Expression of fibronectin splicing variants in organ transplantation: a differential pattern between rat cardiac allografts and isografts

Allograft rejection is associated with infiltration of inflammatory cells and deposition of extracellular matrix proteins. The extent to which diversity in the extracellular matrix regulates inflammatory cell function in transplants remains unclear. One group of extracellular matrix proteins, termed fibronectins (FNs), exhibits inherent diversity as a consequence of alternative splicing in three segments: EIIIA, EIIIB, or V. Although the EIIIA segment has documented functions in mesenchymal cell differentiation, neither this segment nor the EIIIB segment have been tested for effects specific to leukocyte functions. By contrast, the V region can include the CS-1 segment to which leukocytes may adhere through alpha 4 beta 1 integrins. In this study, we demonstrate that EIIIA+, EIIIB+, and V+ FN variants are synthesized, primarily by macrophages in distinct temporal and spatial patterns in two rat cardiac transplant models: either with antigenic challenge, allografts, or without challenge, isografts. The ratio of EIIIA inclusion into FN increases by day 1 in allografts and isografts and remains high until allografts are rejected (approximately 7 days) but falls to normal levels in tolerated isografts (day 6). EIIIB+ FN ratios in allografts peak later than do EIIIA+ FNs (day 4). EIIIB+ FN ratios remain relatively low in isografts. Interestingly, EIIIA+ and EIIIB+ FNs are deposited prominently in the myocardium of rejecting allografts in close association with infiltrating leukocytes, and FN expression and deposition are prominent at sites of infarction. By contrast, these FNs are largely restricted to the epicardium and to a lesser degree in the immediately adjacent myocardium in isografts. CS-1+ FNs increase in allografts and isografts at 3 hours after transplantation but are particularly prominent in allografts 1 to 3 days before rejection. Our data suggest that FN splicing variants have a differential role in the effector functions of leukocytes in allografts and isografts and provide a foundation for testing their function on leukocytes and a rationale for FN-based therapeutics to modulate allograft rejection in transplant recipients.     

Tolerance induction ameliorates allograft vasculopathy in rat aortic transplants. Influence of Fas-mediated apoptosis

Based on successful induction of donor-specific unresponsiveness by alloantigenic stimulation in several animal models of acute rejection, we hypothesized that similar immune manipulations would also inhibit the evolution of chronic rejection and transplant vasculopathy. To induce immune tolerance, DA rats received a PVG heart allograft and were immunosuppressed with cyclosporine for 30 d. At day 100 the animals were challenged with a PVG aortic allograft after either 1 or 18 h of cold ischemia. 8 wk after the aortic transplantation, the grafts were investigated for morphological changes, infiltrating cells, apoptosis, and Fas-Fas ligand expression. Control allografts showed advanced transplant arteriosclerosis, whereas tolerance-induced aortic allografts displayed reduced neointimal formation, less medial atrophy, fewer apoptotic cells, and fewer Fas- and FasL-expressing cells. Prolonged ischemic storage time did not profoundly alter the morphological changes of the allografts. Fas expression was found in T cells, macrophages, vascular smooth muscle cells, and endothelial cells, whereas FasL was expressed mainly by T cells and macrophages. FasL mRNA expression was evident throughout the entire allograft wall. In conclusion, induction of allospecific tolerance can effectively prevent transplant arteriosclerosis. Cold ischemia damage does not abrogate the beneficial effect of tolerance, but creates a separate identity of mainly endothelial lesions. Furthermore, Fas-mediated apoptosis appears to be involved in the pathological lesions seen in chronic rejection.     

SEM evaluation of endothelial changes following end-to-side microvascular anastomoses in rats

A scanning electron microscopic (SEM) study was carried out to evaluate the endothelial changes in carotid end-to-side microsurgical anastomoses. This procedure, associated with occlusion of the brachiocephalic trunk, was performed in 24 Sprague-Dawley rats. The animals were sacrificed at time intervals ranging between 15 minutes and 14 days after surgery. The specimens were collected after perfusion-fixation in vivo and treated for SEM observations. The findings show that the first 45 minutes are the most critical time for thrombus formation. Reendothelialization could be detected at 3 days after the microsurgical procedure. Endothelial regeneration occurred earlier on the suture line than on the stitches, documented by the migration and morphological changes of the endothelial cells advancing from the normal endothelium to cover the defect and the suture material. The repair was usually completed within 14 days. Damage of the inner vascular wall, related to minor failures of the microsurgical technique, appears to be a cause of delayed reendothelialization.     

Morphology and function of preserved microvascular arterial grafts: an experimental study in rats

The aim of this study is to examine the morphology and function and small-caliber, arterial grafts after preservation in the University of Wisconsin solution (UW). Rat carotid arteries were stored in UW (n = 10) or in phosphate-buffered saline (PBS) (n = 10) for 1, 3, 7, and 14 days and were examined with light microscopy (LM) and scanning electron microscopy (SEM). Rat aortic preparations were stored in UW or PBS for 1 hour, 24 hours, 72 hours, 7 days, and 14 days and assessed for functional responses (stimulated contraction and endothelium-dependent relaxation). Segments (5 mm) of rat carotid arteries were stored in UW or PBS for 3 days, 7 days, and 14 days and orthotopically implanted as autografts and allografts. No immunosuppressive or anticoagulant agents were used. After 28 days of implantation, the grafts were assessed for patency and excised for LM and SEM. In UW, the endothelial layer remained intact up to 9 days of storage. In PBS, the endothelial layer showed deterioration after 1 day and was completely lost after 3 days. Functional responses were demonstrated to exist for as long as 7 days storage in UW. In PBS, no responses could be evoked after 24 hours storage. Autografts preserved in UW for 3 days (n = 6), 7 days (n = 6), and 14 days (n = 6) showed patency rates of 83.3%, 66.6%, and 66.6%, respectively, whereas patency rates of allografts were 66.6%, 33.3%, and 33.3%, respectively. Autografts stored in PBS for 3 days (n = 6), 7 days (n = 6), and 14 days (n = 6) showed patency rates of 33.3%, 33.3%, and 50%, respectively, whereas patency rates of allografts were 16.7%, 0%, and 33.3%, respectively. The UW preserved autografts showed normal morphology. All other groups showed vessel wall degeneration which in the allograft groups, were accompanied by lymphocellular infiltration. In conclusion, the endothelial layer and vessel wall of arteries are adequately preserved in UW. Functional responses are retained up to 14 days storage in UW, but, are lost after 24 hours storage in PBS. Autograft implantation studies accordingly show good performance of arterial segments preserved in UW, whereas allografts are subject to degradation as a result of rejection.     

Gas chromatography-mass spectrometry of Alternaria mycotoxins

BACKGROUND: We have previously reported that vascular inducible nitric oxide synthase (iNOS) gene transfer inhibits injury-induced intimal hyperplasia in vitro and in vivo. One mechanism by which NO may prevent intimal hyperplasia is by preserving the endothelium or promoting its regeneration. To study this possibility we examined the effect of iNOS gene transfer on endothelial cell (EC) proliferation and viability. METHODS: An adenoviral vector (AdiNOS) containing the human iNOS cDNA was constructed and used to infect cultured sheep arterial ECs. NO production was measured, and the effects of continuous NO exposure on EC proliferation, viability, and apoptosis were evaluated. RESULTS: AdiNOS-infected ECs produced 25- to 100-fold more NO than control (AdlacZ) infected cells as measured by nitrite accumulation. This increased NO synthesis did not inhibit EC proliferation as reflected by tritiated thymidine incorporation. Chromium 51 release assay revealed that EC viability was also unaffected by AdiNOS infection and NO synthesis. In addition, prolonged exposure to NO synthesis did not induce EC apoptosis. Instead, NO inhibited lipopolysaccharide-induced apoptosis in these cells by reducing caspase-3-like protease activity. CONCLUSIONS: Vascular iNOS gene transfer, while inhibiting smooth muscle cell proliferation, does not impair EC mitogenesis or viability. Augmented NO synthesis may also protect ECs against apogenic stimuli such as lipopolysaccharide. Therefore iNOS gene transfer may promote endothelial regeneration and can perhaps accelerate vascular healing.     

Cell-free arterial grafts: morphologic characteristics of aortic isografts, allografts, and xenografts in rats

PURPOSE: Chronic rejection of arterial allografts and xenografts results in arterial wall dilation and rupture, making them unsuitable for long-term arterial replacement in vascular surgery. In the arterial wall, as in other organs, the cells probably carry major antigenic determinants. Arterial wall cellular components can be removed by detergent treatment to produce a graftable matrix tube. METHODS: We compared the patency and macroscopic and microscopic morphologic changes that occurred in sodium dodecyl sulfate (SDS)-treated and untreated arterial isografts, allografts, and xenografts 2 months after implantation in rats. We quantified elastin, collagen, and nuclear density in the three layers of the graft wall (intima, media, and adventitia) by morphometric methods. The SDS treatment removed endothelial and smooth muscle cells and cells in the adventitia but preserved elastin and collagen extracellular matrix. RESULTS: All arterial xenografts, whether SDS treated or untreated, were aneurysmal 2 months after grafting, with loss of the medial cellular and extracellular components. In allografts, SDS treatment prevented dilation, reduced adventitial inflammatory infiltration, and preserved medial elastin. The SDS-treated allografts had an evenly distributed, noninflammatory intimal thickening that was richer in elastin fibers than that in untreated allografts. CONCLUSIONS: These results suggest an interspecies, but not an intraspecies, graft antigenicity of arterial extracellular matrix. The SDS treatment prevented chronic rejection of the arterial allograft and led to the proliferation of an elastin-rich and adapted intima.     

DNA requirements in vivo for phage T4 packaging

OBJECTIVE: We sought to determine the morphology, mechanisms of deterioration, cellular viability, extracellular matrix integrity, and the role of immune responses in the dysfunction of cryopreserved aortic and pulmonic valve allografts. METHODS: We studied 33 explanted left-sided (n = 20) or right-sided (n = 13) cryopreserved human allograft heart valves explanted several hours to 9 years after operation, 14 nonimplanted allografts, and 16 aortic valves removed from transplanted allograft hearts 2 days to 4 years after operation. Analysis included gross inspection, radiography, light microscopy, electron microscopy, and immunohistochemical studies. RESULTS: Allografts implanted for more than 1 day had progressive collagen hyalinization and loss of normal structural complexity and cellularity, including endothelium and deep connective tissue cells. Inflammatory cells were generally minimal or absent in the allografts. Transmission electron microscopy of long-term cryopreserved allograft valves revealed no viable cells, focal calcification centered around dead cell remnants, and distorted but preserved collagen. In contrast, aortic valves from transplanted hearts showed remarkable structural preservation, including endothelium and abundant deep connective tissue cells; inflammatory infiltrates were generally mild and of no apparent deleterious consequence, including valves from patients who died of fatal rejection. CONCLUSIONS: Cryopreserved allografts are morphologically nonviable; their collagen is flattened but largely preserved. They are unlikely to grow, remodel, or exhibit active metabolic functions, and their usual degeneration cannot be attributed to immunologic responses. In contrast, aortic valves of transplanted hearts maintain near-normal overall architecture and cellularity and do not show apparent immunologic injury, even in the setting of fatal myocardial parenchymal rejection or graft arteriosclerosis.     

Apoptosis within spontaneously accepted mouse liver allografts: evidence for deletion of cytotoxic T cells and implications for tolerance induction

MHC-mismatched liver grafts are accepted spontaneously between many mouse strains. The underlying mechanism(s) is unclear. In the B10 (H2(b)) to C3H (H2(k)) strain combination used in this study, donor T cells within the liver were rapidly replaced within 2 to 4 days of transplantation with those of the recipient. Freshly isolated liver graft-infiltrating cells harvested on days 4 and 7 exhibited strong CTL responses against donor alloantigens. CTL activity was reduced substantially, however, by day 14, although levels of CTL precursors in the spleen and liver remained high. Examination of the liver allografts by in situ terminal deoxynucleotidyltransferase-catalyzed dUTP-digoxigenin nick end labeling on days 4, 7, and 14 after transplantation revealed prominent apoptotic cells dispersed throughout the nonparenchymal cell population. When acute liver allograft rejection was induced by administration of IL-2 from days 0 to 4 post-transplant (median survival time, 5 days), apoptotic activity (day 4) was reduced substantially, whereas CTL activity was enhanced. Nonparenchymal cells isolated from allografts of unmodified recipients 4, 7, and 14 days after transplantation exhibited significantly higher DNA fragmentation after 18-h culture than cells from liver isografts. Moreover, the level was 4 to 5 times higher than that of cells from IL-2-treated mice (on day 4). These observations suggest that T cell deletion, not regulation, may be responsible for spontaneous liver allograft acceptance. The molecular recognition events that cause apoptosis of infiltrating T cells and why this occurs within liver grafts, but not heart or skin grafts, remain to be elucidated.     

Progressive albuminuria and glomerulosclerosis in a rat model of chronic renal allograft rejection

A significant proportion of renal allografts fail within several months or years after transplantation, primarily because of chronic rejection. The etiology and pathophysiology of this condition remain unclear. We studied the renal function, morphology, and immunohistology, in parallel, among F344-to-Lewis allografts (n = 23) and isografts (n = 13) over the course of 24 weeks. Only an initial 10-day course of CsA (5 mg/kg/day) was given to both groups to prevent acute rejection. Hypertension did not develop, although awake systolic blood pressure was significantly higher in allografts at the end of the study. Significant differences in urine albumin excretion (UalbV) between isografts and allografts were evident as early as 4 weeks after engraftment but rose dramatically by 20 weeks (3.3 +/- 0.7 vs. 21.2 +/- 3.7 mg/day, respectively, P < .001). This pattern continued until the conclusion of the study (5.0 +/- 1.1 vs. 53.5 +/- 7.6 mg/day, P < .001). Serum creatinine values were only significantly elevated in allografts at 16 weeks, which temporally corresponded to the dramatic increase in UalbV. However, renal blood flow and glomerular filtration rate, measured by paraaminohippurate and inulin clearances, respectively, were significantly lower in allografted organs, at 24 weeks. The frequency of glomerulosclerosis lesions was significantly increased in allografted kidneys at 24 weeks and correlated with UalbV values. Glomerular localization of mononuclear leukocyte subsets were equivalent between allografts and isografts; however, the numbers of interstitial macrophages, CD8+, and pan-T-cells were all significantly greater in allografts at 24 weeks. The infiltration of significantly greater numbers of macrophages and lymphocytes into the tubulointerstitium of the allograft group suggests a mononuclear leukocyte effector cell mediation of the progressive glomerular abnormalities in this model of chronic renal allograft rejection in the rat.     

Fibrin glue containing fibroblast growth factor type 1 and heparin with autologous endothelial cells reduces intimal hyperplasia in a canine carotid artery balloon injury model

PURPOSE: Intimal hyperplasia plagues all types of vascular intervention. Early confluent re-endothelialization may attenuate the smooth muscle cell (SMC) proliferative response. We previously reported that fibroblast growth factor type 1 (FGF-1) and heparin at relative concentrations of 10 ng/ml:250 U/ml delivered in a fibrin glue (FG) suspension can selectively stimulate endothelial cells (EC) and inhibit SMC proliferation in cell culture. This current study evaluates this surface treatment with and without seeded autologous ECs on intimal hyperplasia in a canine carotid artery balloon injury model. METHODS: Twenty-nine adult dogs underwent bilateral balloon injury to a 6 cm segment of their carotid arteries. The injury resulted in a reproducible removal of the intima and 4 to 6 medial lamellae. Nine dogs were used in part I to determine the percent retention of FGF-1 and EC when applied in a FG suspension to the balloon-injured carotid arteries. Part 2 used the remaining 20 dogs to determine the effect of this surface treatment on intimal hyperplasia. In 10 group I dogs, FG (fibrinogen 32.1 mg/ml and thrombin 0.32 U/ml) containing FGF-1 (11 ng/ml) and heparin (250 U/ml) was applied to the luminal surface of one carotid artery, whereas the contralateral carotid artery underwent balloon injury alone. In 10 group II dogs, an identical FG preparation with FGF-1 and heparin was applied to the surface of one carotid artery, whereas the contralateral carotid artery received FG/FGF-1/heparin that also contained autologous ECs (P3; 5 x 10(4) to 10 x 10(4) cells/cm2). Five dogs from both group I and group II were killed at 10 days and the remaining 10 dogs at 30 days. Histologic analysis and computerized morphometric analysis were used to determine intimal and medial thickness and area, percent endothelialization, and medial SMC proliferative rate. RESULTS: There was no measurable neointima in any 10-day dog. There was no difference in neointimal area between the treatments in group I 30-day dogs. There was a significant decrease in maximal neointimal area, intima/media thickness ratio, and intima/media area ratio in group II 30-day dogs that were treated with FG/FGF-1/heparin plus EC. There was an insignificant increase in percent EC coverage and an insignificant decrease in medial SMC proliferative rate in group II 10-day dogs treated with FG/FGF-1/heparin plus EC. CONCLUSIONS: In this canine carotid model, FG with FGF-1 and heparin did not induce significant intimal or medial thickening after 10 or 30 days when compared with vessels that were only balloon-injured. The seeding of autologous ECs within the FG/FGF-1/heparin suspension caused a reduction in neointima formation with no concomitant medial thickening 30 days after injury. The use of FG to locally deliver FGF-1 and ECs may have clinical relevance in the inhibition of intimal hyperplasia.     

Survey of the effects of smoking in an occupational cohort study. Value and limits of a new method

The arterial wall injury associated with arterial graft implantation causes smooth muscle cells (SMCs) in the media to migrate and proliferate in the intima at the graft-artery junction resulting in anastomotic intimal hyperplasia (AIH). An important step in developing a small-diameter prosthesis may be to stimulate endothelialization and thereby inhibit AIH. In this study, we investigated the effect of coacervated and crosslinked alpha-elastin on proliferation of SMCs and endothelial cells (ECs) in vitro. Coacervation is an important step in the conversion of proelastin to make an elastin fiber in vivo. SMCs and ECs were prepared from porcine aortic media and endothelium, respectively. SMCs and ECs (three to five passages, 4 x 10[4] cells/well) were seeded onto 12 well plates, coated and crosslinked with 0 or 10 mg/mL of coacervated alpha-elastin. After the 1st, 2nd, or 3rd day of cultivation, proliferation was assayed by scintillation counting of [3H]-thymidine incorporation. For the 4th day only, 0, 0.1, 1, 10 mg/mL concentration of coacervated alpha-elastin was coated and crosslinked. SMC proliferation (1st, 2nd day: p<0.005; 3rd, 4th day: p<0.0001) was significantly inhibited over time and dose dependently, eg, 0.1 mg/mL (45.7+/-2.3%: % of control p<0.005), 1 mg/mL (5.9+/-0.7%, p<0.0005), 10 mg/mL (2.8+/-0.4%, p<0.0005). EC proliferation was inhibited over time by 10 mg/mL of coacervated alpha-elastin (2nd, 3rd day: p<0.005; 4th day: p<0.0001), but proliferation (132.8+/-9.9%: % of control p=NS) was stimulated by 0.1 mg/mL of coacervated alpha-elastin. These results suggest that coating and crosslinking a coacervated alpha-elastin into the structure of arterial prosthesis may inhibit AIH and stimulate endothelialization.     

Localization of Six4/AREC3 in the developing mouse retina; implications in mammalian retinal development

BACKGROUND: We designed an antisense phosphorothioate oligodeoxynucleotide (oligo) to specifically inhibit the expression of rat intercellular adhesion molecule-1 (ICAM-1) mRNA (IP-9125). METHODS: IP-9125 oligo was delivered intravenously by osmotic pump alone or in combination with cyclosporine (CsA) to recipients in order to prevent the rejection of kidney or heart allografts. In additional experiments, kidney allografts were perfused with IP-9125 before grafting. RESULTS: IP-9125 inhibited ICAM-1 mRNA and ICAM-1 protein expression in rat aortic endothelial cells; scrambled controls IP-12140 and IP-13944 were ineffective. Untreated ACI (RT1a) recipients rejected Lewis (RT1l) kidney allografts at a mean survival time of 8.5+/-1.1 days. A 14-day intravenous administration of 2.5 mg/kg/day IP-9125 prolonged the survival of kidney allografts to 39.2+/-16.4 days; 5.0 mg/kg/day, to 43.0+/-17.5 days; and 10.0 mg/kg/day, to 50.4+/-21.6 days. In contrast, a scrambled control IP-12140 was not effective. A combination of 10 mg/kg/day IP-9125 and 1.0 mg/kg/day CsA delivered for 14 days synergistically extended kidney allograft survival times 88.5+/-7.5 days. In contrast, the combination of 10.0 mg/kg/day control IP-12140 with CsA was ineffective (20.7+/-3.2 days) when compared with CsA alone (20.2+/-4.0 days). Similar results were obtained for heart transplants in recipients treated with IP-9125 alone or in combination with CsA. Furthermore, in situ immunostaining showed that IP-9125 significantly reduced the expression of ICAM-1 protein in kidney allografts. Finally, perfusion of kidney grafts alone with 20.0 mg per 2 ml of IP-9125 protected kidney allografts from rejection (37.5+/-7.5 days; P < 0.001), whereas perfusion with 20 mg per 2 ml of control IP-12140 was ineffective (12.6+/-5.0 days). CONCLUSIONS: Rat ICAM-1 IP-9125 oligo inhibits ICAM-1 protein expression in vitro and in vivo as well as blocks allograft rejection when used for pretreatment of donors, graft perfusion, or postoperative treatment of recipients.     

Expression of chemokine genes during rejection and long-term acceptance of cardiac allografts

Chemokines are cytokines with chemoattractant properties for leukocytes. They may play a critical role in directing leukocytes to graft sites and in amplifying intragraft inflammation during rejection. Previous studies have tested the intragraft expression of chemokine genes during the rejection of allogeneic skin grafts in mice. In the current study, we used a heterotopic heart transplant model in mice to test the intragraft expression of these genes in nonrejecting cardiac isografts, rejecting cardiac allografts, and cardiac allografts that were accepted due to immunosuppression with gallium nitrate. With the exception of low levels of interleukin-1beta and JE, intragraft expression of the the proinflammatory cytokine genes was not observed in either isografts or native heart. Two distinct patterns of chemokine mRNA were observed in the rejecting cardiac allografts. Intra-allograft expression of interleukin-1beta, interferon-gamma-inducible protein, JE, and KC was prominent by day 3 after transplantation. The expression of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and regulated upon activation, normal T cell expressed and secreted (RANTES) was at low or undetectable levels at day 3 after transplantation but at high levels by day 8 after transplantation. Sixty days after transplantation, intra-allograft expression of chemokines in hearts from gallium nitrate-treated recipients indicated low levels of MIP-1alpha, MIP-1beta, and KC but high levels of interferon-gamma-inducible protein and RANTES.     

Application of coacervated alpha-elastin to arterial prostheses for inhibition of anastomotic intimal hyperplasia

Prevention of anastomotic intimal hyperplasia (AIH) requires inhibition of the migration of smooth muscle cells (SMCs) and promotion of endothelial cell (ECs) growth from the native arterial wall. We investigated the effect of coacervated alpha-elastin on migration of SMCs and ECs in vitro. SMCs and ECs were prepared from porcine aortic media and endothelium. Coacervated alpha-elastin was coated and cross-linked around the perimeter of each 1 cm diameter center of a well in a 12 well plate. SMCs and ECs were placed and cultured within the center of each well. The migration of SMCs and ECs on coacervated alpha-elastin was assayed on the second, third (10 mg/ml), or fourth day (0.1, 1.0, 10.0 mg/ml) of cultivation by measuring the area of migration from the 1 cm diameter center. Coacervated alpha-elastin was then coated and cross-linked on a Dacron graft using 1% glycerol polyglycidyl ether (GPGE) and examined with scanning electron microscopy to determine the feasibility of graft coating. SMC migration was significantly inhibited dose dependently over time (p < 0.005), e.g., 0.1 mg/ml (45.4% +/- 2.7%: % of MES [pH 5] and 1% GPGE without alpha-elastin), 1.0 mg/ml (32.0% +/- 1.4%), 10.0 mg/ml (8.3% +/- 2.9%). EC migration (90.7% +/- 6.2%: p = ns) was not inhibited by 0.1 mg/ml of coacervated alpha-elastin. Cross-linked coacervated alpha-elastin was coated on a dacron graft uniformly. Incorporation of coacervated alpha-elastin into the structure of arterial prostheses offers the possibility of inhibition of SMC hyperplasia without inhibition of EC formation.     

Retinal pigment epithelium cells cultured on synthetic biodegradable polymers

Glycation of proteins of the vessel wall is thought to play an important role in the pathogenesis of vascular complications in diabetes by affecting structure and function of these proteins. Adhesive proteins in the extracellular matrix (ECM) of endothelial cells (ECs) are essential for attachment of ECs to the subintima. In this study, we investigated the effect of glycation of ECM and purified adhesive proteins on EC adhesion and spreading. ECM was incubated with the reactive sugar glucose-6-phosphate (0-500 mmol/l) for different time periods (0-14 days) at 37 degrees C. Degree of glycation, measured in an enzyme-linked immunosorbent assay using a monoclonal antibody specific for advanced glycation end products, increased in a time- and concentration-dependent manner. Glycation of ECM with 50 mmol/l glucose-6-phosphate resulted in increased coverage by ECs as measured in a cell adhesion assay and was the result of an increase in number of adhered cells, while cell size was unaffected. Glycation of ECM with higher concentrations of glucose-6-phosphate resulted in decreased coverage by ECs caused by both a reduction in number of adhered ECs and impaired spreading. Experiments with purified glycated matrix proteins indicate that the decrease in EC adhesion and spreading on glycated ECM may result from glycation of vitronectin. Impaired EC adhesion and spreading caused by vitronectin glycation may result in impaired endothelial function and contribute to vascular disease.     

Identification and characterization of cells infiltrating the graft and aqueous humour in rat corneal allograft rejection

In a rat model of corneal transplantation, Fischer 344 (RT1(lv1)) rats received orthotopic corneal isografts or Wistar-Furth (RT1(u)) donor allografts. Rejection was observed in 25 of 26 allograft recipients, at a median time of 18 days, with all isografts surviving > 100 days. Flow cytometric analysis of aqueous humour identified cellular infiltration of the aqueous at the time of allograft rejection, in contrast to the acellular aqueous found in isografts at corresponding times following transplantation. A higher proportion of CD8+ than CD4+ cells was found at days 1-3 following rejection, whereas there was a higher proportion of CD4+ cells at days 5-8. No changes in peripheral blood T cell subsets were found at the time of rejection. Immunohistochemical analysis of cells infiltrating recipient iris and grafted cornea undertaken at days 1-2, 4 and 7-10 following onset of rejection, demonstrated inflammatory cells in the graft epithelium, stroma and aggregated on the endothelium. Large numbers of macrophages, T cells (CD4+ > CD8+ at all time points), natural killer (NK) cells and neutrophils were detected in graft tissue at days 1-2 and 4, diminishing after that time. Most infiltrating cells expressed MHC class II antigen, and a smaller number expressed IL-2R. Expression of the co-stimulatory marker B7 was identified in a few cells at day 4 in the region of the graft-host wound. The immune response in graft rejection was characterized at day 4 also by expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells of iris and corneal vessels, demonstration of interferon-gamma on mononuclear cells in the peripheral (recipient) cornea, and tumour necrosis factor-alpha on aggregated mononuclear cells on the graft, but not recipient, endothelium. Only sparse cellular infiltrates were found in isograft controls, with inflammation located at the graft-host wound. These findings suggest that inflammatory cells reach a corneal allograft by two routes--from vessels in the peripheral recipient cornea, and from vessels in the recipient iris via the aqueous humour. Different aqueous and intragraft T cell subset proportions were seen early in rejection, although a preponderance of CD4+ cells was found in both aqueous and graft at later times.     

Structural recovery of small arteries following clamp injury: a light and electron microscopic investigation in the rat

Endothelial injuries were induced in the left common iliac arteries (1 mm in diameter) of rats, by the placement of 1 mm Scovell-Lewis microvascular clamps for 5 minutes, to create a lesion in which to quantitate the rate and degree of cellular regeneration. The left (clamp-injured) and right (control) iliac arteries from the 15 rats used in this study were viewed with the electron microscope at 2, 7, and 14 days after clamping, and the clamp sites were analysed morphometrically. At 2 days there was only minimal denudation of the endothelium; most cells were disoriented and showed some signs of traumatic injury. By 7 days there was a completely continuous endothelial lining, but there was also evidence of increased cytoplasmic activity in these cells, as well as a statistically significant simplification in their intercellular junctional morphology. These changes persisted at 14 days after injury, but they were less pronounced. Smooth muscle cells in the media were relatively unaffected by the trauma in the first 2 days after clamping. However, they exhibited a change of phenotype from contractile to synthetic by 7 days after injury. By 14 days most smooth muscle cells had reverted back to the contractile phenotype, with little evidence of residual damage. These studies reveal that the reconstitution and regeneration of the endothelium is very rapid following clamp injury, but that significant residual ultrastructural changes in the interendothelial junctions persist for at least 14 days after injury. These findings indicate that there is potential for subsequent pathological changes in sites of vascular clamp injury.     

Laminar shear stress-induced GRO mRNA and protein expression in endothelial cells

BACKGROUND: The shear stress induced by blood flow may play a pivotal role in the induction or prevention of atherosclerosis by changing endothelial functions. To disclose the mechanisms of this change, we prepared an endothelial cell (EC) cDNA library to select specific clones expressed in response to shear stress. METHODS AND RESULTS: The mRNA of cultured confluent bovine aortic ECs (BAECs) subjected to steady laminar shear stress (30 dyne/cm2) for 4 hours was separated, and a cDNA library was prepared. Nine clones whose expressions were specifically enhanced by the shear stress were selected by use of a differential hybridization method. One clone had 94% homology at the nucleotide sequence level to Oryctolagus cuniculus gro (GRO) mRNA and 79% homology at the amino acid sequence level to human GRO-beta. The GRO mRNA expression was increased in both BAECs and human umbilical vein ECs (HUVECs) after the ECs were subjected to high (30 dyne/cm2) and low (5 dyne/cm2) laminar shear stress. GRO-alpha and/or -beta protein expression also increased after the HUVECs and BAECs were subjected to shear stress. Because GRO protein has been shown to function as an adhesion factor of monocytes on the surface of ECs, we studied whether shear stress-induced monocyte adhesion was caused by GRO protein expression on ECs. The 4-hour shear stress enhanced monocyte adhesion to ECs by 2.5-fold over control levels, and this enhancement was inhibited by 53% by anti-GRO-alpha antibody. CONCLUSIONS: The present study is the first report that shear stress induced the expression of GRO mRNA and protein in ECs and enhanced the monocyte adhesion on ECs via GRO protein. Further investigations of the functions and participation in atherogenesis of this selected clone may clarify the significance of shear stress on atherogenesis.