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

5A Apolipoprotein Mimetic Peptide Promotes Cholesterol Efflux and Reduces Atherosclerosis in Mice

Intravenous administration of apolipoprotein (apo) A-I complexed with phospholipid has been shown to rapidly reduce plaque size in both animal models and humans. Short synthetic amphipathic peptides can mimic the antiatherogenic properties of apoA-I and have been proposed as alternative therapeutic agents. In this study, we investigated the atheroprotective effect of the 5A peptide, a bihelical amphipathic peptide that specifically effluxes cholesterol from cells by ATP-binding cassette transporter 1 (ABCA1). 5A stimulated a 3.5-fold increase in ABCA1-mediated efflux from cells and an additional 2.5-fold increase after complexing it with phospholipid (1:7 mol/mol). 5A-palmitoyl oleoyl phosphatidyl choline (POPC), but not free 5A, was also found to promote cholesterol efflux by ABCG1. When incubated with human serum, 5A-POPC bound primarily to high-density lipoprotein (HDL) but also to low-density lipoprotein (LDL) and promoted the transfer of cholesterol from LDL to HDL. Twenty-four hours after intravenous injection of 5A-POPC (30 mg/kg) into apoE-knockout (KO) mice, both the cholesterol (181%) and phospholipid (219%) content of HDL significantly increased. By an in vivo cholesterol isotope dilution study and monitoring of the flux of cholesterol from radiolabeled macrophages to stool, 5A-POPC treatment was observed to increase reverse cholesterol transport. In three separate studies, 5A when complexed with various phospholipids reduced aortic plaque surface area by 29 to 53% (n = 8 per group; p < 0.02) in apoE-KO mice. No signs of toxicity from the treatment were observed during these studies. In summary, 5A promotes cholesterol efflux both in vitro and in vivo and reduces atherosclerosis in apoE-KO mice, indicating that it may be a useful alternative to apoA-I for HDL therapy

Lipoprotein X Causes Renal Disease in LCAT Deficiency.

Human familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by low HDL, accumulation of an abnormal cholesterol-rich multilamellar particle called lipoprotein-X (LpX) in plasma, and renal disease. The aim of our study was to determine if LpX is nephrotoxic and to gain insight into the pathogenesis of FLD renal disease. We administered a synthetic LpX, nearly identical to endogenous LpX in its physical, chemical and biologic characteristics, to wild-type and Lcat-/- mice. Our in vitro and in vivo studies demonstrated an apoA-I and LCAT-dependent pathway for LpX conversion to HDL-like particles, which likely mediates normal plasma clearance of LpX. Plasma clearance of exogenous LpX was markedly delayed in Lcat-/- mice, which have low HDL, but only minimal amounts of endogenous LpX and do not spontaneously develop renal disease. Chronically administered exogenous LpX deposited in all renal glomerular cellular and matrical compartments of Lcat-/- mice, and induced proteinuria and nephrotoxic gene changes, as well as all of the hallmarks of FLD renal disease as assessed by histological, TEM, and SEM analyses. Extensive in vivo EM studies revealed LpX uptake by macropinocytosis into mouse glomerular endothelial cells, podocytes, and mesangial cells and delivery to lysosomes where it was degraded. Endocytosed LpX appeared to be degraded by both human podocyte and mesangial cell lysosomal PLA2 and induced podocyte secretion of pro-inflammatory IL-6 in vitro and renal Cxl10 expression in Lcat-/- mice. In conclusion, LpX is a nephrotoxic particle that in the absence of Lcat induces all of the histological and functional hallmarks of FLD and hence may serve as a biomarker for monitoring recombinant LCAT therapy. In addition, our studies suggest that LpX-induced loss of endothelial barrier function and release of cytokines by renal glomerular cells likely plays a role in the initiation and progression of FLD nephrosis

Electron microscopic analysis of LpX movement through renal glomerular compartments.

<p>Circulating LpX particles (small arrows in (A, B)) bind to endothelial cell lamellipodia in (A) WT and (B) <i>Lcat</i>^-/- mouse glomerular capillaries (arrowheads), are internalized (long arrows in (A), and degraded (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.s003" target="_blank">S3 Fig</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.s004" target="_blank">S4 Fig</a>). LpX bound to the cell surface (B1), is partially (B2), small arrows in inset) and then completely engulfed (B3). LpX penetrates the glomerular basement membrane (GBM) in WT (C) and <i>Lcat</i>^-/- mice ((D) and, inset in (D), arrowheads), markedly disrupting its structure (C, D; asterisks). The typical intramembranous lesion as found in the peripheral GBM of human FLD is seen in the inset in D, displaying a characteristic lamellar structure within a lucent lacuna in <i>Lcat</i>^-/- mice. In (D), several lamellipodia (arrows) engulf an LpX particle in the GBM. LpX penetrates the glomerular urinary space of both WT (E, G) and <i>Lcat</i>^-/- (F, H) mice. LpX binds to podocyte cell bodies (PCBs) and foot processes (PFPs) at multiple sites (E, F: small arrows; H: arrowheads), and was internalized into PCBs (F; large arrow). Large vacuoles (G, H; large arrows) containing partially degraded LpX particles (G, H; small arrows) as well as numerous small unilamellar vesicles are often observed, consistent with cell-mediated LpX degradation. (I) In WT mice, LpX did not accumulate in the mesangial matrix and occasional foamy mesangial cells were observed. (J) Mesangial cells near the sites of LpX deposition engulf LpX particles. (K) Marked retention of LpX in <i>Lcat</i>^-/- mouse mesangial matrix. The regions near large arrows 1 & 2 in (K) are shown enlarged in K1&2. LpX binds to the mesangial cell prior to engulfment. Scale bars: A, B1, F, H, J = 200 nm; B2, D (inset), K1, K2 = 250 nm; B–E, G, I, K = 500 nm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.s003" target="_blank">S3 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.s004" target="_blank">S4 Fig</a>, for additional examples.</p

LpX metabolism <i>in vivo.</i>

<p>(A) Blood samples from <i>Lcat</i>^-/- and WT mice were collected prior to (“basal”) and, at 1 and 24 hrs after LpX injection. Plasma samples from WT (n = 6) and <i>Lcat</i>^-/- (n = 6) mice were pooled and lipoproteins were separated by FPLC. Phospholipid (PL), Total Cholesterol (TC), and Free Cholesterol (FC) were measured in collected fractions. Prior to LpX injection, TC, PL and FC were abundant in HDL in WT mice, whereas they were absent in <i>Lcat</i>^-/- mice, which have only small amounts of lipids in VLDL/LpX and small HDL. One hour after injection in <i>Lcat</i>^-/- mice, LpX PL and FC are clearly present in a large peak in the VLDL region, whereas in WT mice, the peak is reduced, consistent with our findings using fluorescent PE-tagged LpX (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.g001" target="_blank">Fig 1B</a>). One hour after LpX administration, a new peak in the HDL region (25 ml elution volume) appeared in WT mice; in <i>Lcat</i>^-/- mice, this peak was observed prior to LpX administration and was increased at 1 hr post-injection. At this time, the PL and FC content of the <i>Lcat</i>^-/- peak was increased compared to the <i>Lcat</i>^-/- pre-injection peak, as well as to the WT peak. (B) Characterization of particles eluted at 25 ml using native gradient gel electrophoresis 1 hr post-injection. Native gradient gel electrophoresis confirmed that lipid-containing particles were present in the 25 ml fraction in the 7–8 nm size range. (C) SDS-PAGE (16% acrylamide gel) apoA-I immunoblot of small HDL particles (25 ml elution volume) generated by LpX at I hr. ApoA-I immunostaining confirmed the presence of apoA-I in these particles, which suggests that in the presence of apoA-I, LpX-derived PL, and to a lesser extent FC, increased the pool size of small HDL particles. These findings <i>in vivo</i> are consistent with the apoA-I and LCAT-dependent remodeling of LpX that we observed in vitro (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.g001" target="_blank">Fig 1B–1D</a>). The peak is still visible 24 hours after injection in WT mice, while in <i>Lcat</i>^-/- mice, it returns to basal levels (Fig 4A).</p

LpX remodeling <i>in vitro.</i>

<p>(A) TEM analysis of synthetic LpX particles. <i>Left panel</i>: Low magnification image (Scale bar; 500 nm<i>)</i>. <i>Middle panel</i>: High magnification (Scale bar; 100 nm). <i>Right panel</i>: LpX particle size distribution. Size categories (nm): I (0–50), II (50–100), III (100–150), IV (150–200), and V (200–245). Small unilamellar vesicles as well as small, medium and, large multivesicular vesicles are seen. (B) LpX remodeling by LCAT and apoA-I in vitro. Agarose gel electrophoresis of LpX labeled with both fluorescent PE <i>(red)</i> and cholesterol (<i>blue</i>) incubated with Alexa 647-tagged apoA-I (<i>green</i>) and/or LCAT in vitro and scanned. Colocalization of LpX PE and cholesterol fluorescence is seen as <i>magenta</i> (merged image). Lane 1: ApoA-I; Lane 2: LpX; Lane 3: LpX + ApoA-I; Lanes 4–6: LpX + ApoA-I + 2, 4, or, 6 mg LCAT, respectively; Lane 7: LpX + 6 mg LCAT. (C) FPLC analysis of dual fluorescent PE- and cholesterol-tagged LpX incubated without (<i>left</i>) or with apoA-I and 6 mg LCAT <i>(right</i>). Fractions were analyzed for rhodamine (PE) fluorescence (<i>upper panels</i>) and TopFluor cholesterol fluorescence (<i>lower panels</i>). Note the additional peak (<i>arrows</i>) after incubation with apoA-I and LCAT. (D) LpX is converted to plasma HDL in vitro. Fluorescent PE-tagged LpX was incubated overnight with pooled human plasma. Agarose gels were scanned for PE fluorescence and then stained with Sudan Black. Lane 1: Fluorescent LpX. Lane 2: Pooled human plasma. Lane 3: Pooled human plasma + fluorescent LpX. <i>Arrows</i> indicate origin.</p

Effect of LpX on glomerular cell function <i>in vitro.</i>

<p>(A) LpX compromises the integrity of endothelial cell monolayers. Confluent HUVEC cell monolayers were incubated with PBS alone, or with PBS containing HDL (1 mg/ml), LDL (1 mg/ml), or LpX 5 mg/ml). Following a transient artifactual increase in impedance after the change in medium, LpX markedly decreased impedance. (B,C) LpX alters podocyte cytoskeletal actin organization. Representative confocal images of phalloidin-stained immortalized human podocytes incubated in vitro in the absence (B) or presence of (C) synthetic LpX. Scale bar: 100 μm.(D) LpX endocytosis stimulates mesangial cell IL-6 cytokine secretion in vitro. Confluent cultured mesangial cells were treated with 200 μg/ml fluorescent PE-tagged synthetic LpX, in the absence or presence of 5 μM amiodarone for 18 hrs, and then IL-6 was quantified by ELISA assay. Note that amiodarone further increased IL-6 secretion with or without LpX treatment. *p <0.05; **p = 0.01; unpaired two-tailed t-test.</p

LpX induces nephropathology in <i>Lcat</i>^-/- mice.

<p>(A) Effect of LpX injection on renal function. Albumin to creatinine ratios (μg/mg) in urine (UACR) were measured prior to and then every week after exogenous LpX treatment, starting on week 2. Data are expressed as mean ± SEM. P values of group differences at each time point are reported. (B) Histological analysis. Representative images of PAS-stained sections of kidneys from WT and <i>Lcat</i>^-/- mice treated or not treated with LpX. No histological alterations were present in WT mice. WT mice treated with LpX showed no changes or only mild mesangial matrix expansion. In <i>Lcat</i>^-/- mice, LpX treatment increased mesangial matrix (<i>asterisk</i>) and, occasionally, PAS-positive material in glomerular capillaries (<i>arrow</i>) was observed (Scale bars: 20 μm). (C) Representative images of PAS-stained sections of kidneys from WT (<i>upper panel</i>) and <i>Lcat</i>^-/- (<i>lower panel</i>) mice treated with LpX. Tubular cell vacuolation was present focally in <i>Lcat</i>^-/- mice. (Scale bars: 50 μm). (D) Podocyte effacement revealed by TEM (left, <i>black arrows</i>; Scale bar: 1 μm) and SEM (right, <i>white arrows</i>; Scale bar: 20 μm) in glomeruli of <i>Lcat</i>^-/- mice treated with LpX.</p