From bloodjournal.hematologylibrary.org by guest on June 6, 2013. For personal use only.
Prepublished online January 22, 2004; doi:10.1182/blood-2003-11-4051
Hepatic Low-Density Lipoprotein Receptor-Related Protein Deficiency in Mice Increases Atherosclerosis Independent of Plasma Cholesterol Sonia M Espirito Santo, Nuno M Pires, Lianne S Boesten, Gery Gerritsen, Niels Bovenschen, Ko Willems van Dijk, J W Jukema, Hans M Princen, Andre Bensadoun, Wei-Ping Li, Joachim Herz, Louis M Havekes and Bart J van Vlijmen
Articles on similar topics can be found in the following Blood collections Hemostasis, Thrombosis, and Vascular Biology (2497 articles) Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml
Advance online articles have been peer reviewed and accepted for publication but have not yet appeared in the paper journal (edited, typeset versions may be posted when available prior to final publication). Advance online articles are citable and establish publication priority; they are indexed by PubMed from initial publication. Citations to Advance online articles must include the digital object identifier (DOIs) and date of initial publication.
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.
bloodjournal.hematologylibrary.org by guest on June 6, ForDOI personal use only. Blood First From Edition Paper, prepublished online February 5,2013. 2004; 10.1182/blood-2003-11-4051
Hepatic Low-Density Lipoprotein Receptor-Related Protein Deficiency in Mice Increases Atherosclerosis Independent of Plasma Cholesterol
Short title: Hepatic LRP Deficiency and Atherosclerosis
Sonia M. S. Espirito Santo1,2, Nuno M. M. Pires1,2, Lianne S. M. Boesten1,2, Gery Gerritsen3, Niels Bovenschen1,4, Ko Willems van Dijk3,5, J. Wouter Jukema2, Hans M. G. Princen1, André Bensadoun6, Wei-Ping Li7, Joachim Herz8, Louis M. Havekes1,2,4, and Bart J. M. van Vlijmen1,2.
From the 1TNO-Prevention and Health, Gaubius Laboratory, Leiden; the Departments of 2
Cardiology,
3
Human Genetics, and
5
General Internal Medicine, Leiden University
Medical Center, Leiden; the 4Department of Plasma Proteins, Sanquin Research at CLB, Amsterdam, The Netherlands; the 6Division of Nutritional Sciences, Cornell University, Ithaca, NY; the Departments of 7Cell Biology and 8Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Address correspondence to: Bart J.M. van Vlijmen, PhD, Dept. of Cardiology, Leiden University Medical Center c/o TNO-Prevention and Health, P.O. Box 2215, 2301 CE Leiden, The Netherlands Phone: +31-71-5181537; Fax: +31-71-5181904; E-mail:
[email protected]
Supported by grants from the Royal Netherlands Academy of Arts and Sciences (B.J.M.v.V.), European Union project QLK1-CT-1999-498 (S.M.S.E.S.) and The Netherlands Heart Foundation project 2000.099 (G.G. and K.W.v.D.). Total word count: 3616; Abstract word count: 200 Scientific heading: Hemostasis, Thrombosis, and Vascular Biology.
1 Copyright (c) 2004 American Society of Hematology
From bloodjournal.hematologylibrary.org by guest on June 6, 2013. For personal use only.
Abstract The low-density lipoprotein (LDL) receptor-related protein (LRP) has a wellestablished role in the hepatic removal of atherogenic apolipoprotein (APO) E-rich remnant lipoproteins from plasma. In addition, LRP recognizes multiple distinct pro- and anti-atherogenic ligands in vitro. Here, we investigated the role of hepatic LRP in atherogenesis independent of its role in removal of APOE-rich remnant lipoproteins. Mice that allow inducible inactivation of hepatic LRP were combined with LDL receptor and APOE double deficient mice (MX1Cre+LRPflox/floxLDLR-/-APOE-/-). On a
LDLR-/-
APOE-/- background, hepatic LRP deficiency resulted in decreased plasma cholesterol and triglycerides (cholesterol: 17.1±5.2 vs. 23.4±6.3 mM, P=0.025; triglycerides: 1.1±0.5 vs.
2.2±0.8
mM,
P=0.002;
LRPflox/floxLDLR-/-APOE-/-
mice,
for
MX1Cre+LRPflox/floxLDLR-/-APOE-/-
respectively).
Lower
plasma
and
control
cholesterol
in
MX1Cre+LRPflox/floxLDLR-/-APOE-/- mice coincided with increased plasma lipoprotein lipase (71.2±7.5 vs. 19.1±2.4 ng/ml, P=0.002), coagulation factor VIII (4.4±1.1 vs. 1.9±0.5 U/mL, P=0.001), von Willebrand factor (2.8±0.6 vs. 1.4±0.3 U/mL, P=0.001), and tissue-type plasminogen activator (1.7±0.7 vs. 0.9±0.5 ng/ml, P=0.008) as compared to controls. Strikingly, MX1Cre+LRPflox/floxLDLR-/-APOE-/- mice showed a 2-fold higher atherosclerotic lesion area as compared to controls (408.5±115.1 vs. 219.1±86.0 103µm2, P=0.003). Our data indicate that hepatic LRP plays a clear protective role in atherogenesis independent of plasma cholesterol, possibly due to maintaining low levels of its pro-atherogenic ligands.
2
From bloodjournal.hematologylibrary.org by guest on June 6, 2013. For personal use only.
Introduction The low-density lipoprotein (LDL) receptor-related protein (LRP) is a large cell surface receptor that is ubiquitously expressed in a variety of tissues and is present on a wide range of different cell types, including hepatocytes, monocytes, and smooth muscle cells.1 The LRP, together with the LDL receptor, has a well-established role in the hepatic removal of pro-atherogenic remnants of chylomicrons and very low-density lipoproteins (VLDL) from the circulation.2 Apolipoprotein (APO) E serves as the ligand for LRP mediated hepatic uptake of remnant lipoproteins.3 Recently, it has been demonstrated that LRP not only plays a role in atherosclerosis at the level of the liver by removing atherogenic lipoproteins from the circulation, but also has a clear role in atherosclerosis extrahepatically via controlling smooth muscle cell platelet-derived growth factor (PDGF) receptor activation.4 However, the role of LRP in atherosclerosis may not be restricted to lipoprotein removal and PDGF receptor activation. In vitro studies show that LRP binds a wide range of distinct ligands.5 Hepatic LRP is suggested to be an important determinant of the levels of its ligands in plasma. Some of these ligands have been indicated to play a clear role in modulating atherogenesis, including tissue-type plasminogen activator (t-PA),6 urokinase tissue-type plasminogen activator (u-PA),7 plasminogen activator inhibitor-1 (PAI-1),8 tissue factor pathway inhibitor (TFPI),9 coagulation factor VIII (FVIII),10 and lipoprotein lipase (LPL).11 In the present study, we hypothesize that hepatic LRP may play a role in atherogenesis independent of its well-known role in the plasma removal of atherogenic APOE-rich remnant lipoproteins. To this end, we further investigated the role of LRP in atherogenesis by using mice conditionally lacking hepatic LRP (MX1Cre+LRPflox/flox) on a LDL receptor and APOE deficient background (LDLR-/-APOE-/-). Whereas APOE deficiency will exclude removal of APOE-rich lipoproteins via both LDL receptor and LRP, LDL receptor deficiency will also exclude possible removal of lipoproteins mediated by the APOB100 pathway.
3
From bloodjournal.hematologylibrary.org by guest on June 6, 2013. For personal use only.
Our data show that MX1Cre+LRPflox/floxLDLR-/-APOE-/- mice have slightly though statistically significant lower plasma cholesterol and triglyceride levels as compared to control LRPflox/floxLDLR-/-APOE-/- mice. As expected MX1Cre+LRPflox/floxLDLR-/-APOE-/mice had higher plasma levels of the LRP ligands LPL, FVIII, and t-PA. Nevertheless, despite the lower plasma cholesterol, MX1Cre+LRPflox/floxLDLR-/-APOE-/- mice show increased atherosclerosis. Thus, our data indicate that hepatic LRP plays a clear protective role in atherogenesis independent of plasma cholesterol, possibly due to maintaining low levels of its pro-atherogenic ligands.
4
From bloodjournal.hematologylibrary.org by guest on June 6, 2013. For personal use only.
Methods Transgenic Animals The experimental animals were obtained by crossbreeding LDLR-/-APOE-/- mice12 with MX1Cre+LRPflox/floxLDLR-/- mice2 resulting in mice deficient for LRP, LDLR, and APOE (MX1Cre+LRPflox/floxLDLR-/-APOE-/-) and control littermates that lack both LDLR and APOE (LRPflox/floxLDLR-/-APOE-/-). Mice were genotyped by PCR.2,12 For experiments, six-week old male transgenic MX1Cre+LRPflox/floxLDLR-/-APOE-/- (n=15) and male littermate
control
LRPflox/floxLDLR-/-APOE-/-
mice
(n=16)
were
used.
MX1Cre+LRPflox/floxLDLR-/-APOE-/- (n=12) and control LRPflox/floxLDLR-/-APOE-/- (n=13) were induced for LRP deficiency by intraperitoneal injection of a polyinosinic:polycytidylic ribonucleic acid (pI:pC, Sigma, St. Louis, USA).2 Three mice of each genotype did not receive pI:pC injections and were included as extra uninduced controls. In addition, MX1Cre+LRPflox/flox mice were combined with reporter mice carrying a conditional ßgalactosidase gene that allows monitoring of Cre-recombinase-mediated DNA excisions.13 Transgenic offspring was induced with pI:pC parallel with the experimental animals. All mice were fed a standard chow diet (SRM-A, Hope Farms, The Netherlands). The institutional committee on animal welfare of TNO-PG approved all animal experiments.
Plasma Parameters Blood was collected by tail bleeding. Cholesterol and triglycerides were measured in EDTA-plasma enzymatically using available kits #C0534 and #337-B (Sigma Diagnostics, St. Louis, USA), respectively. Plasma HDL cholesterol was determined using phosphotungstic acid.14 Lipoprotein distribution was determined by fast performance liquid chromatography (FPLC) size fractionation.14 Plasma mouse APOB (B48 and B100) and APOAI concentrations were determined by imunoblotting using polyclonal rabbit-antisera against mouse APOB and APOAI (TNO, Leiden, The Netherlands). Peroxidase-labeled polyclonal goat-anti-rabbit
5
From bloodjournal.hematologylibrary.org by guest on June 6, 2013. For personal use only.
antibody (Nordic, Tilburg, The Netherlands) was used as secondary antibody and bound peroxidase was visualized using BM-blue peroxidase substrate (Roche Diagnostics, GmbH, Mannheim, Germany). Protein bands were scanned and subsequently analyzed using TINA version 2.09 software. LPL mass and activity were determined in EDTA-plasma.15 FVIII activity, vWF antigen and t-PA antigen were determined in citrated plasma.16, 17
Tissue Analysis Tissues of MX1Cre+LRPflox/flox transgenic mice that were combined with Cre reporter mice, were frozen, cryosectioned (10-µm), and stained for ß-galactosidase activity. Livers,
hearts
LRPflox/floxLDLR-/-APOE-/-
and were
aortas fixed
from in
MX1Cre+LRPflox/floxLDLR-/-APOE-/phosphate-buffered
and
4%-formaldehyde,
dehydrated, and embedded in paraffin. In addition, fresh parts of livers (3 random animals per group) were used for detection membrane LRP.2 Cross sections (5-µm) of the descending aorta were stained for LRP and PDGF receptor.4 Hearts were crosssectioned (5-µm) throughout the entire aortic root area. Per mouse, 4 sections with 30µm intervals were used for quantification of atherosclerotic lesion area.18 Sections were routinely stained with hematoxylin-phloxine-saffron (HPS). Lesion area was determined using Leica Qwin image analysis software (EIS, Asbury NJ).18 Sections of the aortic root area were stained with rabbit-anti-mouse macrophages antibody (AIA-31240, 1/3000, Accurate Chemical and Scientific, Westbury, NY) and a monoclonal mouse-anti- -smooth muscle cell actin antibody (clone 1A4, dilution 1:800, Sigma, The Netherlands). Biotinylated donkey-anti-rabbit antibody (dilution 1:300, Vector Laboratories, Burlingame, Ca) and horse-anti-mouse antibody (dilution 1:400, Vector Laboratories, Burlingame, Ca) were used as secondary antibodies, followed by incubation with horseradish peroxidase labeled avidin-biotin complex. Peroxidase activity was visualized with Nova Red (Vector Labs, Burlingame, Ca). Macrophage (AIA-31240
6
From bloodjournal.hematologylibrary.org by guest on June 6, 2013. For personal use only.
positive area) and smooth muscle cell (1A4 positive area) lesion areas were quantified using Leica Qwin image analysis software (EIS, Asbury NJ) at the level of 11 individual lesions overlapping in size ranging from 50,000-200,000 µm2 and expressed as a percentage of the size of individual lesion. Collagen and elastin were stained using Sirius Red and Resorcin-Fuchsin (Chroma-Gesellschaft, Stuttgart, Germany), respectively.
Statistical analysis All data are presented as mean±SD. Data were analyzed using the MannWhitney U-test. P-values less than 0.05 were regarded as statistically significant.
7
From bloodjournal.hematologylibrary.org by guest on June 6, 2013. For personal use only.
Results Induction of LRP Deficiency We15 and others2,19 have demonstrated that the inducible MX1Cre transgene achieves near-complete recombination of conditional alleles mainly in the liver. This was verified by crossbreeding MX1Cre transgenics with a reporter strain that allows monitoring Cre recombinase-mediated DNA excisions in mice. PI:pC treatment of 6week-old offspring and subsequent tissue analysis for ß-galactosidase activity confirmed a complete recombination in the liver 12 weeks after pI:pC induction (Figure 1A). MX1Cre-mediated recombination was, to a lesser extent, also detected at the level of the spleen (Figure 1B). Using this reporter approach, no MX1Cre mediated recombination was found in heart, muscle, fat, stomach, intestine, brain, and importantly also not in the vasculature as analyzed at the level of the descending aorta (Figure 1C), aortic arch, and aortic
root.
Like
pI:pC
induced
MX1Cre+LRPflox/flox
mice,15
pI:pC
induced
MX1Cre+LRPflox/floxLDLR-/-APOE-/- mice had no detectable LRP in liver membrane extracts as determined by immunoblotting with antibodies directed against the 85 KD subunit of LRP (Figure 1D). In the same mice, however, aortic LRP protein could be detected, using a smooth muscle cell-specific immunofluorescent staining (Figure 1, E and F). Moreover, no increased expression of aortic PDGF receptor was observed, a feature typical for vascular LRP deletion4 (Figure 1, G and H). Collectively, these data indicate successful deletion of hepatic LRP in MX1Cre+LRPflox/floxLDLR-/-APOE-/- mice, whereas aortic LRP expression remains unaffected.
Plasma lipid and lipoprotein Since LDL receptor and APOE double deficient mice are unable to clear lipoproteins via the APOE as well as the APOB100 pathway, one would expect no effect of LRP deficiency on plasma lipid levels in MX1Cre+LRPflox/floxLDLR-/-APOE-/- animals as compared to LRPflox/floxLDLR-/-APOE-/- mice. Uninduced MX1Cre+LRPflox/floxLDLR-/-APOE-/and control LRPflox/floxLDLR-/-APOE-/- mice had identical plasma cholesterol, triglyceride
8
From bloodjournal.hematologylibrary.org by guest on June 6, 2013. For personal use only.
levels, and lipoprotein distribution (Table 1; Figure 2, A and B). Four weeks after induction of LRP deficiency by pI:pC, MX1Cre+LRPflox/floxLDLR-/-APOE-/- mice remarkably showed significant lower plasma cholesterol (P=0.025) and plasma triglycerides (P=0.002)
as
compared
to
controls
(Table
1).
Lower
plasma
lipids
in
MX1Cre+LRPflox/floxLDLR-/-APOE-/- mice as compared to controls persisted for at least 12 weeks, i.e. the end of the study (Cholesterol: 17.1±2.9 vs. 24.6±5.0 mM, P=0.001; Triglycerides: 0.8±0.4 vs. 1.5±0.5 mM, P=0.008, respectively). The lowering in plasma cholesterol was mainly confined to the VLDL/LDL-sized lipoprotein fraction (Figure 2, C and D). There was no effect of LRP deficiency on plasma HDL cholesterol (0.45±0.23 mM and 0.38±0.17 mM, P=0.200 for controls and MX1Cre+LRPflox/floxLDLR-/-APOE-/- mice, respectively). In addition, plasma apolipoproteins were not detectably affected by LRP status (relative amounts of plasma: APOB48: 100±18% vs. 119±24%, P=0.240; APOB100: 100±7% vs. 98.9±19.2%, P=0.485; APOAI: 100±43% vs. 107±25%, P=0.320 for controls and MX1Cre+LRPflox/floxLDLR-/-APOE-/- mice, respectively).
Plasma LPL Mass and Activity LPL binds to LRP in vitro.5 To investigate whether LRP plays a role in LPL processing in vivo, we measured both plasma LPL mass and activity levels. Uninduced MX1Cre+LRPflox/floxLDLR-/-APOE-/- and control LRPflox/floxLDLR-/-APOE-/- mice did not differ in plasma LPL levels (Table 2). However, pI:pC induced MX1Cre+LRPflox/floxLDLR-/-APOE/-
mice had a significant (P=0.002) 3.7-fold higher plasma LPL levels as compared to
control LRPflox/floxLDLR-/-APOE-/- mice. Surprisingly, increased LPL levels did not coincide with increased plasma LPL activity (Table 2). MX1Cre+LRPflox/flox LDLR-/-APOE-/- and control mice had similar plasma LPL activity, that were also similar to plasma LPL activity observed in uninduced MX1Cre+LRPflox/floxLDLR-/-APOE-/- and control mice. These data indicate that hepatic LRP deficiency results in accumulation of inactive plasma LPL.
9
From bloodjournal.hematologylibrary.org by guest on June 6, 2013. For personal use only.
Plasma FVIII, VWF and T-PA Among the postulated ligands for LRP is the coagulation factor VIII (FVIII).5 Recently, we observed that FVIII indeed accumulates in the plasma of MX1Cre+LRPflox/flox mice.16 Von Willebrand factor (vWF), the carrier protein of FVIII,20 also accumulates in plasma of these mice.16 In the current study, uninduced MX1Cre+LRPflox/floxLDLR-/-APOE/-
and control LRPflox/floxLDLR-/-APOE-/- mice did not differ in plasma FVIII (P=0.400) and
vWF
(P=1.000)
levels
(Table
2).
However,
4
weeks
after
induction,
MX1Cre+LRPflox/floxLDLR-/-APOE-/- mice had significant 2-fold higher plasma FVIII (P=0.010) and vWF (P=0.001) as compared to LRPflox/floxLDLR-/-APOE-/- mice (Table 2). Elevated levels of FVIII and vWF persisted at least until 12 weeks after pI:pC induction (FVIII: 1.4±0.3 vs. 3.7±0.4 U/ml, P
