Mouse Model of Venous Bypass Graft Arteriosclerosis

Saphenous vein grafts are widely used for treatment of severe atherosclerosis via aortocoronary bypass surgery, a procedure often complicated by later occlusion of the graft vessel. Because the molecular mechanisms of this process remain largely unknown, quantitative models of venous bypass graft arteriosclerosis in transgenic mice could be useful to study this process at the genetic level. We describe herein a new model of vein grafts in the mouse that allows us to take advantage of transgenic, knockout, or mutant animals. Autologous or isogeneic vessels of the external jugular or vena cava veins were end-to-end grafted into carotid arteries of C57BL/6J mice. Vessel wall thickening was observed as early as 1 week after surgery and progressed to 4-, 10-, 15-, and 18-fold original thickness in grafted veins at age 2, 4, 8, and 16 weeks, respectively. The lumen of grafted veins was significantly narrowed because of neointima hyperplasia. Histological and immunohistochemical analyses revealed three lesion processes: marked loss of smooth muscle cells in vein segments 1 and 2 weeks after grafting, massive infiltration of mononuclear cells (CD11b/18+) in the vessel wall between 2 and 4 weeks, and a significant proliferation of vascular smooth muscle cells (α-actin+) to constitute neointimal lesions between 4 and 16 weeks. Similar vein graft lesions were obtained when external jugular veins or vena cava were isografted into carotid arteries of C57BL/6J mice. Moreover, no significant intima hyperplasia in vein-to-vein isografts was found, although there was leukocyte infiltration in the vessel wall. Thus, this model, which reproduces many of the features of human vein graft arteriosclerosis, should prove useful for our understanding of the mechanism of vein graft disease and to evaluate the effects of drugs and gene therapy on vascular diseases. Saphenous vein grafts are widely used for treatment of severe atherosclerosis via aortocoronary bypass surgery, a procedure often complicated by later occlusion of the graft vessel. Because the molecular mechanisms of this process remain largely unknown, quantitative models of venous bypass graft arteriosclerosis in transgenic mice could be useful to study this process at the genetic level. We describe herein a new model of vein grafts in the mouse that allows us to take advantage of transgenic, knockout, or mutant animals. Autologous or isogeneic vessels of the external jugular or vena cava veins were end-to-end grafted into carotid arteries of C57BL/6J mice. Vessel wall thickening was observed as early as 1 week after surgery and progressed to 4-, 10-, 15-, and 18-fold original thickness in grafted veins at age 2, 4, 8, and 16 weeks, respectively. The lumen of grafted veins was significantly narrowed because of neointima hyperplasia. Histological and immunohistochemical analyses revealed three lesion processes: marked loss of smooth muscle cells in vein segments 1 and 2 weeks after grafting, massive infiltration of mononuclear cells (CD11b/18+) in the vessel wall between 2 and 4 weeks, and a significant proliferation of vascular smooth muscle cells (α-actin+) to constitute neointimal lesions between 4 and 16 weeks. Similar vein graft lesions were obtained when external jugular veins or vena cava were isografted into carotid arteries of C57BL/6J mice. Moreover, no significant intima hyperplasia in vein-to-vein isografts was found, although there was leukocyte infiltration in the vessel wall. Thus, this model, which reproduces many of the features of human vein graft arteriosclerosis, should prove useful for our understanding of the mechanism of vein graft disease and to evaluate the effects of drugs and gene therapy on vascular diseases. Autologous vein grafts remain the only surgical alternative for many types of vascular reconstruction, but the failure rate of these grafts after 1 year approaches 20%.1Dalman RL Taylor LM Infrainguinal revascularization procedures.in: Porter JM Taylor LM Basic Data Underlying Clinical Decision Making in Vascular Surgery. Quality Medical Publishers, St Louis1994: 141-143Google Scholar The development of neointima or arteriosclerosis in the grafted vessel often leads to obliterative stenosis. The hallmarks of neointima lesions are mononuclear cell infiltration, smooth muscle cell proliferation, and extracellular matrix deposition.2Davies MG Hagen P-O Pathobiology of intimal hyperplasia.Br J Surg. 1994; 81: 1254-1269Crossref PubMed Scopus (382) Google Scholar The pathogenesis of this disease remains poorly understood, and no successful clinical interventions have been identified. Several animal models manifesting lesions resembling human vein graft arteriosclerosis have been developed3Angelini GD Bryan AJ Williams HMJ Morgan R Newby AC Distention promotes platelet and leukocyte adhesion and reduces short-term patency in pig arteriovenous bypass grafts.J Thorac Cardiovasc Surg. 1990; 99: 433-439PubMed Google Scholar, 4Davies MG Klyachkin ML Dalen H Massey MF Svendsen E Hagen PO The integrity of experimental vein graft endothelium: implications on the etiology of early vein graft failure.Eur J Vasc Surg. 1993; 7: 156-165Abstract Full Text PDF PubMed Scopus (56) Google Scholar, 5Boerboom LE Olinger GN Liu TZ Rodriguez ER Ferrans VJ Kissebah AH Histologic, morphometric, and biochemical evaluation of vein bypass grafts in a nonhuman primate model. I. Sequential changes within the first three months.J Thorac Cardiovasc Surg. 1990; 99: 97-106PubMed Google Scholar, 6Landymore RW Kinley CE Cameron CA Intimal hyperplasia in autogenous vein grafts used for arterial bypass: a canine model.Cardiovasc Res. 1985; 19: 589-592Crossref PubMed Scopus (20) Google Scholar, 7Norman PE House AK The influence of nifedipine on microvascular vein graft intimal thickening.Aust N Z J Surg. 1993; 63: 294-298Crossref PubMed Scopus (6) Google Scholar and have helped address specific interventional issues, but they have not helped to clarify the underlying mechanism of the disease. Attracted by the well-defined genetic systems, a number of investigators have begun to use the mouse as an experimental system for atherosclerosis research.8O'Neill TP Apolipoprotein E-deficient mouse model of human atherosclerosis.Toxicol Pathol. 1997; 25: 20-21Crossref PubMed Scopus (16) Google Scholar, 9Paigen B Morrow A Brandon C Mitchell D Holmes P Variation in susceptibility to atherosclerosis among inbred strains of mice.Atherosclerosis. 1985; 57: 65-73Abstract Full Text PDF PubMed Scopus (497) Google Scholar, 10Zhang SH Reddick RL Burkey B Maeda N Diet-induced atherosclerosis in mice heterozygous and homozygous for apolipoprotein E gene disruption.J Clin Invest. 1994; 94: 937-945Crossref PubMed Scopus (210) Google Scholar, 11Fyfe AI Qiao J Lusis AL Immune-deficient mice develop typical atherosclerotic fatty streaks when fed an atherogenic diet.J Clin Invest. 1994; 94: 2516-2520Crossref PubMed Scopus (138) Google Scholar Hundreds of inbred lines have been established, the genetic map is relatively well defined, and both congenic strains and recombinant strains are available to facilitate genetic experimentation. In just a few years, murine lipoproteins have been characterized, genetic variants of apolipoproteins have been identified,12Lusis AJ Taylor BA Wangenstein RW LeBoruf RC Genetic control of lipid transport.J Biol Chem. 1983; 258: 5071-5077Abstract Full Text PDF PubMed Google Scholar, 13Stolt J Rubin EM Atherogenesis: insights from the study of transgenic and gene-targeted mice.Trends Cardiovasc Med. 1993; 3: 130-134Abstract Full Text PDF PubMed Scopus (18) Google Scholar and genetic variation in susceptibility to atherosclerosis among inbred mouse strains has been demonstrated. The study of vein graft arteriosclerosis in such strains should make it possible to define the specific relations of many genes and cell types to the pathogenesis of this lesion. For example, it has been postulated that hypercholesterolemia is a risk factor for the development of lesions in vein grafts,14The Post-Coronary Artery Bypass Graft Trail Investigators The effect of aggressive lowering of low-density lipoprotein cholesterol levels and low-dose anticoagulation on obstructive changes in saphenous-vein coronary-artery bypass grafts.New Engl J Med. 1997; 336: 153-162Crossref PubMed Scopus (977) Google Scholar and it is now possible to study mice that lack apolipoprotein E-containing lipoproteins or low-density lipoprotein receptors.13Stolt J Rubin EM Atherogenesis: insights from the study of transgenic and gene-targeted mice.Trends Cardiovasc Med. 1993; 3: 130-134Abstract Full Text PDF PubMed Scopus (18) Google Scholar, 15Breslow JL Transgenic mouse models of lipoprotein metabolism and atherosclerosis.Proc Natl Acad Sci USA. 1993; 90: 8314-8318Crossref PubMed Scopus (114) Google Scholar Mice are also available that have deficient macrophage function16Yoshida H Hayashi S Kunisada T Ogawa M Nishikawa S Okamura H Sudo T Shultz LD Nishikawa S The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene.Nature. 1990; 345: 442-444Crossref PubMed Scopus (1543) Google Scholar or lack endothelial adhesion molecules17Tedder TF Steeber DA Pizcueta P L-selectin-deficient mice have impaired leukocyte recruitment into inflammatory sites.J Exp Med. 1995; 181: 2259-2264Crossref PubMed Scopus (399) Google Scholar, 18Sligh JE Ballantyne CM Rich SS Hawkins HK Smith CW Bradley A Beaudet AL Inflammatory and immune responses are impaired in ICAM-1 deficient mice.Proc Natl Acad Sci USA. 1993; 90: 8529-8533Crossref PubMed Scopus (525) Google Scholar or nitric oxide synthases,19Wei X-q Charles IG Smith A Ure J Feng G-j Huang F-p Xu D Muller W Moncada S Liew FY Altered immune responses in mice lacking inducible nitric oxide synthase.Nature. 1995; 375: 408-411Crossref PubMed Scopus (1159) Google Scholar, 20MacMicking JD Nathan C Hom G Chartrain N Fletcher DS Trumbauer M Stevens K Xie Q-w Sokol K Hutchinson N Chen H Mudgett JS Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase.Cell. 1995; 81: 641-650Abstract Full Text PDF PubMed Scopus (1293) Google Scholar which might be important molecules in the development of venous bypass graft arteriosclerosis. In the present study, we describe a simple model wherein external jugular or vena cava veins were auto- or isografted into carotid arteries in C57BL/6J mice. We observed intimal lesions within 14 days that progress to marked stenosis in the grafted vessel within 16 weeks. We demonstrated that inflammatory features appeared in the lesions at the early stages followed by smooth muscle cell proliferation and extracellular matrix deposition in the vein graft neointima. Three month-old male C57BL/6J were purchased from the Charles River Laboratory (Sulzfeld, Germany) and maintained for 1 week on a light/dark (12-hour/12-hour) cycle at 24°C and received food and water ad libitum before experimentation. All procedures were performed according to protocols approved by the Institutional Committee for Use and Care of Laboratory Animals. C57BL/6J mice were used as donors and recipients for vein grafts, because these mice are susceptible to atherosclerosis when a cholesterol-enriched diet is administered.8O'Neill TP Apolipoprotein E-deficient mouse model of human atherosclerosis.Toxicol Pathol. 1997; 25: 20-21Crossref PubMed Scopus (16) Google Scholar, 9Paigen B Morrow A Brandon C Mitchell D Holmes P Variation in susceptibility to atherosclerosis among inbred strains of mice.Atherosclerosis. 1985; 57: 65-73Abstract Full Text PDF PubMed Scopus (497) Google Scholar, 10Zhang SH Reddick RL Burkey B Maeda N Diet-induced atherosclerosis in mice heterozygous and homozygous for apolipoprotein E gene disruption.J Clin Invest. 1994; 94: 937-945Crossref PubMed Scopus (210) Google Scholar, 11Fyfe AI Qiao J Lusis AL Immune-deficient mice develop typical atherosclerotic fatty streaks when fed an atherogenic diet.J Clin Invest. 1994; 94: 2516-2520Crossref PubMed Scopus (138) Google Scholar, 12Lusis AJ Taylor BA Wangenstein RW LeBoruf RC Genetic control of lipid transport.J Biol Chem. 1983; 258: 5071-5077Abstract Full Text PDF PubMed Google Scholar, 13Stolt J Rubin EM Atherogenesis: insights from the study of transgenic and gene-targeted mice.Trends Cardiovasc Med. 1993; 3: 130-134Abstract Full Text PDF PubMed Scopus (18) Google Scholar In addition, many mutant or knockout mice are available with this genetic background. Mice were anesthetized with pentobarbital sodium (50 mg/kg body weight, intraperitoneally). Atropine sulfate (1.7 mg/kg body weight) was administered to maintain the respiratory tract in good condition. The operation was performed under a dissecting microscope (Wild M8, Basel, Switzerland). The mouse was fixed in a supine position with its neck extended. A midline incision was made on the ventral side of the neck from the lower mandible to the sternum. The right cleidomastoid muscle was resected. The vein-grafting procedure is schematically illustrated in Figure 1. The right common carotid artery was mobilized free from the bifurcation in the distal end toward the proximal end as far as possible. The vessel was ligated with an 8-0 silk suture and dissected between the middle ties. The proximal and distal portions of the artery were passed through cuffs made of a polyethylene cannula 0.65 mm in diameter outside and 0.5 mm inside (Portex LTD, London, United Kingdom). The cuff length was 1 mm with a 1-mm handle or extension. The vessel, together with the handle, were fixed by microhemostat clamps (4 mm in length; Martin, Tuttlingen, Germany). The suture at the end of the artery was removed, and a segment of the artery was everted over the cuff body with a stent and fine tweezers and fixed to the cuff with an 8-0 silk suture. Another portion of the artery was similarly prepared (Figure 1, a through d). Three types of vein grafts, autologous external jugular vein, isogeneic jugular, and vena cava veins, were used in the present study. The right external jugular vein was exposed, and three branches were ligated with an electrocoagulator (model SN 54.131; Martin). Both ends were ligated with the suture, and a 1-cm vein segment was removed. For vena cava preparation, a midline abdominal incision was made in the donor animal, and 0.5 ml of saline solution containing 100 units of heparin was injected into the inferior vena cava. After 3 minutes, the anterior thoracic cage was opened from the diaphragm and incised laterally to the internal mammary vessels, and the vena cava (1 cm) was removed. Each vein graft was harvested carefully to avoid mechanical injury during surgical preparation. All grafts were washed with saline solution containing 100 U/ml of heparin. The vein segment was grafted between the two ends of the carotid artery by sleeving the ends of the vein over the artery cuff and ligating them together with the 8-0 suture (Figure 1e). The cuff handle was cut off after completion of the anastomoses, the vascular clamps were removed, and evidence of pulsations was sought in both the grafted and native vessels. If there were no pulsations or pulsations diminished within a few minutes of restoration of blood flow, clot formation or occlusion of output was assumed, and the procedure was considered a surgical failure. If there were vigorous pulsations in the grafted vessel, the skin incision was closed with a 6-0 interrupted suture. One hour was needed to perform the whole operation, and the ischemia time of vein segments was about 15 minutes. For histological analysis, perfusion was performed as described previously.21Carmeliet P Moons L Stassen J-M De Mol M Bouché A van den Oord JJ Kockx MM Collen D Vascular wound healing and neointima formation induced by perivascular electric injury in mice.Am J Pathol. 1997; 150: 761-774PubMed Google Scholar Briefly, mice were anesthetized and perfused with 0.9% NaCl solution via cardiac puncture in the left ventricle, and subsequently perfusion fixed with 4% phosphate-buffered formaldehyde (pH 7.2) for 2 and 5 minutes, respectively. The vein grafts were harvested at 1, 2, 4, 8, and 16 weeks postoperatively (six to eight randomly chosen mice at each time point) by cutting the transplanted segments from the native vessels at the cuff end. Samples were fixed with 4% phosphate-buffered formaldehyde at 4°C for 24 hours. For frozen section preparation, mice were sacrificed by cervical dislocation, and vein grafts were harvested, immediately frozen in liquid nitrogen, and stored at −80°C. After fixation, the grafts were cut in the middle of the vein segments, dehydrated in graded ethanol baths, cleared in xylol, and embedded in paraffin.22Xu Q Dietrich H Steiner HJ Gown AM Schoel B Mikuz G Kaufmann SHE Wick G Induction of arteriosclerosis in normocholesterolemic rabbits by immunization with heat shock protein 65.Arterioscler Thromb. 1992; 12: 789-799Crossref PubMed Scopus (350) Google Scholar Histological sectioning began at the center of the graft to avoid the effects of the cuff. Routinely, 7 μm-thick sections were made throughout the dissected fragments, stained with hematoxylin and eosin (H&E), and examined microscopically (Leitz, Munich, Germany). Because the venous media is very thin (one or two layered cells or 10 to 20 μm thick), the thickness of the normal and lesioned vessel walls was simultaneously measured and calculated microscopically. The intima and media were defined as the region between the lumen and the adventitia. The thickness of the vessel wall was determined by measuring four regions of a section along a cross and recorded in micrometers (means ± SD). Ten cross-sections were obtained by selecting the first of every three sections from each animal. Cell counts in the intima and media were performed on two regions of each section and expressed as the number of nuclei per 100 μm of the vessel wall. The procedure used in the present study was similar to that described previously.23Xu Q Kleindienst R Waitz W Dietrich H Wick G Increased expression of heat shock protein 65 coincides with a population of infiltrating T lymphocytes in atherosclerotic lesions of rabbits specifically responding to heat shock protein 65.J Clin Invest. 1993; 91: 2693-2702Crossref PubMed Scopus (183) Google Scholar Briefly, serial 5 μm-thick frozen sections were cut from cryopreserved tissue blocks, fixed in a cold 1:1 acetone-chloroform mixture for 10 minutes, and washed with phosphate-buffered saline (PBS) for 20 minutes. The sections were subsequently placed in a humidified chamber, where they were overlayered with a rat monoclonal antibody (CD11b/18) against mouse MAC-1 leukocytes (PharMingen, San Diego, CA) and incubated for 1 hour at room temperature. After washing with PBS, sections were incubated with rabbit anti-rat immunoglobulin (Dakopatts, Copenhagen, Denmark) for 1 hour. Sections were washed in PBS three times, incubated with alkaline phosphatase-anti-alkaline phosphatase complex (Dakopatts) for 30 minutes, washed in PBS three times, and developed for 20 minutes at room temperature on a shaker using a substrate solution containing 9.8 ml of Tris buffer (0.1 mol/L, pH 8.2), 0.2 ml of dimethylformamide, 8 mg of naphthol AS-MX phosphate, 3 mg of levamisole, and 10 mg of fast red TR salt (Sigma Chemical Co., St. Louis, MO). A counterstaining with hematoxylin was performed at room temperature for 3 minutes. For smooth muscle cell staining, a mouse monoclonal antibody against α-actin (Sigma) labeled with phosphatase was used. The procedure was similar to that described for CD11b/18 labeling except for omission of the second antibody. Semiquantitive evaluation was performed at 10 × 25 magnification. Positive-stained cells in the intima and media were counted on two regions of each section and expressed as the range of the cell number or the percentage of total nuclei per 100 μm of the vessel wall. Statistical analyses were performed on a Macintosh computer with StatView SE+Graphics software using the Mann-Whitney U test and analysis of variance, respectively. Results are given as means ± standard deviations (SD). A P value <0.05 was considered significant. Figure 1 schematically represents the procedure of grafting a vein segment to the carotid artery using the cuff technique. This method is technically simple, easy to learn, and less traumatic than the suturing technique. Using this method, a total of 57 vein grafts in C57BL/6J mice were studied, with a surgical success rate of about 90%. Fifty-four of 57 animals survived until their designated time of harvest, and all vein grafts were patent at the time of harvest. Three vein grafts were nearly occluded because of thrombosis. All control veins and vein grafts were histologically examined, and five per group were immunohistochemically analyzed. Representative histological sections of control external jugular vein and vein grafts are shown in Figure 2. In the control vein, only two layers of cells, presumably a monolayer each of endothelial and smooth muscle cells, respectively, formed the intima and media, whereas the adventitia was composed of connective tissues, including vasa vasorum (Figure 2A). Interestingly, significant cell loss and vessel wall degeneration in the vein graft was observed 1 week after implantation simultaneous to connective tissue deposition and mononuclear cell infiltration in adventitia (Figure 2B). Concordant with these observations is a report that a loss of endothelial and smooth muscle cells in human saphenous vein bypass grafts was demonstrated 1 to 10 days postoperatively,24Kockx MM Cambier BA Bortier HE de Meyer GR Declercq SC van Cauwelaert PA The modulation of smooth muscle cell phenotype is an early event in human aorto-coronary saphenous vein grafts.Virchows Arch A Pathol Anat Histopathol. 1992; 420: 155-162Crossref PubMed Scopus (51) Google Scholar suggesting that changes in the early stage of grafts in this mouse model are similar to those in humans. By 2 weeks, mononuclear cells infiltrated into the vessel wall from both lumen and adventitia sides (Figure 2C). In late vein grafts, 4-, 8-, and 16-week autograft sections (Figure 2, D through F) showed neointima hyperplasia that thickened rapidly. Obviously, three changes can be found: thickening of the vessel wall up to 10 or 20 layers of cells, declining infiltrated mononuclear cells, and increasing matrix protein accumulation in neointima. The 16-week lesion especially showed a lower cell number and an abundance of matrix proteins (Figure 2F). Neointima hyperplasia significantly narrowed the lumen to 20% of the original size by 16 weeks, and no neovascularization in the lesion was observed (Figure 3). To statistically analyze vein graft remodeling, Figure 4 summarizes data of neointima thickness measured microscopically. Thickening of the vein grafts began as early as 1 week after surgery, although no significant difference was found compared with controls. The thickness of the vessel wall increased significantly in 2-week vein grafts, and neointima hyperplasia progressed throughout all time points. During vein graft remodeling, 4-, 10-, 15-, and 18-fold increases in vessel wall thicknesses were found in 2, 4, 8 and 16 week grafts, respectively (Figure 4B). When cell nuclei in the intima and media of control veins and neointima of grafted vessels were counted in 100 μm length, total cell numbers increased significantly in 2-week grafts, were markedly higher in 4- and 8-week grafts, and then began to decline (Figure 5), observations similar to those in other animal models such as rats, rabbits, and pigs.3Angelini GD Bryan AJ Williams HMJ Morgan R Newby AC Distention promotes platelet and leukocyte adhesion and reduces short-term patency in pig arteriovenous bypass grafts.J Thorac Cardiovasc Surg. 1990; 99: 433-439PubMed Google Scholar, 4Davies MG Klyachkin ML Dalen H Massey MF Svendsen E Hagen PO The integrity of experimental vein graft endothelium: implications on the etiology of early vein graft failure.Eur J Vasc Surg. 1993; 7: 156-165Abstract Full Text PDF PubMed Scopus (56) Google Scholar, 5Boerboom LE Olinger GN Liu TZ Rodriguez ER Ferrans VJ Kissebah AH Histologic, morphometric, and biochemical evaluation of vein bypass grafts in a nonhuman primate model. I. Sequential changes within the first three months.J Thorac Cardiovasc Surg. 1990; 99: 97-106PubMed Google Scholar, 6Landymore RW Kinley CE Cameron CA Intimal hyperplasia in autogenous vein grafts used for arterial bypass: a canine model.Cardiovasc Res. 1985; 19: 589-592Crossref PubMed Scopus (20) Google Scholar, 7Norman PE House AK The influence of nifedipine on microvascular vein graft intimal thickening.Aust N Z J Surg. 1993; 63: 294-298Crossref PubMed Scopus (6) Google ScholarFigure 5Neointima hyperplasia in vein autografts. The procedure for animal models and the preparation of H&E-stained sections are the same as those described in the legend to Figure 2. Total H&E-stained nuclei in 100-μm lengths of (neo)intima and media of veins were counted manually. Two opposite areas from each section were counted, and five sections per animal were selected. The graph shows data (mean ± SD) obtained from six to eight animals per time point. *Significant difference from the control; P < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the role of a specific gene or protein in the development of neointima in vein grafts, an animal model of vein isografts is needed. For instance, the effect of low-density lipoprotein receptors of endothelial and smooth muscle cells on vein graft remodeling in normocholesterolemia can be identified by isografting low-density lipoprotein receptor-deficient vein segments into arteries of wild-type littermates. We therefore established several isograft animal models, including vena cava segments isografted to external jugular veins and vena cava or external jugular vein segments isografted to the arteries. Data shown in Figure 6 indicate the process of vein graft remodeling. The structure and diameter (about 0.9 mm) of the vena cava vessel were similar to those of external jugular veins (Figure 6, A and B). Both vein segments can be used as graft sources in bypass graft models. In addition, intima thickening in vein-to-vein isografts was much less significant than that of vein-to-artery isografts 4 weeks after surgery (Figure 6C), although there was leukocyte infiltration in the vessel wall. The results shown in Figure 6, D through F, provide evidence that the formation of neointima in grafted veins was similar in both vena cava isografts and jugular vein autografts. Likewise, mononuclear cell infiltration and matrix protein deposition in the neointima were demonstrated in all three types of grafts. To statistically compare the three vein bypass grafts, the thickness of neointima was measured as described in Figure 4 and as summarized in Figure 7, indicating that neointima hyperplasia occurred in all three grafts. No significant difference in the lesioned areas of either autografts or isografts was seen, suggesting that comparable results can be obtained from both animal models. There is evidence that lesions of vein graft-induced neointima derived from other animal models and human specimens contain macrophages and smooth muscle cells.25Kockx MM Cambier BA Bortier HE De Meyer GR Declercq SC van Cauwelaert PA Bultinck J Foam cell replication and smooth muscle cell apoptosis in human saphenous vein grafts.Histopathology. 1994; 25: 365-371Crossref PubMed Scopus (67) Google Scholar, 26Ip JH Fuster V Badimon FL Badimon J Taubman MB Chesebro JH Syndromes of accelerated atherosclerosis: role of vascular injury and smooth muscle cell proliferation.J Am Coll Cardiol. 1990; 15: 1667-1687Abstract Full Text PDF PubMed Scopus (703) Google Scholar To analyze the kinetics of cell components in the development of neointima hyperplasia of vein grafts, immunohistochemical staining using monoclonal antibodies against MAC-1+ (CD11b/18) leukocytes and α-actin+ smooth muscle cells was performed on frozen sections. The results shown in Figure 8 indicate MAC-1+ cell infiltration in vein graft lesions. It is known that MAC-1+ cells are monocytes/macrophages, natural killer cells, and granulocytes. We found that the majority of infiltrated cells in neointima were mononuclear cells, ie, monocytes/macrophages. In control veins, MAC-1+ monocytes/macrophages were rarely seen in the intima and media, whereas abundant infiltration of these positive cells were found in intima and/or adventitia of 1- and 2-week vein grafts (Figure 8, B and C). MAC-1+monocytes/macrophages were detected at the luminal surface at 1 and 2 weeks postengraftment and were seen transmurally by 4 and 8 weeks (Figure 8, D and E). MAC-1+ cells were predominant in the neointima of 2- and 4-week grafts (Table 1), were distributed in both luminal and adventitial sites in 8-week vein grafts, and were found occasionally in the lesions of 16-week vein grafts (Figure 8, D through F; Table 1). Thus, the numbers of MAC-1+ monocyte/macrophage infiltration increased during the first 4 weeks and decreased thereafter.Table 1MAC-1+ and α-Actin+ Cells in Media and NeointimaThe number or %/100 μmGraft age (week)MAC-1+ cellsα-actin+ cellsTotal nuclei00–2 (no.)5–9 (no.)11 ± 513–8 (no.)0–3 (no.)13 ± 628–14 (no.)0–1 (no.)29 ± 11450 ± 20 (%)10 ± 8 (%)123 ± 44825 ± 19 (%)58 ± 21 (%)121 ± 29163 ± 5 (%)69 ± 23 (%)94 ± 21The positive-stained cells (red) and total number of nuclei (blue) were counted. In each section, two regions (100 μm/region) were evaluated. Data from groups 0, 1, and 2 weeks represent ranges of t