Junichi Kohmoto

Carbon monoxide protects rat lung transplants from ischemia-reperfusion injury via a mechanism involving p38 MAPK pathway.

Carbon monoxide (CO) provides protection against oxidative stress via anti‐inflammatory and cytoprotective actions. In this study, we tested the hypothesis that a low concentration of exogenous (inhaled) CO would protect transplanted lung grafts from cold ischemia‐reperfusion injury via a mechanism involving the mitogen‐activated protein kinase (MAPK) signaling pathway. Lewis rats underwent orthotopic syngeneic or allogeneic left lung transplantation with 6 h of cold static preservation. Exposure of donors and recipients (1 h before and then continuously post‐transplant) to 250 ppm CO resulted in significant improvement in gas exchange, reduced leukocyte sequestration, preservation of parenchymal and endothelial cell ultrastructure and reduced inflammation compared to animals exposed to air. The beneficial effects of CO were associated with p38 MAPK phosphorylation and were significantly prevented by treatment with a p38 MAPK inhibitor, suggesting that COs efficacy is at least partially mediated by activation of p38 MAPK. Furthermore, CO markedly suppressed inflammatory events in the contralateral naïve lung. This study demonstrates that perioperative exposure of donors and recipients to CO at a low concentration can impart potent anti‐inflammatory and cytoprotective effects in a clinically relevant model of lung transplantation and support further evaluation for potential clinical use.

Ex Vivo Application of Carbon Monoxide in University of Wisconsin Solution to Prevent Intestinal Cold Ischemia/Reperfusion Injury

Carbon monoxide (CO), a byproduct of heme catalysis, was shown to have potent cytoprotective and anti‐inflammatory effects. In vivo recipient CO inhalation at low concentrations prevented ischemia/reperfusion (I/R) injury associated with small intestinal transplantation (SITx). This study examined whether ex vivo delivery of CO in University of Wisconsin (UW) solution could ameliorate intestinal I/R injury. Orthotopic syngenic SITx was performed in Lewis rats after 6 h cold preservation in control UW or UW that was bubbled with CO gas (0.1–5%) (CO‐UW). Recipient survival with intestinal grafts preserved in 5%, but not 0.1%, CO‐UW improved to 86.7% (13/15) from 53% (9/17) with control UW. At 3 h after SITx, grafts stored in 5% CO‐UW showed improved intestinal barrier function, less mucosal denudation and reduced inflammatory mediator upregulation compared to those in control UW. Preservation in CO‐UW associated with reduced vascular resistance (end preservation), increased graft cyclic guanosine monophosphate levels (1 h), and improved graft blood flow (1 h). Protective effects of CO‐UW were reversed by ODQ, an inhibitor of soluble guanylyl cyclase. In vitro culture experiment also showed better preservation of vascular endothelial cells with CO‐UW. The study suggests that ex vivo CO delivery into UW solution would be a simple and innovative therapeutic strategy to prevent transplant‐induced I/R injury.

Mechanisms of Toll-like receptor 4 (TLR4)-mediated inflammation after cold ischemia/reperfusion in the heart.

Background. Toll-Like Receptor 4 (TLR4) signaling mediates early inflammation after cold ischemia-reperfusion (I/R). We hypothesized that the TLR4 coreceptor CD14, the intracellular adaptor proteins myeloid differentiation factor 88 (MyD88) and TIR domain-containing-adaptor inducing IFN&bgr; (TRIF) would be required for cold I/R induced inflammation. High mobility group box 1 (HMGB1) is a putative endogenous activator of TLR4. Therefore, we also assessed the contribution of HMGB1 in cold I/R induced inflammation. Methods. Syngeneic heart transplants were performed in mice deficient in CD14, MyD88, TRIF, or wild-type mice. In other experiments, anti-HMGB1 neutralizing antibody or control IgG was administered at reperfusion. Donor hearts were subjected to 2 hr of cold ischemia and retrieved after 3 hr of reperfusion. Results. After cold I/R, grafts revealed striking translocation of HMGB1 out of the nucleus in cardiac myocytes. Administration of an anti-HMGB1 neutralizing antibody resulted in reduced systemic interleukin (IL)-6, tumor necrosis factor alpha (TNF&agr;), and intercellular adhesion molecule-1 (ICAM-1) messenger RNA (mRNA) levels (P≤0.05). Compared with controls, CD14 knock-out (KO) mice exhibited significantly lower (P≤0.05) systemic IL-6 and JE/monocyte chemotractant protein-1 levels after cold I/R. Intragraft TNF&agr; and IL-1&bgr; mRNA levels were also significantly lower (P≤0.05) in CD14 KO grafts. MyD88 KO mice exhibited significantly lower (P≤0.05) systemic IL-6 levels compared with control mice after cold I/R. Intragraft TNF&agr;, IL-6, and ICAM-1 mRNA levels were also significantly lower (P≤0.05) in MyD88 KO grafts. Significantly lower levels (P≤0.05) of serum IL-6, monocyte chemotractant protein-1 as well as intragraft TNF&agr;, IL-6, IL-1&bgr;, and ICAM-1 were observed after cold I/R in TRIF deficient animals compared with controls. Conclusions. CD14, MyD88, TRIF, and HMGB1 contribute to the inflammatory response that occurs after cold I/R. These results provide insight into the mechanisms of TLR4-mediated inflammation after cold I/R.

Toll-like receptor 4 mediates the early inflammatory response after cold ischemia/reperfusion.

Background. Ischemia/reperfusion (I/R) injury leads to graft dysfunction and may contribute to alloimmune responses posttransplantation. The molecular mechanisms of cold I/R injury are only partially characterized but may involve toll-like receptor (TLR)-4 activation by endogenous ligands. We tested the hypothesis that TLR4 mediates the early inflammatory response in the setting of cold I/R in a murine cardiac transplant model. Methods. Syngeneic heart transplants were performed in mutant mice deficient in TLR4 signaling (C3H/HeJ) and wild-type mice (C3H/HeOuJ). Transplants were also performed between the strains (mutant hearts into wild-type recipients and the converse). Donor hearts were subjected to 2 hr of cold ischemia. The grafts were retrieved at 3 and 24 hr after reperfusion. Serum samples were collected for cytokine analysis. Reverse-transcription polymerase chain reaction and histologic analysis were used to assess intra-graft inflammation. Results. After transplant, serum tumor necrosis factor (TNF), interleukin (IL)-6, JE/monocyte chemotractant protein (MCP)-1, IL-1&bgr;, and troponin I levels, as well as intragraft TNF, IL-1&bgr;, IL-6, early growth response (EGR)-1, intercellular adhesion molecule (ICAM)-1, and inducible nitric oxide synthase (iNOS) mRNA levels, were significantly lower in the mutant→mutant group compared to the wild-type→wild-type group (P≤0.05). Intermediate levels of serum IL-6, JE/MCP-1, as well as intragraft TNF, IL-1&bgr;, IL-6, and ICAM-1 mRNA were observed after transplants in the mutant→wild-type and wild-type→mutant groups. Immunohistochemistry revealed less myocardial nuclear factor-&kgr;B nuclear translocation at and less neutrophil infiltration in the mutant→mutant group compared to the wild-type→wild-type group. Conclusions. These findings demonstrate that TLR4 signaling is central to both the systemic and intragraft inflammatory responses that occur after cold I/R in the setting of organ transplantation and that TLR4 signaling on both donor and recipient cells contributes to this response.

Ex vivo carbon monoxide prevents cytochrome P450 degradation and ischemia/reperfusion injury of kidney grafts

Renal ischemia/reperfusion injury is a major complication of kidney transplantation. We tested if ex vivo delivery of carbon monoxide (CO) to the kidney would ameliorate the renal injury of cold storage that can complicate renal transplantation. Orthotopic syngeneic kidney transplantation was performed in Lewis rats following 24 h of cold preservation in University of Wisconsin solution equilibrated without or with CO (soluble CO levels about 40 microM). Ischemia/reperfusion injury in control grafts resulted in an early upregulation of inflammatory mediator mRNAs and progressive deterioration of graft function. In contrast, the grafts preserved with CO had significantly less oxidative injury and this was associated with improved recipient survival compared to the control group. Renal injury in the control group showed considerable degradation of cytochrome P450 heme proteins, active heme metabolism and increased detrimental intracellular free heme levels. Kidney grafts preserved in CO-equilibrated solution maintained their cytochrome P450 protein levels, had normal intracellular heme levels and had less lipid peroxidation. Our results show that CO-mediated suppression of injurious heme-derived redox reactions offers protection of kidney grafts from cold ischemia/reperfusion injury.

Carbon monoxide ameliorates renal cold ischemia-reperfusion injury with an upregulation of vascular endothelial growth factor by activation of hypoxia-inducible factor.

Background. We have previously shown that carbon monoxide (CO) inhalation at a low concentration provides protection against cold ischemia-reperfusion (I/R) injury after kidney transplantation. As vascular endothelial growth factor (VEGF) may promote the recovery process of impaired vascular endothelial cells during I/R injury, we examined whether protective effects of CO involved VEGF induction and its upstream hypoxia-inducible factor (HIF)-1 activation. Methods. Lewis rat kidney graft, preserved in University of Wisconsin at 4°C for 24 hr, was orthotopically transplanted into syngeneic recipient. Recipients were continuously maintained in air or exposed to CO (250 ppm) for 1 hr before and 24 hr after transplant. Results. Prolonged cold preservation resulted in progressive impairment of kidney graft function with early inflammatory responses. Carbon monoxide significantly protected kidney grafts from cold I/R injury, improved renal function and enhanced recipient survival. Real-time reverse transcriptase-polymerase chain reaction revealed upregulation of HIF-1&agr; and VEGF in the CO-treated kidney grafts as early as 1 hr after reperfusion. Western blot showed CO significantly upregulated VEGF expression 1 to 3 hr after kidney transplantation. Considerably more VEGF-positive cells were observed mainly in tubular epithelial cells in CO-treated, but not air-exposed, kidney grafts at 3 hr after reperfusion. YC-1, HIF-1&agr; inhibitor, completely abrogated the actions of CO on VEGF induction and reversed the protective effects afforded by CO. Nitric oxide production in the grafts was increased by CO, however, abolished by YC-1. Conclusion. These results demonstrate that the protective effect of CO against renal cold I/R injury may involve VEGF upregulation through its upstream signal, HIF-1 activation.

A Single Intraperitoneal Dose of Carbon Monoxide-Saturated Ringer's Lactate Solution Ameliorates Postoperative Ileus in Mice

Treatment with inhaled carbon monoxide (CO) has been shown to ameliorate bowel dysmotility caused by surgical manipulation of the gut in experimental animals. We hypothesized that administration of CO dissolved in lactated Ringers solution (CO-LR) might provide similar protection to that observed with the inhaled gas while obviating some of its inherent problems. Postoperative gut dysmotility (ileus) was induced in mice by surgical manipulation of the small intestine. Some mice were treated with a single intraperitoneal dose of CO-LR immediately after the surgical procedure, whereas other mice received only the LR vehicle. Twenty-four hours later, intestinal transit of a nonabsorbable marker (70-kDa fluorescein isothiocyanate-labeled dextran) was delayed in mice subjected to intestinal manipulation but not the sham procedure. Gut manipulation also was associated with increased expression within the muscularis propria of transcripts for interleukin-1β, cyclooxygenase-2, inducible nitric-oxide synthase, intracellular adhesion molecule-1, and Toll-like receptor-4, as well as infiltration of the muscularis propria with polymorphonuclear leukocytes and activation of mitogen-activated protein kinases and nuclear factor-κB. All of these effects were attenuated by treatment with CO-LR. The salutary effect of CO-LR on gut motility, as well as many of the anti-inflammatory effects of CO-LR, was diminished by treatment with a soluble guanylyl cyclase (sGC) inhibitor, suggesting that the effects of CO are mediated via activation of sGC. These data support the view that a single intraperitoneal dose of CO-LR ameliorates postoperative ileus in mice by inhibiting the inflammatory response in the gut wall induced by surgical manipulation, possibly in a sGC-dependent fashion.

Carbon monoxide-saturated preservation solution protects lung grafts from ischemia-reperfusion injury

OBJECTIVES In previous work we have demonstrated that delivery of low concentrations (250 ppm) of carbon monoxide by means of inhalation to donors, recipients, or both protects transplanted lungs from ischemia-reperfusion injury (improved gas exchange, diminished intragraft and systemic inflammation, and retention of graft vascular endothelial cell ultrastructure). In this study we examined whether delivery of carbon monoxide to lung grafts in the preservation solution could protect against lung ischemia-reperfusion injury. METHODS Orthotopic left lung transplantation was performed in syngeneic Lewis to Lewis rats. Grafts were preserved in University of Wisconsin solution with or without (control solution) carbon monoxide at 4 degrees C for 6 hours. Carbon monoxide gas (5% or 100%) was bubbled into University of Wisconsin solution at 4 degrees C for 5 minutes before use. RESULTS In control animals, ischemia-reperfusion injury resulted in significant deterioration of graft function and was associated with a massive cellular infiltrate 2 hours after reperfusion. Grafts stored in University of Wisconsin solution with carbon monoxide (5%), however, demonstrated significantly better gas exchange and significantly reduced intragraft inflammation (reduced inflammatory mediators and cellular infiltrate). Experiments demonstrated that the protective effects afforded by 100% University of Wisconsin solution with carbon monoxide were not as potent as those of 5% University of Wisconsin solution with carbon monoxide. CONCLUSIONS This study demonstrates that 5% carbon monoxide as an additive to the cold flush/preservation solution can impart potent anti-inflammatory and cytoprotective effects after cold preservation and transplantation of lung grafts. Such ex vivo treatment of lung grafts with carbon monoxide can minimize concerns associated with carbon monoxide inhalation and might offer the opportunity to significantly advance the application of carbon monoxide in the clinical setting.

Nitrite Reduces Acute Lung Injury and Improves Survival in a Rat Lung Transplantation Model

Ischemia/reperfusion injury (IRI) is the most common cause of early mortality following lung transplantation (LTx). We hypothesized that nitrite, an endogenous source of nitric oxide (NO), may protect lung grafts from IRI. Rat lung grafts were stored in preservation solution at 4°C for 6 hours. Both grafts and recipients were treated with nitrite. Nitrite treatment was associated with significantly higher levels of tissue oxygenation, lower levels of cytokines and neutrophil/macrophage infiltration, lower myeloperoxidase activity, reduced oxidative injury and increased cGMP levels in grafts than in the controls. Treatment with either a nitric oxide scavenger or a soluble guanylyl cyclase (sGC) inhibitor diminished the beneficial effects of nitrite and decreased cGMP concentrations. These results suggest that nitric oxide, generated from nitrite, is the molecule responsible for the effects of nitrite via the nitric oxide/sGC/cGMP pathway. Allopurinol, a xanthine oxidoreductase (XOR) inhibitor, abrogated the protective effects of nitrite, suggesting that XOR is a key enzyme in the conversion of nitrite to nitric oxide. In vitro experiments demonstrated that nitrite prevented apoptosis in pulmonary endothelial cells. Nitrite also exhibits longer survival rate in recipients than control. In conclusion, nitrite inhibits lung IRI following cold preservation and had higher survival rate in LTx model.

Low-dose carbon monoxide inhibits progressive chronic allograft nephropathy and restores renal allograft function

Chronic allograft nephropathy (CAN) represents progressive deterioration of renal allograft function with fibroinflammatory changes. CAN, recently reclassified as interstitial fibrosis (IF) and tubular atrophy (TA) with no known specific etiology, is a major cause of late renal allograft loss and remains a significant deleterious factor of successful renal transplantation. Carbon monoxide (CO), an effector byproduct of heme oxygenase pathway, is known to have potent anti-inflammatory and antifibrotic functions. We hypothesized that inhaled CO would inhibit fibroinflammatory process of CAN and restore renal allograft function, even when the treatment was initiated after CAN was established. Lewis rat kidney grafts were orthotopically transplanted into binephrectomized allogenic Brown Norway rats under brief tacrolimus (0.5 mg/kg im, days 0-6). At day 60, CO (20 ppm) inhalation was initiated to recipients and continued until day 150 or animal death. Development of CAN was confirmed at day 60 with decreased creatinine clearance (CCr), significant proteinuria, and histopathological findings of TA, IF, and intimal arteritis. Air-treated control recipients continued to deteriorate with further declines of CCr and increases of urinary protein excretion and died with a median survival of 82 days. In contrast, progression of CAN was decelerated when recipients received CO on days 60-150, showing markedly improved graft histopathology, restored renal function, and improved recipient survival to a median of >150 days. CO significantly reduced intragraft mRNA levels for IFN-gamma and TNF-alpha at day 90. Expression of profibrotic TGF-beta/Smad was significantly suppressed with CO, together with downregulation of ERK-MAPK pathways. Continuous CO (20 ppm) treatment for days 0-30, days 30-60, or days 0-90, or daily 1-h CO (250 ppm) treatment for days 0-90, also showed efficacy in inhibiting CAN. The study demonstrates that CO is able to inhibit progression of fibroinflammatory process of CAN, restore renal allograft function, and improve survival even when the treatment is started after CAN is diagnosed.