The very low- density lipoprotein receptor (Vldlr) and the receptor 2 (apoER2; low- density lipoprotein receptor-related 8; Gene Name: Lrp8) are structurally the most similar to the Ldlr (26). The low-density lipoprotein receptor-related protein 1

(Lrp1), the low-density lipoprotein receptor-related protein 2 (Lrp2), the low-density lipoprotein receptor-related protein 5 (Lrp5), and the low-density lipoprotein receptor- related protein 6 (Lrp6) have significantly larger protein structures than the Ldlr. Due to the significant role these receptors play in regulation of cholesterol homeostasis, lipid metabolism, and other cellular events, perturbation of their function can significantly impact the atherosclerotic disease process (27-32).

The Ldlr is a ubiquitously expressed receptor whose primary function is removal of the cholesterol-rich lipoprotein particles, low-density lipoprotein (LDL), from the circulation (32). Familial hypercholesterolemia (FH) is a disorder caused by impaired function of the Ldlr which results in a significant increase in serum LDL and a much greater risk of developing atherosclerosis (1, 33-35). Homozygosity for in the

LDLR gene has been shown to result in serum LDL levels as high as 800mg/dL in humans (36). There are five functional mutations in the Ldlr which result in the onset of

FH: a defect in the synthesis of the receptor; defective targeting of the receptor to the cell surface; a binding site defect that inhibits receptor-ligand binding; defective internalization of receptor-ligand complexes; and defective receptor recycling to the cell surface following dissociation of the receptor and the ligand (37). Autosomal recessive hypercholesterolemia (ARH) has similar clinical presentations as FH, but is due to mutations in an Ldlr adapter protein that causes impaired internalization of Ldlr (38, 39).

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The increased expression of proprotein convertase subtilisin/kexin type 9 (Pcsk9),

which binds the Ldlr and facilitates its degradation, has also been shown to increase

serum LDL levels and increase the risk for atherosclerosis (1, 40-42). Similarly,

deficiency in the Ldlr in mice, which are highly resistant to atherosclerosis, results in

significant increases in serum LDL and increased risk of atherosclerosis (28). This

enhanced susceptibility to atherosclerosis can be exacerbated with a high fat, high

cholesterol Western-type diet (43-46). For this reason, Ldlr deficient (Ldlr-/-) mice are a commonly used animal model of atherosclerosis.

The Lrp1 is a cargo receptor which recognizes more than 30 ligands and

modulates a number of activities, including lipoprotein clearance, SMC proliferation, and macrophage viability (47-50). The Lrp1 regulates SMC proliferation through its interaction with the PDGF receptor which results in reduced expression of the PDGF receptor on the cell surface (47). Inactivation of the Lrp1 gene (Lrp1-/-) in mice results in a significant increase in SMC proliferation and exacerbates atherosclerosis in Ldlr-/- mice (47). Macrophage-specific deletion of Lrp1 enhanced atherosclerotic disease in

Ldlr-/- mice and Ldlr-/- mice on an apolipoprotein E (Apoe) deficient background fed a

Western-type diet (51, 52). In addition to increased plaque size, enhanced plaque

necrosis was also detected in the Ldlr-/- mice lacking macrophage Lrp1 (52, 53). This

was attributed to increased susceptibility of Lrp1-/- macrophages to cell death and a

reduced ability to phagocytose apoptotic macrophages (53).

Another Ldlr family member that plays an integral role in lipoprotein metabolism

is the Vldlr. This receptor functions primarily in the extrahepatic clearance of very low-

density lipoprotein (VLDL) and (54, 55). Deficiency in Vldlr (Vldlr-/-) in

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mice results in a significant increase in serum levels (55). Additionally,

plaque necrosis in macrophage-rich regions was detected in the lesions of Vldlr-/- Ldlr-/- mice following 8 weeks on a Western-type diet, but not in Ldlr-/- mice at the same time

point (56). Alterations in the expression of Vldlr during monocyte to macrophage

differentiation suggested that this receptor may also play a role in regulating the

differentiation process (57).

The Lrp5 and Lrp6 are structurally and functionally very similar. These receptors

have been shown to participate in Wnt signaling (58). The binding of Wnt proteins to a

receptor and Lrp5/6 results in inactivation of glycogen synthase kinase-3β

(GSK3β), blockage of β- degradation, and upregulation of Wnt target (58,

59). Additionally, Lrp5 plays an important role in the regulation bone mass density and mineralization (60, 61). Interestingly, Lrp5 deficient Apoe-/- (Lrp5-/- Apoe-/-) mice are

protected against atherosclerotic lesion calcification (62). A missense in Lrp6

(Lrp6 R611C) has also been associated with premature CVD and symptoms of

metabolic syndrome including elevated serum LDL, triglyceride, and fasting glucose

levels (63).

In contrast to other members of the Ldlr family, the apoER2 does not play a

pivotal role in lipoprotein clearance and metabolism, but rather functions in the

facilitation of outside-in signaling. The apoER2 is primarily expressed in the brain,

testes, and placenta (26). In the brain, apoER2 is necessary for reelin-stimulated

activation of Akt which is important for proper neuronal migration and layering during

brain development (64-68). In the testes, apoER2 facilitates the internalization of

selenoprotein P (Sepp1) in Sertoli cells of the semniferous epithelium, which is

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necessary for proper sperm maturation (69, 70). The apoER2 is also expressed in a

number of cell types involved in atherosclerosis including endothelial cells, SMCs,

monocytes/macrophages, and platelets (71-75). In endothelial cells, the binding of

apoER2 to antiphospholipid antibody-induced β2-glycoprotein I (β2GPI) dimers results

in inhibition of endothelial nitric oxide synthase (eNOS) activity (76). The binding of

β2GPI to apoER2 and glycoprotein Ibα in platelets initiates platelet activation and clot formation (77). In monocytes, ligation of apoER2 with activated protein C has been shown to inhibit endotoxin-induced production of the pro-coagulant protein tissue factor through a mechanism involving activation of Akt (74). Taken together these studies suggest that apoER2 may play an integral role in the regulation of atherosclerosis since nitric oxide production by endothelial cells and platelet activation can significantly affect atherosclerosis and thrombosis. Furthermore, genetic analyses have identified five single nucleotide polymorphisms (SNPs) in the LRP8 gene that are associated with familial and premature CAD and MI in humans providing further support that apoER2 plays a role in atherosclerosis (78, 79).

Rationale

Cardiovascular disease is the leading cause of death in the U.S.

Atherosclerosis, the underlying cause of CHD, accounts for about half of all deaths from

CVD in the U.S (1). The most significant clinical complication associated with

atherosclerosis is thrombosis due to plaque disruption which can result in MI and stroke.

Unstable plaques, which are more likely to rupture, are associated with distinct

compositional characteristics such as necrotic core formation, increased macrophages,

and reduced SMCs (3, 18). Therefore, understanding the mechanisms that contribute

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to plaque complexity, plaque rupture, and inefficient repair following plaque rupture are

of great clinical significance. Previous studies have shown that mutations in members

of the Ldlr family of receptors can significantly increase atherosclerotic disease risk and

plaque complexity. Five SNPs within the Ldlr family member LRP8 have been

associated with familial and premature CAD and MI suggesting that the protein product

of LRP8, apoER2, has a significant role in the atherosclerotic disease process (78, 79).

The apoER2 is primarily expressed in the brain and testes where it plays a critical role

in neuronal migration and sperm maturation, respectively (64-70). The apoER2 is also

expressed in cells involved in atherosclerosis including monocytes/macrophages and

SMCs where its function is not well understood (74, 75). The objective of this study was

to determine the effect apoER2 has on atherosclerosis. Since previous studies have

shown that apoER2 promotes neuronal cell viability and alterations in macrophage and

SMC viability can promote plaque complexity, plaque instability, and subsequent MI, we

hypothesized that apoER2 deficiency would enhance atherosclerotic lesion severity

through a mechanism involving reduced macrophage and SMC viability (18, 80-82). In order to test this hypothesis, atherosclerotic lesion severity was assessed in Lrp8+/+ Ldlr-

/- and Lrp8-/- Ldlr-/- mice fed a high fat, high cholesterol Western-type diet for 12 and 24

weeks. Furthermore, cell viability and mechanisms underlying alterations in cell viability

were assessed in control and apoER2 deficient macrophages and SMCs in vitro and in

vivo.

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Methods

Mice – The Lrp8-/- mice were previously generated in a mixed genetic background of

C57BL/6 and 129sv (64). The Lrp8-/- mice were backcrossed with FVB/N or C57BL/6J

(Jackson Laboratories) mice for 10 generations to produce Lrp8-/- mice on a congenic

FVB/N or C57BL/6 background, respectively. Female Lrp8-/- mice on a congenic

C57BL/6 background were also bred with male Ldlr-/- mice on the same background

(Jackson laboratories) to obtain Lrp8+/-Ldlr+/- offspring, which were then mated to obtain

Lrp8+/+Ldlr-/- and Lrp8-/-Ldlr-/- littermates (83). Age-matched male mice on congenic

C57BL/6 background were used for all atherosclerosis experiments, blood pressure

measurements, and peritoneal macrophage isolations. Age-matched male mice on

congenic FVB/N background were used for all SMC isolations, whole aorta protein preps, and F2α-isoprostane immunofluorescent staining. Mice were fed a standard

rodent chow diet and were maintained in a specific pathogen-free environment on a 12

hour light/dark cycle. For atherosclerosis studies, mice were fed a high fat (21.2% by

weight, 42% kcal) and high cholesterol (0.15% by weight) Western-type diet (Harlan

TD.88137) for 12 or 24 weeks. All procedures and animal care was approved by the

University of Cincinnati Institutional Animal Care and Use Committee.

En Face Staining of Aortas – Mice were anesthetized and perfused with ice-cold

phosphate-buffered saline (PBS) for 5 minutes. Whole aortas were then excised from

the aortic root to the ileal bifurcation, fixed in 10% formalin, and dissected longitudinally.

The luminal side of the aortas were stained with Oil Red O for 30 minutes and rinsed

with water several times to remove excess staining solution. Images were obtained with

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an Olympus SZ40 microscope and quantification of the aortic lesion area was performed with ImageJ software (NIH) using digitalized images from each mouse aorta.

Plasma Cholesterol Measurements – Blood was collected from mice via tail bleed in ethylenediaminetetraacetic acid (EDTA)-coated Microvette® CB 300 capillary tubes

(Sarstedt) after a 16 hour overnight fast. Following separation by centrifugation, the plasma was removed and cholesterol levels were measured using Infinity™ Total

Cholesterol Reagent (Thermo-Scientific) according to the manufacturer’s instructions.

Plasma Lipoprotein Profile – Blood was collected from mice with ad libitum access to

food via cardiocentesis in EDTA-coated syringes. Following separation by

centrifugation, the plasma was removed and separated via fast performance liquid

chromatography using two tandem superose™ 6 size-exclusion columns. Cholesterol

levels in the elution fractions were measured using Infinity Total Cholesterol Reagent

(Thermo-Scientific) according to the manufacturer’s instructions.

Atherosclerotic Lesion Analysis – Atherosclerotic lesions were assessed in mice after

12 or 24 weeks of feeding the Western type diet according to the procedure as

previously described (84, 85). Briefly, mice were anesthetized and perfused with PBS

for 5 minutes followed by a 5 minute perfusion with 4% paraformaldehyde. Following

dissection, the upper half of the heart and the proximal aorta were stored in 4%

paraformaldehyde for 2 days. Tissues were cryopreserved in 30% sucrose at 4°C for 2

days prior to being embedded in optimum cutting temperature (OCT) compound for

frozen section preparation. Cryosections of 5-μm thickness through the aortic valve region of the aortic root were stained with Oil Red O to measure neutral lipid

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accumulation. Serial sections were also stained with Sirius Red to identify collagen

deposition. The necrotic core area was measured as the hematoxylin and eosin-

negative regions in the intima. The CD68 and α-smooth muscle-actin antigens were

detected in the aortic sections for the cellular composition analysis of the plaques by immunofluorescent staining with anti-CD68 (Abcam) and anti-α-smooth muscle-actin

(Sigma Aldrich) primary antibodies followed by secondary antibodies conjugated to

Alexa594. The p53 and PPARγ antigens were detected in aortic sections by immunofluorescent staining with anti-p53 (Cell Signaling Technology) and anti-PPARγ

(Abcam) primary antibodies followed by secondary antibodies conjugated to Alexa594

(Invitrogen). The CD68 antigen was detected in the double stained sections with anti-

CD68 (Abcam) and anti-rat secondary antibodies conjugated to Alexa488. TUNEL staining was performed according to manufacturer’s instructions with the In Situ Cell

Death Detection Kit (Roche) containing fluorescein conjugated dUTPs. All of the fluorescently labeled sections were counterstained with 4,6-diamidino-2-phenylindole

(DAPI). Images were obtained with an Olympus BX61 microscope and quantitative analysis of lesion areas in digitalized images was performed using ImageJ software

(NIH) with at least two sections per mouse.

Assessment of Circulating Monocytes and Neutrophils – Blood was collected via

submandibular bleeds from mice fasted overnight for 16 hours and 2 hours after an oral

gavage of a mixed meal containing 13.33 mM triolein, 2.6 mM cholesterol, 2.6 mM egg

phosphatidylcholine reconstituted in a 50% glucose solution with saline (86).

Erythrocytes were lysed with 1X RBC Lysis Buffer (eBioscience) for 15 minutes at room temperature. The cells were then washed with flow cytometry staining buffer

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2+ 2+ (Ca /Mg -free Hank’s Balanced Salt Solution (HBSS) with 0.3%NaN3 and 1% bovine

serum albumin (BSA) [fraction V; Sigma]). Non-specific binding to Fc receptors was

blocked by incubating the cells with CD16/32 antibodies (eBioscience). Antibody labeling was performed at a 1:25 dilution in flow cytometry buffer with APC Cy-7- conjugated anti-CD11b, PE-conjugated anti-CD115, PercP-Cy5.5-conjugated anti-

Ly6G, and APC-conjugated anti-Ly6C antibodies at room temperature for 30 minutes in the dark. Subsequent flow cytometry analysis was performed using a Guava easyCyte™ 8HT system (Millipore) and data were analyzed using Guava InCyte software (Millipore) (87).

Macrophage Cell Culture – Primary mouse macrophages were isolated from the

peritoneum of age-matched Lrp8+/+ and Lrp8-/- mice maintained on a chow diet 4 days

after injection of sterile 4% thioglycollate solution into their peritoneal cavities (88). The isolated cells were allowed to adhere to tissue culture plates for at least 4 hour and then washed vigorously with sterile PBS to remove non-adherent cells. The adhering mouse peritoneal macrophages were cultured in RPMI-1640 medium (Thermo Scientific) containing 10% endotoxin-free fetal bovine serum (FBS, Invitrogen), 100 units/ml penicillin 100 μg/ml streptomycin solution (Thermo Scientific), and 2 mM L-glutamine

(Thermo Scientific) at 37°C and 5% CO2 unless noted otherwise. Cell counts were

performed manually by trypan blue exclusion. The peritoneal macrophages were

harvested for further analysis within 4 days of isolation. The mouse macrophage cell

line RAW 264.7 cells (ATCC) were also cultured under similar conditions.

Phosphorylated Akt and total Akt protein levels were assessed in whole cell lysates

obtained from RAW 264.7 cells that were seeded at equal densities, allowed to adhere

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for 16 hour overnight, replaced with serum-free media for 24 hour, and then returned to

fresh media containing 10% FBS for the indicated time.

Inactivation of Lrp8 Gene Expression in RAW 264.7 Cells – Inactivation of the Lrp8 gene in RAW 264.7 cells was accomplished by incubating the cells with a mixture of 5 lentiviral particles expressing short-hairpin RNA (shRNA) that target the mouse Lrp8 gene (MISSION™ lentiviral transduction particles from pLKO.1-puro vectors obtained from Sigma-Aldrich, identification numbers: TRCN0000176508, TRCN0000177833,

TRCN0000178706, TRCN0000176636, TRCN0000177656). Briefly, RAW 264.7 cells were incubated for 16 hours in culture medium containing 8 μg/mL hexadimethrine bromide and all five lentiviral particles, each at a multiplicity of infection of 1 for a total multiplicity of infection of 5. The lentiviral particles were removed the following day and the cells were allowed to recover in fresh culture medium for 24 hours. Following puromycin (2-10 μg/ml) selection for 1-2 weeks, the cells were returned to basal medium and were occasionally cultured in the presence of puromycin in order to maintain selection of transduced cells. The transduction was verified based on lack of

Lrp8 mRNA and apoER2 protein as assessed by quantitative real-time PCR and

Western blot analysis, respectively. Similarly, RAW 264.7 cells were also transduced with lentiviral particles containing an empty vector (MISSION™ pLKO.1-puro control transduction particles #SHC001V) as a control. All cells were cultured in the absence of puromycin for at least one week prior to performing all experiments.

Quantitative Real-Time PCR – Total RNA was isolated from mouse peritoneal

macrophages and RAW 264.7 cells with the RNeasy® Plus Mini Kit (Qiagen) and the

RNeasy® Mini Kit (Qiagen), respectively, according to the manufacturer’s instructions.

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Genomic DNA was removed by treatment with TURBO DNA-free™ (Ambion) according

to the manufacturer’s instructions. The RNA was then quantified by measuring the

absorbance at 260 nm. The complementary DNA (cDNA) was synthesized using the

iScript™ cDNA Synthesis Kit (Bio-Rad) and quantitative real-time PCR (qPCR) was

performed on an iQ™ 4 iCycler (Bio-Rad) using the iQ™ SYBER® Green Supermix (Bio-

Rad) and the sequence specific primers (Table 1) according to the manufacturer’s instructions. The mRNA expression levels relative to the expression of the Gapdh

housekeeping gene were calculated using the ΔCT qPCR data analysis method.

Immunoblot Analysis – The whole aortas of mice were excised following complete

perfusion with 1X PBS and were immediately flash frozen in liquid nitrogen.

Homogenization of individual aortas was performed in RIPA buffer containing 0.05 M

Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 1% igepal, 0.1% sodium

dodecyl sulfate (SDS), 1 mM EDTA, 1X phosphatase inhibitor cocktail 2 (Sigma-

Aldrich), 1X phosphatase inhibitor cocktail 3 (Sigma-Aldrich), and 1X complete protease

inhibitor cocktail (Roche). RAW 264.7 whole cell lysates were prepared in RIPA buffer

containing 0.05 M Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 1%

igepal, 0.1% SDS, 1 mM EDTA, 1X phosphatase inhibitor cocktail 2 (Sigma-Aldrich), 1X

phosphatase inhibitor cocktail 3 (Sigma-Aldrich), and 1X complete protease inhibitor

cocktail (Roche). Nuclear protein extracts were isolated for PPARγ immunoblots from

cultured cells using the Nuclear Extraction Kit (Cayman Chemical) according to

manufacturer’s instructions. The protein concentrations were determined using the

Pierce® BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer’s

instructions. Equal amounts of the protein samples were resolved in 10% SDS-

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polyacrylamide gels in the presence of dithiothreitol (DTT) and were then transferred to

Immun-blot PVDF membrane (Bio-Rad). The membranes were blocked with buffer

containing 0.1% Tween-20 and 5% nonfat dry milk for 1 hour at room temperature.

Primary antibody incubations were performed overnight at 4°C and secondary antibody

incubations were performed for 1 hour at room temperature. The primary antibodies

used were anti-glutathione peroxidase 1 (Abcam), anti-thioredoxin reductase 1

(Abcam), anti-Lrp8 (Sigma-Aldrich), anti-phospho-Akt (Serine 473, Cell Signaling

Technology), anti-Akt (Cell Signaling Technology), anti-phospho-p53 (Serine 15, Cell

Signaling Technology), anti-p53 (Cell Signaling Technology), anti-cathepsin L (R&D

Systems), anti-PPARγ (Cell Signaling Technology), anti-TATA binding protein (TBP,

Cell Signaling Technology), anti-phospho-p38 MAPK (Threonine 180/Tyrosine 182, Cell

Signaling Technology), anti-p38 MAPK (Cell Signaling Technology), anti-phospho-

SAPK/JNK (Threonine 183/Tyrosine 185, Cell Signaling), anti-SAPK/JNK (Cell

Signaling), and anti-tubulin (Fisher Scientific, Abcam). The secondary antibodies used

were HRP-linked anti-rabbit (Cell Signaling Technology, Dako), HRP-linked anti-mouse

(Cell Signaling Technology), HRP-linked anti-rat (Sigma-Aldrich), and HRP-linked anti-

goat (Dako). Immunoreactive bands were visualized by chemiluminescence using

Pierce® ECL Western Blotting Substrate (Thermo Scientific) and an Amersham™ ECL

Advance™ Western Blotting Detection Kit (GE Healthcare). Densitometry analysis was

performed with digitalized images using ImageJ software (NIH).

Copper-Oxidation of LDL – Human plasma was obtained from the Hoxworth Blood

Center at the University of Cincinnati (Cincinnati, OH). The LDL was isolated following

two 24 hour centrifugations at 57,000 x g in KBr between the densities 1.02 and 1.063

15 g/mL. The isolated LDL was dialyzed against PBS. Oxidation of the LDL was performed with 0.0125 mM CuSO4 for a 16 hour overnight incubation while rotating at

100 RPM at 30°C. The oxidized LDL was then dialyzed against PBS or saline and stored at 4°C until use.

In Vitro Neutral Lipid Accumulation – Cells were seeded at equal densities and incubated overnight in the presence or absence of 50 μg/mL copper-oxidized human

LDL. Incubations were performed in media without FBS that contained 5% human lipoprotein deficient serum. Following the overnight incubation, cells were harvested and stained with HCS LipidTOX™ Green Neutral Lipid Stain using a modification of the manufacturer’s protocol. Briefly, the HCS LipidTOX™ Green Neutral Lipid Stain was diluted 1:500 and cell staining was performed for 30 minutes at room temperature

(Invitrogen) (89). Flow cytometry analysis was performed immediately using a Guava easyCyte™ 8HT system (Millipore) and data were analyzed using Guava InCyte software (Millipore).

Flow Cytometry Analysis of Apoptotic Cells – Cells were incubated overnight in the presence or absence of 500 μg/ml copper-oxidized human LDL. Following incubation, the cells were gently removed from the plate and Annexin-V and propidium idodide staining was performed using the Annexin-V-APC Apoptosis Detection Kit (eBioscience) according to the manufacturer’s instructions. Flow cytometry analysis was performed using a Guava easyCyte™ 8HT system (Millipore) and data were analyzed using Guava

InCyte software (Millipore).

16

Macrophage Dichlorodihydrofluorescein Diacetate Flow Cytometry – Cells were seeded at equal densities and were allowed to adhere overnight under standard culture conditions. The cells were detached from the plate, the cells were washed, and incubated with 5-(and -6)-carboxy-2’,7’-dichlorodihydrofluorescein diacetate (DCF-DA,

Invitrogen) for 30 minutes in the dark at 37°C (90, 91). Flow cytometry analysis was performed using a Guava easyCyte™ 8HT system (Millipore) and data were analyzed using Guava InCyte software (Millipore). The corrected median fluorescence intensity

(MFI) was calculated by subtracting the negatively stained MFI from the positively stained MFI.

Microarray – RNA was isolated from equally seeded non-transduced RAW 264.7 cells and RAW 264.7 cells with apoER2 knocked down after an overnight period of serum deprivation to induce synchronization followed by 24 hours in standard culture conditions as previously described for quantitative real-time PCR. After isolation, the

RNA was pooled into five groups per genotype where each group contained three pooled RNA samples. RNA integrity and concentration were analyzed using a Agilent

Bioanalyzer RNA 6000 Pico Kit (Agilent Technologies). Labeling of the RNA was then performed with the Ambion WT Expression Kit labeling system (Ambion) for hybridization to Mouse Gene 1.0 ST Array (Affymetrix). The CEL files were generated by the Affymetrix GeneChip Scanner 3000 7G. Analysis of differentially expressed genes was performed with ToppGene data analysis software (Cincinnati Children’s

Hospital Medical Center) with a Bonferroni correction and P<0.05.

Smooth Muscle Cell Isolation and Culture – Mouse thoracic aortas were excised and

SMCs were isolated via dispersion using a modification of Mimura’s procedure

17

(92, 93). Briefly, aortas were rinsed in sterile HBSS, pre-digested at 37°C for 30

minutes with 1 mg/mL collagenase and 0.5mg/mL elastase followed by a 1 hour

digestion at 37°C with the same digestion solution. Isolated SMCs were rinsed with

sterile HBSS and seeded in media equilibrated in a 37°C and 5% CO2 incubator. All

SMCs were cultured in DMEM with low glucose (Thermo Scientific) containing 10% FBS

(Invitrogen), 100 units/ml penicillin 100 μg/ml streptomycin solution (Thermo Scientific), and 2 mM L-glutamine (Thermo Scientific) at 37°C and 5% CO2 unless noted otherwise.

Population Doubling Measurement – Cell population doublings was measured with a

modification of Hütter’s method (94). Cells were seeded at equal densities and then

every four days for 12 consecutive days the cells were removed from the wells and

viable and non-viable cell counts were performed on both the cells attached to the plate

and those unattached in the media using trypan blue exclusion. The cells were then re- seeded at equal densities and incubated at 37°C and 5% CO2 until the next passage

was performed. Population doublings were calculated using the previously published

equation n = (log10F-log10I)/0.301, where n is the number of population doublings, F is

the number of viable cells at the end of the passage, and I is the number of viable cells

seeded at the beginning of the passage.

Smooth Muscle Cell Dichlorodihydrofluorescein Diacetate Microscopy – Cells were seeded at equal densities on sterile glass coverslips and maintained under standard culture conditions until reaching 70% confluence. The cells were then incubated at

37°C and 5% CO2 for 48 hours in starving media that consisted of DMEM with low

glucose, 0.2% FBS, 0.04% BSA, 100 units/ml penicillin 100 μg/ml streptomycin solution,

and 2 mM L-glutamine. Following removal of the media, the cells were incubated with

18

DCF-DA for 30 minutes in the dark at 37°C (90, 91). The cells were then washed with

HBSS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. The

cells were then washed with HBSS and counterstained with DAPI for 5 minutes at room

temperature. The coverslips were then mounted and images were obtained with an

Olympus BX61 microscope.

F2α-Isoprostane Staining – Briefly, mice were anesthetized and perfused with PBS for 5 minutes followed by a 5 minute perfusion with 4% paraformaldehyde. Following dissection, the upper half of the heart and the aorta were stored in 4%

paraformaldehyde for 2 days. Tissues were cryopreserved in 30% sucrose at 4°C for 2

days prior to being embedded in OCT compound for frozen section preparation.

Cryosections of 5-μm thickness through the aorta were immunofluorescently labeled.

The F2α-isoprostane antigen was detected in aortic sections by immunofluorescent

staining with anti-F2α-isoprostane (Abcam) primary antibodies followed by secondary

antibodies conjugated to Alexa594 (Invitrogen). Counterstaining of the sections with

DAPI was also performed. Images were obtained with an Olympus BX61 microscope and quantitative analysis of lesion areas in digitalized images was performed using

ImageJ software (NIH) with at least two sections per mouse.

NAPDH Oxidase Activity Assay – Mouse whole aortas were homogenized in a glass

dounce homogenizer with homogenization buffer containing 250 mM sucrose, 20 mM

HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, and 1 mM ethylene glycol tetraacetic

acid (EGTA) Proteinase Inhibitor Cocktail at pH 7.4. The homogenate was centrifuged

at 1,000 x g for 10 minutes at 4°C followed by centrifugation of the supernatant at

100,000 x g for 1 hour at 4°C. The pellet was resuspended in membrane fraction

19

suspension buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 10 mM EDTA, 2%

Triton, and Proteinase Inhibitor Cocktail (Roche) and the protein concentration was

determined using a bicinchoninic acid (BCA) assay kit (Pierce) according to the

manufacturer’s instructions. The isolated membrane fraction was added to 5uM

lucigenin (Sigma Aldrich) in HBSS, 0.1mM NADPH was added, and luminescence was measured every 15 seconds for 10 minutes.

Tissue Selenium Measurement – Selenium levels were measured in mouse whole

aortas and macrophages using a method modified from previously published studies

(95, 96). Flash frozen whole aortas were incubated with an acid digestion mix

containing nitric acid and perchloric acid (4:1) at 190°C for 90 minutes. The tissue and

acid mixture was then incubated for an additional 30 minutes at 150°C. The samples

were incubated at 60°C for 30 minutes with 2.5 mM EDTA, 6.3 mM diaminonapthalene

(DAN; 0.1%) in 0.1 M HCl, and cyclohexane. The selenium-DAN extraction was

performed by shaking the samples on an orbital shaker for 5 minutes. The fluorescence

was measured with 364 nm excitation and 523 nm emission.

Blood Pressure Measurement – Blood pressure measurements were taken via tail-cuff

using a CODA-8 system (Kent Scientific) (97). Unused measurements were taken for a

period of five consecutive days in order to acclimate the mice to the machine. This was

followed by a 10 consecutive day period where blood pressure measurements were

taken 20 times a day with very little time between each reading. Only readings

determined by the machine as true readings were used. Mean arterial pressure,

systolic blood pressure, and diastolic blood pressure were determined by the machine

for each reading.

20

Statistical Analysis – Statistical analysis was performed using SigmaPlot software.

Values were expressed as mean ± standard error (SE). A two-tailed Student’s t-test

was used when paired comparisons were made. A one-way ANOVA with Holm-Sidak

post-hoc analysis was used when multiple comparisons were made. Statistical significance was determined as differences with P ≤ 0.05.

21

Results

Inactivation of the Lrp8 Gene Enhances Atherosclerotic Plaque Complexity and

Necrosis Independent of Plasma Lipids

Previous studies showed that five SNPs in the LRP8 gene are associated with

familial and premature CAD and MI in humans suggesting that the protein encoded by

this gene, apoER2, plays a significant role in atherosclerosis (78, 79). In order to

determine if the expression of apoER2 has an effect on atherosclerosis, we generated

Lrp8-/- mice on an Ldlr deficient background (Lrp8-/- Ldlr-/-) in order to enhance their susceptibility to atherosclerosis. The mice were then placed on a high fat, high cholesterol Western-type diet for 12 weeks and 24 weeks in order to assess the early and late stages of atherosclerosis. As anticipated, the Lrp8+/+ Ldlr+/+ and Lrp8-/- Ldlr+/+ mice did not develop atherosclerosis following 24 weeks on a Western-type diet (Figure

1A). The en face analysis of the whole aortas revealed that the percentage of the whole aorta covered in lesions (aortic lesion area) was greater in the Lrp8-/- Ldlr-/- mice compared to the Lrp8+/+ Ldlr-/- mice at the same time point (Figure 1A and 1B). These alterations in lesion area occurred independent of plasma lipids. Plasma cholesterol levels were similar between Lrp8+/+ Ldlr-/- and Lrp8-/- Ldlr-/- mice following 24 weeks on a

Western-type diet (Figure 2A). Additionally, the lipoprotein profiles of the Lrp8+/+ Ldlr-/-

mice and the Lrp8-/- Ldlr-/- mice were not different after 24 weeks on the Western-type

diet (Figure 2B).

We next wanted to assess plaque composition as a measure of atherosclerosis

disease severity. Complex lesions are associated with distinct histological features

22

such as necrotic core formation and leukocyte infiltration (98). After 12 weeks of

Western diet feeding, secondary lesions had developed over previously established

lesions in the Lrp8-/- Ldlr-/- mice, but not in the Lrp8+/+ Ldlr-/- control mice (Figure 3A).

Since secondary lesion formation is characteristic of more complex plaques, these data suggested that inactivation of the Lrp8 gene resulted in the formation of more complex or advanced lesions. Consistent with these results, the Lrp8-/- Ldlr-/- mice had a large

amount of lesion necrosis that resulted in the formation of a necrotic core, while minimal

necrosis was present in the Lrp8+/+ Ldlr-/- lesions following 24 weeks on a Western-type

diet (Figure 3B and 3D). The Lrp8-/- Ldlr-/- lesions also had less lipid-rich area and

more extracellular matrix than Lrp8+/+ Ldlr-/- mice as evidenced by Oil Red O staining

and Sirius Red staining, respectively. Interestingly, plaque size (cross-sectional lesion

area) following 24 weeks on diet was not different between the two genotypes (Figure

3C). These data suggested that although more of the total area of the aorta was

covered in atherosclerotic plaques, the plaques themselves were similar in size with

more complex composition in the absence of apoER2. This is consistent with previous

reports that alterations in plaque composition can significantly impact the stability of a

plaque without effecting lesion size (99).

Further analysis of plaque cellularity revealed that the Lrp8-/- Ldlr-/- aortic root

lesions had cellular compositions associated strongly with plaque instability after 24

weeks of Western diet feeding. Immunofluorscent labeling of the macrophage marker

CD68 demonstrated high levels of CD68-positive area located on the luminal edges of

the plaques in the Lrp8-/- Ldlr-/- mice that was not seen in the Lrp8+/+ Ldlr-/- lesions

(Figure 4). Additionally, immunofluorescent labeling of the SMC marker α-smooth

23

muscle-actin (α-SM-actin) revealed that the luminal edges of the Lrp8-/- Ldlr-/- plaques appeared relatively devoid of SMCs compared to the Lrp8+/+ Ldlr-/- lesions (Figure 4).

Fluorescent TUNEL analysis of the lesions indicated that the Lrp8-/- Ldlr-/- mice had greater areas of apoptotic cells within the plaques compared to the Lrp8+/+ Ldlr-/- mice consistent with the increased necrotic tissue identified in the Lrp8-/- Ldlr-/- mice (Figure

4). Interestingly, the apoptotic cells in the Lrp8-/- Ldlr-/- mice were primarily localized to

the luminal edge of the plaque. Taken together, these data suggest that inactivation of

the Lrp8 gene enhances the progression of atherosclerosis and promotes the formation

of more complex, necrotic lesions independent of plasma lipids.

Lrp8 Deficiency Does Not Alter Circulating Monocytes and Neutrophils

The increased CD68 positive area that appeared to colocalize strongly with the

TUNEL positive area in the Lrp8-/- Ldlr-/- lesions suggested that apoER2 may play a role

in the maintenance of macrophage atherosclerosis-related functions and viability. Since

an increase in lesional macrophages could also be the product of changes in circulating

monocytes and neutrophils, we first wanted to verify that apoER2 deficiency did not

alter the levels of circulating leukocytes (100-103). Flow cytometry analysis was

performed on whole blood collected from Lrp8+/+ and Lrp8-/- mice following an overnight

fast and two hours after an oral gavage of a mixed meal to induce a postprandial state

(86). The percentage of circulating monocytes (CD115 +, CD11b +) was not different

between the Lrp8+/+ and Lrp8-/- mice at both the fasting and postprandial time points

(87) (Figure 5A). The fasting and postprandial levels of circulating neutrophils (Ly6G +,

Ly6C +), which can significantly alter monocyte recruitment to atherosclerotic lesions,

was also unaffected by the inactivation of Lrp8 (87, 102) (Figure 5B). Taken together,

24

these data suggest that the increase in atherosclerotic lesion macrophages was not due

to an apoER2-dependent increase in circulating monocytes and neutrophils.

ApoER2 is Expressed in Mouse Macrophages

Previous studies have identified the expression of apoER2 in the monocytic cell line, U937 cells, suggesting that apoER2 is expressed in macrophages (74). As expected, Lrp8 mRNA was detected in thioglycollate-elicited peritoneal macrophages isolated from Lrp8+/+ mice, but not in the macrophages isolated from Lrp8-/- mice (Figure

6A). The expression of Lrp8 mRNA and its protein product, apoER2, was also detected in the mouse macrophage cell line RAW 264.7 cells (Figure 6A and 6B). The difficulty

in culturing Lrp8-/- primary macrophages and the large number of animals needed for

experiments precluded the use of primary macrophages in our functional analyses. In

lieu of primary mouse macrophages, we utilized the RAW 264.7 cells to determine if

apoER2 regulates macrophage atherosclerosis-related functions and viability. We

generated RAW 264.7 cells with apoER2 successfully knocked down at both the mRNA

and protein levels through expression of shRNA specifically targeting mouse Lrp8

(Figure 6A and 6B). In addition to non-transduced cells, RAW 264.7 cells were also

transduced with an empty vector plasmid for use as controls.

Reduced Expression of ApoER2 Results in Increased Lipid Accumulation in

Macrophages

Macrophages play a critical role in the atherosclerosis disease process through the scavenging of extracellular lipids and foam cell formation (14). Macrophage foam cell death is also known to significantly contribute to plaque necrosis and can result in

25

alterations in the functions of neighboring cells. Therefore, we hypothesized that

apoER2 deficiency alters macrophage neutral lipid accumulation which drives the

enhanced lesion necrosis seen in vivo. We first wanted to determine if the absence of

apoER2 resulted in alterations in macrophage foam cell formation. Non-transduced

RAW 264.7 cells (control RAW 264.7), RAW 264.7 cells transduced with the empty vector control (EV RAW 264.7), and RAW 264.7 cells with apoER2 knocked down (KD

RAW 264.7) were incubated overnight in the presence or absence of human copper- oxidized LDL followed by fluorescent staining for intracellular neutral lipids with HCS

LipidTOX™ Green Neutral Lipid Stain and subsequent flow cytometry analysis. The apoER2 KD RAW 264.7 cells accumulated significantly more intracellular lipids both with and without oxidized LDL (oxLDL) incubation compared to the control RAW 264.7 cells and the EV RAW 264.7 cells (Figure 7). These results indicate that apoER2 deficiency results in heightened macrophage foam cell formation.

ApoER2 Deficient Macrophages are More Susceptible to Stress-Induced Death

Since our previous results indicated that apoER2 protects against macrophage

foam cell formation and the death of macrophage foam cells can markedly increase

lesion necrosis, we hypothesized that apoER2 may also play a role in the maintenance

of macrophage viability. In order to test this hypothesis, control RAW 264.7 cells, EV

RAW 264.7 cells, and apoER2 KD RAW 264.7 cells were incubated overnight with

human copper-oxidized LDL and Annexin-V and propidium iodide staining was

assessed by flow cytometry analysis. Under basal culture conditions without oxLDL, the apoER2 KD RAW 264.7 cells underwent significantly more early apoptosis (Annexin-V

+, PI -) (Figure 8). Although cell death (Annexin-V +, PI +) was significantly higher in

26

the apoER2 KD RAW 264.7 cell population compared to the EV RAW 264.7 cells, it

should be noted that there was no difference detected between the control RAW 264.7 cells and the apoER2 KD RAW 264.7 cells under basal conditions. This discrepancy is

likely due to the fact that the EV RAW 264.7 cells had significantly less cell death

(Annexin-V +, PI+) occurring compared to the control RAW 264.7 cells. The apoER2

KD RAW 264.7 cells treated with oxLDL also underwent significantly more early apoptosis (Annexin-V +, PI -) and cell death (Annexin-V +, PI +) compared to the control

RAW 264.7 cells and the EV RAW 264.7 cells (Figure 8). These data suggest that reduced expression of the apoER2 in macrophages enhances their sensitivity to stress- induced cell death.

Macrophage Oxidative Stress is Enhanced in the Absence of ApoER2

Excessive lipid accumulation in macrophages can result in increased oxidative

stress in addition to increased cell death (104-106). Therefore, we tested the

hypothesis that apoER2 protects macrophages against oxidative stress. Staining for

reactive oxygen species (ROS) with DCF-DA and subsequent flow cytometry analysis of

RAW 264.7 cells cultured under basal conditions revealed that the apoER2 KD RAW

264.7 cells had higher levels of intracellular ROS compared to the control RAW 264.7

cells (Figure 9). Selenium is an essential component of many anti-oxidant proteins, including glutathione peroxidase 1 (Gpx1) and thioredoxin reductase 1 (Txnrd1) (107-

109). Since apoER2 is an essential mediator of selenium uptake in the brain and testes, we hypothesized that the increased oxidative stress present in apoER2 deficient macrophages was due to decreased selenium uptake by these cells (69, 70, 110).

Surprisingly, the macrophage selenium levels were not different between the apoER2

27

KD RAW 264.7 cells and the control RAW 264.7 cells indicating that the increased

oxidative stress occurs in the apoER2 KD RAW 264.7 cells independent of cellular

selenium levels (Figure 10).

ApoER2 Regulates the Expression and Activation of Pro-Survival and Pro-

Apoptotic Proteins

Previous studies demonstrated that the apoER2 in neurons and U937 cells

regulates ligand-stimulated Akt activation (67, 74). Akt is a serine/threonine kinase

whose activation controls a number of cellular processes including cell survival, cell

proliferation, and cell metabolism (111-113). We hypothesized that the increased

susceptibility of apoER2 deficient macrophages to stress-induced cell death was due to defective apoER2-dependent activation of Akt. Consistent with this hypothesis, the

apoER2 KD RAW 264.7 cells exhibited significantly lower levels of serum-induced Akt

activation following a 30 minute incubation compared to control RAW 264.7 cells

(Figure 11). We also detected significantly higher levels of the pro-apoptotic proteins

cathepsin L and p53 in the apoER2 KD RAW 264.7 cells cultured under basal

conditions compared to the control RAW 264.7 cells (114-117) (Figure 12). Not only

were the levels of phosphorylated p53 increased, but total p53 levels were increased as

well, which is consistent with previous reports that of p53 results in

stabilization of the protein (118). Additionally, immunofluorescent staining for p53 and

the macrophage marker CD68 revealed that there was significantly more p53-positive

area present within the atherosclerotic plaques of the Lrp8-/- Ldlr-/- mice compared to the

Lrp8+/+ Ldlr-/- mice following 24 weeks of Western diet feeding (Figure 13). Taken

together, these data suggest that reduced expression of apoER2 in macrophages not

28

only results in defective activation of Akt, but also leads to increases in the abundance

of pro-apoptotic proteins as well.

PPARγ Expression and Activation are Increased in the Absence of ApoER2

Previous studies have shown that activation of the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) inhibits the activation of Akt and leads to increased levels of cathepsin L and p53 (114, 119-123). The activation of PPARγ is also known to upregulate genes important in macrophage lipid accumulation, such as

Cd36, Lrp1, and Abca1 (124-126). Therefore, we hypothesized that apoER2 deficiency resulted in aberrant macrophage PPARγ activation. Consistently, the apoER2 KD RAW

264.7 cells had higher levels of the PPARγ-encoding mRNA, Pparg, and higher nuclear protein levels of both PPARγ 1 and PPARγ 2 compared to control RAW 264.7 and EV

RAW 264.7 cells (Figure 14A). The mRNA expression of the PPARγ-responsive genes

Cd36, Lrp1, and Abca1 were also significantly upregulated in the apoER2 KD RAW

264.7 cells compared to controls (Figure 14B). Additionally, microarray analysis of apoER2 KD RAW 264.7 and control RAW 264.7 cells showed a 2-fold or greater upregulation of 23 additional genes associated with treatment with the PPARγ agonist rosiglitazone in the apoER2 KD RAW 264.7 cells compared to the control RAW 264.7 cells (Table 2). In agreement with these in vitro data, there was also significantly more

PPARγ-positive area within the atherosclerotic plaques of Lrp8-/- Ldlr-/- mice compared

to the Lrp8+/+ Ldlr-/- mice following 24 weeks of Western diet feeding (Figure 15). Taken

together, these data suggest that apoER2 protects against macrophage foam cell

formation and death through a mechanism involving regulation of Akt and PPARγ

activation.

29

ApoER2 Regulates SMC Proliferation and Viability In Vitro

In addition to macrophages, SMCs can also significantly impact plaque

composition and stability. The reduced α-SM-actin staining in the Lrp8-/- Ldlr-/- lesions

after 24 weeks on Western diet coupled with the increased lesion necrosis observed in these mice suggested that apoER2 might also regulate SMC viability in addition to macrophage viability. Since apoER2 expression has previously been reported in SMCs, we tested this hypothesis by measuring the number of population doublings occurring

between passages and the percent viability at each passage in SMCs isolated from the

aortas of chow-fed Lrp8+/+ and Lrp8-/- mice. Population doubling analysis of the SMCs isolated from 6 week old mice showed that the number of population doublings occurring between each passage decreased over time in the Lrp8-/- cells compared to

the Lrp8+/+ with no population doublings occurring in these cells between the second and third passage (Figure 16A). Interestingly, the viability of the Lrp8-/- SMCs also decreased with each passage, with significantly reduced viability occurring at passage 3

(Figure 16B). Taken together, these data suggest that inactivation of the Lrp8 gene results in a reduction in the proliferation and viability of SMCs isolated from the aortas of

6 week old mice.

Since atherosclerosis is a disease that progresses with age, we also wanted to

assess the phenotype of SMCs isolated from older mice to determine if age is a

contributing factor to the in vitro and in vivo phenotypes previously observed.

Interestingly, SMCs isolated from 10 to 12 week old mice displayed a severe inability to

proliferate and maintain viability in vitro. Significant reductions in viability accompanied

with potentially lower proliferation rates resulted in failure of the Lrp8-/- SMCs to double

30

in vitro and actually led to fewer SMCs present at each passage than were originally

seeded (Figure 17). Population doubling analysis of SMCs isolated from 26 to 34 week

old mice revealed that reduced population doublings occurred between passages in the

Lrp8-/- SMCs compared to the Lrp8+/+ SMCs immediately after isolation (Figure 18A).

Interestingly, analysis of the viability of these cells showed that there was no difference

in viability between the two genotypes at each passage suggesting that the reduced

population doublings observed was solely due to reduced SMC proliferation (Figure

18B). Consistent with this, analysis of the non-adherent cells present in the media at

each passage showed that cell adhesion was not altered in these cells by the absence

of apoER2 (Figure 18C and 18D). Taken together, these data suggest that in the

absence of apoER2 younger SMCs display a moderate defect in proliferation and

viability which is severely enhanced with age.

Defective Activation of Akt Occurs in SMCs in the Absence of ApoER2

It has previously been established that apoER2 functions primarily as a signaling

receptor which can regulate the activation of Akt, c-Jun NH2-terminal kinase (JNK), and

p38MAPK signaling pathways (67, 79, 80, 127, 128). All of these signaling pathways have been shown to result in alterations in cell proliferation and/or cell viability.

Therefore, we hypothesized that reduced expression of apoER2 resulted in alterations

in these signaling pathways which led to the reduction in proliferation and viability

observed in the Lrp8-/- SMCs. Analysis of the phosphorylation of the viability protein,

Akt, as an indicator of activation in the homogenates of whole aortas revealed that there

was a trend towards reduced Akt activation in the Lrp8-/- aortas compared to Lrp8+/+

aortas (Figure 19). Interestingly, there was no difference between the two genotypes in

31

the activation of the JNK and the p38MAPK pathways in the whole aortas (Figure 20).

Taken together, these data suggest that a reduction in apoER2 results in defective

activation of Akt but does not affect the activation of JNK and p38MAPK in the aorta.

ApoER2 Protects Against Oxidative Stress in SMCs and the Whole Aorta

Independent of Selenium Uptake

Since oxidative stress has the ability to significantly reduce cell proliferation

and/or cell viability and our previous studies demonstrated that apoER2 protects against

oxidative stress in macrophages, we hypothesized that increased oxidative stress in

Lrp8-/- SMCs resulted in defective proliferation and viability. In vitro staining with DCF-

DA demonstrated greater levels of ROS in the Lrp8-/- SMCs compared to the Lrp8+/+

SMCs (Figure 21). Consistent with this, we observed higher levels of F2α-isoprostane,

a well-established marker of oxidative stress, in both the Lrp8-/- aortic arch and thoracic

aortas compared to Lrp8+/+ aortas isolated from chow-fed mice (129, 130) (Figure 22).

An increase in the mRNA expression of the ROS-producing , NADPH oxidase

2 (Nox2) and NADPH oxidase 4 (Nox4), in the Lrp8-/- whole aortas suggested that the

increased oxidative stress observed in these mice may be due to enhanced production

of superoxide by these enzymes (131) (Figure 23A). However, analysis of NADPH oxidase activity in Lrp8+/+ and Lrp8-/- SMCs in vitro showed that NADPH oxidase

enzyme activity was not affected by the absence of apoER2 (Figure 23B).

Since apoER2 plays an important role in the uptake of selenium in other tissues

and selenium is required for the production of certain anti-oxidants, we hypothesized

that selenium deficiency caused by the lack of apoER2 resulted in enhanced oxidative

32

stress in Lrp8-/- SMCs and aortas (69, 107, 110). Surprisingly, measurement of the

whole tissue selenium levels in Lrp8+/+ and Lrp8-/- mice demonstrated that the selenium

levels were significantly higher in the whole aortas of Lrp8-/- mice compared to the

Lrp8+/+ mice (Figure 24). There was also no difference in the mRNA expression of the

selenium-dependent anti-oxidant, Gpx1, in the whole aortas of Lrp8+/+ and Lrp8-/- mice

(Figure 25A). Alternatively, there was a significant increase in the mRNA expression of another selenium-dependent anti-oxidant, Txnrd1, in the whole aortas Lrp8-/- mice compared to Lrp8+/+ mice (Figure 25B). Immunoblot analysis of the whole aortas of

Lrp8+/+ and Lrp8-/- mice revealed that there was no difference in Gpx1 and Txnrd1

protein abundance between the two genotypes (Figure 25C and 25D). Furthermore,

the enzyme activity of Gpx1 was also not affected by the absence of apoER2 in the

aorta (Figure 25E). Taken together, these data suggest that apoER2 deficiency results

in increased oxidative stress in the aorta and in aortic SMCs in vitro which results in

reduced SMC proliferation and reduced SMC viability. This occurs through a

mechanism involving defective activation of Akt in the absence of apoER2, but is

independent of aortic selenium levels. Since SMCs are one of the primary components

of the atherosclerotic lesion, the altered proliferation and viability observed in SMCs in

the absence of apoER2 is likely a major contributing factor to the increased lesion

complexity identified in the Lrp8-/- Ldlr-/- mice.

Inactivation of the Lrp8 Gene Does Not Affect Blood Pressure in Mice

The major cellular component within the vessel wall is SMCs whose primary

function is to regulate vascular tone. Therefore, alterations in the function and phenotype of vascular SMCs can not only have an effect on the atherosclerotic disease

33

process, but can also significantly impact blood pressure regulation. For this reason,

we hypothesized that apoER2 deficiency would result in changes in vascular tone due

to the previously observed SMC phenotype. Tail-cuff blood pressure analysis was

performed on Lrp8+/+ and Lrp8-/- mice maintained on a standard chow diet. Twenty blood pressure readings were taken daily for ten days following a five day acclimation period. Interestingly, there was no difference in the mean arterial flow, systolic blood pressure, and diastolic blood pressure measurements between the Lrp8+/+ and the Lrp8-

/- mice (Figure 26). These data suggest that the severe phenotype observed in the

Lrp8-/- aortic SMCs and whole aorta is not sufficient to induce a disease state. It is

plausible that a “second hit”, such as a high fat diet, is required to induce the

exacerbated disease state.

34

Discussion

This study identified the apoER2 as a determinant of atherosclerotic disease

severity. The absence of apoER2 in Ldlr-/- mice resulted in the formation of highly necrotic, complex lesions abundant in macrophages and apoptotic cells, but relatively devoid of SMCs. These changes in atherosclerosis occurred independent of plasma lipids. In vitro analysis of apoER2 deficient macrophages demonstrated that apoER2 protects against aberrant lipid accumulation, cell death, and oxidative stress through a mechanism involving activation of the pro-survival protein Akt and regulation of PPARγ activation. Similarly, in vitro analyses of apoER2 deficient SMCs demonstrated that apoER2 promotes cell proliferation and viability and protects against oxidative stress through a mechanism involving activation of the pro-survival protein Akt. Taken together, these data suggest that apoER2 confers protection from the formation of highly complex necrotic atherosclerotic lesions through maintenance of proper macrophage and SMC functions.

A genome-wide linkage analysis previously identified an association between a

SNP in the LRP8 gene (rs5174 [R952Q]) with familial and premature CAD and MI in

humans (79). A follow-up study demonstrated an association of four additional SNPs

within the LRP8 gene (rs7546246, rs2297660, rs3737983, and rs5177) with familial and

premature CAD and MI (78). This study also showed that one haplotype carrying

protective alleles for all five of the LRP8 SNPs conferred protection from premature

CAD and MI. The mechanism by which apoER2 confers protection against familial and

premature CAD and MI is currently unknown. Transfection of the human

megakaryoblast cell line, Meg-01 cells, with the human LRP8 R952Q variant resulted in

35

higher, more sustained levels of oxLDL-induced p38MAPK activation compared to

controls (79). Additionally, platelets isolated from patients expressing the LRP8 R952Q

variant displayed increased platelet aggregation in vitro in response to adenosine

diphosphate (ADP) compared to platelets isolated from patients with wild-type LRP8

(79). Taken together, these data suggest that apoER2 may confer protection against

familial and premature CAD and MI through a mechanism involving regulation of platelet

activation. Since apoER2 is also expressed by endothelial cells, SMCs, and

monocytes/macrophages which can also promote CAD and MI, it is possible that the

expression of LRP8 genetic variants can alter the activity of these cells as well (73-75).

Therefore, further studies are necessary to determine the role apoER2 plays in

atherosclerotic disease processes resulting in CAD and MI.

The full-length apoER2 consists of a ligand binding domain, epidermal growth

factor (EGF) precursor homology domain, O-linked sugar domain, transmembrane

domain, and a cytoplasmic domain (26). The full-length human LRP8 transcript

sequence consists of 19 exons (transcript length 4,506 bps; translation length 963

residues), whereas the full-length murine Lrp8 transcript sequence consists of 20 exons

(transcript length 7,674 bps; translation length 996 residues) (26, 132-134). There are many similarities between the human and murine Lrp8 sequences (26, 132). For example, exon 18 and exon 19 in full-length human and murine Lrp8, respectively, display 95% . Additionally, the sequence encoding the first codon of exon 19 in human LRP8, which has previously been associated with the LRP8 SNP

R952Q and familial and premature CAD and MI, displays 100% sequence homology with the sequence encoding the first codon of exon 20 in murine Lrp8 (79).

36

Alternative splicing of Lrp8 occurs in all tissues in which the receptor is

expressed (73, 132-135). An apoER2 splice variant lacking the 59 insert in

the cytoplasmic tail has been identified in human SMCs and endothelial cells (73). This

59 amino acid insert is encoded by exon 18 in humans and exon 19 in mice, and is

unique to apoER2 (26, 132). The use of Lrp8 knockin mice which constitutively express

an Lrp8 isoform which contains or lacks the 59 amino acid insert has led to the

identification of this region as a necessary component for reelin stimulated

enhancement of long-term potentiation in the adult mouse brain (136). Alternatively, the

presence or absence of this 59 amino acid insert does not affect the internalization of

Sepp1, as apoER2 isoforms with and without this insert internalize Sepp1 similarly

(137). The use of chimeras containing the extracellular and the transmembrane domains of Ldlr fused with the cytoplasmic domain of apoER2 showed that the presence of the 59 amino acid insert in the cytoplasmic tail reduced the association of

the chimeras with the clathrin-coated vesicle component AP2 and enhanced their

association with caveolin-1 (Cav-1), a protein component of the caveolae (138). This

suggests that the presence of the 59 amino acid insert may promote localization of

apoER2 to the caveolae and its function as a signaling receptor. Furthermore, this 59

amino acid insert has been identified as a binding site on apoER2 for adapter proteins,

such as JIP1/2 (127, 139).

The identification of the JIP1/2 binding region located within the 59 amino acid

insert in the cytoplasmic tail suggests that apoER2 may be involved in the regulation of

JNK and/or p38MAPK signaling (127). JIP1/2 are scaffolding proteins which bind JNK

and other related kinases (140-142). JNK is activated by stress stimuli, such as

37

oxidative stress, and is involved in the regulation of cellular processes, such as cell

proliferation. JIP1/2 regulate the activation of JNK suggesting that apoER2 may

regulate JNK signaling through this interaction (140-142). Interestingly, treatment of

murine primary neurons in vitro with Apoe results in an apoER2-mediated decrease in

JNK activation (143). The apoER2 has also been implicated in p38MAPK signaling, which

is activated in response to stress stimuli, such as oxidative stress. JIP-2 is known to

regulate the activation of p38MAPK, in addition to JNK, suggesting that apoER2 may

regulate p38MAPK signaling through this interaction as well (144, 145). The apoER2 is

the only LDLR family member expressed by platelets and, interestingly, the treatment of

platelets with LDL induces the activation of p38MAPK and enhances platelet aggregation

(72, 128). In contrast, treatment with Apoe inhibits platelet aggregation suggesting that

Apoe treatment may also decrease p38MAPK activation (72, 128, 146). Additionally, expression of LRP8 containing SNP R952Q, which is associated with familial and

premature CAD and MI, significantly increases p38MAPK activation and platelet

aggregation compared to cells expressing wild-type LRP8 (79).

Alterations in the activation of the JNK and p38MAPK signaling pathways did not

appear to be the primary mechanism contributing to the phenotype of apoER2 deficient

SMCs identified in this study. We demonstrated that the absence of apoER2 resulted in increased oxidative stress and cell death in both macrophages and SMCs. Since the activation of mitogen-activated protein kinase (MAPK) signaling is regulated by cellular stress and can significantly alter cell viability, and the ligation of apoER2 with Apoe has previously been shown to inhibit MAPK activation, we hypothesized that deficiency in apoER2 would result in enhanced activation of JNK and/or p38MAPK signaling .

38

However, analysis of whole aortas from Lrp8+/+ and Lrp8-/- mice revealed that

inactivation of the Lrp8 gene did not have an effect on the activation of JNK and

p38MAPK suggesting that alterations in these signaling pathways were not underlying the

phenotypes identified in vitro. However, large variation between each sample and the low sample size make it difficult to draw firm conclusions from these data. Although not statistically significant, there was a trend towards increased JNK2 phosphorylation and increased p38MAPK phosphorylation in the Lrp8-/- aortas compared to the Lrp8+/+ controls. Power calculations suggest that increasing the sample size to ten and seven, respectively, may be sufficient to detect significant differences between the two genotypes. Therefore, further analysis of JNK and p38MAPK signaling in the aortas of

Lrp8+/+ and Lrp8-/- mice is necessary before firm conclusions can be drawn from the

results.

In addition to its role in the regulation of MAPK signaling, the apoER2 has also

been shown to be a critical mediator of ligand-dependent activation of Akt. In the brain,

the binding of reelin to apoER2 results in phosphorylation of the adapter protein

disabled-1 (Dab1) and subsequent activation of phosphoinositide-3 kinase (PI3K) and

Akt (67). Activation of the reelin-apoER2 signaling pathway is necessary for proper

neuronal migration and layering during brain development (64, 68). In the human

monocytic cell line U937 cells, ligation of apoER2 with activated protein c was also been

shown to result in the downstream activation of Akt (74). Therefore, it was not

surprising that we observed defective Akt activation in the Lrp8-/- whole aortas and in the

apoER2 KD RAW 264.7 macrophages compared to controls. Although a statistically

significant difference was not detected between the Lrp8+/+ and Lrp8-/- aortas, there was

39 a strong trend towards reduced phosphorylation of Akt in the Lrp8-/- aortas compared to the Lrp8+/+. The lack of statistical significance is likely due to the low sample size and relatively large amount of variation between the Lrp8+/+ samples. Power calculations indicate that the sample size should be increased to at least 11 samples per group in order to achieve statistical significance.

It is likely that reduced apoER2 in macrophages and aortic SMCs inhibits apoER2-dependent activation of Akt. Alternatively, the decreased activation of Akt observed in apoER2 KD RAW 264.7 cells could also be due to the increased activation of PPARγ. The well-established inhibitor of Akt activation, PTEN, has previously been shown to be upregulated in response to PPARγ activation (122, 123). Since it is well established that activation of Akt promotes cell proliferation and viability, it is likely that the inhibition of Akt activation seen in the absence of apoER2 results in the alterations in proliferation and viability observed in macrophages and SMCs (111, 112).

Interestingly, diminished Akt activity has been previously shown to result in the formation of highly complex atherosclerotic lesions due to significant reductions in macrophage and SMC viability (147, 148).

This study is not the first to propose a role for apoER2 in the maintenance of cell viability. In neurons, the absence of apoER2 resulted in increased age-dependent cell death (80). Additionally, the increased neurodegeneration observed in Npc1-/- mice was associated with significantly lower levels of Lrp8 mRNA compared to controls (81).

Furthermore, Pcsk9 potentiated neuronal cell apoptosis through a mechanism involving downregulation of apoER2 (82). Taken together, these data provide compelling evidence that apoER2 plays a critical role in the maintenance of cell viability.

40

Consistent with these data, this study demonstrated that in the absence of apoER2

macrophages and aortic SMCs have enhanced susceptibility to cell death. In vivo analysis of atherosclerotic plaques also showed an increase in TUNEL positive staining and an increase in p53-positive immunostaining in the plaques of Lrp8-/- Ldlr-/- mice

compared to Lrp8+/+ Ldlr-/- mice. We believe that this is at least partially due to defective activation of Akt in apoER2 deficient SMCs and macrophages. Another possible mechanism underlying the reduced viability in these cells is the increased expression and activation of PPARγ in the apoER2 deficient macrophages which resulted in upregulation of the pro-apoptotic protein cathepsin L and increased stabilization of p53 compared to controls (114, 119-121). This study did not assess PPARγ activation in

Lrp8-/- SMCs, but previous studies have indicated that activation of PPARγ inhibits SMC

proliferation and induces SMC apoptosis (120, 149). Therefore we cannot rule out the

possibility that these pro-apoptotic proteins are also upregulated in Lrp8-/- SMCs in

response to increased PPARγ activation.

Selenium is a trace element that is cotranslationally incorporated into

selenoproteins as selenocysteine (150). The primary source of selenium for cells is

circulating Sepp1 which has ten selenocysteine residues (151). Cells use

selenocysteine obtained from the intracellular breakdown of Sepp1 in the biosynthesis

of other anti-oxidant selenoproteins such as Gpx1 and Txnrd1 (152). Previous studies

have demonstrated that reduced selenium uptake leads to lower levels of the anti-

oxidant selenoproteins Gpx1 and Txnrd1 resulting in increased oxidative stress (153,

154). The apoER2 has been shown to play a necessary role in Sepp1 uptake in the

testes and in the brain with reduced expression of apoER2 resulting in tissue selenium

41

deficiency (69, 110). Therefore, we hypothesized that the increased oxidative stress

associated with apoER2 deficiency was due to reduced selenium uptake and a

subsequent reduction in the anti-oxidant selenoproteins Gpx1 and Txnrd1. Surprisingly,

selenium levels were not lower in the Lrp8-/- aortas or in apoER2 KD RAW 264.7

macrophages compared to the controls. There was also no difference in Gpx1 activity

or in the protein levels of Gpx1 and Txnrd1 in the Lrp8+/+ and Lrp8-/- aortas. While

apoER2 is necessary for selenium uptake in the testes and the brain, it was not

essential for selenium uptake in the aorta and in macrophages (69, 110). Another Ldlr

family member, Lrp2, also functions in selenium uptake (155). Therefore, it is likely that

in the aorta and in macrophages other receptors, such as Lrp2, are the primary

facilitators of selenium uptake or are able to compensate for the reduced expression of

apoER2 in these cells. It is important to note that although the selenium levels were

significantly higher in the Lrp8-/- aortas compared to the Lrp8+/+ the power was 0.512

which is below the desired level of 0.800. Therefore, it is necessary to increase the sample size to at least seven samples per group before it can be firmly concluded that the selenium levels are higher in the Lrp8-/- aortas compared to Lrp8+/+ aortas.

However, the important observation is that selenium levels are not decreased in the

Lrp8-/- aortas suggesting that the increased oxidative stress is not due to selenium

deficiency.

Reactive oxygen species have important biological roles in the cell (156).

However, excessive production of ROS or a reduction in functional anti-oxidant proteins

puts cells in a state of oxidative stress that can result in alterations in cell proliferation,

metabolism, and viability (157-160). This study demonstrated that Lrp8-/- SMCs and

42

apoER2 KD RAW 264.7 macrophages were under increased oxidative stress even

under basal culture conditions. Additionally, a larger area of positive immunostaining for

the oxidative stress marker F2α-isoprostane was identified in both the aortic arch and

thoracic aortas of chow-fed Lrp8-/- mice compared to Lrp8+/+ mice (130). Surprisingly,

the increased oxidative stress was not due to reduced uptake of selenium. In fact, the

Lrp8-/- aortas had significantly higher levels of selenium compared to the Lrp8+/+ aortas.

Although this study does not provide a direct link between apoER2 and oxidative stress, we propose that defective activation of Akt in the absence of apoER2 is the underlying cause of the increased oxidative stress seen in the apoER2 deficient SMCs, macrophages, and aortas. One of the many downstream targets of Akt is nuclear factor

κB (NFκB) (161). Inhibition of Akt activation in the brain has been shown to result in a subsequent decrease in NFκB activation and increased oxidative stress (162, 163).

The increase in oxidative stress was attributed to downregulation of the potent anti- oxidant protein superoxide dismutase-1 (SOD1) whose transcription is regulated by

NFκB. Therefore, it is possible that the enhanced oxidative stress identified in the apoER2 deficient SMCs and macrophages was caused by defective activation of NFκB by Akt which lead to downregulation of SOD1 and increased oxidative stress.

The NFκB is a transcription factor whose activation results in increased expression of proinflammatory molecules such as monocyte chemoattractant protein-1

(MCP-1) and toll-like receptors. Due to its pro-inflammatory nature, activation of NFκB has long been thought to have a pro-atherogenic function. Deficiency in the NFκB- activating proteins toll-like receptor 4 or myeloid differentiation factor 88 (MyD88) resulted in reduced atherosclerosis in hypercholesterolemic Apoe-/- mice (164, 165). In

43

contrast to these studies, NFκB activation has also been shown to possess anti-

atherogenic properties. Macrophage specific deletion of IκB kinase 2 (IKK2), an essential component of NFκB activation, resulted in increased atherosclerotic lesion size and increased plaque necrosis in Ldlr-/- mice (166). Additionally, IKK2 deficiency

resulted in increased susceptibility of macrophages to cell death in vitro and in vivo

(166). A polymorphism in A20 that prolongs NFκB activation was also proposed to be anti-atherogenic (167). Furthermore, activation of NFκB during resolution of inflammation led to upregulation of anti-inflammatory genes in rat leukocytes (168).

Taken together, these data suggest that activation of NFκB has beneficial effects which are likely dependent on the cell type, time of activation, and duration of activation.

Therefore, it is possible that defective apoER2-induced activation of Akt results in lower levels of activated NFκB and subsequent pro-apoptotic and pro-atherogenic effects.

The nuclear receptor PPARγ is activated by naturally occurring fatty acid

derivatives, such as 15-deoxy-Δ12,14 prostaglandin J2 (169, 170). Isoprostanes are

prostaglandin-like compounds that are produced non-enzymatically by ROS-catalyzed

peroxidation of arachidonic acid (129). Similar to prostaglandins, isoprostanes have also been shown to activate PPARγ (171). This study showed that aortic SMCs and macrophages were under increased oxidative stress in the absence of apoER2.

-/- Additionally, Lrp8 aortas had higher levels of the oxidative stress marker F2α-

isoprostane. We also demonstrated that a reduction in macrophage apoER2 resulted in

enhanced activation of PPARγ through an unknown mechanism. Therefore, we

propose that the increased activation of PPARγ observed in the apoER2 deficient

44

macrophages is likely a byproduct of the increased oxidative stress observed in these

cells.

Activation of PPARγ in macrophages results in upregulation of receptors

important in lipid uptake, such as CD36 and Lrp1, and proteins important in cholesterol

efflux, such as Abca1 and Abcg1 (124-126, 172). Inactivation of the Pparg gene

reduced macrophage lipid accumulation compared to Pparg+/+ macrophages following

oxLDL incubation due to the inability of the Pparg-/- cells to upregulate Cd36 (173). Our

results show that aberrant activation of PPARγ in the absence of apoER2 significantly

increased lipid accumulation in macrophages. We believe that this is due to the fact

that the scavenger receptor Cd36 was much more highly upregulated than the

cholesterol efflux gene Abca1. Therefore it is likely that the apoER2 KD RAW 264.7

cells were scavenging significantly more lipid than they were able to efflux resulting in

the increased cellular lipid levels observed.

In contrast to our study, previous studies have also suggested that PPARγ

activation is atheroprotective. Atherosclerotic lesion size was reduced in Ldlr-/- mice

treated with the PPARγ agonists rosiglitazone or GW7845 compared to untreated Ldlr-/- mice fed a Western-type diet for 10 weeks (174). Additionally, macrophage-specific inactivation of the Pparg gene in Ldlr-/- mice increased lesion size compared to controls

following 8 and 16 weeks on a Western-type diet (173). In this study, we showed that

reduced expression of apoER2 in macrophages resulted in augmented PPARγ

expression and activation compared to controls causing increased macrophage lipid

accumulation and cell death. This was confirmed in vivo by enhanced PPARγ immunostaining in the atherosclerotic lesions of Lrp8-/- Ldlr-/- mice following 24 weeks on

45

a Western-type diet compared to Lrp8+/+ Ldlr-/- mice. The PPARγ-positive area within these lesions was largely associated with the macrophage marker CD68 suggesting that there are more PPARγ-positive macrophages in the Lrp8-/- Ldlr-/- lesions than in the

Lrp8+/+ Ldlr-/- mice. We believe that the increase in macrophage PPARγ activation resulted in enhanced macrophage susceptibility to stress-induced cell death which greatly contributed to the enhanced lesion complexity and necrosis seen in the Lrp8-/-

Ldlr-/- mice.

The discrepancy between our study and the previous studies is likely due to the

amount of time the mice were on the Western-type diet and the measurement of

atherosclerosis severity that was used (84, 175). The previously mentioned studies

maintained mice on the Western-type diet for 10 weeks and 8 and 16 weeks,

respectively (173, 174). It is possible that the animals were not on the Western-type

diet long enough to fully evaluate the late stages of atherosclerosis (175). In our study,

we evaluated the early stages of atherosclerosis after feeding the mice the Western-

type diet for 12 weeks and evaluated the later stages of atherosclerosis after feeding

the mice the Western-type diet for 24 weeks. Additionally, the previously mentioned

studies only used lesion size as a measurement for atherosclerosis severity. In our

study, we did not see differences in lesion size, but did see significant changes in

plaque composition and complexity. Therefore, it is possible that differences in plaque

composition, such as necrotic area, lipid-rich area, and extracellular matrix, may have

been overlooked in the previous studies.

Systemic activation of PPARγ has been shown to significantly improve insulin sensitivity, reduce blood glucose levels, and lower circulating due to its

46

ability to increase glucose utilization (176). For this reason, PPARγ agonist

thiazolidinedione (TZD) drugs, such as Avandia®, were utilized clinically for the

treatment of type II diabetes (177). Since PPARγ was also shown to reduce

macrophage inflammation, reduce endothelial cell activation, reduce SMC proliferation,

and increase SMC apoptosis, treatment with TZDs was also presumed to be useful for

preventing atherosclerosis (178-182). However, in 2007 a large meta-analysis revealed

that patients treated with rosiglitazone (Avandia®) had a 43% higher risk of MI and a

possible increase in the risk for cardiovascular death (183). This resulted in the Food

and Drug Administration restricting the use of Avandia® to treat type II diabetes to only

patients who were previously being treated with Avandia® and those that are

unresponsive to all other treatments (184). It is still unclear why treatment with

Avandia® resulted in such poor cardiac outcomes and why all patients do not respond

poorly to treatment with Avandia®. Here we show that reduced expression of apoER2 in

macrophages resulted in increased expression and activation of PPARγ. Therefore, we

suggest that LRP8 may be a modifier gene that determines cardiac outcomes in

patients treated with Avandia®.

Difficulties in studying atherosclerosis in humans have resulted in the use of

animal models to study the disease. Although the vasculature of the mouse is not ideal for human comparison, the mouse model has emerged as the leading animal model for

studying atherosclerosis (185). This is primarily due to their fast rate of reproduction,

their ease of use, the large amount of information known about their genetic

background, the ease at which genetic manipulation can be performed, and the

relatively fast rate at which atherosclerotic lesions occur (84, 185). Atherosclerosis

47

studies done in mice are typically diet-induced by feeding of a high fat diet. Additionally,

genetic manipulation of the Apoe or Ldlr genes must also be performed to enhance the

susceptibility of mice to atherosclerosis (28, 186-188). Although the primary location of

atherosclerosis in humans is the coronary arteries, atherosclerotic lesions are rarely

identified in the coronary arteries of mice. The primary site of lesion formation in mice is

the aortic root which is likely due to the increased heart rate observed in mice compared

to humans (185, 189).

In addition to differences in lesion locations, mouse plaques rarely rupture.

There are very few examples of plaque rupture occurring in mice. For this reason,

characterizing plaque instability in the mouse model can be extremely difficult (190).

Interestingly, transluminal adenoviral delivery of the human p53 transgene to the carotid

lesions of Apoe-/- mice resulted in a significant reduction in plaque cap SMCs and led to

rupture of 40% of the lesions following treatment with phenylephrine (191). In our

model, p53 protein levels were higher in apoER2 deficient macrophages in vitro and in the atherosclerotic lesions of Lrp8-/- Ldlr-/- mice compared to controls. The apoER2

deficient plaques also had characteristics similar to unstable plaques including the

presence of a necrotic core, the accumulation of macrophages and apoptotic cells in the plaque cap region, and a reduction in SMCs in the plaque cap region. We attribute these changes in lesion composition to the enhanced susceptibility of apoER2 deficient macrophages to stress-induced death and the reduced proliferative capacity and

viability of apoER2 deficient SMCs seen in vitro. While the Lrp8-/- Ldlr-/- lesions did

appear more complex and had a phenotype similar to unstable plaques, plaque

disruption and plaque thrombosis were not seen in the aortic root lesions even after 24

48

weeks on the Western diet. However, we cannot rule out the possibility of rupture occurring in this model if atherosclerosis were assessed following a longer period of

Western diet feeding and/or assessment of plaques was performed in more humanistic, rupture-prone arteries, such as the innominate artery (190).

Monocytes and macrophages have integral roles in atherosclerosis and in

maintenance of plaque stability (103). During fatty streak formation, activated

endothelial cells and leukocytes recruit monocytes to the site of lesion formation.

Following migration across the endothelium, the monocytes proliferate in the intima and

differentiate into macrophages. These macrophages serve as a mechanism to remove

lipoprotein particles, dead cells, and cellular debris that accumulate in the intima. As

macrophages accumulate large amounts of lipid they form highly lipidated foam cells

(14-16). While macrophages are initially beneficial due to their ability to remove

extracellular lipids from the vasculature, foam cells can further promote atherosclerosis

by secreting inflammatory cytokines that activate neighboring endothelial cells,

leukocytes, and SMCs. They also produce large amounts of matrix-degrading matrix

metalloproteinases and secrete molecules that enhance SMC migration and proliferation and promote apoptosis in neighboring cells (12, 13, 19). For this reason,

complex lesions tend to have larger amounts of lesional macrophages. The localization

of the macrophages tends to occur at the shoulder regions of the plaque and within the

plaque cap, resulting in weakening of the plaque structure (20, 22, 24).

This study demonstrated that apoER2 protects against aberrant lipid

accumulation, oxidative stress, and cell death in mouse macrophages. We also

observed an increase in the p53-positive plaque area in the atherosclerotic lesions of

49

Lrp8-/- Ldlr-/- mice which strongly colocalized with the macrophage marker CD68

suggesting that more apoptotic macrophages were present compared to the Lrp8+/+ Ldlr-

/- lesions. In contrast to this study, previous studies have shown that increased

macrophage death can be beneficial in reducing atherosclerosis (192, 193). We believe

that the apoER2 deficient macrophages require a “second hit” to exert a detrimental

physiological effect. Under basal culture conditions, there were a larger percentage of

pre-apoptotic apoER2 KD macrophages, but not dead cells compared to controls. The

exposure of phosphatidylserine on the exterior of the cell is an early event in the

initiation of apoptosis and can be reversed if the cell is able to recover. Therefore, only

cells staining positive for propidium iodide can actually be considered dead. There was

not a significant difference between the genotypes in the percentage of dead cells under

basal conditions. The enhanced susceptibility of the apoER2 deficient macrophages to

cell death was only seen in the presence of a high concentration of oxLDL. In vivo we

believe that the environment of the fatty streak is not tumultuous enough to promote

excessive macrophage death in the absence of apoER2. Once the plaque has formed

we believe this provides an environment conducive to increased susceptibility to

macrophage death in the Lrp8-/- Ldlr-/- mice. This results in increased macrophage

lipotoxicity, oxidative stress, and cell death that significantly contribute to lesion necrosis

and plaque complexity.

The in vitro analyses of the effects of apoER2 deficiency on macrophage

functions and viability were performed using the mouse macrophage cell line RAW

264.7 cells. The knockdown of Lrp8 was achieved in these cells through stable

expression of shRNA targeting mouse Lrp8. This resulted in significant reductions in

50

Lrp8 mRNA and apoER2 protein. A set of RAW 264.7 cells were also transduced with

an empty vector construct for use as a control. Surprisingly, the transduction with the

empty vector did significantly increase the Lrp8 mRNA levels compared to the non-

transduced control cells. However, apoER2 protein levels were not different between

the non-transduced control and empty vector control cells. Although differences

between the non-transduced control and the empty vector control cells were observed

in the lipid accumulation and the viability analyses, the empty vector cells trended in the

opposite direction of the RAW 264.7 cells with Lrp8 knocked down. This indicates that

the increased lipid accumulation and susceptibility to stress-induced cell death observed in the apoER2 knockdown RAW 264.7 cells is due to the absence of apoER2 and not the transduction process. If anything, the phenotypic alterations induced by the transduction process may have dampened the effects of apoER2 deficiency in these

cells.

Vascular SMCs are a critical component in the atherosclerotic disease process.

Activated endothelial cells, subendothelial lipids, and intimal macrophages promote the

migration of SMCs from the tunica media into the tunica intima and promote SMC

proliferation (12, 13, 194-196). This increase in intimal SMCs results in the formation of

a fibrous plaque rich in SMCs and extracellular matrix proteins produced by the SMCs.

As lesions become more severe, the SMCs form a fibrous cap that covers the plaque

separating the inner constituents of the plaque from the lumen (17). While SMC

proliferation has a detrimental effect on atherosclerosis in the early stages of the

disease, maintenance of SMC viability and proliferation are necessary to reduce clinical

complications in the late stages of the disease (147, 197-199). The SMCs located

51

within the fibrous cap play a crucial role in preventing plaque rupture. Not only do the

cells themselves enhance the stability of the plaque cap, but they also are the primary source for extracellular matrix proteins. Reductions in fibrous cap SMCs results in thinning and weakening of the cap due to loss of SMC cellularity and reduced production of extracellular matrix (21, 22). This reduces plaque stability, significantly increasing the likelihood for plaque rupture and the associated downstream consequences, such as thrombosis resulting in MI (3, 18). In instances of non-fatal MI following plaque rupture, SMC proliferation and maintenance of viability are also critical for efficient repair to occur (200).

This study showed that the absence of apoER2 resulted in significant alterations

in SMC proliferation and viability in vitro. The SMCs isolated from 6 week old Lrp8-/- mice had diminished proliferative capacity and reduced viability over time with each passage in culture compared to controls. Interestingly, the SMCs isolated from 10 to 12 week old mice had a drastically different phenotype than the cells isolated from 6 week old mice. The 10 to 12 week old Lrp8-/- SMCs were not able to double at all in vitro. In

fact, negative population doublings were calculated at each passage due to SMC levels

dropping below those originally seeded. This severe phenotype was attributed to

significant reductions in the percent viability of the Lrp8-/- SMCs at each passage. The

apoER2 deficient SMCs isolated from these mice were not even able to survive until the

third passage in vitro. Similarly, the number of population doublings in the SMCs

isolated from 26 to 34 week old Lrp8-/- mice were significantly lower than Lrp8+/+ SMCs immediately after isolation. In contrast to the other two age groups, there was no change in the viability of these cells indicating that the reductions in population

52

doublings in the older Lrp8-/- SMCs were strictly due to reduced proliferative capacity.

We believe that in the younger 6 week old mice, the SMCs are able to proliferate and

maintain viability somewhat normally. However, as the mice age it is likely that the

SMCs become more susceptible to cell death induced by the absence of apoER2,

which is consistent with the phenotype of the SMCs isolated from 10 to 12 week old

mice. It is likely that the remaining SMCs which do not undergo cell death then enter

into a senescent-like state where they proliferate at a much lower rate consistent with

the phenotype of the Lrp8-/- SMCs isolated from 26 to 34 week old mice. Since previous

studies have shown that SMCs isolated from atherosclerotic plaques replicate at a

much lower rate than those from non-diseased vessels and senescent SMCs have been

identified in both human and animal lesions, we believe that the reduced proliferation of

SMCs induced by the absence of apoER2 is just as detrimental as the reduced viability

observed in these cells (201).

It was surprising that the alterations in SMC proliferation and viability seen in the

Lrp8-/- SMCs in vitro did not result in changes in the blood pressure of these mice. This

can likely be attributed to the fact that, although proliferation and viability were reduced

in the absence of apoER2, a percentage of SMCs were still able to proliferate and

maintain viability. Therefore, it is likely that the alterations in SMC proliferation and

viability were not enough to induce a disease phenotype, such as hypertension, under

otherwise healthy conditions. It is believed that a “second hit”, such as diet-induced

atherosclerosis, is necessary for the Lrp8-/- SMC phenotype to have detrimental effects in vivo. Since the apoER2 deficient SMCs were capable of proliferating, albeit at a

lower rate than Lrp8+/+ SMCs, atherogenesis is thought to have occurred as expected in

53

the Lrp8-/- Ldlr-/- mice. As the disease progressed in the Lrp8-/- Ldlr-/- mice, the lack of

SMC proliferation and enhanced cell death likely exacerbated the severity of the lesion

progression contributing to the formation of highly complex, necrotic lesions.

The interactions that occur between SMCs and macrophages can significantly

impact their function and phenotype (12, 13, 196, 202). Our in vitro studies

demonstrate that when cultured alone SMCs and macrophages have reduced viability

and other dysfunctions associated with reduced apoER2. However, within the vessel

wall interactions between the two cell types could exacerbate the phenotypes observed

in vitro. For example, a large amount of foam cell death likely contributes to reductions

in SMC viability within the Lrp8-/- Ldlr-/- plaques. Additionally, endothelial cells and platelets, both of which participate in atherosclerosis and thrombosis, are also known to express apoER2. Therefore, we cannot rule out that these cells may also contribute to the advanced atherosclerosis and plaque necrosis observed in the Lrp8-/- Ldlr-/- mice.

Maintenance of proper endothelial function is a critical component in the

regulation of vascular tone and atherosclerosis. A reduction in the bioavailability of

nitric oxide as a result of dysfunctional endothelial cells causes defective vasodilation of

the vessel and predisposes the dysfunctional area to atherosclerosis (203). The apoER2 has recently been associated with the regulation of eNOS activity in antiphospholipid syndrome. The binding of antiphospholipid antibodies to β2GPI results

in the formation of a dimerized β2GPI-antibody complex which interacts directly with apoER2 on endothelial cells. The binding of β2GPI to the ligand binding domain of apoER2 results in enhanced activation of the phosphatase PP2A and subsequent inactivation of eNOS through dephosphorylation (76). Additionally, stimulation of

54

endothelial cells in vitro with Apoe has been proposed to stimulate activation of eNOS

through an apoER2-dependent mechanism (204). Since the regulation of nitric oxide

production by endothelial cells can have such a significant impact on atherosclerosis, it

is likely that apoER2 deficiency results in alterations in endothelial cell function

contributing to the advanced atherosclerosis phenotype we observed in the Lrp8-/- Ldlr-/-

mice.

Platelets play a critical role in the coagulation process following plaque rupture.

Additionally, activated platelets promote inflammation through interactions with

circulating leukocytes (205). The apoER2 has previously been shown to regulate

platelet activation. The expression of apoER2 by platelets has been identified as a

necessary component in β2GPI-induced platelet adhesion (77, 206, 207). Ligation of

apoER2 on platelets with LDL has also been shown to result in activation of p38MAPK

indicative of increased platelet activation, whereas ligation of apoER2 with Apoe

inhibited platelet activation (128, 146, 208). Additionally, apoER2 deficiency in mice has

been shown to reduce platelet aggregation and thrombosis (208). Expression of the

LRP8 R952Q genetic variant has also been shown to increase ADP-stimulated platelet

aggregation (79). Taken together, these data suggest that apoER2 deficiency results in

defective platelet activation and thrombosis. Therefore, it is unlikely that the

atherosclerosis phenotype observed in the Lrp8-/- Ldlr-/- mice in this study is due to

alterations in platelet function.

Future studies are necessary in order to determine the contribution of each cell

type to the increased atherosclerotic lesion complexity observed in the Lrp8-/- Ldlr-/- mice. The use of mice with cell-specific inactivation of the Lrp8 gene will allow us to

55

delineate which cells have the greatest impact on the progression of atherosclerosis in

the absence of apoER2. The transplantation of bone marrow from Lrp8-/- Ldlr-/- mice

into irradiated Lrp8+/+ Ldlr-/- mice will allow us to achieve a macrophage-specific deletion of apoER2 within the atherosclerotic lesions (209). Additionally, smooth muscle-specific

Lrp8-/- mice will be generated by crossing mice expressing Cre driven by the sm22

with Lrp8flox/flox mice (47, 210). Following crossing these mice onto an Ldlr

deficient background, atherosclerosis studies will be performed to determine the effects

of the SMC-specific deletion of apoER2 within the atherosclerotic lesion. Additional

studies will also allow for the delineation of the role apoER2 plays in the maintenance of

endothelial cell function and the effects this has on atherosclerosis. Further

investigation should also be performed in order to determine the exact mechanism by

which apoER2 exerts its effects on macrophages and SMCs. This will include

determining if apoER2 mediates Akt activation in macrophages and SMCs through a

Dab1-dependent pathway. Future studies will also be performed to determine why

apoER2 deficiency results in increased activation of PPARγ in macrophages and if a

similar process occurs in apoER2 deficient SMCs.

56

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Tables

Table 1. Primer sequences used for quantitative real-time PCR analysis of experimental and reference genes.

Gene Name Primer Sequence Abca1 Sense 5’-ACCCACCCTACGAACAACATGAGT-3’ Antisense 5’-AAAGTTTCCAACAACACCGGGAGC-3’ Cd36 Sense 5’-CTGTTATTGGTGCAGTCCTGGC -3’ Antisense 5’-TATGTGGTGCAGCTGCTACAGC-3’ Gapdh Sense 5’-GGTGTGAACGGATTTGGCCGTATT-3’ Antisense 5’-GGTCGTTGATGGCAACAATCTCCA-3’ Gpx1 Sense 5’-GACACCAGGAGAATGGCAAGAATG-3’ Antisense 5’-AATTGGGCTCGAACCCGCCAC-3’ Lrp1 Sense 5’-CTGAAGGGCTTTGTGGATGAGCATAC-3’ Antisense 5’-GTAGAAGTTTCCCGTCAGCCAGTC-3’ Lrp8 (apoER2) Sense 5’-TCATCGTGCCCATAGTGGTAATAG-3’ Antisense 5’-TTGGTGTTCTTCCGCTTCCAGTTC-3’ Nox2 Sense 5’-TGCAGCCTGCCTGAATTTCAACTG-3’ Antisense 5’-AGATGTGCAATTGTGTGGATGGCG-3’ Nox4 Sense 5’-AAACACCTCTGCCTGCTCATTTGG-3’ Antisense 5’-AGGTTCAGGACAGATGCAGATGCT-3’ Pparg (PPARγ) Sense 5’-CTGCAGGCCCTGGAACTG-3’ Antisense 5’-CGATCTGCCTGAGGTCTGTCA-3’ Txnrd1 Sense 5’-CCACAAACAGCGAGGAGACCATAG-3’ Antisense 5’-TCGTTTATCTTCACGCCCACGGTC-3’

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Table 2. Rosiglitazone-associated genes upregulated two-fold or greater in RAW 264.7 cells with Lrp8 knocked down compared to non-transduced RAW 264.7 cells.

Gene Fold Name Description Change Cd36 CD36 antigen 46.20 Pcdh7 Protocadherin 7 16.68 Cd86 CD86 antigen 9.27 Timp2 Tissue inhibitor of metalloproteinase 2 8.55 Ccl7 Chemokine (C-C motif) ligand 7 7.52 Lcn2 Lipocalin 2 7.29 Fabp4 Fatty acid binding protein 4, adipocyte 7.09 Plk2 Polo-like kinase 2 6.99 Ccl2 Chemokine (C-C motif) ligand 2 5.84 Ppap2b Phosphatidic acid phosphatase type 2B 5.01 Cnn2 Calponin 2 3.62 Abca1 ATP-binding cassette, sub-family A (ABC1), member 1 3.55 Adamts1 A disintegrin-like and metallopeptidase (reprolysin type) with 3.20 thrombospondin type 1 motif, 1 Lpl Lipoprotein lipase 2.61 Cd300a CD300A antigen 2.52 Rgs2 Regulator of G-protein signaling 2 2.49 Rxra Retinoid X receptor alpha 2.46 Xdh Xanthine dehydrogenase 2.46 Actg2 Actin, gamma 2, smooth muscle, enteric 2.41 Csf1 Colony stimulating factor 1 (macrophage) 2.37 Gdf15 Growth differentiation factor 15 2.35 Hlx H2.0-like homeobox 2.26 Elovl3 Elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, 2.21 yeast)-like 3 Pltp Phospholipid transfer protein 2.06 Dhrs9 Dehydrogenase/reductase (SDR family) member 9 2.03 Hit Count in Query List = 25; Hit Count in Genome = 360; p = 0.00001218

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Figure Legends

Figure 1. Aortic atherosclerotic lesion area in Ldlr+/+ and Ldlr-/- mice with or

without apoER2 expression. Age-matched male Lrp8+/+ Ldlr+/+, Lrp8-/- Ldlr+/+, Lrp8+/+

Ldlr-/-, and Lrp8-/- Ldlr-/- mice were fed a high fat and high cholesterol Western-type diet

for 24 weeks. A, Representative photomicrographs of en face Oil Red O staining of

mouse whole aortas following Western diet feeding. B, Quantification of the

atherosclerotic lesion area in the thoracic aorta (Thoracic), abdominal aorta (Abdom),

and whole aorta (Whole) of Lrp8+/+ Ldlr-/- (black bars) and Lrp8-/- Ldlr-/- (white bars) mice

following Western diet feeding. Data are expressed as ratios of lesion area to whole

area for each section of the aorta. Lrp8+/+ Ldlr-/- n = 10 and Lrp8-/- Ldlr-/- n = 9 per group.

Mean ± SE. ** denotes P<0.05 difference from Lrp8+/+ Ldlr-/-.

Figure 2. Plasma cholesterol levels and lipoprotein profiles in apoER2 deficient

mice after 24 weeks of Western diet feeding. Age-matched male Lrp8+/+ Ldlr+/+, Lrp8-

/- Ldlr+/+, Lrp8+/+ Ldlr-/-, and Lrp8-/- Ldlr-/- mice were fed a high fat and high cholesterol

Western-type diet for 24 weeks. A, Plasma cholesterol levels in Lrp8+/+ Ldlr+/+, Lrp8-/-

Ldlr+/+, Lrp8+/+ Ldlr-/-, and Lrp8-/- Ldlr-/- mice after Western diet feeding. B, Lipoprotein

profiles of Lrp8+/+ Ldlr-/- (black symbols) and Lrp8-/- Ldlr-/- (white symbols) mice after

Western diet feeding. Plasma cholesterol n = 10 per group; lipoprotein profile Lrp8+/+

Ldlr-/- n = 3 samples pooled from 11 mice total and Lrp8-/- Ldlr-/- n = 4 samples pooled

from 5 mice total. Mean ± SE. * denotes P<0.05 from Ldlr+/+ controls.

Figure 3. Atherosclerotic lesion development in Ldlr-/- mice with or without

apoER2 expression after 12 and 24 weeks on Western diet. Age-matched male

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Lrp8+/+ Ldlr-/- (Con, black bars), and Lrp8-/- Ldlr-/- (KO, white bars) mice were fed a high

fat and high cholesterol Western-type diet for 12 or 24 weeks. A, Representative

photomicrographs of aortic root lesions from Lrp8+/+ Ldlr-/- and Lrp8-/- Ldlr-/- mice

following 12 weeks of Western diet feeding. Sections were stained with Oil Red O or

Sirius Red as indicated. The scale bars represent 100 μm. Arrows indicate collagen

layers. B, Representative photomicrographs of aortic root lesions from Lrp8+/+ Ldlr-/- and Lrp8-/- Ldlr-/- mice following 24 weeks of Western diet feeding. Sections were

stained with Oil Red O or Sirius Red as indicated. The scale bars represent 100 μm.

Arrows indicate the necrotic core. C, Morphometric analysis of the aortic root atherosclerotic lesion sizes from mice following 24 weeks on a Western diet. D,

Morphometric analysis of extracellular matrix area (ECM), lipid-rich area (lipid), and necrotic core area (necrotic) in the aortic root lesions of mice following 24 weeks on a

Western diet. Data are expressed as ratios of compositional area to total lesion size.

Lesion size n = 6, ECM n = 4, lipid n = 7, necrotic n = 7 per group. Mean ± SE. * denotes P≤0.05 difference from Lrp8+/+Ldlr-/- mice.

Figure 4. Cellular composition of the atherosclerotic lesions in Ldlr-/- mice with or

without apoER2 expression after 24 weeks on Western diet. Representative

photomicrographs of aortic root lesions from Lrp8+/+ Ldlr-/- and Lrp8-/- Ldlr-/- mice fed a

high fat and high cholesterol Western-type diet for 24 weeks. Sections were labeled

with immunofluorescent antibodies against CD68 (red, DAPI is blue) and α-smooth

muscle actin (α-SM-actin, red, DAPI is blue) to identify macrophages and smooth

muscle cells, respectively. Fluorscent TUNEL labeling (green, DAPI is red) was also

performed to identify apoptotic cells within the lesions. The scale bars represent 100

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μm. CD68 n = 4 per group; α-SM-actin n = 2 per group; TUNEL Lrp8+/+ Ldlr-/- n = 4 and

Lrp8-/- Ldlr-/- n = 3.

Figure 5. Fasting and postprandial circulating monocytes and neutrophils in

Lrp8+/+ and Lrp8-/- mice. Blood was drawn from Lrp8+/+ (WT, black bars) and Lrp8-/-

(KO, white bars) mice following an overnight fast and two hours after an oral gavage of a mixed meal. A, Whole blood labeled with fluorescently labeled antibodies targeting

CD11b and CD115. B, Whole blood labeled with fluorescently labeled antibodies targeting Ly6C and Ly6G. Data are expressed as the percentage of the total cell population staining positive. n = 3 per group. Mean ± SE.

Figure 6. Generation of Lrp8 deficient macrophages. A, Lrp8 mRNA expression in peritoneal macrophages (MPM) isolated from Lrp8+/+ (Control, black bars) and Lrp8-/-

(KO, white bars) mice, non-transduced control RAW 264.7 cells (Control, black bars),

RAW 264.7 cells transduced with an empty vector (Empty Vector, gray bars), and RAW

264.7 cells with Lrp8 knocked down (KD, white bars). Data are expressed relative to the Gapdh housekeeping gene. B, Levels of apoER2 protein in non-transduced control

RAW 264.7 cells (Con, black bar), RAW 264.7 cells transduced with an empty vector

(EV, gray bar), and RAW 264.7 cells with Lrp8 knocked down (KD, white bar). Data are expressed as ratios of apoER2 to the tubulin loading control. Inset shows representative immunoblots. MPM n = 5 per group, RAW 264.7 mRNA n = 9 per group,

RAW 264.7 control and knockdown protein n = 6 per group, RAW 264.7 empty vector protein n = 9. Mean ± SE. * denotes P≤0.05 difference from Lrp8+/+/non-transduced

control RAW 264.7, † denotes P<0.05 difference from empty vector RAW 264.7.

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Figure 7. Neutral lipid accumulation in macrophages lacking apoER2. Non- transduced control RAW 264.7 cells (gray histograms, black bars), RAW 264.7 cells transduced with an empty vector (gray bars), and RAW 264.7 cells with Lrp8 knocked down (white histograms, KD, white bars) were incubated in the presence (oxLDL) or absence (NT) of 50 µg/mL oxLDL overnight. The cells were then analyzed for neutral lipid accumulation by staining with HCS LipidTOX™ Green Neutral Lipid Stain and subsequent flow cytometry analysis. The median fluorescence intensity (MFI) of the cells was determined by analysis of histograms. Top panels show representative

histograms generated by flow cytometry analysis of non-transduced control and knockdown RAW 264.7 cells. RAW 264.7 empty vector n = 6, RAW 264.7 non- transduced control and knockdown n = 3 per group. Mean ± SE. Different letters denote P≤0.05 difference.

Figure 8. Oxidized LDL-induced death in control and apoER2 deficient macrophages. Non-transduced control RAW 264.7 cells (black bars), RAW 264.7 cells

transduced with an empty vector (gray bars), and RAW 264.7 cells with Lrp8 knocked

down (white bars) were incubated overnight with 500 μg/ml oxLDL. The cells were then

stained with APC-conjugated Annexin-V and propidium iodide and analyzed by flow

cytometry. Top panels show representative dot plots generated by flow cytometry

analysis. Region markers were applied to quantify the percentage of Annexin-V positive

pre-apoptotic cells (A+/PI-) and Annexin-V and propidium iodide positive dead cells

(A+/PI+) in the populations. RAW 264.7 empty vector n = 9, RAW 264.7 non-

transduced control and knockdown n = 6 per group. Mean ± SE. * denotes P<0.05

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difference from non-transduced control cells, † denotes P<0.05 difference from empty

vector cells.

Figure 9. Reactive oxygen species levels in control and apoER2 deficient

macrophages. Non-transduced control RAW 264.7 cells (Con, gray histogram, black

bar) and RAW 264.7 cells with Lrp8 knocked down (KD, white histogram, white bar)

were analyzed by flow cytometry following staining with 5-(and -6)-carboxy-2’,7’-

dichlorodihydrofluorescein diacetate. Region markers were applied to determine the

median fluorescence intensity (MFI) of positive staining cells. Left panel shows a

representative histogram generated by flow cytometry analysis. Data (corrected MFI)

are expressed as the differences of the MFI of positively stained cells (right histogram peaks) from the MFI of the negatively stained cells (left histogram peaks). n = 3 per group. Mean ± SE. * denotes P<0.05 difference from control.

Figure 10. Selenium levels in control and apoER2 deficient macrophages. Whole

cell selenium levels in non-transduced control RAW 264.7 cells (Con, black bar) and

RAW 264.7 cells with Lrp8 knocked down (KD, white bar). Data are expressed as ratios

of the amount of selenium to 1 x 106 cells. n = 3 per group. Mean ± SE.

Figure 11. Activated Akt protein levels in control and apoER2 deficient macrophages. Immunoblots of protein extracts from non-transduced control RAW

264.7 (Control, black bars, +) and Lrp8 knockdown RAW 264.7 cells (KD, white bars, -).

Following serum starvation, cells were incubated with fresh media containing 10% FBS

for 0, 0.5, and 24 hours. A, Analysis of Akt activation was performed using antibodies

targeting Akt phosphorylated at serine 473 (P-Akt). B, Analysis of total Akt protein

96 abundance was performed using antibodies targeting Akt. Data are expressed as ratios of target protein to the tubulin loading control. Inset shows representative immunoblots.

Akt control 0 hr n = 9, Akt KD 0 hr n = 8, Akt control and KD 0.5 hr n = 7, Akt control and

KD 24 hr n = 6. Mean ± SE. * denotes P<0.05 difference from non-transduced control cells.

Figure 12. Pro-apoptotic protein levels in control and apoER2 deficient macrophages. Immunoblots of protein extracts from non-transduced control RAW

264.7 (Control, black bars), RAW 264.7 cells transduced with an empty vector (Empty

Vector, gray bars), and Lrp8 knockdown RAW 264.7 cells (KD, white bars). A,

Following 24 hours in standard culture conditions, the analysis of cathepsin L was performed using antibodies targeting the three isoforms of cathepsin L, the pro-form

(Pro), the single chain form (Single), and the heavy chain form (Heavy). B, Following

24 hours in standard culture conditions, the analysis of p53 was performed using antibodies targeting p53 phosphorylated at serine 15 (P-p53, Phos) and total p53. Data are expressed as ratios of target protein to the tubulin loading control. Inset shows representative immunoblots. RAW 264.7 empty vector n = 9, RAW 264.7 non- transduced control and knockdown n = 6 per group. Mean ± SE. * denotes P≤0.05 difference from non-transduced control cells, † denotes P<0.05 difference from empty vector cells.

Figure 13. Atherosclerotic lesion abundance of p53 in Ldlr-/- mice with or without apoER2 expression after 24 weeks on Western diet. A, Representative photomicrographs of aortic root lesions from Lrp8+/+ Ldlr-/- (Con, black bar) and Lrp8-/-

Ldlr-/- (KO, white bar) mice fed a Western diet for 24 weeks labeled with

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immunofluorescent antibodies against p53 (red), CD68 (blue), and counterstained with

DAPI (green). Morphometric analysis of p53-positive area in the aortic root lesions expressed as ratios of positively-stained area to total lesion size. The scale bars represent 100 μm. n = 6 per group. Mean ± SE. * denotes P<0.05 difference from

Lrp8+/+ Ldlr-/- mice.

Figure 14. PPARγ expression and activation in control and apoER2 deficient macrophages. A, The Pparg (PPARγ) mRNA and nuclear PPARγ1/2 protein levels in non-transduced control (Con, black bars), empty vector (EV, gray bars), and Lrp8 knockdown (KD, white bars) RAW 264.7 cells after a 24 hour incubation in standard culture conditions. Inset shows representative immunoblots of nuclear proteins from

RAW 264.7 cells. B, The mRNA levels of the PPARγ-responsive genes Cd36, Lrp1, and Abca1 in non-transduced control (Con, black bars), empty vector (EV, gray bars), and Lrp8 shRNA transduced (KD, white bars) RAW 264.7 cells. The mRNA data are relative to the Gapdh housekeeping gene. Nuclear protein data are expressed as ratios of target protein to the TATA-binding protein (TBP) loading control. mRNA n = 9 per group, RAW 264.7 empty vector protein n = 9, RAW 264.7 non-transduced control and knockdown protein n = 6. Mean ± SE. * denotes P<0.05 difference from non- transduced control, † denotes P<0.05 difference from empty vector cells.

Figure 15. Atherosclerotic lesion abundance of PPARγ in Ldlr-/- mice with or

without apoER2 expression after 24 weeks on Western diet. A, Representative

photomicrographs of aortic root lesions from Lrp8+/+ Ldlr-/- (Con, black bar) and Lrp8-/-

Ldlr-/- (KO, white bar) mice fed a Western diet for 24 weeks labeled with

immunofluorescent antibodies against PPARγ (red), CD68 (blue), and counterstained

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with DAPI (green). Morphometric analysis of PPARγ-positive area in the aortic root

lesions expressed as ratios of positively-stained area to total lesion size. The scale

bars represent 100 μm. n = 6 per group. Mean ± SE. * denotes P<0.05 difference from

Lrp8+/+ Ldlr-/- mice.

Figure 16. Aortic smooth muscle cell population doublings and viability following

isolation from 6 week old chow-fed Lrp8+/+ and Lrp8-/- mice. A, Population

doublings measured between each passage of Lrp8+/+ (WT, black bars) and Lrp8-/- (KO, white bars) mouse aortic smooth muscle cells in vitro. B, Percent viability determined at each passage for Lrp8+/+ (WT, black bars) and Lrp8-/- (KO, white bars) mouse aortic

smooth muscle cells in vitro. n = 3 per group. Mean ± SE. * denotes P<0.05 difference

from Lrp8+/+.

Figure 17. Aortic smooth muscle cell population doublings and viability following

isolation from 10 to 12 week old chow-fed Lrp8+/+ and Lrp8-/- mice. A, Population

doublings measured between each passage of Lrp8+/+ (WT, black bars) and Lrp8-/- (KO, white bars) mouse aortic smooth muscle cells in vitro. B, Percent viability determined at each passage for Lrp8+/+ (WT, black bars) and Lrp8-/- (KO, white bars) mouse aortic

smooth muscle cells in vitro. Lrp8+/+ and Lrp8-/- Passage 1 n = 4 per group; Lrp8+/+

Passage 2 n = 4; Lrp8-/- Passage 2 n = 3. Mean ± SE. * denotes P<0.05 difference

from Lrp8+/+.

Figure 18. Aortic smooth muscle cell population doublings and viability following

isolation from 26 to 34 week old chow-fed Lrp8+/+ and Lrp8-/- mice. A, Population

doublings measured between each passage of adherent Lrp8+/+ (WT, black bars) and

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Lrp8-/- (KO, white bars) mouse aortic smooth muscle cells in vitro. B, Percent viability determined at each passage for adherent Lrp8+/+ (WT, black bars) and Lrp8-/- (KO, white

bars) mouse aortic smooth muscle cells in vitro. C, The total number of non-adherent

Lrp8+/+ (WT, black bars) and Lrp8-/- (KO, white bars) mouse aortic smooth muscle cells

present in the media at each passage. Data is expressed as a ratio of the total number

of non-adherent cells to the number of cells originally seeded. D, Percent viability

determined for the non-adherent Lrp8+/+ (WT, black bars) and Lrp8-/- (KO, white bars)

mouse aortic smooth muscle cells present in the media at each passage. Lrp8+/+

Passage 1 n = 6; Lrp8-/- Passage 1 n = 7; Lrp8+/+ and Lrp8-/- Passage 2 n = 6 per group;

Lrp8+/+ Passage 3 n = 6; Lrp8-/- Passage 3 n = 4. Mean ± SE. * denotes P<0.05

difference from Lrp8+/+.

Figure 19. Akt activation in the whole aortas of chow-fed Lrp8+/+ and Lrp8-/- mice.

Immunoblots of whole tissue homogenates from Lrp8+/+ (WT, black bars) and Lrp8-/-

(KO, white bars) whole aortas were performed using antibodies targeting Akt

phosphorylated at serine 473 (P-Akt) and total Akt. Data are expressed as ratios of the

target protein to the tubulin loading control. Inset shows representative immunoblots. n

= 3 per group. Mean ± SE. * denotes P<0.05 difference from Lrp8+/+.

Figure 20. JNK and p38MAPK activation in the whole aortas of chow-fed Lrp8+/+ and

Lrp8-/- mice. A, Immunoblots of whole tissue homogenates from Lrp8+/+ (WT, black

bars) and Lrp8-/- (KO, white bars) whole aortas were performed using antibodies

targeting JNK 1/2 dually phosphorylated at threonine 183 and tyrosine 185 (P-JNK 1/2)

and total JNK 1/2. B, Immunoblots of whole tissue homogenates from Lrp8+/+ (WT,

black bars) and Lrp8-/- (KO, white bars) whole aortas were performed using antibodies

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targeting p38MAPK dually phosphorylated at threonine 180 and tyrosine 182 (P-p38) and

total p38MAPK. Data are expressed as ratios of the target protein to the tubulin loading

control. Insets show representative immunoblots. n = 3 per group. Mean ± SE.

Figure 21. Reactive oxygen species levels in Lrp8+/+ and Lrp8-/- aortic smooth

muscle cells in vitro. Representative photomicrographs of smooth muscle cells

isolated from the thoracic aortas of chow-fed Lrp8+/+ and Lrp8-/- stained with 5-(and -6)-

carboxy-2’,7’-dichlorodihydrofluorescein diacetate (green) and counterstained with DAPI

(blue). n = 4 per group.

+/+ -/- Figure 22. F2α-isoprostane abundance in the aortas of chow-fed Lrp8 and Lrp8 mice. Representative photomicrographs of the aortic arch and the thoracic aorta immunofluorscently labeled with antibodies targeting F2α-isoprostane (red) and

counterstained with DAPI (blue). The scale bars represent 100 μm. n = 2 per group.

Figure 23. NADPH oxidase mRNA abundance and enzyme activity in Lrp8+/+ and

Lrp8-/- mice. A, mRNA levels of Nox2 and Nox4 in Lrp8+/+ (WT, black bars) and Lrp8-/-

(KO, white bars) whole aorta. Data are expressed relative to the Gapdh housekeeping gene. B, NADPH oxidase enzyme activity levels in aortic smooth muscle cells isolated from Lrp8+/+ (WT, black bars) and Lrp8-/- (KO, white bars) mice. n = 3 per group. Mean

± SE. * denotes P<0.05 difference from Lrp8+/+.

Figure 24. Selenium levels in Lrp8+/+ and Lrp8-/- whole aortas. Tissue selenium levels in the whole aortas of chow-fed Lrp8+/+ (WT, black symbols) and Lrp8-/- (KO,

white symbols) mice. Data are expressed as ratios of the amount of selenium to the

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weight of the tissue. Lrp8+/+ n = 7 and Lrp8-/- n = 3. Mean ± SE. * denotes P<0.05

difference from Lrp8+/+.

Figure 25. Expression and activity of Gpx1 and Txnrd1 in Lrp8+/+ and Lrp8-/- whole

aortas. The mRNA levels for the genes encoding glutathione peroxidase 1 (A, Gpx1)

and thioredoxin reductase 1 (B, Txnrd1) in the whole aortas of chow-fed Lrp8+/+ (WT,

black bars) and Lrp8-/- (KO, white bars). The protein levels of Gpx1 (C) and Txnrd1 (D) in the whole aortas of chow-fed Lrp8+/+ (WT, black bars) and Lrp8-/- (KO, white bars).

Gpx1 enzyme activity (D) in the whole aortas of chow-fed Lrp8+/+ (WT, black bars) and

Lrp8-/- (KO, white bars). The mRNA data are expressed relative to the Gapdh

housekeeping gene. Protein data are expressed as ratios of the target protein to the

tubulin loading control. Insets show representative immunoblots. n = 3 per group.

Mean ± SE. * denotes P<0.05 difference from Lrp8+/+.

Figure 26. Blood pressure measurements from chow-fed Lrp8+/+ and Lrp8-/- mice.

Mean arterial pressure (A), systolic blood pressure (B), and diastolic blood pressure (C) measurements obtained through tail-cuff blood pressure measurements from Lrp8+/+

(WT, black bars) and Lrp8-/- (KO, white bars) mice. Lrp8+/+ n = 3 and Lrp8-/- n = 4.

Mean ± SE.

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