Remodeling of the Vessel Wall after Copper-Induced Injury Is Highly Attenuated in Mice with a Total Deficiency of Plasminogen Activator Inhibitor-1
2001; Elsevier BV; Volume: 158; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)63949-1
ISSN1525-2191
AutoresVictoria A. Ploplis, Ivo Cornelissen, Mayra J. Sandoval-Cooper, Lisa Weeks, Francisco Noria, Francis Castellino,
Tópico(s)Lipoproteins and Cardiovascular Health
ResumoClinical studies have indicated that high plasma levels of fibrinogen, or decreased fibrinolytic potential, are conducive to an increased risk of cardiovascular disease. Other investigations have shown that insoluble fibrin promotes atherosclerotic lesion formation by affecting smooth muscle cell proliferation, collagen deposition, and cholesterol accumulation. To directly assess the physiological impact of an imbalanced fibrinolytic system on both early and late stages of this disease, mice deficient for plasminogen activator inhibitor-1 (PAI-1−/−) were used in a model of vascular injury/repair, and the resulting phenotype compared to that of wild-type (WT) mice. A copper-induced arterial injury was found to generate a lesion with characteristics similar to many of the clinical features of atherosclerosis. Fibrin deposition in the injured arterial wall at early (7 days) and late (21 days) times after copper cuff placement was prevalent in WT mice, but was greatly diminished in PAI-1−/− mice. A multilayered neointima with enhanced collagen deposition was evident at day 21 in WT mice. In contrast, only diffuse fibrin was identified in the adventitial compartments of arteries from PAI-1−/− mice, with no evidence of a neointima. Neovascularization was observed in the adventitia and was more extensive in WT arteries, relative to PAI-1−/− arteries. Additionally, enhanced PAI-1 expression and fat deposition were seen only in the arterial walls of WT mice. The results of this study emphasize the involvement of the fibrinolytic system in vascular repair processes after injury and indicate that alterations in the fibrinolytic balance in the vessel wall have a profound effect on the development and progression of vascular lesion formation. Clinical studies have indicated that high plasma levels of fibrinogen, or decreased fibrinolytic potential, are conducive to an increased risk of cardiovascular disease. Other investigations have shown that insoluble fibrin promotes atherosclerotic lesion formation by affecting smooth muscle cell proliferation, collagen deposition, and cholesterol accumulation. To directly assess the physiological impact of an imbalanced fibrinolytic system on both early and late stages of this disease, mice deficient for plasminogen activator inhibitor-1 (PAI-1−/−) were used in a model of vascular injury/repair, and the resulting phenotype compared to that of wild-type (WT) mice. A copper-induced arterial injury was found to generate a lesion with characteristics similar to many of the clinical features of atherosclerosis. Fibrin deposition in the injured arterial wall at early (7 days) and late (21 days) times after copper cuff placement was prevalent in WT mice, but was greatly diminished in PAI-1−/− mice. A multilayered neointima with enhanced collagen deposition was evident at day 21 in WT mice. In contrast, only diffuse fibrin was identified in the adventitial compartments of arteries from PAI-1−/− mice, with no evidence of a neointima. Neovascularization was observed in the adventitia and was more extensive in WT arteries, relative to PAI-1−/− arteries. Additionally, enhanced PAI-1 expression and fat deposition were seen only in the arterial walls of WT mice. The results of this study emphasize the involvement of the fibrinolytic system in vascular repair processes after injury and indicate that alterations in the fibrinolytic balance in the vessel wall have a profound effect on the development and progression of vascular lesion formation. Among the critical proteins that constitute the fibrinolytic system in mammals are the zymogen, plasminogen, and its activated product, plasmin, a serine protease; plasminogen activators, eg, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator, which are also serine proteases; receptors for these proteins, eg, the uPA receptor (uPAR); serpin-type inhibitors, eg, plasminogen activator inhibitor-1 (PAI-1); and fibrinogen/fibrin. In addition to the clot-dissolving capacity of this system, a number of in vitro and in vivo studies have implicated plasmin as playing an important role in proteolytic processes associated with cell migration, which is a pivotal event in the inflammatory response. These effects have been attributed to the ability of components of the fibrinolytic system to assemble on cell surfaces through interaction with specific receptors, such as α-enolase for plasminogen1Miles LA Dahlberg CM Plescia J Felez J Kato K Plow EF Role of cell-surface lysines in plasminogen binding to cells: identification of α-enolase as a candidate plasminogen receptor.Biochemistry. 1991; 30: 1682-1691Crossref PubMed Scopus (488) Google Scholar, 2Hamanoue M Takemoto N Hattori T Kato K Kohsaka S Plasminogen binds specifically to alpha-enolase on rat neuronal plasma membrane.J Neurochem. 1994; 63: 2048-2057PubMed Google Scholar, 3Arza B Felez J Lopez-Alemany R Miles LA Munoz-Canoves P Identification of an epitope of alpha-enolase (a candidate plasminogen receptor) by phage display.Thromb Haemost. 1997; 78: 1097-1103PubMed Google Scholar and uPAR for uPA.4Dana K Behrendt N Brünner N Ellis V Ploug M Pyke C The urokinase receptor. Protein structure and role in plasminogen activation and cancer invasion.Fibrinolysis. 1994; 8: 189-203Crossref Scopus (295) Google Scholar These functions are supported by in vitro studies that have identified uPA and uPAR at the leading migratory edges of monocytes and smooth muscle cells.5Estreicher A Muhlhauser J Carpentier JL Orci L Vassalli JD The receptor for urokinase type plasminogen activator polarizes expression of the protease to the leading edge of migrating monocytes and promotes degradation of enzyme inhibitor complexes.J Cell Biol. 1995; 111: 783-792Crossref Scopus (410) Google Scholar, 6Okada SS Grobmyer SR Barnathan ES Contrasting effects of plasminogen activators, urokinase receptor, and LDL receptor-related protein on smooth muscle cell migration and invasion.Arterioscler Thromb Vasc Biol. 1996; 16: 1269-1276Crossref PubMed Scopus (90) Google Scholar In addition to the involvement of components of the fibrinolytic system in facilitating cell migration through matrix-degrading processes,7Kenagy RD Vergel S Mattsson E Bendeck M Reidy MA Clowes AW The role of plasminogen, plasminogen activators, and matrix metalloproteinases in primate arterial smooth muscle cell migration.Arterioscler Thromb Vasc Biol. 1996; 16: 1373-1382Crossref PubMed Scopus (95) Google Scholar, 8Seeds NW Friedman G Hayden S Thewke D Haffke S McGuire P Krystosek A Plasminogen activators and their interaction with the extracellular matrix in neural development, plasticity and regeneration.Semin Neurosci. 1996; 8: 405-412Crossref Scopus (24) Google Scholar, 9Reiter LS Spertini O Kruithof EKO Plasminogen activators play an essential role in extracellular-matrix invasion by lymphoblastic T cells.Int J Cancer. 1997; 70: 461-466Crossref PubMed Scopus (15) Google Scholar these agents have been shown to influence normal and pathological cell migratory events by release and activation of a number of inflammatory mediators,10Matsushima K Taguchi M Kovacs EJ Young HA Oppenheim JJ Intracellular localization of human monocyte associated interleukin 1 (IL 1) activity and release of biologically active IL 1 from monocytes by trypsin and plasmin.J Immunol. 1986; 136: 2883-2891PubMed Google Scholar, 11Lyons RM Gentry LE Purchio AF Moses HL Mechanism of activation of latent recombinant transforming growth factor beta 1 by plasmin.J Cell Biol. 1990; 110: 1361-1367Crossref PubMed Scopus (672) Google Scholar, 12Sitrin RG Shollenberger SB Strieter RM Gyetko MR Endogenously produced urokinase amplifies tumor necrosis factor-alpha secretion by THP-1 mononuclear phagocytes.J Leukoc Biol. 1996; 59: 302-311PubMed Google Scholar as well as by chemotactic processes,13Stepanova V Bobik A Bibilashvily R Belogurov A Rybalkin I Domogatsky S Little PJ Goncharova E Tkachuk V Urokinase plasminogen activator induces smooth muscle cell migration: key role of growth factor-like domain.FEBS Lett. 1997; 414: 471-474Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 14Blasi F The urokinase receptor. A cell surface, regulated chemokine.APMIS. 1999; 107: 96-101Crossref PubMed Scopus (84) Google Scholar, 15Poliakov AA Mukhina SA Traktouev DO Bibilashvily RS Gursky YG Minashkin MM Stepanova VV Tkachuk VA Chemotactic effect of urokinase plasminogen activator: a major role for mechanisms independent of its proteolytic or growth factor domains.J Recept Signal Transduct Res. 1999; 19: 939-951Crossref PubMed Scopus (26) Google Scholar integrin-mediated signaling,16Yebra M Goretzki L Pfeifer M Mueller BM Urokinase-type plasminogen activator binding to its receptor stimulates tumor cell migration by enhancing integrin-mediated signal transduction.Exp Cell Res. 1999; 250: 231-240Crossref PubMed Scopus (106) Google Scholar and other potentially novel mechanisms.17Kanse SM Benzakour O Kanthou C Kost C Lijnen HR Preissner KT Induction of vascular SMC proliferation by urokinase indicates a novel mechanism of action in vasoproliferative disorders.Arterioscler Thromb Vasc Biol. 1997; 17: 2848-2854Crossref PubMed Scopus (77) Google Scholar Plasmin has also been shown to act directly as a chemoattractant for human peripheral monocytes.18Syrovets T Tippler B Rieks M Simmet T Plasmin is a potent and specific chemoattractant for human peripheral monocytes acting via a cyclic guanosine monophosphate-dependent pathway.Blood. 1997; 89: 4574-4583Crossref PubMed Google Scholar Alternatively, PAI-1 has also been implicated in cell migration through its ability to disrupt uPA/uPAR-matrix protein interactions, thus facilitating cell detachment.19Deng G Curriden SA Wang SJ Rosenberg S Loskutoff DJ Is plasminogen activator inhibitor-1 the molecular switch that governs urokinase receptor-mediated cell adhesion and release?.J Cell Biol. 1996; 134: 1563-1571Crossref PubMed Scopus (432) Google Scholar Indeed, macrophage colony-stimulating factor (or CSF-1) and granulocyte-macrophage colony-stimulating factor have been shown to increase both PAI-1 and PAI-2 expression in human monocytes,20Hamilton JA Whitty GA Wojta J Gallichio M McGrath K Ianches G Regulation of plasminogen activator inhibitor-1 levels in human monocytes.Cell Immunol. 1993; 151: 7-17Crossref Scopus (34) Google Scholar potentially implicating these proteins in processes involved in inflammation and tissue remodeling.21Hamilton JA Whitty GA Stanton H Wojta J Gallichio M McGrath K Ianches G Macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor stimulate the synthesis of plasminogen-activator inhibitors by human monocytes.Blood. 1993; 82: 3616-3621PubMed Google Scholar Atherosclerosis is a chronic inflammatory disease in which the fibrinolytic system plays a major role. For example, several studies have indicated that high plasma levels of fibrinogen and decreased fibrinolytic activity, ie, increased PAI-1, lead to an increased risk for cardiovascular disease.22Meade TW Ruddock V Stirling Y Chakrabarti R Miller GJ Fibrinolytic activity, clotting factors, and long-term incidence of ischaemic heart disease in the Northwick Park Heart Study.Lancet. 1993; 342: 1076-1079Abstract PubMed Scopus (735) Google Scholar, 23Gensini GF Comeglio M Colella A Classical risk factors and emerging elements in the risk profile for coronary heart disease.Eur Heart J. 1998; 19: A53-A61PubMed Google Scholar Additionally, components of the fibrinolytic system have been identified in atherosclerotic lesion tissue.24Falkenberg M Tjarnstrom J Ortenwall P Olausson M Risberg B Localization of fibrinolytic activators and inhibitors in normal and atherosclerotic vessels.Thromb Haemost. 1996; 75: 933-938PubMed Google Scholar, 25Robbie LA Booth NA Brown AJ Inhibitors of fibrinolysis are elevated in atherosclerotic plaque.Arterioscler Thromb Vasc Biol. 1996; 16: 539-545Crossref PubMed Scopus (125) Google Scholar Other studies have indicated that insoluble fibrin may promote atherosclerotic lesion formation by affecting smooth muscle cell proliferation and migration, collagen deposition, and cholesterol accumulation.26Nomura H Naito M Iguchi A Thompson WD Smith EB Fibrin gel induces the migration of smooth muscle cells from rabbit aortic explants.Thromb Haemost. 1999; 82: 1347-1352PubMed Google Scholar The generation of mice deficient for components of the fibrinolytic system has resulted in the development of valuable resources for directly assessing the physiological impact of an imbalanced fibrinolytic system on both early and late stages of a number of inflammation-based diseases. Diminished inflammatory responses have been identified in uPA-deficient (UPA−/−) and plasminogen-deficient (PG−/−) mice challenged with a number of different agents.27Gyetko MR Chen GH McDonald RA Goodman R Huffnagle GB Wilkinson CC Fuller JA Toews GB Urokinase is required for the pulmonary inflammatory response to Cryptococcus neoformans. A murine transgenic model.J Clin Invest. 1996; 97: 1818-1826Crossref PubMed Scopus (168) Google Scholar, 28Moons L Shi C Ploplis V Plow E Haber E Collen D Carmeliet P Reduced transplant arteriosclerosis in plasminogen-deficient mice.J Clin Invest. 1998; 102: 1788-1797Crossref PubMed Scopus (76) Google Scholar, 29Ploplis VA French EL Carmeliet P Collen D Plow EF Plasminogen deficiency differentially affects recruitment of inflammatory cell populations in mice.Blood. 1998; 91: 2005-2009Crossref PubMed Google Scholar Murine models for vascular injury/repair are extremely valuable for the study of early and late stage inflammatory disease because the role of genetic factors in inflammation can be investigated effectively using gene-targeted animals. In the current study, a copper-induced model of inflammation has been characterized in WT mice, and applied to mice deficient in the PAI-1 gene (PAI-1−/−). This model is based on the finding that increased plasma levels of copper have been associated with cardiovascular disease.30Reunanen A Knekt P Marniemi J Maki J Maatela J Aromaa A Serum calcium, magnesium, copper and zinc and risk of cardiovascular death.Eur J Clin Nutr. 1996; 50: 431-437PubMed Google Scholar, 31Marniemi J Jarvisalo J Toikka T Raiha I Ahotupa M Sourander L Blood vitamins, mineral elements and inflammation markers as risk factors of vascular and non-vascular disease mortality in an elderly population.Int J Epidemiol. 1998; 27: 799-807Crossref PubMed Scopus (60) Google Scholar The results of this study are presented herein. Prosthetic silicone elastomer, MDX4-4210, (Factor II, Inc., Lakeside, AZ) and copper powder, spherical, −100 + 325 mesh, (Alfa Aesar, Ward Hill, MA) were used to construct the cuffs, which have an inside diameter of 0.028" and an outside diameter of 0.062". The construction was a major modification of a similar cuff used in rats.32Volker W Dorszewski A Unruh V Robenek H Breithardt G Buddecke E Copper-induced inflammatory reactions of rat carotid arteries mimic restenosis/arteriosclerosis-like neointima formation.Atherosclerosis. 1997; 130: 29-36Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar The two-part silicone product was combined with the copper dust and then degassed under vacuum. Stainless steel molds, which mimic the size of the diameter of a mouse carotid artery, were used in the construction. The copper/silicone mixture was spread over both halves of the mold, after which stainless steel rods (0.028" outside diameter/22 gauge) were placed in each of the 15 slots of the mold (0.042" outside diameter of the complete mold slot). The mold halves were combined, pressed closed, and baked at 80°C for ∼8 hours. The copper/silicone-coated rods were then inserted in one-half of a larger mold (0.062" outside diameter of the complete mold slot), coated with silicone alone, pressed closed with the other one-half of the similarly coated mold, and then baked as described above. Male and female C57 BL/6J wild-type (WT) mice (8 to12 weeks of age) and mice totally deficient for the PAI-1 gene (PAI-1−/−) were used in this study. All animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and experimental protocols were approved by the Institutional Animal Care and Use Committee. The left carotid artery of mice anesthetized with an intraperitoneal injection of rodent cocktail (0.015 mg xylazine/0.075 mg ketamine/0.0025 mg aceprozamine/g weight of animal) was surgically exposed via a midline incision over an area from the chin to the sternum that had been sterilized with a 1% iodine solution. The salivary gland was separated and the artery dissected proximal to the bifurcation. A copper/silicone cuff, 1 to 1.5 mm in length, was placed around the periphery of the artery proximal to the bifurcation. The surgical site was then closed with a 6-0 nylon suture and the mice allowed to recover. At 7- and 21-days after implantation, the mice were again anesthetized and the left carotid artery re-exposed to remove the cuffed artery. The contralateral right artery served as a negative control. Arteries were either fixed in 10% neutral-buffered formalin for processing and paraffin embedding or immersed in 20% sucrose and then frozen in Tissue-Tek OCT (Sakura Fine Tek Co., Torrance, CA) compound for cryosectioning. Paraffin-embedded arteries were sectioned between 3 μm and 4 μm. Hematoxylin 2 and eosin Y (H&E; Richard Allen Scientific, Kalamazoo, MI) stains were performed to assess cellular morphology. Masson's trichrome stain33Masson P Trichrome stainings and their preliminary technique.J Tech Methods. 1929; 12: 75-90Google Scholar was used to identify collagen in the arterial wall and Verhoeff's Van Gieson staining34Sheehan D Hrapchak B Connective tissue and muscle fiber stains.in: Theory and Practice of Histotechnology. Battelle Press, Columbus1987: 196-197Google Scholar was used to identify elastica laminae and to perform morphometric measurements. Cryotomy sections (8 μm) were used for Oil Red O staining to identify fat deposits in the injured arteries.35Lillie RD Ashburn LL Super-saturated solutions of fat stains in dilute isopropanol for demonstration of acute fatty degenerations not shown by Herxheimer technique.Arch Pathol. 1943; 36: 432Google Scholar A polyclonal goat anti-mouse fibrin(ogen) antibody (Accurate Chemicals, Westbury, NY) was used for immunohistochemical identification of fibrin. The slides were incubated with rabbit serum and then with the anti-fibrin antibody, followed by secondary rabbit anti-goat IgG (DAKO, Carpinteria, CA) and goat peroxidase anti-peroxidase (DAKO). Peroxidase activity was detected with the substrate, 3-amino,9-ethylcarbazole (AEC) (Biomeda, Foster City, CA). A hematoxylin counterstain (Biomeda) was used for all immunohistochemistry. Antigen retrieval was performed under high temperature and pressure with citrate buffer, pH 6.0 (Zymed, South San Francisco, CA), followed by endogenous peroxidase blocking with Peroxoblock (Zymed). CD45 rat anti-mouse monoclonal antibody (Pharmingen, San Diego, CA) was used to identify leukocytes. The secondary antibody consisted of a biotin-conjugated goat anti-rat antibody (DAKO), which was followed by a solution of streptavidin-conjugated horseradish peroxidase (Biogenex). Detection with AEC, antigen retrieval with citrate buffer, and blocking of endogenous peroxidase were as above. A similar procedure was used for detection of smooth muscle cells with an anti-α-actin monoclonal antibody (Sigma Chemical Co., St. Louis, MO), except that antigen retrieval was accomplished with a limited trypsin treatment. Immunohistochemical identification of von Willebrand factor (vWF)-positive cell types (endothelial cells, megakaryocytes, and platelets) was accomplished using an EPOS anti-human vWF antibody conjugated to peroxidase (DAKO). Development was accomplished with AEC. Antigen retrieval was performed with limited trypsin digestion. Blocking of endogenous peroxidase was as above. PAI-1 expression in the vessel wall was evaluated by immunohistochemistry using a rabbit anti-murine PAI-1 polyclonal antibody (Molecular Innovations, Inc., Royal Oak, MI). The second antibody was biotin-conjugated porcine anti-rabbit IgG (DAKO), after which streptavidin-conjugated horseradish peroxidase (Biogenex) was added. Antigen retrieval with citrate buffer, detection with AEC, and blocking of endogenous peroxidase were as above. Mice were injected intraperitoneally with 50 mg/kg bromodeoxyuridine (BrdU) in physiological saline at times of 24, 16, and 1 hour before sacrificing. A mouse monoclonal anti-human antibody to BrdU was used to identify proliferating cells in the vascular wall. The secondary antibody consisted of a biotin-conjugated rabbit anti-mouse IgG antibody (DAKO), after which was added streptavidin-conjugated horseradish peroxidase, followed by development with AEC. Antigen retrieval was accomplished by incubation in 1 mol/L HCl at 37°C for 10 minutes followed by limited trypsin digestion. Endogenous peroxidase activity was blocked with Peroxoblock. Total cells in the vascular wall compartments were counted and the percentage of BrdU-positive cells was determined for equally spaced sections within the cuffed area of the artery. Five sections per artery, separated by ∼50 μm/section, of 200 to 300 μm length of injured artery were analyzed. The average values per artery for each genotype were used to determine the mean ±SEM. Morphometric measurements of cross-sectional areas were performed on transverse sections of the artery using a computer-assisted image analysis system (Bioquant True Color Windows software; Biometrics, Nashville, TN) on equally spaced sections within the cuffed area of the artery. The number of sections and length of injured artery analyzed were similar to that described for cell proliferation analyses. The average values per artery for each genotype were used to determine the mean ±SEM. Neovascularization was determined by counting the number of vWF-positive vessels in the adventitial compartment of injured arteries and expressed as the number of vessels per mm2area. Counts were made over equally spaced sections (4 to 5 fields/vessel) within the cuffed area of the artery and average values per artery for each genotype were used to determine the mean ±SEM. Ultrastructural analyses were performed on 3-day injured arteries. The arteries were perfused and fixed with Karnovsky solution36Hopwood D "Fixative" In Electron Microscopy in Biology: A Practical Approach.in: Harris JR Oxford University Press, Oxford1991: 7Google Scholar and postfixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol solutions, and embedded in epoxy resins (Polysciences, Warrington, PA). Ultrathin sections (90 nm) were cut and stained in 2% uranyl acetate and Reynolds lead stain.37Hincherick FR Transmission electron microscopy.in: Prophet EB Mills B Arrington JB Sobin LH Laboratory Methods in Histotechnology. American Registry of Pathology, Washington DC1994: 262-263Google Scholar Sections were viewed and photographed using a transmission electron microscope (Hitachi H 600; Hitachi, Tokyo, Japan) at 75 kV accelerating voltage. Where appropriate, values were expressed as mean ±SEM. Comparisons were made using Student's t-test and P values <0.05 were considered significant. H&E stains of WT carotid arteries 7 days after injury demonstrated an increase in cellularity and reactivity in medial and adventitial compartments relative to uninjured arteries, with evidence of a small neointima (Figure 1A). Intimal, medial, and adventitial compartments were significantly increased in size in arteries from WT mice relative to arteries from PAI-1−/− mice (0.011 ± 0.001 mm2 versus 0.004 ± 0.0004 mm2, P < 0.001, respectively, for the intimal compartment; 0.046 ± 0.004 mm2 versus 0.020 ± 0.001 mm2, P = 0.003, respectively, for the medial compartment; and 0.108 ± 0.009 mm2 versus 0.075 ± 0.008 mm2, P = 0.021, respectively, for the adventitial compartment; Table 1). Despite the smaller adventitia in arteries from PAI-1−/− mice relative to WT mice, the proliferative index, as measured by BrdU uptake, was significantly increased in the adventitia in arteries from PAI-1−/− mice compared to arteries from WT mice (16.7 ± 3.3% versus 3.5 ± 1.9%, P = 0.0269, respectively; Table 2). Medial compartments in arteries from both WT and PAI-1−/− mice exhibited a smooth muscle cell-enriched region (Figure 1, C and D). The neointima in WT-injured arteries consisted primarily of leukocytes (Figure 1, E and F). Additionally, fibrin deposits were significantly enhanced in the medial and adventitial compartments (Figure 1G). This is in sharp contrast to that observed in injured arteries from PAI-1−/− mice, wherein no fibrin was observed in the vessel wall (Figure 1H).Table 1Morphometric Analyses of Carotid Arteries from WT and PAI-1−/− MiceTime pointLumenIntimaMediaAdventitiaDAY 7 (mm2)*P value comparisons between days 7 and 21, within each genotype, indicated that statistically significant differences (P < 0.05) were observed in the intimal and medial compartments in WT mice. Luminal and adventitial compartments were not significantly altered. In PAI-1−/−mice significant differences were observed in the lumen, medial, and adventitial but not in the intimal compartment of the vessel. WT (n = 6)0.057 ± 0.0060.011 ± 0.0010.046 ± 0.0040.108 ± 0.009 PAI-1−/−(n = 6)0.059 ± 0.0080.004 ± 0.00040.020 ± 0.0010.075 ± 0.008 P values0.846<0.0010.0030.021DAY 21 (mm2)*P value comparisons between days 7 and 21, within each genotype, indicated that statistically significant differences (P < 0.05) were observed in the intimal and medial compartments in WT mice. Luminal and adventitial compartments were not significantly altered. In PAI-1−/−mice significant differences were observed in the lumen, medial, and adventitial but not in the intimal compartment of the vessel. WT (n = 4)0.042 ± 0.0130.036 ± 0.0050.063 ± 0.0060.087 ± 0.009 PAI-1−/− (n = 8)0.023 ± 0.0050.007 ± 0.0020.024 ± 0.0040.182 ± 0.020 P values0.125<0.00010.00030.0093* P value comparisons between days 7 and 21, within each genotype, indicated that statistically significant differences (P < 0.05) were observed in the intimal and medial compartments in WT mice. Luminal and adventitial compartments were not significantly altered. In PAI-1−/−mice significant differences were observed in the lumen, medial, and adventitial but not in the intimal compartment of the vessel. Open table in a new tab Table 2Percent BrdU(+) Cells in Vessel Wall in WT and PAI-1−/−ArteriesTime pointIntimaMediaAdventitiaDAY 7 WT (n = 3)18.5± 7.56.3± 2.13.5± 1.9 PAI-1−/−(n = 3)10.1± 4.15.4± 2.216.7± 3.3 P valueN.S.N.S.0.0269DAY 21 WT (n = 3)5.9± 2.310.1± 3.910.6± 3.3 PAI-1−/− (n = 3)3.7± 0.92.3± 0.314.4± 3.8 P valueN.S.0.1168N.S. Open table in a new tab H&E stains of carotid arteries from WT mice 21 days after injury revealed an enlarged multilayered neointima (0.036 ± 0.005 mm2 for day 21 versus 0.011 ± 0.001 mm2 for day 7, P = 0.0003; Figure 2A and Table 1), consisting of smooth muscle cells that appeared to be primarily confined to the luminal edges of the neointima (Figure 2C). These arteries also presented enhanced collagen deposition (Figure 2E). WT neointima was also significantly larger than PAI-1−/−neointima at day 21 (0.036 ± 0.005 mm2versus 0.007 ± 0.002 mm 2, P < 0.0001, respectively; Table 1). Cell proliferation in the medial compartment of arteries from WT mice compared to PAI-1−/− arteries were also enhanced (10.1 ± 3.9% versus 2.3 ± 0.3%, P = 0.1168, respectively; Table 2). This is consistent with a lack of neointima formation in arteries from PAI-1−/−mice, which is derived primarily from proliferating smooth muscle cells from the medial compartment (Figure 2, B, D, and F). Although there did not seem to be any degradation of the elastica laminae in injured arteries from WT mice, as visualized by Verhoeff's Van Gieson staining, stretching and thinning were evident (Figure 3A). In contrast, the elastica laminae appeared unaffected in injured arteries from PAI-1−/− mice (Figure 3B). Immunostaining of vWF in carotid arteries from both WT and PAI-1−/− mice indicated the presence of a normal intact contiguous single-layer intima of endothelial cells adjacent to the lumen of the vessel (Figure 3, C and D). Fibrin deposits in the injured WT arteries remained unresolved, primarily in the adventitial compartment (Figure 4A), most likely because of continued injury to the vessel wall by copper. Injured arteries from PAI-1−/− mice demonstrated little, if any, fibrin in the vessel wall (Figure 4B). Enhanced PAI-1 expression was also evident in carotid arteries from WT mice (Figure 4C) in the same vascular compartments that fibrin deposits were found, but not in the uninjured contralateral artery (Figure 4D). Fat deposition in the arterial wall, most likely because of copper ion-induced oxidation of low-density lipoprotein (LDL), was evident in arteries from WT mice, primarily confined to the medial layer adjacent to the elastic lamina (Figure 4E), but not in arteries from PAI-1−/− mice (Figure 4F). At day 21, neovascularization in the adventitia was more evident in injured arteries from WT mice than in PAI-1−/− mice (159 ± 6/mm2 versus 105 ± 8/mm2 vessels, P = 0.0057; Figure 5). Neovascularization of the adventitial compartment in PAI-1−/− arteries was relatively unchanged in day 21 arteries compared to those analyzed at day 7 (105 ± 8/mm2 versus 106 ± 5/mm2, respectively).Figure 3Histological analysis of carotid arteries from WT and PAI-1−/− mice 21 days after implantation of the copper cuff. Verhoeff's Van Gieson stain (black) of WT carotid artery
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