Type VIII Collagen Mediates Vessel Wall Remodeling after Arterial Injury and Fibrous Cap Formation in Atherosclerosis
2013; Elsevier BV; Volume: 182; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2013.02.011
ISSN1525-2191
AutoresJoshua Lopes, Eser Adiguzel, Steven Gu, Shu‐Lin Liu, Guangpei Hou, Scott P. Heximer, Richard K. Assoian, Michelle P. Bendeck,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoCollagens in the atherosclerotic plaque signal regulation of cell behavior and provide tensile strength to the fibrous cap. Type VIII collagen, a short-chain collagen, is up-regulated in atherosclerosis; however, little is known about its functions in vivo. We studied the response to arterial injury and the development of atherosclerosis in type VIII collagen knockout mice (Col8−/− mice). After wire injury of the femoral artery, Col8−/− mice had decreased vessel wall thickening and outward remodeling when compared with Col8+/+ mice. We discovered that apolipoprotein E (ApoE) is an endogenous repressor of the Col8a1 chain, and, therefore, in ApoE knockout mice, type VIII collagen was up-regulated. Deficiency of type VIII collagen in ApoE−/− mice (Col8−/−;ApoE−/−) resulted in development of plaques with thin fibrous caps because of decreased smooth muscle cell migration and proliferation and reduced accumulation of fibrillar type I collagen. In contrast, macrophage accumulation was not affected, and the plaques had large lipid-rich necrotic cores. We conclude that in atherosclerosis, type VIII collagen is up-regulated in the absence of ApoE and functions to increase smooth muscle cell proliferation and migration. This is an important mechanism for formation of a thick fibrous cap to protect the atherosclerotic plaque from rupture. Collagens in the atherosclerotic plaque signal regulation of cell behavior and provide tensile strength to the fibrous cap. Type VIII collagen, a short-chain collagen, is up-regulated in atherosclerosis; however, little is known about its functions in vivo. We studied the response to arterial injury and the development of atherosclerosis in type VIII collagen knockout mice (Col8−/− mice). After wire injury of the femoral artery, Col8−/− mice had decreased vessel wall thickening and outward remodeling when compared with Col8+/+ mice. We discovered that apolipoprotein E (ApoE) is an endogenous repressor of the Col8a1 chain, and, therefore, in ApoE knockout mice, type VIII collagen was up-regulated. Deficiency of type VIII collagen in ApoE−/− mice (Col8−/−;ApoE−/−) resulted in development of plaques with thin fibrous caps because of decreased smooth muscle cell migration and proliferation and reduced accumulation of fibrillar type I collagen. In contrast, macrophage accumulation was not affected, and the plaques had large lipid-rich necrotic cores. We conclude that in atherosclerosis, type VIII collagen is up-regulated in the absence of ApoE and functions to increase smooth muscle cell proliferation and migration. This is an important mechanism for formation of a thick fibrous cap to protect the atherosclerotic plaque from rupture. Collagens are the most abundant constituents of the extracellular matrix in atherosclerotic and restenotic vascular lesions and can be detrimental by contributing to lesion bulk and by signaling regulation of the migration and proliferation of vascular cells. However, collagens can also provide tensile strength to the fibrous cap and protect against plaque rupture, a devastating complication of atherosclerosis.1Adiguzel E. Ahmad P.J. Franco C. Bendeck M.P. Collagens in the progression and complications of atherosclerosis.Vasc Med. 2009; 14: 73-89Crossref PubMed Scopus (179) Google ScholarType VIII collagen, a member of the short-chain nonfibrillar collagen family, is comprised of α1 and α2 collagen chains. It is present in small amounts in normal arteries; however, synthesis is dramatically increased after injury and during development of atherosclerosis in experimental animals and humans.2Bendeck M.P. Regenass S. Tom W.D. Giachelli C.M. Schwartz S.M. Hart C. Reidy M.A. Differential expression of alpha 1 type VIII collagen in injured platelet-derived growth factor-BB–stimulated rat carotid arteries.Circ Res. 1996; 79: 524-531Crossref PubMed Scopus (57) Google Scholar, 3Sibinga N.E. Foster L.C. Hsieh C.M. Perrella M.A. Lee W.S. Endege W.O. Sage E.H. Lee M.E. Haber E. Collagen VIII is expressed by vascular smooth muscle cells in response to vascular injury.Circ Res. 1997; 80: 532-541Crossref PubMed Scopus (77) Google Scholar, 4Sinha S. Kielty C.M. Heagerty A.M. Canfield A.E. Shuttleworth C.A. Upregulation of collagen VIII following porcine coronary artery angioplasty is related to smooth muscle cell migration not angiogenesis.Int J Exp Pathol. 2001; 82: 295-302Crossref PubMed Scopus (24) Google Scholar, 5Yasuda O. Zhang S.H. Miyamoto Y. Maeda N. Differential expression of the alpha1 type VIII collagen gene by smooth muscle cells from atherosclerotic plaques of apolipoprotein-E-deficient mice.J Vasc Res. 2000; 37: 158-169Crossref PubMed Scopus (20) Google Scholar, 6Plenz G. Dorszewski A. Breithardt G. Robenek H. Expression of type VIII collagen after cholesterol diet and injury in the rabbit model of atherosclerosis.Arterioscler Thromb Vasc Biol. 1999; 19: 1201-1209Crossref PubMed Scopus (24) Google Scholar, 7Weitkamp B. Cullen P. Plenz G. Robenek H. Rauterberg J. Human macrophages synthesize type VIII collagen in vitro and in the atherosclerotic plaque.FASEB J. 1999; 13: 1445-1457Crossref PubMed Scopus (61) Google Scholar, 8MacBeath J.R. Kielty C.M. Shuttleworth C.A. Type VIII collagen is a product of vascular smooth-muscle cells in development and disease.Biochem J. 1996; 319: 993-998Crossref PubMed Scopus (45) Google Scholar, 9Plenz G. Dorszewski A. Völker W. Ko Y.S. Severs N.J. Breithardt G. Robenek H. Cholesterol-induced changes of type VIII collagen expression and distribution in carotid arteries of rabbit.Arterioscler Thromb Vasc Biol. 1999; 19: 2395-2404Crossref PubMed Scopus (11) Google Scholar, 10Qiu H. Depre C. Ghosh K. Resuello R.G. Natividad F.F. Rossi F. Peppas A. Shen Y.T. Vatner D.E. Vatner S.F. Mechanism of gender-specific differences in aortic stiffness with aging in nonhuman primates.Circulation. 2007; 116: 669-676Crossref PubMed Scopus (77) Google Scholar In vitro studies have revealed that type VIII collagen is produced by both macrophages and smooth muscle cells (SMCs) and is stimulated by some atherogenic growth factors and cytokines.3Sibinga N.E. Foster L.C. Hsieh C.M. Perrella M.A. Lee W.S. Endege W.O. Sage E.H. Lee M.E. Haber E. Collagen VIII is expressed by vascular smooth muscle cells in response to vascular injury.Circ Res. 1997; 80: 532-541Crossref PubMed Scopus (77) Google Scholar, 11Plenz G. Deng M.C. Robenek H. Völker W. Vascular collagens: spotlight on the role of type VIII collagen in atherogenesis.Atherosclerosis. 2003; 166: 1-11Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 12Cherepanova O.A. Pidkovka N.A. Sarmento O.F. Yoshida T. Gan Q. Adiguzel E. Bendeck M.P. Berliner J. Leitinger N. Owens G.K. Oxidized phospholipids induce type VIII collagen expression and vascular smooth muscle cell migration.Circ Res. 2009; 104: 609-618Crossref PubMed Scopus (99) Google Scholar, 13Garvey S.M. Sinden D.S. Schoppee Bortz P.D. Wamhoff B.R. Cyclosporine up-regulates Krüppel-like factor-4 (KLF4) in vascular smooth muscle cells and drives phenotypic modulation in vivo.J Pharmacol Exp Ther. 2010; 333: 34-42Crossref PubMed Scopus (38) Google Scholar Type VIII collagen can act as a haptotactic factor for SMCs.14Hou G. Mulholland D. Gronska M.A. Bendeck M.P. Type VIII collagen stimulates smooth muscle cell migration and matrix metalloproteinase synthesis after arterial injury.Am J Pathol. 2000; 156: 467-476Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar Moreover, SMCs derived from type VIII collagen knockout mice migrate and proliferate less and express less matrix metalloproteinase (MMP) activity than do cells from wild-type mice.15Adiguzel E. Hou G. Mulholland D. Hopfer U. Fukai N. Olsen B.R. Bendeck M.P. Migration and growth are attenuated in vascular smooth muscle cells with type VIII collagen-null alleles.Arterioscler Thromb Vasc Biol. 2006; 26: 56-61Crossref PubMed Scopus (45) Google Scholar Type VIII collagen-null SMCs were unable to overcome strong adhesion to interstitial type I collagen; however, adding exogenous type VIII collagen to the cultures rescued migration and proliferation. We concluded that type VIII collagen functioned as a provisional matrix masking the interstitial matrix and enabling SMCs to move in response to injury. Despite these in vitro data, there are few functional studies of type VIII collagen in vivo. After discontinuing a high-fat diet in a rabbit model of atherosclerosis, Plenz et al9Plenz G. Dorszewski A. Völker W. Ko Y.S. Severs N.J. Breithardt G. Robenek H. Cholesterol-induced changes of type VIII collagen expression and distribution in carotid arteries of rabbit.Arterioscler Thromb Vasc Biol. 1999; 19: 2395-2404Crossref PubMed Scopus (11) Google Scholar demonstrated a correlation between plaque regression, decreased type VIII collagen expression, and decreased macrophage accumulation in the adventitia. However, those investigators did not determine cause-effect relationships between cholesterol, type VIII collagen, and macrophage accumulation, and they did not explore the underlying mechanisms. In current studies, we use knockout mice to investigate the functions of type VIII collagen in vivo using models of mechanical vascular injury and the apolipoprotein E (ApoE)–null mouse model of atherosclerosis.ApoE is associated with very low-, intermediate-, and high-density lipoproteins and facilitates their clearance from plasma, and also mediates cholesterol efflux from vessel wall macrophages.16Zhu Y. Bellosta S. Langer C. Bernini F. Pitas R.E. Mahley R.W. Assmann G. von Eckhardstein A. Low-dose expression of a human apolipoprotein E transgene in macrophages restores cholesterol efflux capacity of apolipoprotein E-deficient mouse plasma.Proc Natl Acad Sci USA. 1998; 95: 7585-7590Crossref PubMed Scopus (64) Google Scholar Loss-of-function mutations in apoE have been linked to atherosclerosis in humans, and extensive experimental studies in ApoE-null mice have revealed important functions for this apolipoprotein in atherosclerosis, vascular remodeling, and restenosis. There is also evidence for cholesterol-independent effects of ApoE on SMCs, which could protect against atherosclerosis. For example ApoE can inhibit SMC proliferation through up-regulation of nitric oxide17Hui D.Y. Basford J.E. Distinct signaling mechanisms for apoE inhibition of cell migration and proliferation.Neurobiol Aging. 2005; 26: 317-323Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar and the production of prostacyclin18Kothapalli D. Fuki I. Ali K. Stewart S.A. Zhao L. Yahil R. Kwiatkowski D. Hawthorne E.A. FitzGerald G.A. Phillips M.C. Lund-Katz S. Puré E. Rader D.J. Assoian R.K. Antimitogenic effects of HDL and APOE mediated by Cox-2-dependent IP activation.J Clin Invest. 2004; 113: 609-618PubMed Google Scholar; however, few studies have documented protective effects in vivo.In present studies, we show that vessel wall thickening and outward remodeling of vessel diameter are attenuated after wire injury of the femoral artery in type VIII collagen knockout mice. We report here for the first time a direct effect of ApoE in suppression of type VIII collagen gene expression by SMCs; consequently, type VIII collagen gene expression is substantially increased in the Apoe−/− mouse. Deficiency of type VIII collagen in the Apoe−/− mouse results in impaired SMC proliferation and migration and decreased type I collagen accumulation, resulting in thinning of the fibrous cap. These studies point to an important role for type VIII collagen in mediating fibrous cap formation, which stabilizes atheromas.Materials and MethodsFemoral Artery Injury in Col8+/+ and Col8−/− MiceMice with targeted deletion of both the col8a1 and col8a2 genes (Col8−/− mice) backcrossed more than 10 generations in the C57BL/6 strain were generated in the laboratory of Dr. Bjorn Olsen (Harvard Medical School), as previously described,19Hopfer U. Fukai N. Hopfer H. Wolf G. Joyce N. Li E. Olsen B.R. Targeted disruption of Col8a1 and Col8a2 genes in mice leads to anterior segment abnormalities in the eye.FASEB J. 2005; 19: 1232-1244Crossref PubMed Scopus (90) Google Scholar and were generously provided for these experiments. Animal experiments were approved by the local animal care committee at the University of Toronto in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publ No. 85-23, revised 1996). The University of Toronto is compliant with the NIH guide (A5013-01). Before surgery, male mice were injected subcutaneously with 0.1 mg/kg buprenorphine, then anesthetized via inhalation of 1.5% to 2% isofluorane in oxygen, 1.5 L/min. Anesthesia was monitored by observation of breathing rate and pinching between the toes on the paw. Wire injury of the femoral artery was performed by introducing a 0.38-mm diameter straight spring wire into a small branch artery of the femoral artery and advancing it through the femoral artery >5 mm toward the iliac artery. The wire was left in place for 1 minute to denude the endothelium and dilate the artery.20Sata M. Maejima Y. Adachi F. Fukino K. Saiura A. Sugiura S. Aoyagi T. Imai Y. Kurihara H. Kimura K. Omata M. Makuuchi M. Hirata Y. Nagai R. A mouse model of vascular injury that induces rapid onset of medial cell apoptosis followed by reproducible neointimal hyperplasia.J Mol Cell Cardiol. 2000; 32: 2097-2104Abstract Full Text PDF PubMed Scopus (247) Google Scholar Mice were sacrificed at either 7 or 21 days after injury via intraperitoneal injection of ketamine, 333 mg/kg body weight (Ayerst Veterinary Laboratories; Guelph, ON, Canada), and xylazine, 67 mg/kg body weight (Bayer, Inc., Toronto, ON, Canada). The entire circulatory system was perfused at constant physiologic pressure via a catheter placed in the left ventricle, first with 0.9% saline solution (Baxter Corp., Mississauga, ON, Canada) and then with 4% paraformaldehyde for 10 minutes. The femoral artery, extending from the iliac artery to the ligated small branch artery, was removed, placed in 4% paraformaldehyde for 2 hours, then transferred to PBS. The vessels were bisected, then paraffin embedded. Cross-sections (4 μm) were cut from each bisected half to obtain an accurate representation of injury along the length of the femoral artery, and analysis was performed on cross-sections from the middle of the femoral arteries. Tissue processing was performed by the Centre for Modeling Human Disease Pathology Core, the Toronto Centre for Phenogenomics (Toronto, ON, Canada). With use of digital imaging (Simple PCI software version 5.3; Compix, Inc., Mars PA), cross-sectional areas of the neointima and media and vessel wall cell numbers and vessel diameters were measured in sections obtained at 21 days after injury. Sections from uninjured control femoral arteries served as controls. Cell proliferation was detected by immunostaining for Ki-67, and apoptosis by TUNEL in sections obtained at 7 days after injury. Values from Col8−/− mice were compared with those from Col8+/+ control mice.Ki-67 is a nuclear antigen associated with proliferation and is present during the cell cycle but absent during the resting G0 phase. Sections were stained with a 1:200 dilution of rabbit anti–Ki-67 antibody (No. RM-9106–S; Lab Vision Corp., Fremont, CA), then with biotin-conjugated goat anti-rabbit IgG secondary antibody (No. BA-1000; Vector Laboratories, Inc., Burlingame, CA), and were visualized with 3,3′-diaminobenzidine and counterstained with hematoxylin. The percentage of Ki-67–labeled nuclei was measured in the medial layer of the vessel using an Eclipse E600 microscope (Nikon Corp., Tokyo, Japan), a camera (Hamamatsu Photonics KK, Hamamatsu City, Japan), and Simple PCI software (Compix).A TUNEL assay was performed to measure the percentage of apoptotic cells, using a kit from Millipore (Canada), Ltd. (Etobicoke, ON, Canada). Tissues were deparaffinized in a series of xylene washes and rehydrated in ethanol. The tissue was digested with 0.02 mg/mL proteinase K to inactivate nucleases. The slides were then pretreated with an equilibration buffer for 15 minutes, followed by incubation for 1 hour at 37°C in the terminal deoxynucleotidyl transferase enzyme reaction mixture. Sections were treated with stop/wash buffer for 25 minutes, incubated with a fluorescein-tagged anti-digoxigenin antibody for 30 minutes, and counterstained with 0.5 μg/mL propidium iodide. The percentage of TUNEL-positive cells in the medium was determined using an Eclipse E600 microscope, DS-Fi1 camera, and NIS-Elements software (all from Nikon).mRNA Isolation from Male C57BL/6 or Apoe−/− Mouse AortasAortas were isolated from 24-week-old male C57BL/6 or Apoe−/− mice. Aortas were dissected, and extraneous tissue from the adventitial side was carefully removed. Aortas were divided into arch (ascending) and thoracic (descending) regions and stabilized by submerging the tissues in RNAlater (Qiagen GmbH, Hilden, Germany). Before isolating the total RNA, the aortas were weighed, and approximately 10 mg aortic tissue was manually homogenized and treated with 10 mg/mL proteinase K (Qiagen) at 55°C for 10 minutes. The homogenate was clarified via centrifugation, and total RNA was isolated from the supernatant using RNeasy mini columns (Qiagen).Cell Culture and Lipoprotein TreatmentPrimary murine aortic SMCs were isolated from C57BL/6 mice and used between passages 2 and 5. The SMCs were grown to 80% to 90% confluence and were serum-starved via incubation in Dulbecco's Minimum essential's medium containing 1 mg/mL heat-inactivated fatty acid–free bovine serum albumin for 48 hours. The quiescent cells were stimulated with 10% fetal bovine serum in the absence or presence of 2 μmol/L recombinant apolipoprotein or 50 μg/mL lipoprotein (generous gift of Drs. Michael C. Phillips and Sissel Lund-Katz, Children's Hospital of Philadelphia) for 24 hours. Total RNA was isolated from cells lysed in Trizol reagent (Invitrogen Corp., Carlsbad, CA) and analyzed using real-time quantitative RT-PCR (RT-qPCR), as outlined in the following section.Real-Time RT-qPCRReal-time RT-qPCR was performed as described,21Klein E.A. Yung Y. Castagnino P. Kothapalli D. Assoian R.K. Cell adhesion, cellular tension, and cell cycle control.Methods Enzymol. 2007; 426: 155-175Crossref PubMed Scopus (44) Google Scholar using 50 to 100 ng for reverse transcription of total RNA isolated from cultured SMCs or aortas. A 10% aliquot of cDNA was prepared using SYBR Green QPCR Master Mix (Applied Biosystems, Inc., Foster City, CA) to qPCR with 900 nmol/L of the primer-probe sets mCOL8A1 (forward, 5′-AGAGTGCACCCAGCCCCAGT-3′; reverse, 5′-TGGGTGGCACAGCCATCACATTT-3′) and mCOL8A2 (forward, 5′-CCTGCAGGCTCTGCCTGTCC-3′; reverse, 5′-CACTCTTGGCCCACACCCCA-3′). RT-qPCR results were calculated using 18S rRNA as the reference for mRNAs. To detect mouse 18S rRNA, we used TaqMan Universal PCR Master Mix (Applied Biosystems) with forward primer 5′-CCTGGTTGATCCTGCCAGTAG-3′, reverse primer 5′-CCGTGCGTACTTAGACATGCA-3′, and probe 5′-VIC-TGCTTGTCTCAAAGATTA-MGB-NFQ-3′. Each sample was analyzed in duplicate PCR reactions, and mRNA expression was quantified against a standard curve using ABI PRISM 7000 SDS software (Applied Biosystems). Mean quantities and SD were calculated from duplicate PCR reactions.Generation of Col8−/−;Apoe−/− MiceCol8−/− mice were bred with Apoe−/− mice (both on C57BL/6 background) to generate mice that were Col8−/−;Apoe−/−. These were compared with either littermate control Col8+/+;Apoe−/− mice or Apoe−/− mice (purchased from The Jackson Laboratory, Bar Harbor, ME). Genomic DNA was extracted from ear clips, and genotyping was performed via PCR amplification using the following primers: Col8a1 wild type: sense, 5′-CGGGAGTAGGAAAACCAGGAGTGA-3′, and antisense, 5′-GGCCCAAGAACCCCAGGAACA-3′; Col8a1 knockout: sense, 5′-GTGGGGGTGGGGTGGGATTAGATA-3′, and antisense, 5′-CTCGGCCCAAGAACCCCAGGAAC-3; Col8a2 wild type: sense, 5′-CCGGTAAAGTATGTGCAGC-3′, and antisense, 5′-CAAGTCCATTGGCAGCATC-3′; Col8a2 knockout: sense, 5′-CAGCGCATCGCCTTCTATCGC-3′, and antisense identical to wild-type Col8a2; Apoe wild type: sense, 5′-GCCTAGCCGAGGGAGAGCCG-3′, and antisense, 5′-TGTGACTTGGGAGCTCTGCAGC-3′; Apoe knockout: sense, same as wild type, and antisense, 5′-GCCGCCCCGACTGCATCT-3′. Beginning at age 8 to 12 weeks, male and female mice of both genotypes were fed an atherogenic diet containing 40% kcal fat and 1.25% cholesterol by weight (D12108; Research Diets, Inc., New Brunswick, NJ) for 6 or 12 weeks. On the day when sacrificed, mice were euthanized via CO2 asphyxiation. The left ventricle was cannulated, and animals were perfused at physiologic pressure (100 mmHg), first with sterile saline solution and then with 4% paraformaldehyde for 5 to 10 minutes. The aortic arch and descending aorta to the iliac bifurcation were isolated, cleared of fat and surrounding tissues, and used as described below to measure oil-red-o staining, immunohistochemistry, matrix staining and plaque architecture, and in situ zymography. For analyses that required fresh-frozen tissues, mice were perfused with sterile saline solution, and arterial tissues were immediately dissected in PBS, embedded in OCT, and snap frozen in liquid nitrogen.Mean Arterial PressureMean arterial pressure was measured after 6 and 12 weeks of the atherogenic diet via catheterization of the right common carotid artery using a 1.4F blood pressure probe (Millar Instruments, Inc., Houston, TX). Mice were anesthetized using 3% isofluorane, the carotid artery was catheterized, and blood pressure was allowed to stabilize for 3 minutes with 1% isofluorane. Measurement of blood pressure was performed for 1 minute, and mean arterial pressure was calculated using the following formula: Mean arterial pressure = Diastolic blood pressure + 1/3 Pulse pressure.Plasma Lipid AnalysisWhole blood samples were collected at sacrifice via right ventricle puncture and placed in heparinized 1.5-mL tubes. Blood samples were spun at 14,800 × g for 5 minutes at 4°C and were stored at 4°C for a maximum of 2 days before analysis. Plasma samples were analyzed using a multiplate spectrophotometer (Microskan; Titertek Instruments, Inc., Huntsville, AL) at 492 nm using enzymatic assays (Synchron; Beckman Coulter, Inc., Brea, CA) for triglycerides (kit No. 445850) and total cholesterol (kit No. 467825).Oil Red O StainingAtherosclerotic plaque burden in the descending aorta (downstream of the left subclavian artery to the iliac bifurcation) was determined via Oil Red O staining. Aortas were incised longitudinally, pinned en face to black silicone plates using 0.1-mm minuten pins, rinsed with isopropanol, and stained with 18 mg/mL Oil Red O for 30 minutes at room temperature on a shaker. Stained aortas were washed three or four times in 70% isopropanol and imaged using a CoolPix digital camera (Nikon). The percentage of Oil Red O–positive plaque per total aortic surface area was quantified using digital image analysis (NIS-Elements Basic Research; Nikon).Immunohistochemistry, Matrix Staining, and Plaque ArchitectureParaformaldehyde-fixed, paraffin-embedded longitudinal sections of the mouse aortic arch including the brachiocephalic and left carotid artery branchpoints were deparaffinized in xylenes, rehydrated in an ethanol series, and blocked using 0.3% H2O2 in cold methanol, followed by a 1% bovine serum albumin blocking solution (kit No. D12287; Invitrogen). Primary antibodies directed against mouse α-smooth muscle actin raised in goat (1:500) (A2547; Sigma; St. Louis, MO), mouse Mac-2 raised in rat (1:100) (CL8942AP; Cedarlane Laboratories USA, Inc., Burlington, NC), or type I collagen raised in rabbit (1:200) (ab21286; Abcam plc, Cambridge, MA) were used to stain for SMCs, macrophages, and type I collagen, respectively. Negative controls included sections incubated without primary antibody. Slides were then incubated with species-specific biotinylated secondary antibodies including anti-goat (1:4000) (B2763; Invitrogen), anti-rat (1:200) (E0468; DakoCytomation Inc., Carpinteria, CA), and anti-rabbit (1:1000) (B2770; Invitrogen), followed by incubation in steptavidin–horseradish peroxidase–conjugated solution following the manufacturer's instructions (kit No. D12287; Invitrogen). Sections were then incubated in 3,3′-diaminobenzidine (kit No. D12287; Invitrogen), a chromogenic substrate, followed by counterstaining with hematoxylin. Serial sections were stained using Picrosirius Red (PSR) for collagen and either Verhoeff-Van Geisin stain or Movat's pentachrome stain for elastin. Whole plaques located on the lesser curvature of the arch and in the brachiocephalic and left carotid artery branchpoints were measured. Plaque area was defined as the region extending from the internal elastic lamina to the luminal edge of the plaque. Thresholding of maximum and minimum color intensity was conducted using NIS-Elements Basic Research software (Nikon), with positively stained regions expressed as a percentage of plaque area. Fibrous cap thickness and necrotic core area were assessed using PSR-stained longitudinal sections of lesser curvature plaques. Relative fibrous cap thickness was calculated by dividing fibrous cap thickness by the entire height of the plaque (from the internal elastic lamina to the luminal edge). Necrotic core area was measured as a percentage of total plaque area. The percentage of area stained positive for SMCs and collagen was also assessed independently in the plaque fibrous cap. Ki-67 and TUNEL assays were performed as described (see Femoral Artery Injury in Col8+/+ and Col8−/− Mice). PSR- and Movat's pentachrome–stained plaque sections from the lesser curvature were ranked according to criteria established by Virmani et al.22Virmani R. Kolodgie F.D. Burke A.P. Farb A. Schwartz S.M. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions.Arterioscler Thromb Vasc Biol. 2000; 20: 1262-1275Crossref PubMed Scopus (3182) Google Scholar The percentage of samples with plaques in each of the five categories (no plaques, intimal xanthoma, pathologic intimal thickening, fibrous cap atheroma, and thin fibrous cap atheroma) was plotted for Col8+/+;Apoe−/− and Col8−/−;Apoe−/− mice.Fluorescence in Situ ZymographyIn situ examination of gelatinase activity was performed using fluorescence-quenched gelatin (DQ-Gelatin, No. D12054; Invitrogen). Gelatinase-catalyzed hydrolysis of the molecule relieves the quenching, and the magnitude of the resultant fluorescence is proportional to the extent of proteolytic digestion. Longitudinal cryosections (8 μm long) of mouse aortic arch were washed in ISZ incubation buffer [50 mmol/L Tris-HCl (pH 7.8), 150 mmol/L NaCl, 5 mmol/L CaCl2, and 0.2 mmol/L NaN3] and incubated overnight at 4°C in incubation buffer. The sections were then counterstained for 10 minutes with Hoechst 33258 diluted 1:10,000 in incubation buffer. A prewarmed solution of incubation buffer containing 0.1% agarose and 0.1 mg/mL fluorescence-quenched gelatin was applied to the sections, which were then coverslipped and incubated for 30 minutes at 37°C. Images of the lesser curvature plaque were acquired using an E600 Epifluorescence Microscope using a filter set with excitation of 465 to 495 nm and emission of 515 to 555 nm, and a DS-Fi1 camera with NIS-Elements software (all from Nikon), with exposure time set to 2 seconds and gain set to 9.60. All sections were imaged under the same conditions. Samples were ranked for gelatinase activity on a scale of 1 to 4.Statistical AnalysisAll animal experiments were performed with the experimenter blinded to the genotype of the mice (J.L., E.A., G.H. and M.P.B.). Data were analyzed using Student's t-test (comparing two groups) or analysis of variance (comparing multiple groups). After analysis of variance, Student-Newman-Keuls post hoc tests were used to determine statistically significant differences between groups, with a significance level of P ≤ 0.05. For data that did not fit a normal distribution and ranked data, the nonparametric U-test was used to compare the means between two groups.ResultsVessel Wall Thickening and Outward Remodeling Is Reduced in Col8−/− Mice after Femoral Artery InjuryIn previous studies, we and others have shown that type VIII collagen expression was up-regulated after endothelial denuding injury2Bendeck M.P. Regenass S. Tom W.D. Giachelli C.M. Schwartz S.M. Hart C. Reidy M.A. Differential expression of alpha 1 type VIII collagen in injured platelet-derived growth factor-BB–stimulated rat carotid arteries.Circ Res. 1996; 79: 524-531Crossref PubMed Scopus (57) Google Scholar, 3Sibinga N.E. Foster L.C. Hsieh C.M. Perrella M.A. Lee W.S. Endege W.O. Sage E.H. Lee M.E. Haber E. Collagen VIII is expressed by vascular smooth muscle cells in response to vascular injury.Circ Res. 1997; 80: 532-541Crossref PubMed Scopus (77) Google Scholar, 6Plenz G. Dorszewski A. Breithardt G. Robenek H. Expression of type VIII collagen after cholesterol diet and injury in the rabbit model of atherosclerosis.Arterioscler Thromb Vasc Biol. 1999; 19: 1201-1209Crossref PubMed Scopus (24) Google Scholar; however, these studies merely described a correlation and did not address the functions of type VIII collagen in the injured vessel. To investigate, we used a wire to denude the endothelium in the femoral arteries of Col8+/+ and Col8−/− mice. In the absence of injury, there were no significant differences in medial SMC number, medial area, or vessel diameter between Col8+/+and Col8−/− mice (Table 1). The response to arterial injury involves thickening of both intimal and medial layers and outard remodeling of vessel diameter.20Sata M. Maejima Y. Adachi F. Fukino K. Saiura A. Sugiura S. Aoyagi T. Imai Y. Kurihara H. Kimura K. Omata M. Makuuchi M. Hirata Y. Nagai R. A mouse model of vascular injury that induces rapid
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