In Situ Detection of Tissue Factor within the Coronary Intima in Rat Cardiac Allograft Vasculopathy
1999; Elsevier BV; Volume: 154; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)65267-4
ISSN1525-2191
AutoresHans Hölschermann, Rainer M. Bohle, Hagen Zeller, Heiko Schmidt, U. Stahl, Ludger Fink, Helmut Grimm, Harald Tillmanns, Werner Haberbosch,
Tópico(s)Platelet Disorders and Treatments
ResumoCardiac allograft vasculopathy is a major cause of morbidity and mortality of cardiac transplant recipients. The underlying cause of this disease remains unclear. Histological studies have implicated accelerated hemostasis and intravascular fibrin deposition in its pathogenesis. In the present study a defined model of this disease in the rat was used to elucidate the implication of tissue factor in the production of the hypercoagulable state observed in cardiac allograft vessels. Tissue factor protein and mRNA expression were studied in rat heart allografts developing allograft vasculopathy resembling human disease. Immunohistochemistry demonstrated tissue-factor-positive cells present in the allograft coronary intima and adventitia. Significant staining for tissue factor was detected in the endothelium lining coronary lesions in cardiac allografts and in interstitial mononuclear cells, respectively. Both transplant coronary endothelial cells and mononuclear cells contained tissue factor mRNA as indicated by oligo-cell reverse transcription polymerase chain reaction after laser-assisted cell picking. In contrast, tissue factor mRNA and protein were not or negligibly dectectable within the coronary intima of nontransplanted control hearts. Thus, the present study clearly demonstrates that aberrant tissue factor expression occurs within the coronary intima after cardiac transplantation. Tissue factor, activating downstream coagulation mechanisms, may account for the intravascular clotting abnormalities observed in cardiac allografts and may represent a key factor in transplant atherogenesis. Cardiac allograft vasculopathy is a major cause of morbidity and mortality of cardiac transplant recipients. The underlying cause of this disease remains unclear. Histological studies have implicated accelerated hemostasis and intravascular fibrin deposition in its pathogenesis. In the present study a defined model of this disease in the rat was used to elucidate the implication of tissue factor in the production of the hypercoagulable state observed in cardiac allograft vessels. Tissue factor protein and mRNA expression were studied in rat heart allografts developing allograft vasculopathy resembling human disease. Immunohistochemistry demonstrated tissue-factor-positive cells present in the allograft coronary intima and adventitia. Significant staining for tissue factor was detected in the endothelium lining coronary lesions in cardiac allografts and in interstitial mononuclear cells, respectively. Both transplant coronary endothelial cells and mononuclear cells contained tissue factor mRNA as indicated by oligo-cell reverse transcription polymerase chain reaction after laser-assisted cell picking. In contrast, tissue factor mRNA and protein were not or negligibly dectectable within the coronary intima of nontransplanted control hearts. Thus, the present study clearly demonstrates that aberrant tissue factor expression occurs within the coronary intima after cardiac transplantation. Tissue factor, activating downstream coagulation mechanisms, may account for the intravascular clotting abnormalities observed in cardiac allografts and may represent a key factor in transplant atherogenesis. The development of an unusually accelerated form of coronary artery disease, termed cardiac allograft vasculopathy, has become the major cause of long-term morbidity and mortality in the increasing number of heart transplant survivors.1Weis M von Scheidt W Cardiac allograft vasculopathy.Circulation. 1997; 96: 2069-2077Crossref PubMed Scopus (401) Google Scholar With no real solution to understanding its pathogenesis or to preventing its development, cardiac graft vasculopathy is the predominant cause of graft loss and retransplantation after the first year after transplantation and one of the most discouraging aspects of heart transplantation today.2Kaye MP The Registry of the International Society for Heart and Lung Transplantation: Tenth Official Report, 1993.J Heart Lung Transplant. 1993; 12: 541-548PubMed Google ScholarThe pathology of cardiac allograft vasculopathy is characterized by an obliterative myointimal proliferation that affects the whole length of the coronary arteries, including epicardial arteries and small penetrating intramyocardial branches. However, the pathophysiological mechanisms responsible for the development of transplant vasculopathy are incompletely understood. Although it has been postulated that this panvasculitis may emerge in response to alloreactive immune-mediated damage of the vessel intima, the role of immune mechanisms in the pathogenesis remains highly speculative.3Gao SZ Schroeder JS Alderman EL Hunt SA Valantine HA Wiederhold V Stinson EB Prevalence of accelerated coronary artery disease in heart transplant survivors: comparison of cyclosporine and azathioprine regimens.Circulation. 1989; 80: 100-105Google Scholar In fact, despite the better control of immune responses and fewer acute rejection episodes with the introduction of current immunosuppressive therapy, there has been no concomitant reduction in the development of transplant vasculopathy.3Gao SZ Schroeder JS Alderman EL Hunt SA Valantine HA Wiederhold V Stinson EB Prevalence of accelerated coronary artery disease in heart transplant survivors: comparison of cyclosporine and azathioprine regimens.Circulation. 1989; 80: 100-105Google Scholar, 4Hakim M Wallwork J English T Cyclosporin A in cardiac transplantation: medium-term results in 62 patients.Ann Thorac Surg. 1988; 46: 495-501Abstract Full Text PDF PubMed Scopus (24) Google Scholar Thus, with regard to the presence of an intact endothelial monolayer seen on vascular lesions of this disease,5Billingham ME Cardiac transplant atherosclerosis.Transplant Proc. 1987; 19: 19-25PubMed Google Scholar the absence of a true vasculitic picture6Hruban RH Beschorner WE Baumgartner WA Augustine SM Reitz BA Hutchins GM Accelerated arteriosclerosis in heart transplant recipients: an immunopathology study of 22 transplanted hearts.Transplant Proc. 1991; 223: 1230Google Scholar, 7Louie HW Pang M Lewis W Drinkwater DC Laks H Immunhistochemical analysis of accelerated graft atherosclerosis in cardiac transplantation.Curr Surg. 1989; 46: 479PubMed Google Scholar, 8Paavonen T Mennander A Lautenschlager I Mattila S Hayry P Endothelialitis and accelerated arteriosclerosis in human heart transplant coronaries.J Heart Lung Transplant. 1993; 12: 117PubMed Google Scholar and the lack of standard atherogenic risk factors,9Wenke K Meiser B Thiery J Nagel D Scheidt Wv Steinbeck G Seidel D Reichart B Simvastatin reduces graft vessel disease and mortality after heart transplantation.Circulation. 1997; 96: 1398-1402Crossref PubMed Scopus (444) Google Scholar, 10Gao SZ Alderman EL Schroeder JS Silverman JF Hunt SA Accelerated coronary vascular disease in the heart transplant patient: coronary arteriographic findings.J Am Coll Cardiol. 1988; 12: 334-340Abstract Full Text PDF PubMed Scopus (427) Google Scholar respectively, transplant-induced vasculopathy may occur by mechanisms different from that of conventional atherosclerotic events or classic cell-mediated/humoral allograft rejection. Histological examinations characterizing early pathological changes in the coronary artery of allografted hearts suggest that intravascular activation of the coagulation system might be closely involved in the pathogenesis of the disease and that the clotting abnormalities observed within the coronary system of cardiac allografts may have a more significant role in the induction of cardiac graft vasculopathy than was hitherto considered.11Faulk WP Labarrere CA Vascular immunopathology and atheroma development in human allografted organs.Arch Pathol Lab Med. 1992; 116: 1337-1344PubMed Google Scholar, 12Labarrere CA Pitts D Nelson DR Faulk W Page vascular tissue plasminogen activator and the development of coronary artery disease in heart-transplant recipients.N Engl J Med. 1995; 333: 1111-1116Crossref PubMed Scopus (92) Google Scholar Intraluminal and intralesional thrombus formation as well as fibrin deposition along the vessel intima commonly observed in coronary arteries after cardiac transplantation13Arbustini E Roberts WC Morphological observations in the epicardial coronary arteries and their surroundings late after cardiac transplantation.Am J Cardiol. 1996; 78: 814-820Abstract Full Text PDF PubMed Scopus (48) Google Scholar may be directly involved in the initiation and progression of transplant vasculopathy by stimulating migration of smooth muscle cells from the inner media to intima and proliferation in the intima.14Rabbani LE Loscalzo J Recent observations on the role of hemostatic determinants in the development of the atherothrombotic plaque.Atherosclerosis. 1994; 105: 1-7Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 15Weiss RH Maduri M The mitogenic effect of thrombin in vascular smooth muscle cells is largely due to basic fibroblast growth factor.J Biol Chem. 1993; 268: 5724-5727Abstract Full Text PDF PubMed Google Scholar, 16Smith EB Thompson WD Fibrin as a factor in atherogenesis.Thromb Res. 1994; 73: 1-19Abstract Full Text PDF PubMed Scopus (105) Google ScholarGiven its role as the primary initiator of the coagulation protease cascade, tissue factor (TF), an integral 47-kd cell membrane-bound glycoprotein, may contribute to the intravascular thrombus and fibrin formation observed in cardiac allografts.17Nemerson Y Tissue factor and hemostasis.Blood. 1988; 71: 71-78PubMed Google Scholar TF, normally absent in circulating blood cells and endothelium and functionally sequestered from circulating blood by predominant expression in the vascular adventitia,18Wilcox JN Smith KM Schwartz SM Gordon D Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque.Proc Natl Acad Sci USA. 1989; 86: 2839-2843Crossref PubMed Scopus (1069) Google Scholar, 19Drake TA Morrissey JH Edgington TS Selective cellular expression of tissue factor in human tissues.Am J Pathol. 1989; 134: 1089-1097Google Scholar is inducible expressed in a number of cell types, including endothelial cells,20Scarpati EM Sadler JE Regulation of endothelial cell coagulation properties.J Biol Chem. 1989; 264: 20705-20713Abstract Full Text PDF PubMed Google Scholar, 21Nawroth PP Handley DA Esmon CT Stern DM Interleukin 1 induces endothelial cell procoagulant activity while suppressing cell-surface anticoagulant activity.Proc Natl Acad Sci USA. 1986; 83: 3460-3464Crossref PubMed Scopus (452) Google Scholar, 22Bierhaus A Zhang Y Deng Y Mackman N Quehenberger P Haase M Luther T Müller M Böhrer H Greten J Eike M Bauerle P Waldherr R Kisiel W Ziegler R Stern DM Nawroth PP Mechanism of the TNFα mediated induction of endothelial tissue factor.J Biol Chem. 1995; 270: 26419-26432Crossref PubMed Scopus (124) Google Scholar monocytes,23Edgington TS Mackman N Brand K Ruf W The structural biology of expression and function of tissue factor.Thromb Haemost. 1991; 66: 67-79Crossref PubMed Scopus (509) Google Scholar, 24Fan S-T Mackman N Cui M-Z Edgington TS Integrin regulation of an inflammatory effector gene: direct induction of the tissue factor promoter by engagement of β1 or α4 integrin chains.J Immunol. 1995; 154: 3266-3274PubMed Google Scholar, 25Gregory SA Kornbluth RS Helin H Remold HG Edgington TS Monocyte pro-coagulant inducing factor: a lymphokine involved in the T-cell instructed monocyte pro-coagulant response to antigen.J Immunol. 1986; 141: 3231-3239Google Scholar fibroblasts,26Bloem LJ Chen L Konigsberg WH Bach R Serum stimulation of quiescent human fibroblasts induces the synthesis of TF mRNA followed by the appearance of tissue factor antigen and procoagulant activity.J Cell Physiol. 1989; 139: 418-423Crossref PubMed Scopus (52) Google Scholar and vascular smooth muscle cells27Taubman MB Marmur JD Rosenfield C-L Guha A Nichtberger S Nemerson Y Agonist-mediated tissue factor expression in cultured vascular smooth muscle cells: role of Ca2+ mobilization and protein kinase C activation.J Clin Invest. 1993; 91: 547-552Crossref PubMed Scopus (162) Google Scholar in response to a variety of inflammatory and immunological stimuli. It is tempting to speculate that the development of intimal hyperplasia after cardiac transplantation involves the induction of TF in the intima or media of the affected vessels. The purpose of the present study therefore was to determine the presence of TF expression in the vessel wall of cardiac allografts. The distribution, extent, and cellular localization of TF expression in allograft vasculopathy lesions was assessed in a rat heterotopic heart transplantation model.Materials and MethodsAnimalsAdult, inbred male Lewis (RT1) and F-344 (RT1) rats weighing 250 to 300 g were purchased from Charles River Laboratories (Kingston, NY). All rats were housed under conventional conditions and maintained on standard laboratory rat chow and water ad libitum.Heterotopic Heart TransplantationLewis rats served as donors and F-344 rats as recipients. Heterotopic heart transplantation was performed using a cuff anastomosis technique as described previously.28Heron I A technique for accessory cervical heart transplantation in rabbits and rats.Acta Pathol Scand. 1973; 79: 366Google Scholar The recipients were anesthetized with phentanylcitrate, 0.315 mg/kg body weight, given intramuscularly. The abdomen was opened with a midline incision, the left kidney was removed, and the kidney vessels were cuffed as described elsewhere.28Heron I A technique for accessory cervical heart transplantation in rabbits and rats.Acta Pathol Scand. 1973; 79: 366Google Scholar The donors were anesthetized with pentobarbital, 60 mg/kg body weight intraperitoneally, and 300 IU of heparin was injected intravenously before harvesting the heart. The grafts were flushed with cold Ringer lactate solution containing 50 IU of heparin/ml. Immediately after harvesting, the graft was anastomosed with the cuffed vessels; the cold ischemic times did not exceed 10 minutes. Cardiac allograft survival was monitored by daily palpation of the graft. Allografts were followed for 120 days after transplantation. The recipients were treated with daily intraperitoneal cyclosporine A (Sandoz, Basel, Switzerland) beginning on the day of transplantation. The initial dose of 2 mg/kg body weight/day was reduced to 0.5 mg/kg body weight/day on postoperative day 80.Histological ExaminationAll hearts continued to beat throughout the study period. Rats were killed after 120 days by deep phenobarbital anesthesia. The heart allografts, the recipients' own hearts, and other pertinent tissues were removed. Native nontransplanted donor hearts served as controls. Hearts were promptly sectioned into five slices: 1) basal (included the atria and the base of the ventricles), 2 to 4) middle (included the midportion of the ventricles), and 5) apical (included the ventricular apex). Slices 1, 2, 4, and 5 were embedded in isopentane-precooled OCT compound (Miles, Elkhart, IN), quick-frozen in liquid nitrogen, and stored at −80°C for future processing. Frozen sections were cut at 5 μm, fixed in acetone, and immunostained using the alkaline phosphatase and anti-alkaline-phosphatase (APAAP) method.29Cordell JL Falini B Erber WN Gosh AK Abdulaziz Z MacDonald S Pulford KAF Stein H Mason DY Immunoenzymatic labelling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes).J Histochem Cytochem. 1984; 32: 219-229Crossref PubMed Scopus (2872) Google Scholar Slice 3 was fixed in 4% buffered formaldehyde solution for 24 hours and embedded in paraffin. The sections were routinely stained with hematoxylin and eosin (H&E) and elastic van Gieson for regular histopathological analysis. The sections were examined by standard light microscopy and scored for both the severity of rejection and cardiac allograft vasculopathy (Table 1, Table 2;Figure 1), using previously described scoring systems.30Sarris GE Mitchell RS Billingham ME Glasson JR Cahill PD Miller DC Inhibition of accelerated cardiac allograft arteriosclerosis by fish oil.J Thorac Cardiovasc Surg. 1989; 97: 841-855PubMed Google Scholar, 31Lurie KG Billingham ME Jamieson SW Harrison DC Reitz BA Pathogenesis and prevention of graft atherosclerosis in an rat experimental heart transplantation model.Transplantation. 1981; 31: 41-47Crossref PubMed Scopus (126) Google Scholar Large vessels were defined as arteries with more than two smooth muscle cell layers; small vessels were defined as arteries/arterioles with two or fewer smooth muscle cell layers.Table 1Histological Grading Scale for the Assessment of Degree of RejectionGradeHistological appearance0No rejection1Mild rejection with scanty mononuclear cell infiltrate; minimal or no fibrosis2Moderate rejection with moderate mononuclear cell infiltrate3Severe rejection with diffuse and severe mononuclear cell infiltrate, focal hemorrhage, and necrosis Open table in a new tab Table 2Histological Grading Scale for the Assessment of Degree of Cardiac Allograft VasculopathyGradeHistological appearance0Vessel unaffected1Accumulation of inflammatory cells along intimal surfaces but with <10% occlusion of the lumen2More advanced lesion including definite intimal proliferation and thickening with 50% of its lumen4100% vessel occlusion of lumen Open table in a new tab ImmunostainingImmunostaining was performed by the highly sensitive alkaline phosphatase (APAAP) technique, using a slightly modified method of Cordell.29Cordell JL Falini B Erber WN Gosh AK Abdulaziz Z MacDonald S Pulford KAF Stein H Mason DY Immunoenzymatic labelling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes).J Histochem Cytochem. 1984; 32: 219-229Crossref PubMed Scopus (2872) Google Scholar Briefly, freshly prepared, acetone-fixed cryostat sections (5 μm) were incubated for 30 minutes with primary antibodies at room temperature, followed by a 30-minute incubation at room temperature with rabbit anti-mouse immunoglobuline (rabbit "link", 1:40; Dako, Hamburg, Germany) supplemented with pooled rat serum (1:750; Sigma, Deisenhofen, Germany) to inhibit nonspecific cross-reactivity, and APAAP complex (1:50; Dako), respectively. The "link" and APAAP steps were repeated for 10 minutes. The samples were thoroughly washed in Tris/HCL-buffered saline (pH 7.6) between the "link" and APAAP steps. Alkaline phosphate substrate reaction with new fuchsin (100 μg/ml) and levamisole (400 μg/ml) was performed for 30 minutes at room temperature. Immunohistology for TF antigen was performed using the murine monoclonal rabbit TF antibody AP-1 (0.375 μg/ml, kindly provided by Dr. Michael D. Ezekowitz, Yale University New Haven, CT), the production, purification, and characterization of which are reported elsewhere.32Pawashe AB Paolo G Ambrosio G Migliaccio F Ragni M Immacolata P Chiariello M Bach R Garen A Konigsberg WK Ezekowitz MD A monoclonal antibody against rabbit tissue factor inhibits thrombus formation in stenotic injured rabbit carotid arteries.Circ Res. 1994; 74: 56-63Crossref PubMed Scopus (152) Google Scholar The ability of the antibody to cross-react with rat TF apoprotein was assessed by immunoblotting with rat brain thromboplastin. Two hundred micrograms of rat brain acetone salt (Sigma) was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (10% gel), transferred to nitrocellulose, and incubated with the mouse anti-rabbit TF antibody (0.375 μg/ml) for 1.5 hours at room temperature after blocking with 5% milk protein for 1 hour at room temperature. After washing in PBS with 0.1% Tween 20, peroxidase-conjugated sheep anti-mouse secondary antibody (0.3 μg/ml; Amersham, Freiburg, Germany) was added. Final bands were detected on x-ray film using the enhanced chemiluminescent system (Amersham). As a positive control for TF, immunohistochemistry was performed on sections of rat kidney. To confirm the identity of cells positive for TF staining, murine monoclonal antibody to rat monocytes/macrophages (clone ED1, 0.22 μg/ml; Camon, Wiesbaden, Germany) and to rat endothelial cell antigen-1/pan-endothelium (clone HIS 52, 1:100; Camon) were used. Negative controls were performed using mouse anti-rabbit immunoglobulin (clone MR 12/53, 0.425 μg/ml; Dako). Sections were counterstained with hematoxylin and mounted in gelatin.Laser-Assisted Cell Picking and Tissue Factor RT-PCRCryosections (5 μm) of transplanted (n = 10) and nontransplanted control hearts (n = 5) were prepared. Three sections of each heart were collected into a microcentrifuge tube and stored at −80°C. Afterwards, tissue was homogenized (Fisher Scientific, Nidderau, Germany) using the RNAzol-B (WAK, Bad Homburg, Germany) method for RNA extraction. For cell picking, three sections of each heart were mounted on glass slides (thickness, 0.17 mm) and stored at −20°C for several hours until further investigation. In both preparations, Kwok's criteria for avoidance of contamination were meticulously obeyed.33Kwok S Procedures to minimize PCR-product carryover.in: Innis MA Gelfand DH Snisky JJ White TJ PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego1990: 142-145Google Scholar Histological sections were stained with hemalaun (Merck, Darmstadt, Germany) according to standard procedures, subsequently immersed in 70%, 96%, and 100% ethanol, and stored up to 2 hours in 100% ethanol until cell picking.Laser-Assisted Cell Picking and Cell LysisOligo-cell samples of endothelial cells and monocytes/macrophages (five to seven cells), respectively, of intramyocardial arteries from transplanted and control hearts were collected by cell picking. In brief, a sterile syringe needle (30 gauge, 1/2 inch) on a micromanipulator, a computer-controlled microscope stage, and an ultraviolet (UV)-laser microbeam (P.A.L.M., Wolfratshausen, Germany) connected with an inverse microscope (Zeiss Axiovert 135, Zeiss, Jena, Germany), a CCD camera, and a frame grabber were used as described elsewhere34Becker I Becker HF Röhrl HH Höfler H Laser-assisted preparation of single-cells from stained histological slides.Histochem Cell Biol. 1997; 108: 448-451Crossref Scopus (33) Google Scholar (depicted in Figure 4). The highly precise UV-laser system is able to produce gaps of 1 μm in histological sections,35Schütze K Lahr G Identification of expressed genes by laser-mediated manipulation of single cells.Nature Biotechnol. 1998; 16: 737-742Crossref Scopus (371) Google Scholar allowing the separation of a single endothelial cell layer from the adjacent media. At ×400 magnification, media cells and adventitia cells were photolysed by UV-laser pulses. From normal intima, endothelia were picked in microclusters of two cells, and from intimal proliferations, clusters of three to five cells were picked and transferred into a precooled 0.5-ml reaction tube, containing 10 μl of freshly prepared FSB buffer (50 mM Tris/HCl, pH 8.3, 75 mmol/L KCl, 3 mmol/L MgCl2, 4% RNAse inhibitor (20 U/μl; Perkin Elmer, Weiterstadt, Germany), and 0.5% Igepal CA-630 (Sigma)).36Brady G Iscove NN Construction of cDNA libraries from single cells.Methods Enzymol. 1993; 225: 661-673Google Scholar After incubation on ice for 5 minutes, samples were snap-frozen in liquid nitrogen and stored at −80°C.Figure 4Demonstration of laser-assisted cell picking in cardiac allograft vasculopathy. A: Advanced occlusion of an intramyocardial coronary artery (hemalaun-stained frozen section). B: Intimal proliferations were dissected free from media by UV-laser microbeam. C: Subdivision of intimal proliferations into oligo-cellular clusters and picking of a cluster with a syringe needle. The picked cell cluster is adhesive to the needle and is lifted under microscopic control. D: Section after removal of the intima cell cluster. Now the next cluster can be picked. Original magnification, ×20.View Large Image Figure ViewerDownload Hi-res image Download (PPT)cDNA Synthesis and RT-PCRSamples were incubated for 10 minutes at 70°C for RNA denaturing and then rapidly cooled on ice. For cDNA synthesis, 1 μl of random hexamers (50 μmol/L; Perkin Elmer), 0.5 μl of RNAse inhibitor (20 U/μl; Perkin Elmer), 1 μl of dNTPs (10 mmol/L each; Eurobio, Raunheim, Germany), 50 U of MuLV reverse transcriptase (Perkin Elmer), and 4 μl of H2O were added. First-strand reactions, 10 minutes at 20°C, 60 minutes at 43°C, and 5 minutes at 99°C, were performed using a TRIO Thermoblock (Biometra, Göttingen, Germany). cDNA was stored at −20°C until used for PCR. One-half of cDNA was used for porphobilinogen deaminase (PBGD) housekeeping gene detection (L. Fink, U. Stahl, L. Ermert, W. Kummer, W. Seeger, and R.M. Bohle, manuscript submitted). The second half was used for TF PCR together with 10 pmol of reverse (5′-TCCAAGTTAAATTAAACGCTTTCCC-3′) and forward (5′-CCACCTTTCTC-GGCTTCCTT-3′) primer sequence (accession number U07619), 1 μl of dNTPs (10 mmol/L each; Eurobio), and 2.5 U of AmpliTaq Gold (Perkin Elmer) in a total volume of 50 μl. Reaction buffer (Perkin Elmer) contained 1.5 mmol/L MgCl2. PCR conditions (TRIO Thermoblock, Biometra) for cell-picking templates were 94°C for 2 minutes 45 seconds; 60 cycles of 94°C for 45 seconds, 62°C for 45 seconds, and 73°C for 45 seconds; and 73°C for 5 minutes. PCR conditions for complete heart slices were 94°C for 2 minutes 45 seconds; 35 cycles of 94°C for 45 seconds, 62°C for 45 seconds, and 73°C for 45 seconds; and 73°C for 5 minutes. Fifteen percent of each PCR reaction was electrophoresed together with a ϕ X 174 HinfI size marker on a 2.5% agarose/Tris-buffered ethanolamine gel and stained with ethidium bromide.Statistical AnalysisSignificance of differences between transplanted and nontransplanted hearts was determined by the two-tailed Fisher's exact test. Statistical significance was indicated by P < 0.05.ResultsHistopathological ObservationsAt the time of sacrifice (day 120 after transplantation) all of the cardiac allografts were beating. The histological score evaluating the degree of cellular interstitial rejection at the time of sacrifice was 1.5 ± 0.53 (median, 1.5). Allografts exhibited mild to moderate myocardial rejection with focal areas of mononuclear cell infiltration, cardiomyocytolysis, and some degree of endomyocardial fibrosis and interstitial fibrosis. The nontransplanted native donor hearts generally demonstrated an entirely normal morphological structure with no signs of vascular obliteration or endocardial/myocardial damage.A total of 646 artery cross sections in 15 rats (10 transplanted and 5 nontransplanted) were available for the study. We found that up to 90% (89.7 ± 10.3) of vessels were affected by transplant vasculopathy in all allografts removed at day 120 after transplantation (average lesion grade, 2.9), thereby confirming previous studies performed in the same donor-recipient graft model.37Adams DH Tilney NL Collins JJJ Karnovsky MJ Experimental graft arteriosclerosis.Transplantation. 1992; 53: 1115-1119Crossref PubMed Scopus (130) Google Scholar The nontransplanted native hearts had morphologically normal coronary arteries.Rat cardiac allografts developed coronary vascular lesions indistinguishable in appearance from human accelerated graft vasculopathy (shown in Figure 1). Whereas early lesions with less than 10% occlusion demonstrated patchy endothelial protrusions, endothelial swelling and adherence of a few mononuclear cells along the vessel intima (Figure 1B), advanced lesions with more than 50% occlusion were characterized by a marked cellular expansion of the intima and an inflammatory infiltrate typically found within the internal elastica lamina and in a halo zone exterior to the adventitia (Figure 1D). Occasionally, the internal elastic membrane was stretched and focally disrupted in advanced lesions. Diffuse fibrointimal thickening that markedly compromised the lumen resulted in a virtually complete occlusion of some coronary arteries.TF Antigen Detection in Coronary Vessels of Nontransplanted HeartsTF antigen detection in cryosections of transplanted and nontransplanted hearts was performed using the anti-rabbit TF monoclonal antibody AP-1. The ability of AP-1 to cross-react with rat TF was established by detection of the 47,000 molecular weight band of reduced TF by immunoblotting with rat brain acetone salt (Figure 2A). As a positive control for TF, immunostaining was performed on sections of rat kidney, demonstrating TF-specific staining of the kidney glomeruli when AP-1 was used for immunohistochemistry (Figure 2B). There was no or negligible TF detectable in the coronary system of nontransplanted control hearts by immunohistochemistry (Figure 3A). In some rare intramyocardial nontransplant vessels, focal areas of vascular endothelium stained weakly for TF (2.1% of sections analyzed).Figure 2Recognition of rat tissue factor by AP-1. A: The ability of the mouse anti-rabbit TF antibody AP-1 to cross-react with rat TF was established by Western blot analysis. AP-1 recognizes the 47-kd band of TF in rat brain acetone salt (source of TF). B: As a positive control for TF, immunohistochemical staining was performed on sections of rat kidney, demonstrating anti-TF staining of a rat kidney glomerulus. Original magnification, ×40.View Large Image Figure ViewerDownload Hi-res
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