Patricia G. Yancey

Inactivation of Macrophage Scavenger Receptor Class B Type I Promotes Atherosclerotic Lesion Development in Apolipoprotein E–Deficient Mice

Background—Scavenger receptor class B type I (SR-BI) is expressed in macrophages, where it has been proposed to facilitate cholesterol efflux. However, direct evidence that the expression of macrophage SR-BI is protective against atherosclerosis is lacking. In this study, we examined the in vivo role of macrophage SR-BI in atherosclerotic lesion development in the apolipoprotein (apo) E–deficient mouse model. Methods and Results—ApoE-deficient mice with (n=16) or without (n=15) expression of macrophage SR-BI were created by transplanting lethally irradiated apoE-deficient mice with bone marrow cells collected from SR-BI−/− apoE−/− mice or SR-BI+/+ apoE−/− mice. The recipient mice were fed a chow diet for 12 weeks after transplantation for analysis of atherosclerosis. Quantification of macrophage SR-BI mRNA by real-time reverse transcription–polymerase chain reaction indicated successful engraftment of donor bone marrow and inactivation of macrophage SR-BI in recipient mice reconstituted with SR-BI−/− apoE−/− bone marrow. There were no significant differences in plasma lipid levels, lipoprotein distributions, and HDL subpopulations between the 2 groups. Analysis of the proximal aorta demonstrated an 86% increase in mean atherosclerotic lesion area in SR-BI−/− apoE−/− → apoE−/− mice compared with SR-BI+/+ apoE−/− → apoE−/− mice (109.50±18.08 versus 58.75±9.58×103 &mgr;m2; mean±SEM, P =0.017). No difference in cholesterol efflux from SR-BI+/+ apoE−/− or SR-BI−/− apoE−/− macrophages to HDL or apoA-I discs was detected. Conclusions—Expression of macrophage SR-BI protects mice against atherosclerotic lesion development in apoE-deficient mice in vivo without influencing plasma lipids, HDL subpopulations, or cholesterol efflux. Thus, macrophage SR-BI plays an antiatherogenic role in vivo, providing a new therapeutic target for the design of strategies to prevent and treat atherosclerosis.

Conditional Knockout of Macrophage PPARγIncreases Atherosclerosis in C57BL/6 and Low-Density Lipoprotein Receptor–Deficient Mice

Objective—Peroxisome proliferator-activated receptor gamma (PPARγ) is highly expressed in macrophage-derived foam cells of atherosclerotic lesions, and its expression may have a dramatic impact on atherosclerosis. Methods and Results—To investigate the contribution of macrophage PPARγ expression on atherogenesis in vivo, we generated macrophage-specific PPARγ knockout (MacPPARγKO) mice. C57BL/6 and low-density lipoprotein (LDL) receptor–deficient (LDLR−/−) mice were reconstituted with MacPPARγKO or wild-type marrow and challenged with an atherogenic diet. No differences were found in serum lipids between recipients reconstituted with MacPPARγKO and wild-type marrow. In contrast, both C57BL/6 and LDLR−/− mice transplanted with MacPPARγKO marrow had significantly larger atherosclerotic lesions than control recipients. In addition, MacPPARγKO→LDLR−/− mice had higher numbers of macrophages in atherosclerotic lesions compared with controls. Peritoneal macrophages isolated from the MacPPARγKO mice had decreased uptake of oxidized but not acetylated LDL and showed no changes in either cholesterol efflux or inflammatory cytokine expression. Macrophages from MacPPARγKO mice had increased levels of migration and CC chemokine receptor 2 (CCR2) expression compared with wild-type macrophages. Conclusion—Thus, macrophage PPARγ deficiency increases atherosclerosis under conditions of mild and severe hypercholesterolemia, indicating an antiatherogenic role for PPARγ, which may be caused, at least in part, by modulation of CCR2 expression and monocyte recruitment.

Dysfunctional High-Density Lipoprotein in Patients on Chronic Hemodialysis

OBJECTIVES This study examined the functionality of high-density lipoprotein (HDL) in individuals with end-stage renal disease on dialysis (ESRD-HD). BACKGROUND The high rate of cardiovascular disease (CVD) in chronic kidney disease is not explained by standard risk factors, especially in patients with ESRD-HD who appear resistant to benefits of statin therapy. HDL is antiatherogenic because it extracts tissue cholesterol and reduces inflammation. METHODS Cellular cholesterol efflux and inflammatory response were assessed in macrophages exposed to HDL of patients with ESRD-HD or controls. RESULTS HDL from patients with ESRD-HD was dramatically less effective than normal HDL in accepting cholesterol from macrophages (median 6.9%; interquartile range [IQR]: 1.4% to 10.2%) versus control (median 14.9%; IQR: 9.8% to 17.8%; p < 0.001). The profound efflux impairment was also seen in patients with ESRD-HD and diabetes compared with patients with diabetes without renal disease (median 8.1%; IQR: 3.3% to 12.9%) versus control (median 13.6%; IQR: 11.0% to 15.9%; p = 0.009). In vitro activation of cellular cholesterol transporters increased cholesterol efflux to both normal and uremic HDL. HDL of patients with ESRD-HD had reduced antichemotactic ability and increased macrophage cytokine response (tumor necrosis factor-alpha, interleukin-6, and interleukin-1-beta). HDL of patients with ESRD-HD on statin therapy had reduced inflammatory response while maintaining impaired cholesterol acceptor function. Interestingly, impaired HDL-mediated efflux did not correlate with circulating C-reactive protein levels or cellular inflammatory response. CONCLUSIONS These findings suggest that abnormal HDL capacity to mediate cholesterol efflux is a key driver of excess CVD in patients on chronic hemodialysis and may explain why statins have limited effect to decrease CV events. The findings also suggest cellular cholesterol transporters as potential therapeutic targets to decrease CV risk in this population.

Serum Proprotein Convertase Subtilisin/Kexin Type 9 and Cell Surface Low-Density Lipoprotein Receptor Evidence for a Reciprocal Regulation

Background— Proprotein convertase subtilisin/kexin type 9 (PCSK9) modulates low-density lipoprotein (LDL) receptor (LDLR) degradation, thus influencing serum cholesterol levels. However, dysfunctional LDLR causes hypercholesterolemia without affecting PCSK9 clearance from the circulation. Methods and Results— To study the reciprocal effects of PCSK9 and LDLR and the resultant effects on serum cholesterol, we produced transgenic mice expressing human (h) PCSK9. Although hPCSK9 was expressed mainly in the kidney, LDLR degradation was more evident in the liver. Adrenal LDLR levels were not affected, likely because of the impaired PCSK9 retention in this tissue. In addition, hPCSK9 expression increased hepatic secretion of apolipoprotein B–containing lipoproteins in an LDLR-independent fashion. Expression of hPCSK9 raised serum murine PCSK9 levels by 4.3-fold in wild-type mice and not at all in LDLR−/− mice, in which murine PCSK9 levels were already 10-fold higher than in wild-type mice. In addition, LDLR+/− mice had a 2.7-fold elevation in murine PCSK9 levels and no elevation in cholesterol levels. Conversely, acute expression of human LDLR in transgenic mice caused a 70% decrease in serum murine PCSK9 levels. Turnover studies using physiological levels of hPCSK9 showed rapid clearance in wild-type mice (half-life, 5.2 minutes), faster clearance in human LDLR transgenics (2.9 minutes), and much slower clearance in LDLR−/− recipients (50.5 minutes). Supportive results were obtained with an in vitro system. Finally, up to 30% of serum hPCSK9 was associated with LDL regardless of LDLR expression. Conclusions— Our results support a scenario in which LDLR represents the main route of elimination of PCSK9 and a reciprocal regulation between these 2 proteins controls serum PCSK9 levels, hepatic LDLR expression, and serum LDL levels.

Human C-reactive protein promotes oxidized low density lipoprotein uptake and matrix metalloproteinase-9 release in Wistar rats

C-reactive protein (CRP) is present in the atherosclerotic plaques and appears to promote atherogenesis. Intraplaque CRP colocalizes with oxidized low density lipoprotein (OxLDL) and macrophages in human atherosclerotic lesions. Matrix metalloproteinase-9 (MMP-9) has been implicated in plaque rupture. CRP promotes OxLDL uptake and MMP induction in vitro; however, these have not been investigated in vivo. We examined the effect of CRP on OxLDL uptake and MMP-9 production in vivo in Wistar rats. CRP significantly increased OxLDL uptake in the peritoneal and sterile pouch macrophages compared with human serum albumin (huSA). CRP also significantly increased intracellular cholesteryl ester accumulation compared with huSA. The increased uptake of OxLDL by CRP was inhibited by pretreatment with antibodies to CD32, CD64, CD36, and fucoidin, suggesting uptake by both scavenger receptors and Fc-γ receptors. Furthermore, CRP treatment increased MMP-9 activity in macrophages compared with huSA, which was abrogated by inhibitors to p38 mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), and nuclear factor (NF)-κB but not Jun N-terminal kinase (JNK) before human CRP treatment. Because OxLDL uptake by macrophages contributes to foam cell formation and MMP release contributes to plaque instability, this study provides novel in vivo evidence for the role of CRP in atherosclerosis.

Self-Association of Human PCSK9 Correlates with Its LDLR-Degrading Activity

Genetic studies have demonstrated an important role for proprotein convertase subtilisin/kexin type 9 (PCSK9) as a determinant of plasma cholesterol levels. However, the underlying molecular mechanism is not completely understood. To this end, we have generated a mammalian cell expression system for human PCSK9 and its mutants and produced transgenic mice expressing human PCSK9. HEK293T cells transfected with the human PCSK9 DNA construct expressed and secreted PCSK9 and displayed decreased LDLR levels; functional PCSK9 protein was purified from the conditioned medium. In vitro studies showed that PCSK9 self-associated in a concentration-, temperature-, and pH-dependent manner. A mixture of PCSK9 monomers, dimers, and trimers displayed an enhanced LDLR degrading activity compared to monomeric PCSK9. A gain-of-function mutant, D374Y, displayed greatly increased self-association compared to wild-type PCSK9. Moreover, we demonstrated that the catalytic domain of PCSK9 is responsible for the self-association. Self-association of PCSK9 was enhanced by incubation with mouse apoE-/- VLDL and inhibited by incubation with both human and mouse HDL. When PCSK9 protein was incubated with total serum, it partially associated with LDL and HDL but not with VLDL. In transgenic mice, PCSK9 also associated with LDL and HDL but not with VLDL. We conclude that self-association is an intrinsic property of PCSK9, correlated to its LDLR-degrading activity and affected by plasma lipoproteins. These results provide a basis for developing strategies to manipulate PCSK9 activity in the circulation for the treatment of hypercholesterolemia.

Macrophage LRP-1 Controls Plaque Cellularity by Regulating Efferocytosis and Akt Activation

Objective—The balance between apoptosis susceptibility and efferocytosis of macrophages is central to plaque remodeling and inflammation. LRP-1 and its ligand, apolipoprotein E, have been implicated in efferocytosis and apoptosis in some cell types. We investigated the involvement of the macrophage LRP-1/apolipoprotein E axis in controlling plaque apoptosis and efferocytosis. Method and Results—LRP-1−/− macrophages displayed nearly 2-fold more TUNEL positivity compared to wild-type cells in the presence of DMEM alone or with either lipopolysaccharide or oxidized low-density lipoprotein. The survival kinase, phosphorylated Akt, was barely detectable in LRP-1−/− cells, causing decreased phosphorylated Bad and increased cleaved caspase-3. Regardless of the apoptotic stimulation and degree of cell death, LRP-1−/− macrophages displayed enhanced inflammation with increased IL-1&bgr;, IL-6, and tumor necrosis factor-&agr; expression. Efferocytosis of apoptotic macrophages was reduced by 60% in LRP-1−/− vs wild-type macrophages despite increased apolipoprotein E expression by both LRP-1−/− phagocytes and wild-type apoptotic cells. Compared to wild-type macrophage lesions, LRP-1−/− lesions had 5.7-fold more necrotic core with more dead cells not associated with macrophages. Conclusion—Macrophage LRP-1 deficiency increases cell death and inflammation by impairing phosphorylated Akt activation and efferocytosis. Increased apolipoprotein E expression in LRP-1−/− macrophages suggests that the LRP-1/apolipoprotein E axis regulates the balance between apoptosis and efferocytosis, thereby preventing necrotic core formation.

Macrophage Expression of Peroxisome Proliferator–Activated Receptor-α Reduces Atherosclerosis in Low-Density Lipoprotein Receptor–Deficient Mice

Background— The peroxisome proliferator–activated receptor-&agr; (PPAR&agr;) plays important roles in lipid metabolism, inflammation, and atherosclerosis. PPAR&agr; ligands have been shown to reduce cardiovascular events in high-risk subjects. PPAR&agr; expression by arterial cells, including macrophages, may exert local antiatherogenic effects independent of plasma lipid changes. Methods and Results— To examine the contribution of PPAR&agr; expression by bone marrow–derived cells in atherosclerosis, male and female low-density lipoprotein receptor–deficient (LDLR−/−) mice were reconstituted with bone marrow from PPAR&agr;−/− or PPAR&agr;+/+ mice and challenged with a high-fat diet. Although serum lipids and lipoprotein profiles did not differ between the groups, the size of atherosclerotic lesions in the distal aorta of male and female PPAR&agr;−/−→LDLR−/− mice was significantly increased (44% and 46%, respectively) compared with controls. Male PPAR&agr;−/−→LDLR−/− mice also had larger (44%) atherosclerotic lesions in the proximal aorta than male PPAR&agr;+/+→LDLR−/− mice. Peritoneal macrophages from PPAR&agr;−/− mice had increased uptake of oxidized LDL and decreased cholesterol efflux. PPAR&agr;−/− macrophages had lower levels of scavenger receptor B type I and ABCA1 protein expression and an accelerated response of nuclear factor-&kgr;B–regulated inflammatory genes. A laser capture microdissection analysis verified suppressed scavenger receptor B type I and increased nuclear factor-&kgr;B gene expression levels in vivo in atherosclerotic lesions of PPAR&agr;−/−→LDLR−/− mice compared with the lesions of control PPAR&agr;+/+→LDLR−/− mice. Conclusions— These data demonstrate that PPAR&agr; expression by macrophages has antiatherogenic effects via modulation of cell cholesterol trafficking and inflammatory activity.

A Pathway-Dependent on ApoE, ApoAI, and ABCA1 Determines Formation of Buoyant High-Density Lipoprotein by Macrophage Foam Cells

Objective—ABCA1-dependent and ABCA1-independent pathways may operate in high-density lipoprotein formation by macrophages secreting apolipoprotein (apo) E. We examined the impact of ABCA1 on apoE-mediated efflux from cholesterol-enriched macrophages. Methods and Results—Without acceptors, wild-type, ABCA1−/−, and apoE−/− macrophages released 5.7%±0.3%, 1.8%±0.1%, and 2.3%±0.2% of their cholesterol, and the LXR agonist, TO-901317, enhanced efflux by 137%, 10%, and 20%. Although similar amounts of apoE were secreted from ABCA1−/− and wild-type cells, apoE from ABCA1−/− cells was only partially phospholipidated and floated at density >1.21g/mL, whereas apoE from wild-type cells floated at density of 1.09 to 1.17g/mL and paralleled the density of cholesterol. With apoAI, LXR stimulation increased efflux by 139% and 86% from wild-type and apoE−/− cells, resulting in a large difference in efflux (29.5%±0.2% versus 17.0%±0.5%). The density of apoE and cholesterol from wild-type cells did not change with apoAI, and most apoAI floated at density ≥1.17g/mL. In apoE−/− cells, apoAI and cholesterol floated at similar density, but the peak fraction only contained 4 &mgr;g cholesterol/mg protein versus 18 in WT cells. Conclusions—Macrophage apoE requires ABCA1 for formation of high-density lipoprotein. ApoAI facilitates association of apoE with more buoyant high-density lipoprotein, suggesting that apoE, plasma apoAI, and ABCA1 operate together to optimize mobilization of macrophage cholesterol, a process critical to limiting plaque development.

Macrophage Polarization by Angiotensin II-Type 1 Receptor Aggravates Renal Injury-Acceleration of Atherosclerosis

Objective—Angiotensin II is a major determinant of atherosclerosis. Although macrophages are the most abundant cells in atherosclerotic plaques and express angiotensin II type 1 receptor (AT1), the pathophysiologic role of macrophage AT1 in atherogenesis remains uncertain. We examined the contribution of macrophage AT1 to accelerated atherosclerosis in an angiotensin II-responsive setting induced by uninephrectomy (UNx). Methods and Results—AT1−/− or AT1+/+ marrow from apolipoprotein E deficient (apoE−/−) mice was transplanted into recipient apoE−/− mice with subsequent UNx or sham operation: apoE−/−/AT1+/+→apoE−/−+sham; apoE−/−/AT1+/+ →apoE−/−+UNx; apoE−/−/AT1−/−→apoE−/−+sham; apoE−/−/AT1−/−→apoE−/−+UNx. No differences in body weight, blood pressure, lipid profile, and serum creatinine were observed between the 2 UNx groups. ApoE−/−/AT1+/+ →apoE−/−+UNx had significantly more atherosclerosis (16907±21473 versus 116071±8180 &mgr;m2, P<0.05). By contrast, loss of macrophage AT1 which reduced local AT1 expression, prevented any effect of UNx on atherosclerosis (77174±9947 versus 75714±11333 &mgr;m2, P=NS). Although UNx did not affect total macrophage content in the atheroma, lesions in apoE−/−/AT1−/−→apoE−/−+UNx had fewer classically activated macrophage phenotype (M1) and more alternatively activated phenotype (M2). Further, UNx did not affect plaque necrosis or apoptosis in apoE−/−/AT1−/−→apoE−/− whereas it significantly increased both (by 2- and 6-fold, respectively) in apoE−/−/AT1+/+ →apoE−/− mice. Instead, apoE−/−/AT1−/−→apoE−/− had 5-fold–increase in macrophage-associated apoptotic bodies, indicating enhanced efferocytosis. In vitro studies confirmed blunted susceptibility to apoptosis, especially in M2 macrophages, and a more efficient phagocytic function of AT1−/− macrophages versus AT1+/+. Conclusion—AT1 receptor of bone marrow-derived macrophages worsens the extent and complexity of renal injury-induced atherosclerosis by shifting the macrophage phenotype to more M1 and less M2 through mechanisms that include increased apoptosis and impaired efferocytosis.