Human apoC-III Research Articles

Hypertriglyceridemia in human apoC-III transgenic mice promotes metabolic activation of macrophages which can be reversed by fenofibrate treatment

Hypertriglyceridemia in human apoC-III transgenic mice promotes metabolic activation of macrophages which can be reversed by fenofibrate treatment

Molecular modeling of apoE in complexes with Alzheimer's amyloid-β fibrils from human brain suggests a structural basis for apolipoprotein co-deposition with amyloids.

Apolipoproteins co-deposit with amyloids, yet apolipoprotein-amyloid interactions are enigmatic. To understand how apoE interacts with Alzheimer's amyloid-β (Aβ) peptide in fibrillary deposits, the NMR structure of full-length human apoE was docked to four structures of patient-derived Aβ1-40 and Aβ1-42 fibrils determined previously using cryo-electron microscopy or solid-state NMR. Similar docking was done using the NMR structure of human apoC-III. In all complexes, conformational changes in apolipoproteins were required to expose large hydrophobic faces of their amphipathic α-helices for sub-stoichiometric binding to hydrophobic surfaces on sides or ends of fibrils. Basic residues flanking the hydrophobic helical faces in apolipoproteins interacted favorably with acidic residue ladders in some amyloid polymorphs. Molecular dynamics simulations of selected apoE-fibril complexes confirmed their stability. Amyloid binding via cryptic sites, which became available upon opening of flexibly linked apolipoprotein α-helices, resembled apolipoprotein-lipid binding. This mechanism probably extends to other apolipoprotein-amyloid interactions. Apolipoprotein binding alongside fibrils could interfere with fibril fragmentation and secondary nucleation, while binding at the fibril ends could halt amyloid elongation and dissolution in a polymorph-specific manner. The proposed mechanism is supported by extensive prior experimental evidence and helps reconcile disparate reports on apoE's role in Aβ aggregation. Furthermore, apoE domain opening and direct interaction of Arg/Cys158 with amyloid potentially contributes to isoform-specific effects in Alzheimer's disease. In summary, current modeling supported by prior experimental studies suggests similar mechanisms for apolipoprotein-amyloid and apolipoprotein-lipid interactions; explains why apolipoproteins co-deposit with amyloids; and helps reconcile conflicting reports on the chaperone-like apoE action in Aβ aggregation.

Enrichment of Triglyceride-Rich Lipoproteins with Apolipoprotein C-I Is Positively Associated with Their Delayed Plasma Clearance Independently of Other Transferable Apolipoproteins in Postmenopausal Overweight and Obese Women

Background: The role of plasma apolipoprotein (apo) C-I in cardiometabolic risk in humans is unclear. However, in vitro studies showed a dual role for apoC-I, both protective and harmful, depending on the carrier lipoprotein.Objective: We tested the hypothesis that triglyceride (TG)-rich lipoprotein (TRL) apoC-I, not total or HDL apoC-I, is associated with delayed postprandial plasma clearance of TRLs, independently of apoC-II, apoC-III, and apoE.Methods: This cross-sectional study examines the plasma clearance of a 13C-triolein–labeled high-fat meal (68% fat energy) in 20 postmenopausal overweight and obese women [body mass index (in kg/m2) ≥27; aged 45–74 y] as the increment change in area under the 6-h postprandial curves (iAUC6h) of TRL parameters. Lipoproteins were fractionated by fast-protein LC. Transferable apolipoproteins were measured by ELISA. TRL enrichment with apolipoproteins was calculated by dividing their TRL concentrations by TRL apoB. The effects of human apoC-I and apoC-III on the hydrolysis and storage of 3H-triolein–labeled TRLs were tested in 3T3-L1 adipocytes.Results: TRL apoC-I was positively associated with plasma apo B-48 and total and non-HDL TGs, cholesterol, and apoB (r = 0.52–0.97) and negatively with HDL cholesterol (r = −0.52) and LDL diameter (r = −0.91) (P < 0.05). Total and HDL apoC-I were correlated only with total (r = 0.62) and HDL (r = 0.75) cholesterol. Women with high fasting TRL enrichment with apoC-I (99–365 μmol apoC-I/μmol apoB), but not apoC-II, apoC-III, or apoE, had higher iAUC6h for TGs (+195%), 13C-TGs (+319%), and apo B-48 (+186%) than those with low enrichment (14–97 μmol apoC-I/μmol apoB). The 4-h postprandial increase in TRL apoC-I was associated with a 4-h increase in TRL TGs and iAUC6h for TGs, 13C-TGs, and apo B-48 (r = 0.74–0.86, P < 0.001), independently of 4-h changes in TRL apoB, apoC-II, apoC-III, or apoE. ApoC-I and apoC-III inhibited 3H-TRL clearance by adipocytes by >75% (P < 0.001).Conclusions: TRL enrichment with apoC-I is positively associated with postprandial hypertriglyceridemia and remnant accumulation in postmenopausal overweight and obese women, independently of apoC-II, apoC-III, or apoE, which may be due to inhibiting TRL clearance by adipocytes. Reducing TRL apoC-I may ameliorate delayed postprandial plasma clearance of TRLs and associated risks in humans.

Human apoCIII transgenic mice with epicardial adipose tissue inflammation and PRESERVATION of the cardiac plexus.

Hypertriglyceridemia is a result of the increase in the serum levels of lipoproteins, which are responsible for the transport of triglycerides and can be caused by genetic and/or metabolic factors. Animal models which either express or lack genes related to changes in the lipoproteins profile are useful to understand lipid metabolism. Apolipoprotein CIII (apoCIII) is an important modulator of hepatic production and peripheral removal of triglycerides. Mice that overexpress the apoCIII gene become hypertriglyceridemic, showing high concentrations of free fatty acids in the blood. Since hypertriglyceridemia is related to atherosclerosis, and the latter refers to cardiac alterations, this study aimed at evaluating the morphological, morphometric and quantitative profiles of the cardiac plexus, as well as the morphometric and histopathological aspects of the epicardial adipose tissue in human apoCIII transgenic mice. Therefore, 8-12-month-old male C57BL/6 mice that overexpressed human apoCIII (CIII) and their respective controls were used. Our results showed that overexpression of human apoCIII did not modify morphological or quantitative parameters of cardiac plexus neurons; however, age increased both, the area and the number of such cells. Furthermore, there was a direct correlation of this dyslipidemia to the thickening of periganglionar type 1 collagens. On the other hand, this overexpression caused epicardial adipose tissue inflammation and an increase in the area of the adipocytes, thus, favoring the recruitment of inflammatory cells in this tissue. In conclusion, this overexpression is harmful since it is related to an increase in cardiac adiposity, as well as to a predisposition to an inflammatory environment in the epicardial fat and to the incidence of cardiovascular diseases.

APOC3-rs2854116 polymorphism related to hypercholesterolemia in primary school children in Hanoi

APOC3-rs2854116 polymorphism related to hypercholesterolemia in primary school children in Hanoi

Intestinal basolateral lipid substrate transport is linked to chylomicron secretion and is regulated by apoC-III

Chylomicron metabolism is critical for determining plasma levels of triacylglycerols (TAGs) and cholesterol, both of which are risk factors for CVD. The rates of chylomicron secretion and remnant clearance are controlled by intracellular and extracellular factors, including apoC-III. We have previously shown that human apoC-III overexpression in mice (apoC-IIITg mice) decreases the rate of chylomicron secretion into lymph, as well as the TAG composition in chylomicrons. We now find that this decrease in chylomicron secretion is not due to the intracellular effects of apoC-III, but instead that primary murine enteroids are capable of taking up TAG from TAG-rich lipoproteins (TRLs) on their basolateral surface; and via Seahorse analyses, we find that mitochondrial respiration is induced by basolateral TRLs. Furthermore, TAG uptake into the enterocyte is inhibited when excess apoC-III is present on TRLs. In vivo, we find that dietary TAG is diverted from the cytosolic lipid droplets and driven toward mitochondrial FA oxidation when plasma apoC-III is high (or when basolateral substrates are absent). We propose that this pathway of basolateral lipid substrate transport (BLST) plays a physiologically relevant role in the maintenance of dietary lipid absorption and chylomicron secretion. Further, when apoC-III is in excess, it inhibits BLST and chylomicron secretion.

Apolipoprotein C-II Mimetic Peptide Promotes the Plasma Clearance of Triglyceride-Rich Lipid Emulsion and the Incorporation of Fatty Acids into Peripheral Tissues of Mice.

Aim Plasma apolipoprotein C-II (apoC-II) activates lipoprotein lipase (LPL) and thus lowers plasma triglycerides (TG). We previously reported that a human apoC-II mimetic peptide (C-II-a) decreased plasma TG in apoC-II mutant mice, as well as in apoE-knockout mice. Because it is unknown what tissues take up free fatty acids (FFAs) released from TG after C-II-a peptide administration, we investigated in mice TG plasma clearance and tissue incorporation, using 3H-triolein as a tracer, with and without C-II-a treatment. Methods and Results Intralipid® fat emulsion was labeled with 3H-triolein and then mixed with or without C-II-a. Addition of the peptide did not alter mean particle size of the lipid emulsion particles (298 nm) but accelerated their plasma clearance. After intravenous injection into C57BL/6N mice, the plasma half-life of the 3H-triolein for control and C-II-a treated emulsions was 18.3 ± 2.2 min and 14.8 ± 0.1 min, respectively. In apoC-II mutant mice, the plasma half-life of 3H-triolein for injected control and C-II-a treated emulsions was 30.1 ± 0.1 min and 14.8 ± 0.1 min, respectively. C57BL/6N and apoC-II mutant mice at 120 minutes after the injection showed increased tissue incorporation of radioactivity in white adipose tissue when C-II-a treated emulsion was used. Higher radiolabeled uptake of lipids from C-II-a treated emulsion was also observed in the skeletal muscle of C57BL/6N mice only. In case of apoC-II mutant mice, decreased uptake of radioactive lipids was observed in the liver and kidney after addition of C-II-a to the lipid emulsion. Conclusions C-II-a peptide promotes the plasma clearance of TG-rich lipid emulsions in wild type and apoC-II mutant mice and promotes the incorporation of fatty acids from TG in the lipid emulsions into specific peripheral tissues.

Abstract 562: Foamy Monocytes in Hypertriglyceridemia

Objective: Hypertriglyceridemia (HTG) increases risk for atherosclerotic cardiovascular disease, but the mechanisms remain poorly defined. Foamy monocytes are lipid-loaded monocytes in circulation that contribute to atherosclerosis under hypercholesterolemia. Human study has proved that HTG is associated with formation of foamy monocytes. Our study is to examine formation of foamy monocytes in HTG and their potential contribution to atherosclerosis in mouse models. Approach and results: In vivo mouse models of HTG included wild-type C57BL/6 mice on high fat diet (HFD) injected intraperitoneally with LPL inhibitor, Poloxamer 407 (P407, 0.25mg/g, every two days), as a chemically-induced model and mice with transgenic overexpression of human ApoCIII (ApoCIIItg) as a genetic model. Based on CD11c and CD36, monocytes were identified as CD36 - CD11c - , CD36 + CD11c - and CD36 + CD11c + subsets. In the first model, at 24h of the first injection, triglyceride levels increased to 367 ±84 mg/dL, higher than that of control group with saline injection (60 ±22 mg/dL, n=4, P&lt;0.001). Meanwhile, the side scatter (SSC, representing cell granularity) values and Nile red staining for lipids of CD36 + CD11c + monocytes increased significantly, indicating formation of foamy monocytes, in mice with HTG. Furthermore, CD11c mean fluorescence intensity of CD36 + CD11c + foamy monocytes increased significantly at 2 weeks of P407 injection. In ApoCIIItg mice fed HFD (5 weeks), the percentage and SSC value of CD36 + CD11c + monocytes increased significantly (37%±5%, 247±8), also indicating elevated granularity and lipid accumulation of these monocytes, compared to wild-type mice (26%±3%, p&lt;0.05; 226±8, p&lt;0.05, n=4-6). In vitro treatment with human triglyceride-rich lipoprotein (hTGRL) for 24h increased the granularity and Nile red staining intensity of THP-1 monocytes, indicating foamy monocyte formation. hTGRL treatment also increased THP-1 monocyte expression of CD36, with greater uptake of cholesteryl ester-rich lipoprotein. Conclusion: High triglyceride promotes foamy monocyte formation and induces monocyte phenotypic changes in mice and tissue culture, with increased expression of CD11c and CD36, which may contribute to development of atherosclerosis under HTG.

The apolipoprotein C-III (Gln38Lys) variant associated with human hypertriglyceridemia is a gain-of-function mutation

Recent cell culture and animal studies have suggested that expression of human apo C-III in the liver has a profound impact on the triacylglycerol (TAG)-rich VLDL1 production under lipid-rich conditions. The apoC-III Gln38Lys variant was identified in subjects of Mexican origin with moderate hypertriglyceridemia. We postulated that Gln38Lys (C3QK), being a gain-of-function mutation, promotes hepatic VLDL1 assembly/secretion. To test this hypothesis, we expressed C3QK in McA-RH7777 cells and apoc3-null mice to contrast its effect with WT apoC-III (C3WT). In both model systems, C3QK expression increased the secretion of VLDL1-TAG (by 230%) under lipid-rich conditions. Metabolic labeling experiments with C3QK cells showed an increase in de novo lipogenesis (DNL). Fasting plasma concentration of TAG, cholesterol, cholesteryl ester, and FA were increased in C3QK mice as compared with C3WT mice. Liver of C3QK mice also displayed an increase in DNL and expression of lipogenic genes as compared with that in C3WT mice. These results suggest that C3QK variant is a gain-of-function mutation that can stimulate VLDL1 production, through enhanced DNL.

A human APOC3 missense variant and monoclonal antibody accelerate apoC-III clearance and lower triglyceride-rich lipoprotein levels.

Recent large-scale genetic sequencing efforts have identified rare coding variants in genes in the triglyceride-rich lipoprotein (TRL) clearance pathway that are protective against coronary heart disease (CHD), independently of LDL cholesterol (LDL-C) levels. Insight into the mechanisms of protection of these variants may facilitate the development of new therapies for lowering TRL levels. The gene APOC3 encodes apoC-III, a critical inhibitor of triglyceride (TG) lipolysis and remnant TRL clearance. Here we report a detailed interrogation of the mechanism of TRL lowering by the APOC3 Ala43Thr (A43T) variant, the only missense (rather than protein-truncating) variant in APOC3 reported to be TG lowering and protective against CHD. We found that both human APOC3 A43T heterozygotes and mice expressing human APOC3 A43T display markedly reduced circulating apoC-III levels. In mice, this reduction is due to impaired binding of A43T apoC-III to lipoproteins and accelerated renal catabolism of free apoC-III. Moreover, the reduced content of apoC-III in TRLs resulted in accelerated clearance of circulating TRLs. On the basis of this protective mechanism, we developed a monoclonal antibody targeting lipoprotein-bound human apoC-III that promotes circulating apoC-III clearance in mice expressing human APOC3 and enhances TRL catabolism in vivo. These data reveal the molecular mechanism by which a missense variant in APOC3 causes reduced circulating TG levels and, hence, protects from CHD. This protective mechanism has the potential to be exploited as a new therapeutic approach to reduce apoC-III levels and circulating TRL burden.

Enrichment of Triglyceride-Rich Lipoproteins with Apolipoprotein C-I Is Positively Associated with Their Delayed Plasma Clearance Independently of Other Transferable Apolipoproteins in Postmenopausal Overweight and Obese Women

Background: The role of plasma apolipoprotein (apo) C-I in cardiometabolic risk in humans is unclear. However, in vitro studies showed a dual role for apoC-I, both protective and harmful, depending on the carrier lipoprotein.Objective: We tested the hypothesis that triglyceride (TG)-rich lipoprotein (TRL) apoC-I, not total or HDL apoC-I, is associated with delayed postprandial plasma clearance of TRLs, independently of apoC-II, apoC-III, and apoE.Methods: This cross-sectional study examines the plasma clearance of a 13C-triolein–labeled high-fat meal (68% fat energy) in 20 postmenopausal overweight and obese women [body mass index (in kg/m2) ≥27; aged 45–74 y] as the increment change in area under the 6-h postprandial curves (iAUC6h) of TRL parameters. Lipoproteins were fractionated by fast-protein LC. Transferable apolipoproteins were measured by ELISA. TRL enrichment with apolipoproteins was calculated by dividing their TRL concentrations by TRL apoB. The effects of human apoC-I and apoC-III on the hydrolysis and storage of 3H-triolein–labeled TRLs were tested in 3T3-L1 adipocytes.Results: TRL apoC-I was positively associated with plasma apo B-48 and total and non-HDL TGs, cholesterol, and apoB (r = 0.52–0.97) and negatively with HDL cholesterol (r = −0.52) and LDL diameter (r = −0.91) (P < 0.05). Total and HDL apoC-I were correlated only with total (r = 0.62) and HDL (r = 0.75) cholesterol. Women with high fasting TRL enrichment with apoC-I (99–365 μmol apoC-I/μmol apoB), but not apoC-II, apoC-III, or apoE, had higher iAUC6h for TGs (+195%), 13C-TGs (+319%), and apo B-48 (+186%) than those with low enrichment (14–97 μmol apoC-I/μmol apoB). The 4-h postprandial increase in TRL apoC-I was associated with a 4-h increase in TRL TGs and iAUC6h for TGs, 13C-TGs, and apo B-48 (r = 0.74–0.86, P < 0.001), independently of 4-h changes in TRL apoB, apoC-II, apoC-III, or apoE. ApoC-I and apoC-III inhibited 3H-TRL clearance by adipocytes by >75% (P < 0.001).Conclusions: TRL enrichment with apoC-I is positively associated with postprandial hypertriglyceridemia and remnant accumulation in postmenopausal overweight and obese women, independently of apoC-II, apoC-III, or apoE, which may be due to inhibiting TRL clearance by adipocytes. Reducing TRL apoC-I may ameliorate delayed postprandial plasma clearance of TRLs and associated risks in humans.

Abstract 17: APOC3 A43T Variant Promotes ApoC-III Catabolism and Accelerates TG-rich Lipoprotein Clearance in Mice and Humans

Humans with loss-of-function (LoF) variants in APOC3 , the gene encoding apolipoprotein C-III (apoC-III), have significantly reduced plasma triglycerides (TG) and protection from coronary disease. These findings suggest that apoC-III may be a viable therapeutic target for decreasing vascular risk through TG reduction, and that elucidation of the protective mechanism of APOC3 LoF variants would inform such strategies. We report here the protective mechanism of the APOC3 A43T missense variant, one of four recently identified CAD-protective variants. By genotyping &gt;8,000 human participants with low TG, we identified 17 APOC3 A43T carriers and phenotyped 6 carriers and 54 matched controls. A43T heterozygotes demonstrate a significant reduction in apoC-III levels relative to non-carriers (50% reduction, P&lt;0.05), resulting in decreased plasma TG (50% reduction, P&lt;0.05). We generated viral vectors expressing WT or A43T apoC-III and expressed these in humanized mouse models to further explore the mechanism of reduced apoC-III levels due to the A43T variant. Mice expressing human CETP and the apoC-III A43T variant exhibit reduced plasma apoC-III (50% reduction, P&lt;0.0001) despite equal hepatic expression and secretion relative to controls expressing WT human apoC-III. These mice also exhibit reduced plasma TG and VLDL-C, and increased HDL-C relative to WT-expressing mice, fully recapitulating the protective lipoprotein profile of the human A43T carriers. Radioisotope-labeled apoC-III turnover studies showed that the A43T mutation causes a &gt;3-fold higher apoC-III clearance rate in vivo (P&lt;0.0001) due to defective integration into lipoprotein particles and accelerated renal catabolism (40% increase, P&lt;0.01). This results in increased lipoprotein lipase (LPL) activity (27% increase, P&lt;0.01) and faster chylomicron-TG clearance (97% increase, P&lt;0.01) in vivo . We are currently performing analogous studies of WT vs. A43T apoC-III turnover and VLDL clearance in human APOC3 A43T carriers. Collectively, our results support the rationale for therapeutic efforts to target circulating apoC-III through disruption of its binding to lipoproteins, mirroring the genetics-driven approaches for targeting PCSK9 that have recently yielded novel therapies.

Apolipoprotein CIII regulates lipoprotein-associated phospholipase A2 expression via the MAPK and NFκB pathways.

Apolipoprotein CIII (apo CIII), a small glycoprotein that binds to the surfaces of certain lipoproteins, is associated with inflammatory and atherogenic responses in vascular cells. Lipoprotein-associated phospholipase A2 (Lp-PLA2) has been proposed as an inflammatory biomarker and potential therapeutic target for cardiovascular disease (CVD). Here, we report that apo CIII increases Lp-PLA2 mRNA and protein levels in dose- and time- dependent manner in human monocytic THP-1 cells, and the increase can be abolished by MAPK and NFκB pathway inhibitors. Lp-PLA2 inhibitor, 1-linoleoyl glycerol attenuates the inflammation induced by apo CIII. In turn, exogenous Lp-PLA2 expression upregulates apo CIII and the upregulation can be inhibited by 1-linoleoyl glycerol in HepG2 cells. Moreover, plasma Lp-PLA2 level is correlated with apo CIII expression in pig liver. In vivo, Lp-PLA2 expression in monocytes and its activity in serum were significantly increased in human apo CIII transgenic porcine models compared with wild-type pigs. Our results suggest that Lp-PLA2 and apo CIII expression level is correlated with each other in vitro and in vivo.

Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia.

ABSTRACTApolipoprotein C-II (APOC2) is an obligatory activator of lipoprotein lipase. Human patients with APOC2 deficiency display severe hypertriglyceridemia while consuming a normal diet, often manifesting xanthomas, lipemia retinalis and pancreatitis. Hypertriglyceridemia is also an important risk factor for development of cardiovascular disease. Animal models to study hypertriglyceridemia are limited, with no Apoc2-knockout mouse reported. To develop a genetic model of hypertriglyceridemia, we generated an apoc2 mutant zebrafish characterized by the loss of Apoc2 function. apoc2 mutants show decreased plasma lipase activity and display chylomicronemia and severe hypertriglyceridemia, which closely resemble the phenotype observed in human patients with APOC2 deficiency. The hypertriglyceridemia in apoc2 mutants is rescued by injection of plasma from wild-type zebrafish or by injection of a human APOC2 mimetic peptide. Consistent with a previous report of a transient apoc2 knockdown, apoc2 mutant larvae have a minor delay in yolk consumption and angiogenesis. Furthermore, apoc2 mutants fed a normal diet accumulate lipid and lipid-laden macrophages in the vasculature, which resemble early events in the development of human atherosclerotic lesions. In addition, apoc2 mutant embryos show ectopic overgrowth of pancreas. Taken together, our data suggest that the apoc2 mutant zebrafish is a robust and versatile animal model to study hypertriglyceridemia and the mechanisms involved in the pathogenesis of associated human diseases.

Joint Linkage and Association Analysis with Exome Sequence Data Implicates SLC25A40 in Hypertriglyceridemia

Hypertriglyceridemia (HTG) is a heritable risk factor for cardiovascular disease. Investigating the genetics of HTG may identify new drug targets. There are ~35 known single-nucleotide variants (SNVs) that explain only ~10% of variation in triglyceride (TG) level. Because of the genetic heterogeneity of HTG, a family study design is optimal for identification of rare genetic variants with large effect size because the same mutation can be observed in many relatives and cosegregation with TG can be tested. We considered HTG in a five-generation family of European American descent (n = 121), ascertained for familial combined hyperlipidemia. By using Bayesian Markov chain Monte Carlo joint oligogenic linkage and association analysis, we detected linkage to chromosomes 7 and 17. Whole-exome sequence data revealed shared, highly conserved, private missense SNVs in both SLC25A40 on chr7 and PLD2 on chr17. Jointly, these SNVs explained 49% of the genetic variance in TG; however, only the SLC25A40 SNV was significantly associated with TG (p = 0.0001). This SNV, c.374A>G, causes a highly disruptive p.Tyr125Cys substitution just outside the second helical transmembrane region of the SLC25A40 inner mitochondrial membrane transport protein. Whole-gene testing in subjects from the Exome Sequencing Project confirmed the association between TG and SLC25A40 rare, highly conserved, coding variants (p = 0.03). These results suggest a previously undescribed pathway for HTG and illustrate the power of large pedigrees in the search for rare, causal variants.

In This Issue

HomeCirculation ResearchVol. 112, No. 11In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Originally published24 May 2013https://doi.org/10.1161/RES.0b013e31829a38b3Circulation Research. 2013;112:1401Smooth Muscle Cells from Reprogrammed Fibroblasts (p 1433)Karamariti et al devise a new method for generating smooth muscle cells in vitro.Download figureDownload PowerPointSmooth muscle cells (SMCs) are an essential component of blood vessels, and are thus required to engineer functional blood vessels in culture. Tissue engineers need a reliable and plentiful supply of SMCs. However, the methods currently used to obtain these cells - such as retrieving them from adult arteries and expanding them in vitro, or differentiating them from embryonic stem cells - can be inefficient, arduous, or both. Karamariti and colleagues propose a new approach, which involves direct reprogramming of fibroblasts into SMCs, by transfecting human fibroblasts with factors used for making induced pluripotent stem (iPS) cells. But instead of fully dedifferentiating the cells, they plated partially dedifferentiated cells, called PiPS, on collagen IV and induced them to become SMCs. The resulting cells exhibited SMC morphology, expressed SMC-specific markers and were capable of contracting like native SMCs. Through a careful monitoring of the reprogramming process, the researchers also discovered a novel factor, called DKK3, which activated the expression of an important SMC marker. The reprogrammed cells were then used with endothelial cells to build tissue-engineered vessels that functioned like their native counterparts when transplanted into mice. Overall, this new method could improve current approaches to vessel engineering and, as a result, help in the development of vessel grafts for patients.Macrophage ABCA1/G1 Deletion Enhance Atherogenesis (p 1456)Macrophages lacking cholesterol transporters make atherosclerosis much worse, say Westerterp et al.Download figureDownload PowerPointHigh HDL levels in blood are associated with a lower risk of heart disease. The protective effects of HDL have been linked, in part, to its ability to promote cholesterol efflux from macrophages. However, it is unclear whether this efflux is indeed protective. Cholesterol efflux from macrophages is controlled by two transporter proteins, ABCA1 and ABCG1, so Westerterp and colleagues specifically deleted these proteins from the macrophages of atherosclerosis-prone mice. They found that this procedure led to an increase in the lipid content of the macrophages and increased atherogenesis. And while a deficiency in the cell’s cholesterol efflux did play a role in these changes, it was not the only contributing factor. In fact, in mice lacking ABCA1- and ABCG1 transporters in macrophages there was also an increase in monocytes and neutrophils, which led to increased inflammation in atherosclerotic plaques. It turned out that the ABCA1- and ABCG1-deficient macrophages were producing high levels of the cytokines G-CSF and M-CSF, which promote the production of neutrophils and monocytes, respectively. These findings suggest that strategies aimed at promoting cholesterol efflux will prevent the pro-inflammatory and pro-atherogenic potential of macrophages, and furthermore, that monitoring G-CSF and M-CSF levels could help assess the success of such strategies.Antisense Inhibition of ApoC-III (p 1479)Inhibition of ApoC-III by antisense RNA reduces triglyceride levels in both animals and humans.Download figureDownload PowerPointElevated levels of triglycerides in the blood are a well-known risk factor for coronary heart disease. And, therefore, reducing triglyceride levels might reduce that risk. To lower dangerously high triglyceride levels, doctors currently prescribe statins, niacin, fibrates and omega-3-fatty acids, but in many patients, achieving the recommended level is difficult. More effective remedies are therefore required. The glycoprotein ApoC-III is an important regulator of plasma triglyceride, and high levels of ApoC-III are associated with high levels of triglycerides. High ApoC-III levels have also been independently associated with metabolic disorder and type 2 diabetes. Thus, Graham and colleagues investigated lowering ApoC-III levels with antisense nucleotides directed against human and rodent ApoC-III mRNA. The antisense nucleotides successfully reduced plasma levels of both ApoC-III and triglycerides in rats, mice, transgenic mice carrying the human ApoC-III gene, rhesus monkeys, and most importantly, in healthy humans. Although this phase-I trial was performed in just 33 subjects, the results were significant and there were no adverse safety indications. The findings suggest that larger-scale phase-II clinical trials are warranted for this first-of-its-kind antisense ApoC-III therapy. Previous Back to top Next FiguresReferencesRelatedDetails May 24, 2013Vol 112, Issue 11 Advertisement Article InformationMetrics © 2013 American Heart Association, Inc.https://doi.org/10.1161/RES.0b013e31829a38b3 Originally publishedMay 24, 2013 PDF download Advertisement

Abstract 2: Overexpression of Human Apolipoprotein C-III in Mice Decreases Intestinal Transport of Triglyceride Into Lymph

INTRODUCTION Apolipoprotein C-III (apoC-III), synthesized by the liver and intestine, is an inhibitor of LPL-mediated lipolysis and hepatic clearance of triglyceride-rich lipoproteins. ApoC-III overproduction is linked with hypertriglyceridemia and atherosclerosis. ApoC-III may also play an intracellular role in hepatic VLDL assembly/secretion. Little is known about the role of apoC-III in the intestine, although it is secreted on chylomicrons coincident with triglyceride absorption. HYPOTHESIS Given that overexpression of apoC-III results in high plasma triglyceride levels, we hypothesized that it might also stimulate intestinal triglyceride transport, thereby exacerbating plasma hypertriglyceridemia in human apoC-III transgenic (h-apoC-III tg) mice. METHODS 28-30 gram male h-apoC-III tg (on a C57BL/6J background) were fitted with both a mesenteric lymph cannula and a duodenal feeding tube, and received a continuous intraduodenal infusion of triglyceride (6 μmol of 3[H]-Triolein in 0.3ml of phosphate-buffered saline) for 6 hours with hourly lymph samples collected (n=11-12). At the end of the infusion period, luminal and mucosal contents and tissue samples were isolated. An advantage of this lymph fistula model is the ability to sample lymph continuously throughout the triglyceride infusion period while avoiding confounding effects of anesthesia and stomach emptying. RESULTS h-apoC-III tg mice had a decrease in lymph flow and a 43% reduction in lymphatic 3[H]-triglyceride transport compared to WT mice. The h-apoC-III tg mice had 10.0±2.3% of the total dose of 3[H]-triglyceride remaining in intestinal lumen; which was significantly higher than the 3.1±0.4% observed in WT mice. Thin layer chromatographic analysis of the luminal contents showed that h-apoC-III tg mice, as opposed to WT controls, had a significantly higher percentage of fatty acid. There were no significant differences in the luminal triglyceride, diglyceride, and monoglyceride composition between groups. CONCLUSION Our studies reveal a novel role for apoC-III in decreasing intestinal triglyceride transport distinct from its extracellular roles in plasma on lipoprotein lipase, and its intracellular role in hepatic VLDL synthesis and secretion.

Antisense Oligonucleotide Inhibition of Apolipoprotein C-III Reduces Plasma Triglycerides in Rodents, Nonhuman Primates, and Humans

Elevated plasma triglyceride levels have been recognized as a risk factor for the development of coronary heart disease. Apolipoprotein C-III (apoC-III) represents both an independent risk factor and a key regulatory factor of plasma triglyceride concentrations. Furthermore, elevated apoC-III levels have been associated with metabolic syndrome and type 2 diabetes mellitus. To date, no selective apoC-III therapeutic agent has been evaluated in the clinic. To test the hypothesis that selective inhibition of apoC-III with antisense drugs in preclinical models and in healthy volunteers would reduce plasma apoC-III and triglyceride levels. Rodent- and human-specific second-generation antisense oligonucleotides were identified and evaluated in preclinical models, including rats, mice, human apoC-III transgenic mice, and nonhuman primates. We demonstrated the selective reduction of both apoC-III and triglyceride in all preclinical pharmacological evaluations. We also showed that inhibition of apoC-III was well tolerated and not associated with increased liver triglyceride deposition or hepatotoxicity. A double-blind, placebo-controlled, phase I clinical study was performed in healthy subjects. Administration of the human apoC-III antisense drug resulted in dose-dependent reductions in plasma apoC-III, concomitant lowering of triglyceride levels, and produced no clinically meaningful signals in the safety evaluations. Antisense inhibition of apoC-III in preclinical models and in a phase I clinical trial with healthy subjects produced potent, selective reductions in plasma apoC-III and triglyceride, 2 known risk factors for cardiovascular disease. This compelling pharmacological profile supports further clinical investigations in hypertriglyceridemic subjects.

Human Apolipoprotein C-III – A New Intrahepatic Protein Factor Promoting Assembly and Secretion of Very Low Density Lipoproteins

Apolipoprotein (apo) C-III is a small protein (79 amino acids) and a component of triacylglycerol (TAG)-rich very low density lipoproteins (VLDL) and high density lipoproteins. We have unraveled a new intracellular role of apoCIII in promoting hepatic VLDL(1) (Sf > 100) assembly/secretion under lipid-rich conditions. Feeding apoc3-null mice with a high fat diet for two weeks or palm oil gavage failed to stimulate VLDL(1) production in vivo. Reconstitution of apoC-III expression using adenovirus encoding human apoC-III resulted in robust production of VLDL(1) containing apoB-100 or apoB-48. The stimulatory effect of human apoC-III on the assembly and secretion of VLDL(1) was recapitulated ex vivo in McA-RH7777 cells cultured in lipid-rich media. Metabolic labeling experiments showed that apoC-III plays a central role in (i) the formation of lumenal lipid droplets (LLD) rich in TAG, and (ii) promoting bulk TAG incorporation during VLDL(1) assembly. Structure-function analysis of naturally occurring apoC-III variants (Ala23Thr and Lys58Glu) defined two functional domains that play respective roles in LLD formation and VLDL(1) assembly. Unraveling the intracellular role of apoC-III in the atherogenic TAG-rich VLDL(1) production provides new insights into the strong influence of the APOA5-A4-C3-A1 gene locus on plasma TAG concentrations and premature atherosclerosis.

Statins suppress apolipoprotein CIII-induced vascular endothelial cell activation and monocyte adhesion

AimsActivation of vascular endothelial cells (ECs) contributes importantly to inflammation and atherogenesis. We previously reported that apolipoprotein CIII (apoCIII), found abundantly on circulating triglyceride-rich lipoproteins, enhances adhesion of human monocytes to ECs in vitro. Statins may exert lipid-independent anti-inflammatory effects. The present study examined whether statins suppress apoCIII-induced EC activation in vitro and in vivo.Methods and resultsPhysiologically relevant concentrations of purified human apoCIII enhanced attachment of the monocyte-like cell line THP-1 to human saphenous vein ECs (HSVECs) or human coronary artery ECs (HCAECs) under both static and laminar shear stress conditions. This process mainly depends on vascular cell adhesion molecule-1 (VCAM-1), as a blocking VCAM-1 antibody abolished apoCIII-induced monocyte adhesion. ApoCIII significantly increased VCAM-1 expression in HSVECs and HCAECs. Pre-treatment with statins suppressed apoCIII-induced VCAM-1 expression and monocyte adhesion, with two lipophilic statins (pitavastatin and atorvastatin) exhibiting inhibitory effects at lower concentration than those of hydrophilic pravastatin. Nuclear factor κB (NF-κB) mediated apoCIII-induced VCAM-1 expression, as demonstrated via loss-of-function experiments, and pitavastatin treatment suppressed NF-κB activation. Furthermore, in the aorta of hypercholesterolaemic Ldlr−/− mice, pitavastatin administration in vivo suppressed VCAM-1 mRNA and protein, induced by apoCIII bolus injection. Similarly, in a subcutaneous dorsal air pouch mouse model of leucocyte recruitment, apoCIII injection induced F4/80+ monocyte and macrophage accumulation, whereas pitavastatin administration reduced this effect.ConclusionsThese findings further establish the direct role of apoCIII in atherogenesis and suggest that anti-inflammatory effects of statins could improve vascular disease in the population with elevated plasma apoCIII.