Development of Abdominal Aortic Aneurysm Is Decreased in Mice with Plasma Phospholipid Transfer Protein Deficiency
2013; Elsevier BV; Volume: 183; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2013.05.018
ISSN1525-2191
AutoresValérie Deckert, Bénjamin Kretz, Ahmed Habbout, Kawtar Raghay, Jérôme Labbé, Nicolas Abello, Catherine Desrumaux, Thomas Gautier, Stéphanie Lemaire‐Ewing, Guillaume Maquart, Naïg Le Guern, David Masson, Éric Steinmetz, Laurent Lagrost,
Tópico(s)Infectious Aortic and Vascular Conditions
ResumoPlasma phospholipid transfer protein (PLTP) increases the circulating levels of proatherogenic lipoproteins, accelerates blood coagulation, and modulates inflammation. The role of PLTP in the development of abdominal aortic aneurysm (AAA) was investigated by using either a combination of mechanical and elastase injury at one site of mouse aorta (elastase model) or continuous infusion of angiotensin II in hyperlipidemic ApoE-knockout mice (Ang II model). With the elastase model, complete PLTP deficiency was associated with a significantly lower incidence and a lesser degree of AAA expansion. With the Ang II model, findings were consistent with those in the elastase model, with a lower severity grade in PLTP-deficient mice, an intermediate phenotype in PLTP-deficient heterozygotes, and a blunted effect of the PLTP-deficient trait when restricted to bone marrow–derived immune cells. The protective effect of whole-body PLTP deficiency in AAA was illustrated further by a lesser degree of adventitia expansion, reduced elastin degradation, fewer recruited macrophages, and less smooth muscle cell depletion in PLTP-deficient than in wild-type mice, as evident from comparative microscopic analysis of aorta sections. Finally, cumulative evidence supports the association of PLTP deficiency with reduced expression and activity levels of matrix metalloproteinases, known to degrade elastin and collagen. We conclude that PLTP can play a significant role in the pathophysiology of AAA. Plasma phospholipid transfer protein (PLTP) increases the circulating levels of proatherogenic lipoproteins, accelerates blood coagulation, and modulates inflammation. The role of PLTP in the development of abdominal aortic aneurysm (AAA) was investigated by using either a combination of mechanical and elastase injury at one site of mouse aorta (elastase model) or continuous infusion of angiotensin II in hyperlipidemic ApoE-knockout mice (Ang II model). With the elastase model, complete PLTP deficiency was associated with a significantly lower incidence and a lesser degree of AAA expansion. With the Ang II model, findings were consistent with those in the elastase model, with a lower severity grade in PLTP-deficient mice, an intermediate phenotype in PLTP-deficient heterozygotes, and a blunted effect of the PLTP-deficient trait when restricted to bone marrow–derived immune cells. The protective effect of whole-body PLTP deficiency in AAA was illustrated further by a lesser degree of adventitia expansion, reduced elastin degradation, fewer recruited macrophages, and less smooth muscle cell depletion in PLTP-deficient than in wild-type mice, as evident from comparative microscopic analysis of aorta sections. Finally, cumulative evidence supports the association of PLTP deficiency with reduced expression and activity levels of matrix metalloproteinases, known to degrade elastin and collagen. We conclude that PLTP can play a significant role in the pathophysiology of AAA. Plasma phospholipid transfer protein (PLTP) is a cardiovascular risk factor that has been shown to modulate atherogenesis and its thromboembolic complications.1Tzotzas T. Desrumaux C. Lagrost L. 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Indeed, like atherosclerosis, AAA is a complex, multifactorial disease, and reported risk factors in humans include tobacco smoking, male sex, increasing age, hypertension, hyperlipidemia, and a family history of the disorder.9Sakalihasan N. Limet R. Defawe O.D. Abdominal aortic aneurysm.Lancet. 2005; 365: 1577-1589Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar, 10Golledge J. Muller J. Daugherty A. Norman P. Abdominal aortic aneurysm: pathogenesis and implications for management.Arterioscler Thromb Vasc Biol. 2006; 26: 2605-2613Crossref PubMed Scopus (466) Google Scholar, 11Forsdahl S.H. Singh K. Solberg S. Jacobsen B.K. 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Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms.J Clin Invest. 2000; 105: 1641-1649Crossref PubMed Scopus (702) Google Scholar, 23Daugherty A. Manning M.W. Cassis L.A. Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice.J Clin Invest. 2000; 105: 1605-1612Crossref PubMed Scopus (1073) Google Scholar, 24Daugherty A. Cassis L.A. Mouse models of abdominal aortic aneurysms.Arterioscler Thromb Vasc Biol. 2004; 24: 429-434Crossref PubMed Scopus (385) Google Scholar We found that whole-body PLTP deficiency was associated with a reduction in AAA formation and progression, compared with wild-type (WT) mice, with outcomes consistent between models. Overall, compared with the features of AAA in WT mice, AAA in PLTP-deficient mice showed lesser degrees of elastin degradation, smooth muscle cell (SMC) depletion, and macrophage infiltration, as well as lower expression and release of MMPs in the aneurysmal tissue. Therefore, the present study suggests that in addition to its previously recognized role in atherogenesis, PLTP might be a risk factor in AAA formation. All experiments were performed in accordance with institutional guidelines and were approved by the University of Burgundy's Ethics Committee on the Use of Laboratory Animals (protocol numbers 5006 and 7309). PLTP-deficient (PLTP−/−) mice were generated as described by Jiang et al.25Jiang X.C. Bruce C. Mar J. Lin M. Ji Y. Francone O.L. Tall A.R. Targeted mutation of plasma phospholipid transfer protein gene markedly reduces high-density lipoprotein levels.J Clin Invest. 1999; 103: 907-914Crossref PubMed Scopus (322) Google Scholar All of the mice were on a homogeneous C57BL6/J background for at least eight generations. ApoE-deficient mice (ApoE−/−) were purchased from Charles River Laboratories International (L'Arbresle, France). ApoE−/−/PLTP−/− and ApoE−/−/PLTP+/− mice were obtained by crossbreeding and were compared with ApoE−/− littermates. All mice were fed a standard chow diet (A03 diet: SAFE, Augy, France). In the hyperlipidemic Ang II model, the chow diet contained a PLTP inducer (0.2% fenofibrate; Sigma-Aldrich, Lyon, France)26Bouly M. Masson D. Gross B. Jiang X.C. Fievet C. Castro G. Tall A.R. Fruchart J.C. Staels B. Lagrost L. Luc G. Induction of the phospholipid transfer protein gene accounts for the high density lipoprotein enlargement in mice treated with fenofibrate.J Biol Chem. 2001; 276: 25841-25847Crossref PubMed Scopus (84) Google Scholar, 27Tu A.Y. Albers J.J. Functional analysis of the transcriptional activity of the mouse phospholipid transfer protein gene.Biochem Biophys Res Commun. 2001; 287: 921-926Crossref PubMed Scopus (29) Google Scholar, 28Lie J. Lankhuize I.M. Gross B. van Gent T. van Haperen R. Scheek L. Staels B. de Crom R. van Tol A. Fenofibrate reverses the decline in HDL cholesterol in mice overexpressing human phospholipid transfer protein.Biochim Biophys Acta. 2005; 1738: 48-53Crossref PubMed Scopus (9) Google Scholar. The animals had free access to water and food. Transient elastase perfusion was used to induce experimental AAA, as described previously.22Pyo R. Lee J.K. Shipley J.M. Curci J.A. Mao D. Ziporin S.J. Ennis T.L. Shapiro S.D. Senior R.M. Thompson R.W. Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms.J Clin Invest. 2000; 105: 1641-1649Crossref PubMed Scopus (702) Google Scholar In brief, 4- to 5-month-old male WT and PLTP−/− mice were anesthetized with an intraperitoneal injection of 50 mg/kg of pentobarbital and underwent laparotomy under sterile conditions. With the help of an operating microscope (Wild Heerbrugg M650; Leica Microsystems, Wetzlar, Germany), the abdominal aorta was isolated at day 0 and the preperfusion aortic diameter was measured with a calibrated ocular grid. Temporary 6-0 ligatures were placed around the proximal and distal aorta. The proximal ligature was closed to interrupt proximal flow. An aortotomy was performed at the inferior ligature using the tip of a 30-gauge needle, and a microcatheter (MRE-033/010; Bioseb, Chaville, France) was inserted into the lumen. The aortic lumen was perfused for 5 minutes at 100 mmHg with a saline solution containing 0.25 U/mL type 1 porcine pancreatic elastase (Sigma-Aldrich). Control animals were perfused under the same conditions with saline solution. The microcatheter was then removed and the aortotomy was repaired without constriction of the lumen to restore the flow to the lower extremities. The postperfusion diameter was measured 5 minutes later. At day 14, a second laparotomy was performed under anesthesia, and the perfused segment of the aorta was re-exposed. The final aortic diameter was measured in situ. The mice were then sacrificed via intracardiac blood puncture, and the aortic tissue was harvested. AAA was defined as an increase in aortic diameter of at least 100% from the preperfusion diameter.22Pyo R. Lee J.K. Shipley J.M. Curci J.A. Mao D. Ziporin S.J. Ennis T.L. Shapiro S.D. Senior R.M. Thompson R.W. Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms.J Clin Invest. 2000; 105: 1641-1649Crossref PubMed Scopus (702) Google Scholar AAAs were induced in 6-month-old male ApoE−/− and ApoE−/−/PLTP−/− mice by a 28-day continuous infusion of Ang II (Sigma Aldrich, France), as described previously.23Daugherty A. Manning M.W. Cassis L.A. Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice.J Clin Invest. 2000; 105: 1605-1612Crossref PubMed Scopus (1073) Google Scholar Alzet osmotic minipumps (Alzet 2004; Durect, Cupertino, CA) were loaded with either saline solution or Ang II to ensure the delivery of Ang II at 1 μg/kg per minute. The pumps were incubated in normal saline at 37°C for 24 hours before subcutaneous implantation in the interscapular region. After 28 days of infusion, the mice were anesthetized with sodium pentobarbital, blood was drawn by intracardiac puncture, and the aorta was irrigated with cold PBS at physiological pressure. With the help of an operating microscope (Wild Heerbrugg M650), the abdominal aorta was exposed, carefully dissected, and placed on a black wax board. Images were obtained with a Coolpix camera (Nikon, Tokyo, Japan) attached to the microscope; these images were later used to measure the maximal aortic diameter. Measurements were determined, using Photoshop software version 5.0 (Adobe Systems, Mountain View, CA), by a single observer masked to the genotype and treatment of the mice. In this model, aneurysm was defined as commonly described (ie, as a 50% or greater increase in the external width of the suprarenal aorta, compared with that in saline-infused mice).29Satoh K. Nigro P. Matoba T. O'Dell M.R. Cui Z. Shi X. Mohan A. Yan C. Abe J. Illig K.A. Berk B.C. Cyclophilin A enhances vascular oxidative stress and the development of angiotensin II-induced aortic aneurysms.Nat Med. 2009; 15: 649-656Crossref PubMed Scopus (301) Google Scholar, 30Liu O. Jia L. Liu X. Wang Y. Wang X. Qin Y. Du J. Zhang H. Clopidogrel, a platelet P2Y12 receptor inhibitor, reduces vascular inflammation and angiotensin II induced-abdominal aortic aneurysm progression.PLoS One. 2012; 7: e51707Crossref PubMed Scopus (54) Google Scholar Aneurysm severity was rated according to a five-point scale, as described previously31Daugherty A. Manning M.W. Cassis L.A. Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis.Br J Pharmacol. 2001; 134: 865-870Crossref PubMed Scopus (242) Google Scholar, 32Manning M.W. Cassis L.A. Huang J. Szilvassy S.J. Daugherty A. Abdominal aortic aneurysms: fresh insights from a novel animal model of the disease.Vasc Med. 2002; 7: 45-54Crossref PubMed Scopus (128) Google Scholar: type 0, no aneurysm; type I, dilated lumen in the suprarenal region of the aorta with no thrombus; type II, remodeled aneurysmal tissue in the suprarenal region of the aorta that frequently contains thrombus; type III, a pronounced bulbous form of type II that contains thrombus; and type IV, a form in which there are multiple aneurysms containing thrombus, some overlapping, in the suprarenal area of the aorta. Bone marrow transplantation was performed as described previously.7Valenta D.T. Ogier N. Bradshaw G. Black A.S. Bonnet D.J. Lagrost L. Curtiss L.K. Desrumaux C.M. Atheroprotective potential of macrophage-derived phospholipid transfer protein in low-density lipoprotein receptor-deficient mice is overcome by apolipoprotein AI overexpression.Arterioscler Thromb Vasc Biol. 2006; 26: 1572-1578Crossref PubMed Scopus (58) Google Scholar ApoE−/− recipient mice were irradiated with a single dose of 9 Gy (40 Gy/hour) from a cobalt source. Bone marrow–derived cells were isolated from the tibias and femurs of ApoE−/−/PLTP+/+ or ApoE−/−/PLTP−/− donors and were injected into the tail vein of recipient ApoE−/− mice (3 × 106 cells per mouse). The mice were maintained on antibiotic water (1 g/L neomycin; Sigma-Aldrich) for 4 weeks after irradiation. At 5 weeks after transplantation, the mice were implanted with osmotic minipumps containing Ang II, and the experiments described above were performed. Abdominal aortic tissues were fixed with 10% neutral-buffered formalin and embedded in paraffin. Serial cross sections (5 μm thick) of the aortic tissue were stained with H&E, Verhoeff–Van Gieson's stain (for elastin), or Masson's trichrome (for collagen). Immunohistochemical staining of CD4+ and CD8+ T cells, macrophages, and α-actin was performed using mouse anti-mouse CD4+ antibody (1/200; mAb51312; Abcam, Cambridge, UK), rabbit anti-mouse CD8+ antibody (1/250; AJ1170c; Abgent, San Diego, CA), rat monoclonal anti-mouse Mac-3 antibody (1/100; Sc19991; Santa Cruz Biotechnology, Santa Cruz, CA), and anti–α-actin antibody (1/300; Sigma-Aldrich) as primary antibodies, respectively. Immunohistochemistry was performed after deparaffinization, rehydration, and unmasking by heating in 10 mmol/L sodium citrate buffer (pH 6). Nonspecific sites were saturated with bovine serum albumin, and endogenous peroxidase activity was blocked using 3% hydrogen peroxide (Merck, Darmstadt, Germany). For CD4+ T-cell immunostaining, endogenous nonspecific binding sites of biotin/avidin (SP2001; Vector Laboratories, Burlingame, CA) and endogenous mouse Ig (MKB-2213; Vector Laboratories) were blocked before incubation with primary antibodies. This was followed by sequential application of horseradish peroxidase–conjugated dextran polymer containing anti-rabbit and anti-mouse IgG (Dako EnVision; Agilent Technologies, Santa Clara, CA) for 30 minutes for actin slides, HRP-conjugated goat polyclonal anti-rat IgG [heavy and light chains (H&L)] (1/100; GenScript, Piscataway, NJ) for 3 hours for Mac-3 slides, HRP-conjugated donkey anti-rabbit IgG (H&L) (1/250; 6440-05; SouthernBiotech, Birmingham, AL) for 45 minutes for CD8 slides, or biotinylated goat anti-mouse IgG (H&L) (1/250; 1031-08; SouthernBiotech) and streptavidin–horseradish peroxidase (1/100; Life Technologies–Invitrogen, Carlsbad, CA) for 45 minutes for CD4 slides. The immunoreaction was revealed by incubation with either 3,3′-diamino-benzidine tetrahydrochloride (Dako) for α-actin and Mac-3 or Nova Red (SK4800; Vector Laboratories) for CD4 and CD8. All sections were counterstained with hematoxylin. Negative controls were generated by omitting the primary antibody. Elastin degradation and SMC content were graded independently by two masked investigators according to the four grading keys. The degree of macrophage infiltration in the elastase-induced model of AAA was evaluated independently by two masked investigators on a scale from 0 to 4, with 0 representing no inflammation and 4 representing a severe transmural inflammatory response. Quantification of macrophage infiltration in the Ang II–induced model of AAA, and quantification of CD4+ and CD8+ T-cell infiltration in both the elastase-induced and Ang II–induced models of AAA, were determined by a masked observer and were estimated as an index using computer-assisted analysis software version 4.8.2 (AxioVision; Carl Zeiss Microscopy, Jena, Germany). Expression levels of MMP-2, MMP-9, TNF-α, IL-6, MCP-1, IFN-γ, AT1 receptor, and AT2 receptor in the aortic wall and expression level of PLTP in the liver were measured by quantitative real-time RT-PCR (qRT-PCR). In brief, total RNA was extracted from tissues using TRIzol reagent (Life Technologies–Invitrogen). One microgram of RNA was reverse-transcribed into cDNA using SuperScript Reverse Transcriptase (Life Technologies–Invitrogen) and random primers (Eurogentec, Liege, Belgium). qPCR was performed using a SYBR Green real-time PCR kit (Life Technologies–Invitrogen) on a LightCycler 2.0 detection system (Roche Diagnostics, Meylan, France). Values were normalized to GAPDH and expressed relative to WT mice. The following gene-specific primers were used: mPLTP sense 5′-TCGGCGGAGGGTGTGTCCAT-3′ and mPLTP antisense 5′-CATGGCAGAGTCAAAGAAGA-3′; mIL-6 sense 5′-TGCAAGAGACTTCCATCCAGTTGCC-3′ and mIL-6 antisense 5′-TGTGAAGTAGGGAAGGCCGTGGT-3′; mTNF-α sense 5′-TGTGGGCCTCTCATGCACCA-3′ and mTNF-α antisense 5′-AGGCAACCTGACCACTCTCCC-3′; mMCP-1 sense 5′-CTGGATCGGAACCAAATGAG-3′ and mMCP-1 antisense 5′-AAGGCATCACAGTCCGAGTC-3′; mIFN-γ sense 5′-CGCTACACACTGCATCTTGG-3′ and mIFN-γ antisense 5′-GTCACCATCCTTTTGCCAGT-3′; mMMP-2 sense 5′-GATAACCTGGATGCTGTC-3′ and mMMP-2 antisense 5′-CCAACCTTCACGCTCTTG-3′; mMMP-9 sense 5′-ACAATCCTTGCAATGTGGATGTT-3′ and mMMP-9 antisense 5′-CGCCCTGGATCTCAGGAATA-3′; mAT2 receptor sense 5′-CCCTAAAAAGGTGTCCAGCA-3′ and mAT2 receptor antisense 5′-GCACATCACAGGTCCAAAAA-3′; and mGAPDH sense 5′-CATTGTGGAAGGGCTCATGA-3′ and mGAPDH antisense 5′-TCTTCTGGGTGGCAGTGATG-3′. Standard gelatin zymography was performed to measure the activity of MMPs in mouse aorta. Tissue homogenates (15 μg protein) were separated in 10% SDS-PAGE containing 1 mg/mL gelatin (Ready Gel zymogram gels; Bio-Rad Laboratories, Hercules, CA) under nonreducing conditions. After electrophoresis, the gels were washed with 2.5% Triton X-100 at room temperature and then were incubated in a developing buffer (100 mmol/L Tris-HCl, 5 mmol/L CaCl2, 0.05% Brij-35, 0.001% NaN3, pH 7.4) overnight at 37°C. After incubation, the gels were stained with a solution of 0.1% Coomassie Blue R-250 in 20% isopropanol and 10% acetic acid and were subsequently destained with isopropanol/acetic acid solution. The gelatinolytic products were determined using standard proteins (Gelatinase Zymography Standards; AbCys, Paris, France). The gels were analyzed using a GS-800 Bio-Rad densitometer with Bio-Rad Quantity One software version 4.2. Blood samples were collected in heparin-containing tubes, which were then centrifuged at 6300 × g for 10 minutes at 4°C. The plasma samples were stored at −80°C. PLTP activity was measured using a commercially available fluorescence activity assay (Roar Biomedical, New York, NY) as described previously.33Masson D. Drouineaud V. Moiroux P. Gautier T. Dautin G. Schneider M. Fruchart-Najib J. Jauhiainen M. Ehnholm C. Sagot P. Gambert P. Jimenez C. Lagrost L. Human seminal plasma displays significant phospholipid transfer activity due to the presence of active phospholipid transfer protein.Mol Hum Reprod. 2003; 9: 457-464Crossref PubMed Google Scholar Changes in fluorescence were monitored using a Victor3 fluorescence counter (PerkinElmer, Waltham, MA). PLTP activity values were calculated using the initial slope of the phospholipid transfer curve, and were expressed as the increase in fluorescence arbitrary units (AU) per minute. The statistical significance of the differences between two data means was determined with nonparametric U-test. Differences between multiple groups were analyzed by Kruskal–Wallis nonparametric test followed by Dunn's multiple comparison test. Differences were considered statistically significant at P < 0.05. Data are expressed as means ± SEM, except that counts of categorical responses between groups (ie, incidence or severity of AAA) were performed by χ2 analyses based on actual animal numbers. Elastase was perfused transiently by aortotomy into a temporarily isolated aortic segment at day 0. A second laparotomy was performed at day 14 to re-expose the aorta (Figure 1A). Neither preperfusion baseline aortic diameter nor the aortic dilation observed at day 0 immediately after perfusion of the elastase-containing solution differed significantly between WT and PLTP−/− mice (Figure 1B). In contrast, at 14 days after the initial elastase perfusion the final mean aortic diameter was significantly greater in WT than in PLTP−/− mice (P = 0.02), with mean increases of 82 ± 9% and 47 ± 9% in aortic diameter, respectively (P = 0.01) (Figure 1B). Finally, and after 14 days, 53% of WT mice but only 8% of PLTP−/− mice had developed AAA, defined as at least a 100% increase in the mean aortic diameter (P = 0.01) (Figure 1B). Mice receiving a perfusion of saline solution developed no aneurysms, with mean aortic diameters of 0.98 ± 0.03 mm (preperfusion), 1.20 ± 0.04 mm (postperfusion), and 1.05 ± 0.06 mm (final). In addition to the increased aortic diameter, the development of AAA in the WT mouse was characterized by thickening of the aortic wall, destruction of the medial elastic lamellae, and collagenous fibrosis in the adventitia (Figure 2). To further assess the severity of AAA, serial cross sections of the aortic tissue were stained with Verhoeff–Van Gieson's stain to observe the elastin lamellae, or with monoclonal anti–α-actin antibodies to observe SMCs. The lower incidence of AAA in PLTP−/− mice was associated with better preservation of medial elastin; t
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