Vascular cell adhesion molecule-1 is expressed in human coronary atherosclerotic plaques. Implications for the mode of progression of advanced coronary atherosclerosis.

Endothelial attachment is the initial step in leukocyte recruitment into developing atherosclerotic lesions. To determine whether vascular cell adhesion molecule-1 (VCAM-1) expression may play a role in inflammatory cell recruitment into human atherosclerotic lesions, immunohistochemistry was performed with a polyclonal rabbit antisera, raised against recombinant human VCAM-1, on 24 atherosclerotic coronary plaques and 11 control coronary segments with nonatherosclerotic diffuse intimal thickening from 10 patients. Immunophenotyping was performed on adjacent sections to identify smooth muscle cells, macrophages, and endothelial cells. To confirm VCAM-1-expressing cell types, double immunostaining with VCAM-1 antisera and each of the cell-specific markers and in situ hybridization were performed. All atherosclerotic plaques contained some VCAM-1, compared to 45% of control segments. VCAM-1 was found infrequently on endothelial cells at the arterial lumen din both plaques (21%) and in control segments (27%), but was prevalent in areas of neovascularization and inflammatory infiltrate in the base of plaques. Double immunostaining and in situ hybridization confirmed that most VCAM-1 was expressed by subsets of plaque smooth muscle cells and macrophages. The results document the presence of VCAM-1 in human atherosclerosis, demonstrate VCAM-1 expression by human smooth muscle cells in vivo, and suggest that intimal neovasculature may be an important site of inflammatory cell recruitment into advanced coronary lesions.

Endothelial loss and regeneration in a model of transplant arteriosclerosis.

Early endothelial injury may play a role in the development of transplant arteriosclerosis. The present study documents early endothelial changes using a rat aortic graft model. Abdominal aortic allografts from PVG rats were orthotopically transplanted to DA rats. Controls were DA to DA transplants. Endothelial cell (EC) injury, regeneration, and leukocyte infiltration in the intima were evaluated using scanning electron microscopy and histological and immunocytochemical techniques. Nontransplanted aortic segments showed partial loss of ECs after 1 or 2 hr of preservation. Control isografts demonstrated extensive EC denudation and neutrophil adherence to residual ECs at 1 day post-transplantation. After 3 days, isografts showed continued regeneration of ECs in the central area and ingrowth of endothelium from both clamped sites in the recipient aorta. Reendothelialization was complete by day 14. Allografts showed similar findings to isografts up to day 3. In contrast to isografts, however, there was a secondary EC loss beginning at day 7. Monocytes/macrophages and T cells were noted to be adherent to residual ECs in 7- and 14-day allografts. At 20 days, ECs were absent from the luminal surface in the center of allografts. Endothelium did extend from clamped sites toward the midgraft region as in isografts. By 60 days allografts were completely reendothelialized. These results demonstrate that in both isografts and allografts there is initial EC loss due to mechanical trauma and ischemia/reperfusion injury, followed by partial reendothelialization. This latter process continues unabated in isografts, whereas in allografts the secondary EC loss occurs due to an allogenic response. This is followed by complete reendothelialization that occurs during the concurrent development of significant intimal hyperplasia.

Antibody to CD18 reduces neutrophil and T lymphocyte infiltration and vascular cell adhesion molecule-1 expression in cardiac rejection.

Most antirejection therapies target immune activation but may not reduce leukocyte infiltration into the graft. The leukocyte integrin CD18 has been shown to be important for leukocyte migration in vitro. We postulated that antibody blockade of CD18 might reduce the migration of different leukocyte subpopulations into myocardium during rejection. Using a rabbit model, we evaluated the effect of a monoclonal antibody to CD18 on the infiltration of neutrophils (polymorphonuclear leukocytes [PMNs]), T lymphocytes, and macrophages into cardiac grafts. In addition, vascular cell adhesion molecule-1 (VCAM-1) expression was assessed to determine the relationship between leukocyte infiltration and VCAM-1 expression, an unblocked alternate adhesion pathway. Donor hearts from Stauffland rabbits were transplanted heterotopically into the cervical position of New Zealand White recipients. Recipient rabbits received either monoclonal antibody to CD18 daily without other immunosuppression (n=51), saline injections as placebo controls (n=52), or nonfunctional isotype-matched antibody (n=4). Recipient rabbits were killed at 1 hr, 6 hr, 24 hr, 3 days, and 7 days after transplantation (10-12 rabbits per group at each time point). PMNs, T lymphocytes, and macrophages were differentiated by routine staining and immunocytochemistry, respectively, and quantified as the number of cells per standardized field. VCAM-1 expression was examined immunocytochemically in 30 treated and 30 control transplanted hearts. Monoclonal antibody to CD18 significantly reduced the infiltration of PMNs and T lymphocytes into myocardium during rejection, but did not affect the infiltration of macrophages. Blocking the CD18/intercellular adhesion molecule-1 adhesion pathway also resulted in a decrease in VCAM-1 expression, which correlated in time with the reduction in T lymphocyte infiltration.

Gene therapy for donor hearts: ex vivo liposome-mediated transfection.

OBJECTIVE: Liposomes may be an appropriate transfection vehicle for transplanted hearts, avoiding the use of viruses in immunosuppressed hosts and allowing transfection of nondividing cells. To study whether liposome-mediated transfection could be accomplished during transplantation, we used a liposome-reporter gene system in a rabbit model of allograft cardiac transplantation. METHODS: After aortic crossclamping, Stauffland donor hearts were injected with 10 ml Stanford cardioplegic solution; then a 1.3 to 2.0 mg/kg dose of chloramphenicol acetyl transferase in 1:1 deoxyribonucleic acid-liposome complexes was injected proximal to the aortic crossclamp for coronary artery perfusion. The hearts were transplanted into New Zealand White rabbit recipients in the heterotopic cervical position (n = 11 transplants). Recipients were sacrificed at 24 hours. Myocardial specimens (right and left ventricles) and vascular specimens (epicardial coronary artery, aortic root, and coronary sinus) from both the transplanted and native hearts were analyzed for chloramphenicol acetyl transferase protein by means of the enzymatic liquid scintillation assay (counts per minute per milligram of total protein). RESULTS: In the recipient, myocardial chloramphenicol acetyl transferase activity was significantly greater in treated donor hearts (mean 4.6 x 10(5) cpm/mg +/- 1.1 x 10(5) [standard error]) than in native hearts (mean 4.1 x 10(2) cpm/mg +/- 72 [standard error], p < 0.01, Mann-Whitney U test). In treated donor hearts, right and left ventricular specimens, as well as apical and basal myocardial specimens, were transfected equally. Chloramphenicol acetyl transferase activity in vascular specimens also indicated transfection (mean 5.4 x 10(5) cpm/mg +/- 2.5 x 10(5) [standard error]). Chloramphenicol acetyl transferase activity in the coronary sinus was comparable with that in the coronary arteries, which suggests that liposomes can transverse the coronary capillary beds. CONCLUSIONS: These findings demonstrate that ex vivo transfection of donor hearts with a liposome-reporter gene system results in significant in vivo expression of the transfected gene product after cardiac transplantation. Genetic alteration of myocardium and cardiac vasculature has potential clinical applications in the prevention of posttransplantation rejection, ischemia-reperfusion injury, and both transplant and nontransplant coronary artery disease.

Transplant arterial vasculopathy: evidence for a dual pattern of endothelial injury and the source of smooth muscle cells in lesions of intimal hyperplasia.

With use of this straight artery model of transplantation for studying TGVD, we have shown the following: 1. Intimal thickening occurs in a time-dependent predictable manner only in allografts. 2. Endothelial injury as a result of procurement, preservation, and reperfusion by itself does not result in the development of intimal thickening. Additional endothelial injury associated with the presence of an early mononuclear infiltrate is necessary for the development of TGVD. 3. The development of intimal hyperplasia is initially cellular, consisting mainly of macrophages and a smaller proportion of lymphocytes. The macrophages are later replaced by SMCs. These findings are summarized in Figure 16. 4. The source of the SMCs in intimal lesions is most likely the recipient media.

A reduction in accelerated graft coronary disease and an improvement in cardiac allograft survival using low molecular weight heparin in combination with cyclosporine.

Heparin compounds are a complex mixture of mucopolysaccharides that, in addition to their anticoagulant properties, have immunosuppressive activities and affect the reparative aspects of the response to arterial injury. Heparin inhibits smooth muscle cell migration and proliferation and can alter the accumulation of the components of the extracellular matrix after arterial injury. Heparin also interacts specifically with the endothelium. Our hypothesis was that if heparin compounds do affect the immune system, coagulation, smooth muscle cell proliferation, and the endothelium, then heparin combined with cyclosporine should improve allograft survival, reduce rejection, and prevent accelerated graft coronary disease. Low molecular-weight heparins are derived from the larger molecular-weight unfractionated heparin. We chose to use low molecular-weight heparins because of their longer half-life, better bioavailability, and decreased incidence of induced thrombocytopenia and bleeding. With a rat intraabdominal heterotopic model of heart transplantation (Lewis-Brown Norway to Lewis), low molecular-weight heparin in combination with a low dose of cyclosporine (0.5 mg/kg/day) significantly improved allograft survival compared to controls and either low molecular-weight heparin or cyclosporine alone. Histologically, smooth muscle cells are an important cellular component of the lesions of accelerated graft coronary disease. In comparison to animals treated with cyclosporine alone (2 mg/kg/day), the addition of low molecular-weight heparin to cyclosporine (2 mg/kg/day) reduced the frequency and the severity of accelerated graft coronary disease and the extent of parenchymal rejection.

Biocompatibility and bioabsorption of microfibrillar collagen hemostat in experimental animals.

The effects of Microfibrillar Collagen Hemostat (MCH) and Gelfoam after surgical implantation into incision sites of the liver, kidney, and brain were studied in beagle dogs, rabbits, and beagle dogs, respectively. The results of these experimental animal studies suggest that MCH is comparable to Gelfoam with respect to biocompatibility, rate of bioassimilation, and a lack for adverse systemic effects. The brain, liver, and kidney tissues responded comparably to MCH and Gelfoam with a mild to moderate infiltration of macrophages and mononuclear cells. Most of the hemostatic compound had disappeared from the incision sites by Day 28 and completely disappeared by Day 84. The tissue degree response was interpreted as a factor in the process of bioassimilation of the two hemostatic materials. Both hemostatic compounds contributed to adhesion formation in the experimental models. The incidence of adhesions was somewhat lower for MCH than for Gelfoam, but both produced more adhesions than were found at the control sites. The adhesions were only to the adjacent structures and always localized to the surgical site. When MCH or Gelfoam is used under conditions similar to those in the present experimental study, where tissue approximation is impaired, and where growth of granulation tissue is stimulated by the physical presence of the hemostatic compound, there is the possibility for increased incidence of adhesion formation. However, when an intraperitoneal absorbable hemostatic compound is desired, the present studies in experimental animals suggest that MCH will be safe by exhibiting minimal tissue reaction.

Comparative toxicity of two-dexamethasone derivatives following topical ocular instillation to rabbits. II. Systemic histopathological changes.

Dexamethasone-21-tertiary butyl acetate (dexamethasone TBA) and dexamethasone alcohol at concentrations of 0.2, 0.1, 0.01, and 0.001% were topically instilled into the right eyes of groups of 10 rabbits for 21 consecutive days. Vehicle control and untreated control groups were used for comparative evaluation. The dose-dependent changes for each compound were (1) lipid and glyocogen infiltration of liver, (2) hydropic changes of liver, (3) vacuolation and multifocal hepatic necrosis of liver, (4) atrophy of Peyer's patches of intestines, (5) white pulp atrophy of the spleen and (6) atrophy of the adrenal cortex. No pathological changes were noted for other tissues including the eye. In conclusion, the results indicate that systemic histopathologic changes for both steroids were comparable and typical for steroids.

Reduction in cellular and vascular rejection by blocking leukocyte adhesion molecule receptors.

Whether antibody blockage of leukocyte receptors for intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 would prevent cardiac graft rejection was studied in a rabbit heterotopic transplant model. Monoclonal antibody 60.3, anti-CD18 (intercellular adhesion molecule-1 receptor, Group 1, n = 10) and monoclonal antibody HP1/2, anti-VLA-alpha 4 (vascular cell adhesion molecule-1 receptor, Group 2, n = 10) were administered to transplanted unimmunosuppressed animals. At 7 days, donor heart histology was compared to transplanted untreated controls (Group 3, n = 11). Peripheral white blood cell counts on postoperative day 2 were significantly higher in both treatment groups than controls. Significant increases in circulating neutrophils occurred in Group 1 (P < or = 0.05); lymphocytes predominated in Group 2 (P < or = 0.05). A significant reduction in cellular rejection was seen in Group 1 (P < or = 0.05) but not Group 2 hearts. Group 1 hearts demonstrated localization of lymphocytes to perivenular collections, whereas Group 2 hearts evidenced diffuse interstitial infiltration. Both treatment groups demonstrated a reduction in transplant arteritis compared to controls. Results suggest that monoclonal antibody 60.3 (anti-CD18) may hold promise as a therapeutic agent for both cellular and vascular rejection. Monoclonal antibody HP1/2 (anti-VLA-alpha 4) may reduce vascular rejection disproportionate to cellular rejection.

Transplant arteriosclerosis in a rat aortic model.

Transplant arteriosclerosis (TA) has emerged as an obstacle to the long-term survival of transplanted organs, especially cardiac transplants. The animal models that have been used to study TA have not been fully characterized with regard to features such as the time course of cell proliferation and the sequence of cell types arriving in the developing intimal lesion. We present a model of TA based on a transplanted segment of abdominal aorta that helps address these questions. Two strains of rats (PVG x DA) underwent orthotopic aortic transplantation without immunosuppression and were killed at 14, 20, 40, and 60 days after transplantation. The within-strain control group displayed minimal evidence of cellular rejection with minimal to absent intimal lesions. In contrast, the allograft group showed a linearly increasing intimal lesion, up through 60 days after transplantation. The mechanism of intimal thickening was by an increase in cell number at the earlier time points with the later deposition of extracellular matrix. The early intimal lesion consisted mostly of mononuclear inflammatory cells (45%) with gradually increasing presence of smooth muscle cells (SMC) in the intima between 20 and 60 days. Conversely, the media showed gradual infiltration by macrophage-type cells with virtual loss of all SMC from the media by 40 days. The proliferative index showed a peak of 6% and 8% at 20 days in both the intima and media, respectively, and was preceded by the presence of macrophages. In fact, most of the proliferating cells at the earlier time points were either monocytes/macrophages, or were immediately adjacent to monocyte-/macrophage-rich regions. This straight artery segment model of transplant arteriosclerosis provides an easily quantifiable system in which the effects of different interventions (e.g., immunosuppressive regimens) can be tested.

Leukocyte CD18 receptors may be a better target than ICAM-1 ligands for reducing histologic evidence of cellular and vascular rejection in the rabbit.

Building evidence suggests that blocking the ICAM-1/CD18 interaction may affect the course of graft rejection. Treatment with monoclonal antibody (mAb) to CD18 was compared to antibody to ICAM-1 in a rabbit heterotopic heart transplant model to determine whether blocking the leukocyte receptor for ICAM-1, CD18, was more effective than antibody targeting of the ligand for ICAM-1. Following transplantation, 28 recipient rabbits were randomized to receive either placebo, mAb to CD18, or mAb to ICAM-1 for 7 days until sacrifice. The cellular rejection grade and percentage of arteries with vascular rejection were significantly lower in animals treated with anti-CD18 than with anti-ICAM-1. As assessed by histology, antibody treatment was more effective in reducing both cellular and vascular rejection when directed at the leukocyte receptor CD18 than the ICAM-1 ligand. These findings suggest that other ICAM ligands may play an active role in the immune response and that CD18 may be important for migration of lymphocytes through myocardium.

E-selectin expression in human cardiac grafts with cellular rejection.

BACKGROUND: E-selectin expression has recently been documented to occur with lymphocytic infiltration in the skin and synovium. The question of whether E-selectin is expressed in the context of cardiac graft rejection was addressed in this study. METHODS AND RESULTS: One hundred ninety-five human posttransplant cardiac biopsy specimens were immunoreacted with antibodies to E-selectin and VCAM-1, and endothelial expression of both adhesion molecules was recorded as present or absent. Cardiac graft rejection was graded in blinded fashion. The frequency of E-selectin expression was 11% in biopsies without rejection, 36% in mild rejection, and 58% in moderate rejection, a significant correlation (P < .001). VCAM-1 expression was present in 11% of biopsies with no rejection, 37% with mild rejection, and 85% with moderate rejection, corroborating the previously reported strong correlation between VCAM-1 expression and graft rejection (P < .0001). In 71% of specimens, E-selectin expression coincided with VCAM-1 expression. In the remaining 29% of specimens in which E-selectin and VCAM-1 expression were not both present, isolated E-selectin expression was found more frequently in biopsies with early, increasing rejection, whereas isolated VCAM-1 expression was found more frequently in specimens with established moderate rejection and later, resolving rejection. CONCLUSIONS: E-selectin is expressed in cardiac allograft rejection and may play a role in recruitment of lymphocytes into the graft. Rejection trend analysis suggests that E-selectin expression may be prominent early in the course of rejection.

Neovascular expression of E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte content.

BACKGROUND: Leukocyte recruitment is an early event in atherogenesis, and the leukocyte adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) recently have been detected in human atherosclerosis. However, no previous study has evaluated either the distribution of these three molecules at different sites within the arterial intima or their relation to plaque leukocyte content. METHODS AND RESULTS: Immunohistochemistry was performed on 99 coronary artery segments (34 controls and 65 with atherosclerotic plaque) to identify E-selectin, ICAM-1, VCAM-1, macrophages, smooth muscle cells, and T lymphocytes. For each segment, the presence or absence of adhesion molecule was determined at the arterial lumen, on intimal neovasculature, and on intimal nonendothelial cells. Each segment was scored for intimal macrophage and T-lymphocyte densities on a semiquantitative scale of 0 to 3. In atherosclerotic plaques, the prevalences of E-selectin, ICAM-1, and VCAM-1 on plaque neovasculature were twofold higher than their prevalences on arterial luminal endothelium. E-selectin was the only adhesion molecule for which expression on arterial luminal endothelial cells was more prevalent in plaques than in control segments. Increased plaque intimal macrophage density was associated with expression of VCAM-1 on neovasculature (P < .01) and on nonendothelial cells (P < .01). Increased plaque intimal T-lymphocyte density was associated with the presence of both ICAM-1 and VCAM-1 on neovasculature (both P < .01) and on nonendothelial cells (both P < .01). CONCLUSIONS: In atherosclerotic plaques, the expression of all three leukocyte adhesion molecules was more prevalent on intimal neovasculature than on arterial luminal endothelium. Further, the presence on neovasculature and nonendothelial cells of VCAM-1 and ICAM-1 was strongly associated with increased intimal leukocyte accumulation. These findings suggest that leukocyte recruitment through and/or activation of intimal neovasculature may play important roles in the pathogenesis of human atherosclerosis.

Apolipoprotein E localization in human coronary atherosclerotic plaques by in situ hybridization and immunohistochemistry and comparison with lipoprotein lipase.

Apolipoprotein E (apo E) mediates both lipid accumulation by and removal from cells and may be secreted by both macrophages and smooth muscle cells in vitro, but its cellular source in atherosclerotic plaques is not known. Lipoprotein lipase (LPL) also enhances cell lipid accumulation and is synthesized by macrophage foam cells in atherosclerotic plaques. To determine the cellular source of apo E in human coronary atherosclerotic lesions and its relationship to LPL synthesis, in situ hybridization and immunohistochemistry were performed on 12 atherosclerotic plaques and six nondiseased coronary artery segments from 10 cardiac transplant recipients. Apo E messenger RNA was localized to both non-foam cell and foam cell macrophages in plaques, but not to other cell types, and was not detected in nonatherosclerotic arteries. Half of the regions with non-foam cell macrophages expressed neither apo E nor LPL messenger RNA, whereas 86% of macrophage foam cell-containing regions contained both messenger RNAs. Polyclonal antisera raised against human apo E localized apo E protein to the surface of macrophages and surrounding matrix in plaques but not in control coronary segments. An LPL-specific monoclonal antibody demonstrated that, similar to apo E, LPL protein on foam cell and non-foam cell macrophages was detected in atherosclerotic lesions, but LPL was also localized to intimal muscle smooth muscle cells and was not distributed as widely in association with matrix as was apo E. The expression of both apo E and LPL in atherosclerotic lesions but not in normal intima suggest that these molecules play a role in lipid metabolism in atherosclerosis.