John L. Blakemore

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.

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.

Quantum dot mediated imaging of atherosclerosis

The progression of atherosclerosis is associated with leukocyte infiltration within lesions. We describe a technique for the ex vivo imaging of cellular recruitment in atherogenesis which utilizes quantum dots (QD) to color-code different cell types within lesion areas. Spectrally distinct QD were coated with the cell-penetrating peptide maurocalcine to fluorescently-label immunomagnetically isolated monocyte/macrophages and T lymphocytes. QD-maurocalcine bioconjugates labeled both cell types with a high efficiency, preserved cell viability, and did not perturb native leukocyte function in cytokine release and endothelial adhesion assays. QD-labeled monocyte/macrophages and T lymphocytes were reinfused in an ApoE(-/-) mouse model of atherosclerosis and age-matched controls and tracked for up to four weeks to investigate the incorporation of cells within aortic lesion areas, as determined by oil red O (ORO) and immunofluorescence ex vivo staining. QD-labeled cells were visible in atherosclerotic plaques within two days of injection, and the two cell types colocalized within areas of subsequent ORO staining. Our method for tracking leukocytes in lesions enables high signal-to-noise ratio imaging of multiple cell types and biomarkers simultaneously within the same specimen. It also has great utility in studies aimed at investigating the role of distinct circulating leukocyte subsets in plaque development and progression.

Low-Density Lipoprotein Receptor–Related Protein 1 Prevents Early Atherosclerosis by Limiting Lesional Apoptosis and Inflammatory Ly-6Chigh Monocytosis Evidence That the Effects Are Not Apolipoprotein E Dependent

Background— We previously demonstrated that macrophage low-density lipoprotein receptor (LDLR)–related protein 1 (LRP1) deficiency increases atherosclerosis despite antiatherogenic changes including decreased uptake of remnants and increased secretion of apolipoprotein E (apoE). Thus, our objective was to determine whether the atheroprotective effects of LRP1 require interaction with apoE, one of its ligands with multiple beneficial effects. Methods and Results— We examined atherosclerosis development in mice with specific deletion of macrophage LRP1 (apoE−/− M&PHgr;LRP1−/−) and in LDLR−/− mice reconstituted with apoE−/− M&PHgr;LRP1−/− bone marrow. The combined absence of apoE and LRP1 promoted atherogenesis more than did macrophage apoE deletion alone in both apoE-producing LDLR−/− mice (+88%) and apoE−/− mice (+163%). The lesions of both mouse models with apoE−/− LRP1−/− macrophages had increased macrophage content. In vitro, apoE and LRP1 additively inhibit macrophage apoptosis. Furthermore, there was excessive accumulation of apoptotic cells in lesions of both LDLR−/− mice (+110%) and apoE−/− M&PHgr;LRP1−/− mice (+252%). The apoptotic cell accumulation was partially due to decreased efferocytosis as the ratio of free to cell-associated apoptotic nuclei was 3.5-fold higher in lesions of apoE−/− M&PHgr;LRP1−/− versus apoE−/− mice. Lesion necrosis was also increased (6 fold) in apoE−/− M&PHgr;LRP1−/− versus apoE−/− mice. Compared with apoE−/− mice, the spleens of apoE−/− M&PHgr;LRP1−/− mice contained 1.6- and 2.4-fold more total and Ly6-Chigh monocytes. Finally, there were 3.6- and 2.4-fold increases in Ly6-Chigh and CC-chemokine receptor 2–positive cells in lesions of apoE−/− M&PHgr;LRP1−/− versus apoE−/− mice, suggesting that accumulation of apoptotic cells enhances lesion development and macrophage content by promoting the recruitment of inflammatory monocytes. Conclusion— Low-density lipoprotein receptor protein 1 exerts antiatherogenic effects via pathways independent of apoE involving macrophage apoptosis and monocyte recruitment.

Macrophage SR-BI mediates efferocytosis via Src/PI3K/Rac1 signaling and reduces atherosclerotic lesion necrosis

Macrophage apoptosis and efferocytosis are key determinants of atherosclerotic plaque inflammation and necrosis. Bone marrow transplantation studies in ApoE- and LDLR-deficient mice revealed that hematopoietic scavenger receptor class B type I (SR-BI) deficiency results in severely defective efferocytosis in mouse atherosclerotic lesions, resulting in a 17-fold higher ratio of free to macrophage-associated dead cells in lesions containing SR-BI−/− cells, 5-fold more necrosis, 65.2% less lesional collagen content, nearly 7-fold higher dead cell accumulation, and 2-fold larger lesion area. Hematopoietic SR-BI deletion elicited a maladaptive inflammatory response [higher interleukin (IL)-1β, IL-6, and TNF-α lower IL-10 and transforming growth factor β]. Efferocytosis of apoptotic thymocytes was reduced by 64% in SR-BI−/− versus WT macrophages, both in vitro and in vivo. In response to apoptotic cells, macrophage SR-BI bound with phosphatidylserine and induced Src phosphorylation and cell membrane recruitment, which led to downstream activation of phosphoinositide 3-kinase (PI3K) and Ras-related C3 botulinum toxin substrate 1 (Rac1) for engulfment and clearance of apoptotic cells, as inhibition of Src decreased PI3K, Rac1-GTP, and efferocytosis in WT cells. Pharmacological inhibition of Rac1 reduced macrophage efferocytosis in a SR-BI-dependent fashion, and activation of Rac1 corrected the defective efferocytosis in SR-BI−/− macrophages. Thus, deficiency of macrophage SR-BI promotes defective efferocytosis signaling via the Src/PI3K/Rac1 pathway, resulting in increased plaque size, necrosis, and inflammation.

Lentiviral Transduction of ApoAI Into Hematopoietic Progenitor Cells and Macrophages Applications to Cell Therapy of Atherosclerosis

Objective—We used genetically engineered mouse hematopoietic progenitor cells (HPCs) to investigate the therapeutic effects of human apoAI on atherosclerosis in apoE−/− mice. Methods and Results—Lentiviral constructs expressing either human apoAI (LV-apoAI) or green fluorescent protein (LV-GFP) cDNA under a macrophage specific promoter (CD68) were generated and used for ex vivo transduction of mouse HPCs and macrophages. The transduction efficiency was >25% for HPCs and >70% for macrophages. ApoAI was found in the macrophage culture media, mostly associated with the HDL fraction. Interestingly, a significant increase in mRNA and protein levels for ATP binding cassette A1 (ABCA1) and ABCG1 were found in apoAI-expressing macrophages after acLDL loading. Expression of apoAI significantly increased cholesterol efflux in wild-type and apoE−/− macrophages. HPCs transduced with LV-apoAI ex vivo and then transplanted into apoE−/− mice caused a 50% reduction in atherosclerotic lesion area compared to GFP controls, without influencing plasma HDL-C levels. Conclusions—Lentiviral transduction of apoAI into HPCs reduces atherosclerosis in apoE−/− mice. Expression of apoAI in macrophages improves cholesterol trafficking in wild-type apoE-producing macrophages and causes upregulation of ABCA1 and ABCG1. These novel observations set the stage for a cell therapy approach to atherosclerosis regression, exploiting the cooperation between apoE and apoAI to maximize cholesterol exit from the plaque.

Macrophage apoAI protects against dyslipidemia-induced dermatitis and atherosclerosis without affecting HDL

Tissue cholesterol accumulation, macrophage infiltration, and inflammation are features of atherosclerosis and some forms of dermatitis. HDL and its main protein, apoAI, are acceptors of excess cholesterol from macrophages; this process inhibits tissue inflammation. Recent epidemiologic and clinical trial evidence questions the role of HDL and its manipulation in cardiovascular disease. We investigated the effect of ectopic macrophage apoAI expression on atherosclerosis and dermatitis induced by the combination of hypercholesterolemia and absence of HDL in mice. Hematopoietic progenitor cells were transduced to express human apoAI and transplanted into lethally irradiated LDL receptor−/−/apoAI−/− mice, which were then placed on a high-fat diet for 16 weeks. Macrophage apoAI expression reduced aortic CD4+ T-cell levels (−39.8%), lesion size (−25%), and necrotic core area (−31.6%), without affecting serum HDL or aortic macrophage levels. Macrophage apoAI reduced skin cholesterol by 39.8%, restored skin morphology, and reduced skin CD4+ T-cell levels. Macrophage apoAI also reduced CD4+ T-cell levels (−32.9%) in skin-draining lymph nodes but had no effect on other T cells, B cells, dendritic cells, or macrophages compared with control transplanted mice. Thus, macrophage apoAI expression protects against atherosclerosis and dermatitis by reducing cholesterol accumulation and regulating CD4+ T-cell levels, without affecting serum HDL or tissue macrophage levels.

Serum PCSK9 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.

Serum Proprotein Convertase Subtilisin/Kexin Type 9 and Cell Surface Low-Density Lipoprotein ReceptorClinical Perspective

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.

Low-Density Lipoprotein Receptor–Related Protein 1 Prevents Early Atherosclerosis by Limiting Lesional Apoptosis and Inflammatory Ly-6Chigh Monocytosis

Background— We previously demonstrated that macrophage low-density lipoprotein receptor (LDLR)–related protein 1 (LRP1) deficiency increases atherosclerosis despite antiatherogenic changes including decreased uptake of remnants and increased secretion of apolipoprotein E (apoE). Thus, our objective was to determine whether the atheroprotective effects of LRP1 require interaction with apoE, one of its ligands with multiple beneficial effects. Methods and Results— We examined atherosclerosis development in mice with specific deletion of macrophage LRP1 (apoE−/− M&PHgr;LRP1−/−) and in LDLR−/− mice reconstituted with apoE−/− M&PHgr;LRP1−/− bone marrow. The combined absence of apoE and LRP1 promoted atherogenesis more than did macrophage apoE deletion alone in both apoE-producing LDLR−/− mice (+88%) and apoE−/− mice (+163%). The lesions of both mouse models with apoE−/− LRP1−/− macrophages had increased macrophage content. In vitro, apoE and LRP1 additively inhibit macrophage apoptosis. Furthermore, there was excessive accumulation of apoptotic cells in lesions of both LDLR−/− mice (+110%) and apoE−/− M&PHgr;LRP1−/− mice (+252%). The apoptotic cell accumulation was partially due to decreased efferocytosis as the ratio of free to cell-associated apoptotic nuclei was 3.5-fold higher in lesions of apoE−/− M&PHgr;LRP1−/− versus apoE−/− mice. Lesion necrosis was also increased (6 fold) in apoE−/− M&PHgr;LRP1−/− versus apoE−/− mice. Compared with apoE−/− mice, the spleens of apoE−/− M&PHgr;LRP1−/− mice contained 1.6- and 2.4-fold more total and Ly6-Chigh monocytes. Finally, there were 3.6- and 2.4-fold increases in Ly6-Chigh and CC-chemokine receptor 2–positive cells in lesions of apoE−/− M&PHgr;LRP1−/− versus apoE−/− mice, suggesting that accumulation of apoptotic cells enhances lesion development and macrophage content by promoting the recruitment of inflammatory monocytes. Conclusion— Low-density lipoprotein receptor protein 1 exerts antiatherogenic effects via pathways independent of apoE involving macrophage apoptosis and monocyte recruitment.