Fibronectin-α4ß1 interactions in hepatic cold ischemia and reperfusion injury: regulation of MMP-9 and MT1-MMP via the p38 MAPK pathway.

Liver ischemia-reperfusion injury (IRI) remains a challenging problem in clinical settings. The expression of fibronectin (FN) by endothelial cells is a prominent feature of the hepatic response to injury. Here we investigate the effects of the connecting segment-1 (CS-1) peptide therapy, which blocks FN-α4ß1 integrin leukocyte interactions, in a well-established model of 24-h cold liver IRI. CS-1 peptides significantly inhibited leukocyte recruitment and local release of proinflammatory mediators (COX-2, iNOS and TNF-α), ameliorating liver IRI and improving recipient survival rate. CS1 therapy inhibited the phosphorylation of p38 MAPK, a kinase linked to inflammatory processes. Moreover, in addition to downregulating the expression of matrix metalloproteinase-9 (MMP-9) in hepatic IRI, CS-1 peptide therapy depressed the expression of membrane type 1-matrix metalloproteinase (MT1-MMP/MMP-14) by macrophages, a membrane-tethered MMP important for focal matrix proteolysis. Inhibition of p38 MAPK activity, with its pharmacological antagonist SB203580, downregulated MMP-9 and MT1-MMP/MMP-14 expressions by FN-stimulated macrophages, suggesting that p38 MAPK kinase pathway controls FN-mediated inductions of MMP-9 and MT1-MMP/MMP-14. Hence, this study provides new insights on the role of FN in liver injury, which can potentially be applied to the development of new pharmacological strategies for the successful protection against hepatic IRI.

Cytoprotective effects of a cyclic RGD peptide in steatotic liver cold ischemia and reperfusion injury.

The serious need for expanding the donor population has attracted attention to the use of steatotic donor livers in orthotopic liver transplantation (OLT). However, steatotic livers are highly susceptible to hepatic ischemia-reperfusion injury (IRI). Expression of fibronectin (FN) by endothelial cells is an important feature of hepatic response to injury. We report the effect of a cyclic RGD peptide with high affinity for the alpha5beta1, the FN integrin receptor, in a rat model of steatotic liver cold ischemia, followed by transplantation. RGD peptide therapy ameliorated steatotic IRI and improved the recipient survival rate. It significantly inhibited the recruitment of monocyte/macrophages and neutrophils, and depressed the expression of pro-inflammatory mediators, such as inducible nitric oxide synthase (iNOS) and interferon (IFN)-gamma. Moreover, it resulted in profound inhibition of metalloproteinase-9 (MMP-9) expression, a gelatinase implied in leukocyte migration in damaged livers. Finally, we show that RGD peptide therapy reduced the expression of the 17-kDa active caspase-3 and the number of apoptotic cells in steatotic OLTs. The observed protection against steatotic liver IRI by the cyclic RGD peptides with high affinity for the alpha5beta1 integrin suggests that this integrin is a potential therapeutic target to allow the successful utilization of marginal steatotic livers in transplantation.

Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury.

We examined the effects of upregulation of heme oxygenase-1 (HO-1) in steatotic rat liver models of ex vivo cold ischemia/reperfusion (I/R) injury. In the model of ischemia/isolated perfusion, treatment of genetically obese Zucker rats with the HO-1 inducer cobalt protoporphyrin (CoPP) or with adenoviral HO-1 (Ad-HO-1) significantly improved portal venous blood flow, increased bile production, and decreased hepatocyte injury. Unlike in untreated rats or those pretreated with the HO-1 inhibitor zinc protoporphyrin (ZnPP), upregulation of HO-1 by Western blots correlated with amelioration of histologic features of I/R injury. Adjunctive infusion of ZnPP abrogated the beneficial effects of Ad-HO-1 gene transfer, documenting the direct involvement of HO-1 in protection against I/R injury. Following cold ischemia/isotransplantation, HO-1 overexpression extended animal survival from 40% in untreated controls to about 80% after CoPP or Ad-HO-1 therapy. This effect correlated with preserved hepatic architecture, improved liver function, and depressed infiltration by T cells and macrophages. Hence, CoPP- or gene therapy-induced HO-1 prevented I/R injury in steatotic rat livers. These findings provide the rationale for refined new treatments that should increase the supply of usable donor livers and ultimately improve the overall success of liver transplantation.

Fibronectin-alpha4beta1 integrin interactions modulate p42/44 MAPK phosphorylation in steatotic liver cold ischemia-reperfusion injury.

We investigated the effects of the connecting segment-1 (CS1) peptide, which blocks fibronectin (FN)-alpha4beta1 integrin interactions upon cell signaling, leukocyte migration, and secretion of proinflammatory cytokines, in a well-established steatotic rat liver model using ex vivo cold ischemia followed by isotransplantation. In this model, CS1 peptides were administered through the portal vein of steatotic Zucker rat livers prior and after cold ischemic storage. Lean Zucker recipients of fatty orthotopic liver transplantation (OLT) received an additional 3-day course of CS1 peptides post-OLT. CS1 peptide-treated steatotic OLTs harvested at 1, 3, and 7 days showed moderated levels of p42/44 mitogen-activated protein kinase (MAPK) phosphorylation, comparable to those observed in steatotic naive livers. In contrast, p42/44 MAPK phosphorylation was found up-regulated in 1- to 3-day damaged control OLTs. However, 7-day control OLTs were characterized by virtually lack of p42/44 MAPK phosphorylation. Lack of p42/44 MAPK phosphorylation in 7-day control OLTs was correlated with massive presence of leukocytes in the grafts and elevated levels of proinflammatory cytokines. CS1 peptide-treated OLTs at 7 days showed a profound decrease in T-cell (10 +/- 3 vs 56 +/- 20, P < .03) and monocyte/macrophage (+/++ vs +++) infiltration and significantly reduced levels of cytokine expression, such as IL-2 (approximately sixfold), and IFN-gamma (approximately three- to fourfold), as compared with controls.

Cyclic RGD peptides with high affinity for alpha5beta1 integrin protect genetically fat Zucker rat livers from cold ischemia/reperfusion injury.

We tested a hypothesis that interactions between fibronectin (FN), a key extracellular matrix component, and its integrin alpha5beta1 receptor are important in the development of ischemia/reperfusion (I/R) injury of steatotic liver transplants. We examined the effect of a cyclic RGD peptide (cRGD), with high affinity for alpha5beta1 integrin, in a well-established steatotic rat liver model of ex vivo cold ischemia followed by isotransplantation. In this model, cRGD peptides were administered through the portal vein of steatotic Zucker rat livers prior to and after cold ischemic storage. Lean Zucker recipients of fatty orthotopic liver transplants (OLTs) received an additional course of cRGD peptides 1 hour posttransplantation. cRGD peptide therapy significantly inhibited the recruitment of monocyte/macrophages, and repressed the expression of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Moreover, it resulted in selective inhibition of inducible nitric oxide synthase (iNOS), and MMP-9 expression. Importantly, cRGD peptide therapy improved the function and histologic preservation of steatotic liver grafts, extending their 14-day survival in lean recipients from 50% in untreated to 100% in cRGD-treated OLTs. Thus, cRGD peptide-mediated blockade of FN-alpha5beta1 interaction protects against severe I/R injury otherwise experienced by steatotic OLTs.

Blockade of fibronectin-alpha4beta1 adhesive interactions down-regulates cyclooxygenase-2 inducible nitric oxide synthase and prolongs recipient survival in a 24-hour model of cold hepatic ischemia-reperfusion injury.

We investigated the effects of the connecting segment-1 (CS1) peptide, which blocks fibronectin (FN)-alpha4beta1 integrin interactions, upon recipient survival and extent of tissue injury in a well-established rat liver model of ex vivo 24-hour cold ischemia followed by isotransplantation. In this model, CS1 peptides were administered through the portal vein of rat livers prior to and after cold ischemic storage. In addition, recipients of orthotopic liver transplants (OLT) received a dose of CS1 peptides 1 hour post-OLT. CS1 peptide therapy significantly inhibited the intragraft recruitment of T lymphocytes and neutrophil activation/infiltration, and repressed important mediators of inflammation, such as cyclooxygenase-2, and inducible nitric oxide synthase expression. Importantly, CS1 peptide therapy improved function/histological preservation of liver grafts and extended their 14-day survival from 50% in control to 100% in CS1-treated OLTs. Thus, CS1 peptide-mediated blockade of FN-alpha4beta1 interaction protects against severe ischemia-reperfusion injury experienced otherwise by OLTs. These novel findings document the potential of targeting FN-alpha4beta1 in vivo interaction for improving OLT outcomes.

The expression of extracellular matrix proteins during accelerated rejection of cardiac allografts in sensitized rats.

Extracellular matrix (ECM) proteins exert comitogenic effects on T cell activation in vitro, and may play a role in selective lymphocyte migration and tissue positioning in vivo. However, putative roles of ECM in host immune responses leading to graft rejection remain largely obscure. The present study evaluates intragraft expression of prominent ECM components, laminin (LN) and fibronectin (FN), and analyzes their interactions with host cellular repertoire in a well-defined cardiac allograft model in sensitized rats. (LEW x BN)F1 cardiac allografts are rejected within 24 hr in sensitized LEW rats. Immunohistochemical analysis has revealed that this brisk rejection response was associated with an early increase in the intermyocyte and endothelial deposition of LN and FN at the graft site, with their peak at 6 hr after transplantation. The upregulation of ECM preceded intragraft cellular infiltration, which at 6 hr consisted primarily of monocytes and macrophages. The infiltrating cells localized selectively in FN-rich cardiac interstitial and perivascular areas, as documented by two-color staining and laser scanning confocal microscopy. Marginal and transient increase in ECM expression was noted within control isografts. Next, we tested the role of ECM proteins in systemic lymphocyte recirculation. Specifically sensitized lymph node lymphocytes (LNL) were labelled in vitro with a DNA-binding fluorochrome, H33342, and injected i.v. into secondary engrafted recipients, which were sacrificed 6 hr later. These LNL were detected within host cervical lymph nodes in close association with FN deposits. Moreover, LNL labeled in vitro with a cationic membrane-binding fluorochrome, Di "I", were traced in hosts the lymph nodes of which were analyzed for simultaneous detection of LN and FN. Again, transferred LNL migrated selectively to FN-containing compartments, as shown by laser scanning confocal microscopy. Thus, ECM proteins should be regarded as active and important participants in host immune responses leading to graft rejection. FN may act as an ECM component "signal" for graft-infiltrating cells, and may play a key role in selective homing and specific tissue positioning of recirculating specifically sensitized lymphocytes in host peripheral lymphoid tissues.

Blockade of very late antigen-4 integrin binding to fibronectin in allograft recipients. II. Treatment with connecting segment-1 peptides prevents chronic rejection by attenuating arteriosclerotic development and suppressing intragraft T cell and macrophage activation.

BACKGROUND: Chronic rejection remains the leading obstacle to long-term allograft survival. We have shown that treatment of sensitized rats with rapamycin (RPM) does not prevent progressive chronic-type cardiac allograft failure. Having documented the role of fibronectin (FN) in the allograft rejection cascade, we hypothesized that treatment with synthetic peptides that specifically block adhesive interactions between the connecting segment-1 (CS1)-binding domain of FN and alpha4beta1 integrin on circulating cells may prevent the development of chronic rejection in transplant recipients. METHODS AND RESULTS: Lewis rats were sensitized with Brown Norway skin grafts (day -7), followed by transplantation of LBNF1 hearts (day 0). Experimental animals were treated with RPM (day -7 to -1; 0.25 mg/kg/day i.p.), or RPM + CS1 peptides (day +7 to +13; 4 mg/kg/day i.v.), and euthanized at day 60. Unlike cardiac allografts in rats undergoing RPM monotherapy, those after adjunctive CS1 peptides had well preserved myocardial architecture and were free of arteriosclerotic lesions. Moreover, reverse transcription-polymerase chain reaction-based intragraft expression of transcripts for CD3, interferon-gamma, interleukin-12, monocyte chemoattractant protein-1, and transforming growth factor-beta were diminished in the CS1 group when compared with levels in the RPM group. The corresponding expression of cytokine proteins, as determined by immunoperoxidase labeling, was also depressed and correlated with decreased infiltration by T cells and macrophages. CONCLUSION: CS1 peptide-facilitated blockage of alpha4beta1-FN interactions prevents the development of chronic rejection and depresses the expression of key T cell- and macrophage-associated cytokines/chemoattractants. Hence, local synthesis of FN is an ongoing feature of, and adhesive FN-alpha4beta1 associations are critical for, the development of chronic transplant rejection.

Fas ligand gene transfer prolongs rat renal allograft survival and down-regulates anti-apoptotic Bag-1 in parallel with enhanced Th2-type cytokine expression.

BACKGROUND: Fas ligand (FasL) induces apoptosis of cells bearing Fas receptor, and may play a role in the acquisition of immune privilege. We have previously shown that adenovirus (Ad)-mediated FasL gene transfer significantly prolongs survival in a strongly major histocompatibility complex-incompatible rat kidney allograft model. This study analyzes putative mechanisms of FasL-mediated effects, with particular emphasis on Th1 and Th2 immune activation and Bag-1 expression, a Bcl-2-binding anti-apoptotic protein. METHODS: Kidney transplants were performed in Wistar-Furth to Lewis rat combination. Donor kidneys were perfused in situ with Ad-FasL or Ad-beta-Gal, and then transplanted. Kidney allografts were harvested at days 2, 7, and 56 and were evaluated by hematoxylin and eosin and immunohistochemical staining. The expression of FasL, Bag-1, and Th1/Th2 cytokine genes was assessed by Northern blots, Western blots, and competitive template reverse-transcriptase polymerase chain reaction, respectively. RESULTS: Intragraft expression of FasL was enhanced, whereas that of anti-apoptotic Bag-1 gene was diminished throughout, in Ad-FasL-transduced well-functioning renal allografts, compared with Ad-beta-Gal-treated rejecting controls. In parallel, the expression of mRNA coding for IL-2 and IFN-gamma remained depressed, whereas that of IL-4 and IL-10 reciprocally and progressively increased in the Ad-FasL animal group. CONCLUSIONS: Prolonged survival in Ad-FasL-transduced rat renal allograft model correlates with down-regulation of Bag-1, a novel anti-apoptotic gene, and preferential Th2-type cytokine elaboration profile at the graft site. Because Th1-like cells are sensitive to FasL-mediated cytotoxic effects, T-cell apoptosis may preferentially spare Th2-like cells, with resultant prolonged graft survival.

Blockade of very late antigen-4 integrin binding to fibronectin in allograft recipients: I. Treatment with connecting segment-1 peptides prevents acute rejection by suppressing intragraft mononuclear cell accumulation, endothelial activation, and cytokine expression.

BACKGROUND: Allograft rejection is associated with infiltration of inflammatory cells and local deposition of fibronectin (FN). This study was carried out to examine the hypothesis that peptides known to specifically block adhesive interactions between the connecting segment-1 (CS1)-binding domain of FN and alpha4beta1 integrin on circulating cells may interfere with the immune cascade, which would lead to acute rejection in transplant recipients. METHODS AND RESULTS: Cardiac allografts from Lewis x Brown Norway F1 hybrids were rejected in 7+/-1 days in Lewis rats. Treatment with bioactive CS1 peptides (4 mg/kg/day i.v. for 7 days) abrogated acute rejection and prolonged cardiac allograft survival to 13+/-1 days (P<0.001). This effect correlated with decreased expression of total fibronectin and cell adhesion molecules, such as alpha4beta1, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, as well as reduced infiltration by CD4+ and CD8+ T cells at the graft site. Treatment with CS1 peptides decreased alloantigen activation, as evidenced by decreased intragraft infiltration by CD25+ cells, and diminished expression of mRNA coding for Th1 (interleukin [IL]-2, interferon-gamma)- and Th2 (IL-4, IL-5, IL-6)-type cytokines. CS1-mediated immunosuppressive effects could be reversed and acute rejection recreated after adjunctive treatment of rats with recombinant IL-2. CONCLUSION: Our data are consistent with the model in which in vivo interaction between the alpha4beta1 integrin receptor and the cell-associated CS1 motif of FN is critical for rejection cascade. The novel therapeutic approach of selectively blocking the alpha4beta1-FN activation pathway with CS1 peptides prevents acute allograft rejection by inhibiting expansion of antigen-specific T cells and inducing a transient state of cytokine-responsive anergy in the residual T-cell population.

Allochimeric class I MHC molecules prevent chronic rejection and attenuate alloantibody responses.

BACKGROUND: We have shown that treatment with molecularly engineered, allochimeric [alpha1 hl/u]-RT1.Aa class I MHC antigens bearing donor-type Wistar-Furth (WF, RT1.Au) amino acid substitutions for host-type ACI (RTI.Aa) sequences in the alpha1-helical region induces donor-specific tolerance to cardiac allografts in rat recipients. This study examined the effect of allochimeric molecules on the development of chronic rejection. METHODS: Allochimeric [alpha1 hl/u]-RT1.Aa class I MHC antigenic extracts (1 mg) were administered via the portal vein into ACI recipients of WF hearts on the day of transplantation in conjunction with subtherapeutic oral cyclosporine (CsA, 10 mg/kg/day, days 0-2). Control groups included recipients of syngeneic grafts and ACI recipients of WF heart allografts treated with high-dose CsA (10 mg/kg/day, days 0-6). RESULTS: WF hearts in ACI rats receiving 7 days of CsA exhibited myocardial fibrosis, perivascular inflammation, and intimal hyperplasia at day 80. At day 120, these grafts displayed severe chronic rejection with global architectural disorganization, ventricular fibrosis, intimal hyperplasia, and progressive luminal narrowing. In contrast, WF hearts in rats treated with [alpha1 hl/u]-RT1.Aa molecules revealed only mild perivascular fibrosis, minimal intimal thickening, and preserved myocardial architecture. Alloantibody analysis demonstrated no IgM alloantibodies in all groups. An attenuated, but detectable, anti-WF IgG response was present in recipients receiving allochimeric molecules, with IgG1 and IgG2a subclasses predominating. Immunohistochemical analysis of allografts demonstrated minimal T cell infiltration and IgG binding to vascular endothelium. CONCLUSION: Treatment with allochimeric molecules prevents the development of chronic rejection. Such effect may be in part caused by deviation of host alloantibody responses.

Extracellular matrix proteins: bystanders or active participants in the allograft rejection cascade?

The role extracellular matrix (ECM) proteins may play in the host immune cascade triggered by organ transplantation is largely unknown. We have employed well-defined rat cardiac allograft model to test the hypothesis that the expression of ECM components, such as fibronectin (FN) or laminin (LN), represents an integral part of the host rejection response in transplant recipients. Indeed, treatment of rats with anti-LN Ab profoundly affected lymphocyte recirculation pathways, and selectively decreased deposition of alloreactive lymphocytes at the graft site and in host peripheral lymph nodes. Moreover, cardiac rejection was accompanied by markedly increased intragraft expression of both LN and FN. The infiltrating mononuclear cells, however, accumulated selectively in FN-rather than LN-rich cardiac areas. We also observed distinct temporal expression and spatial distribution patterns of FN variants in rat cardiac allografts and isografts. The local synthesis of FN, in both allografts and isografts was increased as early as at 3 h post-transplant. The expression of FN in cardiac allografts but not in isografts, remained high during later intervals to the point of ultimate rejection. The newly synthesized FNs derived from graft infiltrating macrophages and arterioles, and included EIIIA +, EIIIB +, and CS-I + splicing variants. The EIIIA was preferentially enriched in the earlier, whereas EIIIB and CS-I were selectively enhanced in the later, rejection phases. Finally, treatment of rat recipients with synthetic FN-CS-I peptides prevented the incidence of early (acute) and late (chronic) rejection, supporting the notion that distinct FNs may play a fundamental role in the host immune cascade triggered by organ transplantation. These data offer potential novel sites for intervention in the control of transplant rejection, and contribute to the development of refined therapeutic strategies based upon new concepts of host immunosuppression.