Christina Christoffersen

Endothelium-protective sphingosine-1-phosphate provided by HDL-associated apolipoprotein M

Protection of the endothelium is provided by circulating sphingosine-1-phosphate (S1P), which maintains vascular integrity. We show that HDL-associated S1P is bound specifically to both human and murine apolipoprotein M (apoM). Thus, isolated human ApoM+ HDL contained S1P, whereas ApoM− HDL did not. Moreover, HDL in Apom−/− mice contains no S1P, whereas HDL in transgenic mice overexpressing human apoM has an increased S1P content. The 1.7-Å structure of the S1P–human apoM complex reveals that S1P interacts specifically with an amphiphilic pocket in the lipocalin fold of apoM. Human ApoM+ HDL induced S1P1 receptor internalization, downstream MAPK and Akt activation, endothelial cell migration, and formation of endothelial adherens junctions, whereas apoM− HDL did not. Importantly, lack of S1P in the HDL fraction of Apom−/− mice decreased basal endothelial barrier function in lung tissue. Our results demonstrate that apoM, by delivering S1P to the S1P1 receptor on endothelial cells, is a vasculoprotective constituent of HDL.

Isolation and characterization of human apolipoprotein M-containing lipoproteins

Apolipoprotein M (apoM) is a novel apolipoprotein with unknown function. In this study, we established a method for isolating apoM-containing lipoproteins and studied their composition and the effect of apoM on HDL function. ApoM-containing lipoproteins were isolated from human plasma with immunoaffinity chromatography and compared with lipoproteins lacking apoM. The apoM-containing lipoproteins were predominantly of HDL size; ∼5% of the total HDL population contained apoM. Mass spectrometry showed that the apoM-containing lipoproteins also contained apoJ, apoA-I, apoA-II, apoC-I, apoC-II, apoC-III, paraoxonase 1, and apoB. ApoM-containing HDL (HDLapoM+) contained significantly more free cholesterol than HDL lacking apoM (HDLapoM−) (5.9 ± 0.7% vs. 3.2 ± 0.5%; P < 0.005) and was heterogeneous in size with both small and large particles. HDLapoM+ inhibited Cu2+-induced oxidation of LDL and stimulated cholesterol efflux from THP-1 foam cells more efficiently than HDLapoM−. In conclusion, our results suggest that apoM is associated with a small heterogeneous subpopulation of HDL particles. Nevertheless, apoM designates a subpopulation of HDL that protects LDL against oxidation and stimulates cholesterol efflux more efficiently than HDL lacking apoM.

Effect of Apolipoprotein M on High Density Lipoprotein Metabolism and Atherosclerosis in Low Density Lipoprotein Receptor Knock-out Mice

To investigate the role of apoM in high density lipoprotein (HDL) metabolism and atherogenesis, we generated human apoM transgenic (apoM-Tg) and apoM-deficient (apoM–/–) mice. Plasma apoM was predominantly associated with 10–12-nm α-migrating HDL particles. Human apoM overexpression (11-fold) increased plasma cholesterol concentration by 13–22%, whereas apoM deficiency decreased it by 17–21%. The size and charge of apoA-I-containing HDL in plasma were not changed in apoM-Tg or apoM–/– mice. However, in plasma incubated at 37 °C, lecithin:cholesterol acyltransferase-dependent conversion of α- to pre-α-migrating HDL was delayed in apoM-Tg mice. Moreover, lecithin: cholesterol acyltransferase-independent generation of pre-β-migrating apoA-I-containing particles in plasma was increased in apoM-Tg mice (4.2 ± 1.1%, p = 0.06) and decreased in apoM–/– mice (0.5 ± 0.3%, p = 0.03) versus controls (1.8 ± 0.05%). In the setting of low density lipoprotein receptor deficiency, apoM-Tg mice with ∼2-fold increased plasma apoM concentrations developed smaller atherosclerotic lesions than controls. The effect of apoM on atherosclerosis may be facilitated by enzymatic modulation of plasma HDL particles, increased cholesterol efflux from foam cells, and an antioxidative effect of apoM-containing HDL.

Familial Hypercholesterolemia and Atherosclerosis in Cloned Minipigs Created by DNA Transposition of a Human PCSK9 Gain-of-Function Mutant

A transgenic pig model of familial hypercholesterolemia can be used for translational atherosclerosis research. A Model of We hope to inherit our parents’ good features, like blue eyes or musical talent, but not their high cholesterol. Familial hypercholesterolemia, which is passed down in families, results in high levels of “bad” cholesterol [low-density lipoprotein (LDL)] and early onset of cardiovascular disease. To further translational research in this area, Al-Mashhadi and coauthors created a large-animal model of this genetic disease, showing that these pigs develop hypercholesterolemia and atherosclerosis much like people do. The D374Y gain-of-function mutation in the PCSK9 gene (which is conserved between pig and human) causes a severe form of hypercholesterolemia and, ultimately, atherosclerosis. Al-Mashhadi and colleagues engineered transposon-based vectors to express D374Y-PCSK9. After confirming function in human liver cancer cells, the authors cloned minipigs that expressed the mutant gene. On a low-fat diet, these pigs had higher total and LDL cholesterol than their wild-type counterparts. Breeding the male transgenic pigs with wild-type sows produced offspring that also had higher plasma LDL levels compared with normal, healthy pigs. A high-fat, high-cholesterol diet induced severe hypercholesterolemia in these animals as well as accelerated development of atherosclerosis that has human-like lesions. Other large-animal models only develop hypercholesterolemia when placed on the right diet, and small-animal models cannot recapitulate human-like pathology. The PCSK9 transgenic pigs created by Al-Mashhadi et al. develop hypercholesterolemia even on low-fat diets, and thus reflect the inherited human disease. This large-animal model will be important for better understanding the pathogenesis of familial hypercholesterolemia and for testing new therapeutics and imaging modalities before moving into human trials. Lack of animal models with human-like size and pathology hampers translational research in atherosclerosis. Mouse models are missing central features of human atherosclerosis and are too small for intravascular procedures and imaging. Modeling the disease in minipigs may overcome these limitations, but it has proven difficult to induce rapid atherosclerosis in normal pigs by high-fat feeding alone, and genetically modified models similar to those created in mice are not available. D374Y gain-of-function mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene cause severe autosomal dominant hypercholesterolemia and accelerates atherosclerosis in humans. Using Sleeping Beauty DNA transposition and cloning by somatic cell nuclear transfer, we created Yucatan minipigs with liver-specific expression of human D374Y-PCSK9. D374Y-PCSK9 transgenic pigs displayed reduced hepatic low-density lipoprotein (LDL) receptor levels, impaired LDL clearance, severe hypercholesterolemia, and spontaneous development of progressive atherosclerotic lesions that could be visualized by noninvasive imaging. This model should prove useful for several types of translational research in atherosclerosis.

The signal peptide anchors apolipoprotein M in plasma lipoproteins and prevents rapid clearance of apolipoprotein M from plasma.

Lipoproteins consist of lipids solubilized by apolipoproteins. The lipid-binding structural motifs of apolipoproteins include amphipathic α-helixes and β-sheets. Plasma apolipoprotein (apo) M lacks an external amphipathic motif but, nevertheless, is exclusively associated with lipoproteins (mainly high density lipoprotein). Uniquely, however, apoM is secreted to plasma without cleavage of its hydrophobic NH2-terminal signal peptide. To test whether the signal peptide serves as a lipoprotein anchor for apoM in plasma, we generated mice expressing a mutated apoMQ22A cDNA in the liver (apoMQ22A-Tg mice (transgenic mice)) and compared them with mice expressing wild-type human apoM (apoM-Tg mice). The substitution of the amino acid glutamine 22 with alanine in apoMQ22A results in secretion of human apoM without a signal peptide. The human apoM mRNA level in liver and the amount of human apoM protein secretion from hepatocytes were similar in apoM-Tg and apoMQ22A-Tg mice. Nevertheless, human apoM was not detectable in plasma of apoMQ22A-Tg mice, whereas it was easily measured in the apoM-Tg mice. To examine the plasma metabolism, recombinant apoM lacking the signal peptide was produced in Escherichia coli and injected into wild-type mice. The apoM without signal peptide did not associate with lipoproteins and was rapidly cleared in the kidney. Accordingly, ligation of the kidney arteries in apoMQ22A-Tg mice resulted in rapid accumulation of human apoM in plasma. The data suggest that hydrophobic signal peptide sequences, if preserved upon secretion, can anchor plasma proteins in lipoproteins. In the case of apoM, this mechanism prevents rapid loss by filtration in the kidney.

Chamber-Dependent Expression of Brain Natriuretic Peptide and Its mRNA in Normal and Diabetic Pig Heart

Brain natriuretic peptide (BNP) is produced in cardiac myocytes, and increased secretion is closely associated with cardiac dysfunction. However, several fundamental aspects of BNP expression in the myocardium have not yet been resolved. In the present study, we report the presence of a precursor BNP mRNA transcript and a mature BNP mRNA transcript in normal porcine hearts. In normal pigs, the amount of precursor BNP mRNA was similar in atrial and ventricular myocardium, whereas the mature BNP transcript was 10- to 50-fold more abundant in atrial than in ventricular myocardium. Quantitation of proBNP in normal porcine hearts by radioimmunoassay disclosed abundant proBNP in the atria, whereas proBNP was undetectable in the ventricles. Laser confocal microscopy revealed proBNP in secretory granules of atrial but not in the ventricular myocardium of normal pigs. Mild streptozotocin-induced diabetes doubled the expression of BNP mRNA in porcine atrial myocardium (P =0.03), but was without effect on BNP mRNA in the ventricular myocardium. The data suggest that BNP mRNA processing and proBNP storage differ between the atrial and ventricular myocardium. The results also imply that diabetes increases cardiac BNP expression in a chamber-dependent manner.

Induction of Atherosclerosis in Mice and Hamsters Without Germline Genetic Engineering

Rationale: Atherosclerosis can be achieved in animals by germline genetic engineering, leading to hypercholesterolemia, but such models are constrained to few species and strains, and they are difficult to combine with other powerful techniques involving genetic manipulation or variation. Objective: To develop a method for induction of atherosclerosis without germline genetic engineering. Methods and Results: Recombinant adeno-associated viral vectors were engineered to encode gain-of-function proprotein convertase subtilisin/kexin type 9 mutants, and mice were given a single intravenous vector injection followed by high-fat diet feeding. Plasma proprotein convertase subtilisin/kexin type 9 and total cholesterol increased rapidly and were maintained at high levels, and after 12 weeks, mice had atherosclerotic lesions in the aorta. Histology of the aortic root showed progression of lesions to the fibroatheromatous stage. To demonstrate the applicability of this method for rapid analysis of the atherosclerosis susceptibility of a mouse strain and for providing temporal control over disease induction, we demonstrated the accelerated atherosclerosis of mature diabetic Akita mice. Furthermore, the versatility of this approach for creating atherosclerosis models also in nonmurine species was demonstrated by inducing hypercholesterolemia and early atherosclerosis in Golden Syrian hamsters. Conclusions: Single injections of proprotein convertase subtilisin/kexin type 9–encoding recombinant adeno-associated viral vectors are a rapid and versatile method to induce atherosclerosis in animals. This method should prove useful for experiments that are high-throughput or involve genetic techniques, strains, or species that do not combine well with current genetically engineered models.

18F-FDG PET Imaging of Murine Atherosclerosis: Association with Gene Expression of Key Molecular Markers

Aim To study whether 18F-FDG can be used for in vivo imaging of atherogenesis by examining the correlation between 18F-FDG uptake and gene expression of key molecular markers of atherosclerosis in apoE−/− mice. Methods Nine groups of apoE−/− mice were given normal chow or high-fat diet. At different time-points, 18F-FDG PET/contrast-enhanced CT scans were performed on dedicated animal scanners. After scans, animals were euthanized, aortas removed, gamma counted, RNA extracted from the tissue, and gene expression of chemo (C-X-C motif) ligand 1 (CXCL-1), monocyte chemoattractant protein (MCP)-1, vascular cell adhesion molecule (VCAM)-1, cluster of differentiation molecule (CD)-68, osteopontin (OPN), lectin-like oxidized LDL-receptor (LOX)-1, hypoxia-inducible factor (HIF)-1α, HIF-2α, vascular endothelial growth factor A (VEGF), and tissue factor (TF) was measured by means of qPCR. Results The uptake of 18F-FDG increased over time in the groups of mice receiving high-fat diet measured by PET and ex vivo gamma counting. The gene expression of all examined markers of atherosclerosis correlated significantly with 18F-FDG uptake. The strongest correlation was seen with TF and CD68 (p<0.001). A multivariate analysis showed CD68, OPN, TF, and VCAM-1 to be the most important contributors to the uptake of 18F-FDG. Together they could explain 60% of the 18F-FDG uptake. Conclusion We have demonstrated that 18F-FDG can be used to follow the progression of atherosclerosis in apoE−/− mice. The gene expression of ten molecular markers representing different molecular processes important for atherosclerosis was shown to correlate with the uptake of 18F-FDG. Especially, the gene expressions of CD68, OPN, TF, and VCAM-1 were strong predictors for the uptake.

Opposing Effects of Apolipoprotein M on Catabolism of Apolipoprotein B–Containing Lipoproteins and Atherosclerosis

Rationale: Plasma apolipoprotein (apo)M is mainly associated with high-density lipoprotein (HDL). HDL-bound apoM is antiatherogenic in vitro. However, plasma apoM is not associated with coronary heart disease in humans, perhaps because of a positive correlation with plasma low-density lipoprotein (LDL). Objective: We explored putative links between apoM and very-low-density (VLDL)/LDL metabolism and the antiatherogenic potential of apoM in vivo. Methods and Results: Plasma apoM was increased ≈2.1 and ≈1.5 fold in mice lacking LDL receptors (Ldlr−/−) and expressing dysfunctional LDL receptor–related protein 1 (Lrp1n2/n2), respectively, but was unaffected in apoE-deficient (ApoE−/−) mice. Thus, pathways controlling catabolism of VLDL and LDL affect plasma apoM. Overexpression (≈10-fold) of human apoM increased (50% to 70%) and apoM deficiency decreased (≈25%) plasma VLDL/LDL cholesterol in Ldlr−/− mice, whereas apoM did not affect plasma VLDL/LDL in mice with intact LDL receptors. Moreover, plasma clearance of apoM-enriched VLDL/LDL was slower than that of control VLDL/LDL in mice lacking functional LDL receptors and LRP1, suggesting that apoM impairs the catabolism of VLDL/LDL that occurs independently of the LDL receptor and LRP1. ApoM overexpression decreased atherosclerosis in ApoE−/− (60%) and cholate/cholesterol-fed wild-type mice (70%). However, in Ldlr−/− mice the antiatherogenic effect of apoM was attenuated by its VLDL/LDL-raising effect. Conclusion: The data suggest that defect LDL receptor function leads to increased plasma apoM concentrations, which in turn, impairs the removal of VLDL/LDL from plasma. This mechanism opposes the otherwise antiatherogenic effect of apoM.

ApoM: gene regulation and effects on HDL metabolism.

The recently discovered apolipoprotein M (apoM) is a plasma protein of the lipocalin family associated with the lipoproteins (mainly high-density lipoproteins, or HDLs). Expression of the apoM gene in the liver is regulated by transcription factors that control key steps in hepatic lipid and glucose metabolism. Although the concentration of plasma apoM correlates with that of cholesterol, apoM was not identified as a risk factor for cardiovascular disease in two prospective studies. In genetically modified mice, however, changes in plasma apoM concentration caused quantitative and qualitative changes in HDLs, and overexpression of the apoM gene reduced atherosclerosis. In conclusion, it seems that apoM plays a part in lipoprotein metabolism; however, the biological impact of apoM in humans remains to be determined.