Systemic application of carbon monoxide-releasing molecule 3 protects skeletal muscle from ischemia-reperfusion injury.

OBJECTIVE: Ischemia-reperfusion (IR) is a limb- and life-threatening complication of acute limb ischemia and musculoskeletal trauma. Carbon monoxide-releasing molecules (CORMs) have recently been shown to protect microvascular perfusion and to reduce inflammation and injury in various ischemic animal models. The purpose of this study was to examine the effects of water-soluble CORM-3 on the extent of IR-induced muscle injury. METHODS: Wistar rats were randomized into three groups: sham (no ischemia), IR + CORM-3 (10 mg/kg intraperitoneally), and IR + inactive CORM-3 (iCORM-3; 10 mg/kg intraperitoneally). No-flow ischemia was induced by the application of a tourniquet to the hind limb for 2 hours; tourniquet release commenced the reperfusion phase. Both CORM-3 and iCORM-3 were injected immediately after tourniquet release. Temporal changes in microvascular perfusion, cellular tissue injury (ethidium bromide and bisbenzimide staining), and inflammatory response (leukocyte recruitment) within the extensor digitorum longus muscle were assessed using intravital video microscopy every 15 minutes for a total of 90 minutes after initiation of reperfusion. Systemic levels of tumor necrosis factor-α were also measured. RESULTS: Hind limb IR resulted in (1) a significant no-reflow phenomenon followed by progressive increase in microvascular perfusion deficit (21% ± 2% continuously perfused capillaries in IR vs 76% ± 4% in sham [P < .001]; 52% ± 8% nonperfused capillaries in IR vs 13% ± 2% in sham at 90 minutes of reperfusion [P < .001]), (2) tissue injury (ethidium bromide and bisbenzimide staining of 0.52 ± 0.07 in IR vs 0.05 ± 0.03 in sham at 90 minutes of reperfusion [P < .001]), (3) leukocyte recruitment (13.7 ± 0.9 adherent leukocytes/30 seconds/1000 µm2 in IR vs 1.8 ± 0.5 adherent leukocytes/30 seconds/1000 µm2 in sham at 90 minutes of reperfusion [P < .001]), and (4) an increase in circulating tumor necrosis factor-α levels. Systemic administration of CORM-3 (but not of iCORM-3) effectively reduced the IR-associated skeletal muscle perfusion deficits, tissue injury, and inflammatory activation. CONCLUSIONS: CORM-3 displays potent protective and anti-inflammatory effects in an experimental model of hind limb IR, suggesting a potential therapeutic application of CORMs in treatment of ischemic conditions.

Pretreatment of human cerebrovascular endothelial cells with CO-releasing molecule-3 interferes with JNK/AP-1 signaling and suppresses LPS-induced proadhesive phenotype.

OBJECTIVE: Exogenously administered CO interferes with PMN recruitment to the inflamed organs. The mechanisms of CO-dependent modulation of vascular proadhesive phenotype, a key step in PMN recruitment, are unclear. METHODS: We assessed the effects/mechanisms of CO liberated from a water-soluble CORM-3 on modulation of the proadhesive phenotype in hCMEC/D3 in an in vitro model of endotoxemia. To this end, hCMEC/D3 were stimulated with LPS (1 µg/mL) for six hours. In some experiments hCMEC/D3 were pretreated with CORM-3 (200 µmol/L) before LPS-stimulation. PMN rolling/adhesion to hCMEC/D3 were assessed under conditions of laminar shear stress (0.7 dyn/cm(2) ). In parallel, expression of adhesion molecules E-selectin, ICAM-1, and VCAM-1 (qPCR), activation of transcription factors, NF-κB and AP-1 (ELISA), and MAPK-signaling (expression/phosphorylation of p38, ERK1/2, and JNK1/2; western blot) were assessed. RESULTS: The obtained results indicate that CORM-3 pretreatment reduces PMN rolling/adhesion to LPS-stimulated hCMEC/D3 (p < 0.05). Decreased PMN rolling/adhesion to hCMEC/D3 was associated with CORM-3-dependent inhibition of MAPK JNK1/2 activation (Tyr-phosphorylation), inhibition of transcription factor, AP-1 (c-Jun phosphorylation), and subsequent suppression of VCAM-1 expression (p < 0.05). CONCLUSIONS: These findings indicate that CORM-3 pretreatment interferes with JNK/AP-1 signaling and suppresses LPS-induced upregulation of the proadhesive phenotype in hCMEC/D3.

The severity of microvascular dysfunction due to compartment syndrome is diminished by the systemic application of CO-releasing molecule-3.

OBJECTIVES: To examine the protective effects of carbon monoxide (CO), liberated from a novel CO-releasing molecule (CORM-3), on the function of compartment syndrome (CS)-challenged muscle in a rodent model, thus providing for a potential development of a pharmacologic adjunctive treatment for CS. METHODS: Wistar rats were randomized into 4 groups: sham (no CS), CS, CS with inactive CORM-3 (iCORM-3), and CS + CORM-3 (10 mg/kg intraperitoneally). CS was induced by elevation of intracompartmental pressure to 30 mm Hg through an infusion of isotonic saline into the anterior compartment of the hind limb for 2 hours. Both CORM-3 and iCORM-3 were injected immediately after fasciotomy. Microvascular perfusion, cellular tissue injury, and inflammatory response within the extensor digitorum longus muscle were assessed using intravital video microscopy 45 minutes after fasciotomy. Systemic levels of tumor necrosis factor alpha (TNF-α) were also measured. RESULTS: Elevation of intracompartmental pressure resulted in significant microvascular perfusion deficits (23% ± 2% continuously perfused capillaries in CS vs. 76% ± 4% in sham, P < 0.0001; 55% ± 2% nonperfused capillaries in CS vs. 13% ± 2% in sham, P < 0.0001), significant increase in tissue injury (ethidium bromide/bisbenzimide of 0.31 ± 0.05 in CS vs. 0.05 ± 0.03 in sham, P < 0.0001) and adherent leukocytes (13.7 ± 0.9 in CS vs. 1.8 ± 0.5 in sham, P < 0.0001), and a progressive rise in systemic TNF-α. CORM-3 (but not iCORM-3) treatment restored the number of continuously perfused capillaries (57% ± 5%, P < 0.001), diminished tissue injury (ethidium bromide/bisbenzimide of 0.07 ± 0.01, P < 0.001), reversed the CS-associated rise in TNF-α, and decreased leukocyte adherence (0.6 ± 0.3, P < 0.001). CONCLUSIONS: CORM-3 displays a potent protective/anti-inflammatory action in an experimental model of CS, suggesting a potential therapeutic application to patients at risk of developing CS.

Carbon monoxide-releasing molecule 3 inhibits myeloperoxidase (MPO) and protects against MPO-induced vascular endothelial cell activation/dysfunction.

Polymorphonuclear leukocyte (PMN)-derived myeloperoxidase (MPO) contributes to the pathophysiology of numerous systemic inflammatory disorders through: (1) direct peroxidation of targets and (2) production of strong oxidizing compounds, e.g., hypohalous acids, particularly hypochlorous acid, which furthers oxidant damage and contributes to the propagation of inflammation and tissue injury/dysfunction. Carbon monoxide-releasing molecules (CORMs) offer potent anti-inflammatory effects; however, the mechanism(s) of action is not fully understood. This study assessed the potential of MPO activity inhibition by a water-soluble CORM, CORM-3. To this end, we used in vitro assays to study CORM-3-dependent modulation of MPO activity with respect to: (1) the inhibition of MPO's catalytic activity generally and (2) the specific inhibition of MPO's peroxidation and halogenation (i.e., production of hypochlorous acid) reactions. Further, we employed primary human umbilical vein endothelial cells (HUVECs) to investigate MPO-dependent cellular activation and dysfunction by measuring intracellular oxidant stress (DHR-123 oxidation) and HUVEC permeability (flux of Texas red-dextran), respectively. The results indicate that CORM-3 significantly inhibits MPO activity as well as MPO's peroxidation and hypohalous acid cycles specifically (p<0.05 vs uninhibited MPO). In addition, CORM-3 significantly decreases PMN homogenate- or rhMPO-induced intracellular DHR-123 oxidation in HUVECs and rhMPO-induced HUVEC monolayer permeability (p<0.05 vs untreated). In all assays the inactivated CORM-3 was significantly less effective than CORM-3 (p<0.05). Taken together our findings indicate that CORM-3 is a novel MPO inhibitor and mitigates inflammatory damage at least in part through a mechanism involving the inhibition of neutrophilic MPO activity.

The biological effects of lung-derived mediators on the liver.

Despite the use of lung-protective mechanical ventilation (MV), the mortality of patients with acute lung injury remains at 30 to 40%, predominantly due to multiorgan failure. The objective of this study was to determine the biological significance of lung-derived mediators on peripheral organ inflammation. The authors utilized an isolated perfused mouse lung model of lipopolysaccharide (LPS)-induced lung inflammation and protective MV to collect lung-derived mediators. Aliquots of perfusate from these animals (or appropriate controls) were then injected intravenously into a cohort of normal animals whose livers were subsequently assessed in vivo using intravital video microscopy. Perfusate from LPS-inflamed lungs contained significantly higher concentrations of inflammatory mediators than perfusate from saline-instilled lungs. Assessment of livers in the second cohort of animals 120 minutes after perfusate injection revealed decreased sinusoidal blood flow, leukocytosis, and increased cell death in those receiving perfusate from LPS-inflamed lungs compared to perfusate from saline controls. There were no differences between control animals that received pure perfusate or pure LPS mixed with perfusate. These results showed that lung-derived mediators had a significant biological effect on nonpulmonary organs within a short period of time after administration. Therapies targeting these mediators may prevent multiorgan failure and death in patients with acute lung injury.

Carbon monoxide-releasing molecule CORM-3 suppresses vascular endothelial cell SOD-1/SOD-2 activity while up-regulating the cell surface levels of SOD-3 in a heparin-dependent manner.

The role of CO in the modulation of antioxidant enzyme function has not been investigated, yet. In this study we assessed the effects and potential mechanisms of the ruthenium-based water-soluble CO-releasing molecule CORM-3 in the modulation of superoxide dismutase (SOD) activity/binding in vascular endothelial cells (HUVECs). To this end, HUVECs were treated with CORM-3 (100 µM) and assessed for total SOD activity in cell lysates (cell-associated SOD activity) and cell culture supernatants (soluble SOD). In parallel, release/binding of extracellular SOD (SOD-3) in the absence or presence of heparin (1-10 IU/ml), a key factor regulating SOD-3 cell-surface binding, was investigated. In addition, the effects of CORM-3 on the modulation of purified SOD-1 and SOD-2 activity in a cell-free system were also assessed. The results obtained indicate that CORM-3 effectively suppresses the activity of both purified SOD-1 and SOD-2. These findings were accompanied by CORM-3-dependent attenuation of total cell-associated SOD activity (without affecting SOD-1/SOD-2 protein expression) and a subsequent increase in ROS production (DHR123 oxidation) in HUVECs. In parallel, a concomitant increase in soluble-SOD activity (due to increased SOD-3 release from the cell surface) was observed in the cell culture supernatants. However, in the presence of heparin, total cell-associated SOD activity was significantly increased by CORM-3, because of increased binding of SOD-3 to HUVECs. Taken together these findings indicate for the first time that CORM-3 modulates both the activity of intracellular SOD (i.e., SOD-1 and SOD-2) and the binding of extracellular SOD (SOD-3) to the cell surface.

Carbon monoxide liberated from CO-releasing molecule (CORM-2) attenuates ischemia/reperfusion (I/R)-induced inflammation in the small intestine.

CORM-released CO has been shown to be beneficial in resolution of acute inflammation. The acute phase of intestinal ischemia-reperfusion (I/R) injury is characterized by oxidative stress-related inflammation and leukocyte recruitment. In this study, we assessed the effects and potential mechanisms of CORM-2-released CO in modulation of inflammatory response in the small intestine following I/R-challenge. To this end mice (C57Bl/6) small intestine were challenged with ischemia by occluding superior mesenteric artery (SMA) for 45 min. CORM-2 (8 mg/kg; i.v.) was administered immediately before SMA occlusion. Sham operated mice were injected with vehicle (0.25% DMSO). Inflammatory response in the small intestine (jejunum) was assessed 4 h following reperfusion by measuring tissue levels of TNF-alpha protein (ELISA), adhesion molecules E-selectin and ICAM-1 (Western blot), NF-kappaB activation (EMSA), along with PMN tissue accumulation (MPO assay) and leukocyte rolling/adhesion in the microcirculation of jejunum (intravital microscopy). The obtained results indicate that tissue levels of TNF-alpha, E-selectin and ICAM-1 protein expression, activation of NF-kappaB, and subsequent accumulation of PMN were elevated in I/R-challenged jejunum. The above changes were significantly attenuated in CORM-2-treated mice. Taken together these findings indicate that CORM-2-released CO confers anti-inflammatory effects by interfering with NF-kappaB activation and subsequent up-regulation of vascular pro-adhesive phenotype in I/R-challenged small intestine.

CORM-3-derived CO modulates polymorphonuclear leukocyte migration across the vascular endothelium by reducing levels of cell surface-bound elastase.

Recently, it has been shown that carbon monoxide (CO)-releasing molecule (CORM)-released CO can suppress inflammation. In this study, we assessed the effects and potential mechanisms of a ruthenium-based water-soluble CO carrier [tricarbonylchloroglycinate-ruthenium(II) (CORM-3)] in the modulation of polymorphonuclear leukocyte (PMN) inflammatory responses in an experimental model of sepsis. Sepsis in mice was induced by cecal ligation and puncture. CORM-3 (3 mg/kg iv) was administered 15 min after the induction of cecal ligation and puncture. PMN accumulation in the lung (myeloperoxidase assay), bronchoalveolar lavage (BAL) fluid, and lung vascular permeability (protein content in BAL fluid) were assessed 6 h later. In in vitro experiments, human PMNs were primed with LPS (10 ng/ml) and subsequently stimulated with formyl-methionyl-leucylphenylalanine (fMLP; 100 nM). PMN production of ROS (L-012/dihydrorhodamine-123 oxidation), degranulation (release of elastase), and PMN rolling, adhesion, and migration to/across human umbilical vein endothelial cells (HUVECs) were assessed in the presence or absence of CORM-3 (1-100 muM). The obtained results indicated that systemically administered CORM-3 attenuates PMN accumulation and vascular permeability in the septic lung. Surprisingly, in in vitro experiments, treatment of PMNs with CORM-3 further augmented LPS/fMLP-induced ROS production and the release of elastase. The latter effects, however, were accompanied by an inability of PMNs to mobilize elastase to the cell surface (plasma membrane), an event required for efficient PMN transendothelial migration. The CORM-3-induced decrease in cell surface levels of elastase was followed by decreased PMN rolling/adhesion to HUVECs and complete prevention of PMN migration across HUVECs. In contrast, treatment of HUVECs with CORM-3 had no effect on PMN transendothelial migration. Taken together, these findings indicate that, in sepsis, CORM3-released CO, while further amplifying ROS production and degranulation of PMNs, concurrently reduces the levels of cell surface-bound elastase, which contributes to suppressed PMN transendothelial migration.

Hindlimb ischemia/reperfusion-induced remote injury to the small intestine: role of inducible nitric-oxide synthase-derived nitric oxide.

Systemic inflammatory response syndrome, as a consequence of ischemia/reperfusion (I/R), negatively influences the function of the affected organs. The objective of this study was to assess the role of nitric oxide (NO) in remote intestinal inflammatory response elicited by hindlimb I/R. To this end, C57BL/6 (wild type; WT) and inducible nitric-oxide synthase (iNOS)-deficient mice were subjected to bilateral hindlimb ischemia (1 h) followed by 6 h of reperfusion. Some WT mice were injected with iNOS inhibitor N-[3-(aminomethyl)benzyl] acetamidine (1400W) (5 mg/kg s.c.) immediately before reperfusion, and proinflammatory response was assessed 6 h later. Hindlimb I/R resulted in dysfunction of the small intestine as assessed by the increase in permeability [blood-to-lumen clearance of Texas Red-dextran (molecular mass 3 kDa)] and an increase in the luminal levels of tumor necrosis factor (TNF)-alpha protein and nitrate/nitrite (NO(2)(-)/NO(3)(-)). The above-mentioned changes were accompanied by up-regulation of the proinflammatory phenotype in the mucosa of small intestine with respect to 1) an increase in TNF-alpha and iNOS protein expression, 2) leukocyte accumulation, 3) formation of edema, 4) an increase in leukocyte rolling/adhesion in the submucosal microvasculature, and 5) activation of transcription factor nuclear factor-kappaB and up-regulation of adhesion molecule expression. Interestingly, the most profound changes with respect to intestinal dysfunction were found in jejunum and ileum, whereas duodenum was affected the least. Interfering with iNOS activity (1400W and iNOS-deficient mice) significantly attenuated hindlimb I/R-induced inflammatory response and dysfunction of the small intestine with respect to the above-mentioned markers of inflammation. The obtained results indicate that hindlimb I/R induces remote inflammatory response in the small intestine through an iNOS-derived NO-dependent mechanism.

Low-dose inhaled carbon monoxide attenuates the remote intestinal inflammatory response elicited by hindlimb ischemia-reperfusion.

Heme oxygenase (HO) represents the rate-limiting enzyme in the degradation of heme into carbon monoxide (CO), iron, and biliverdin. Recent evidence suggests that several of the beneficial properties of HO, may be linked to CO. The objectives of this study were to determine if low-dose inhaled CO reduces remote intestinal leukocyte recruitment, proinflammatory cytokine expression, and oxidative stress elicited by hindlimb ischemia-reperfusion (I/R). Male mice underwent 1 h of hindlimb ischemia, followed by 3 h of reperfusion. Throughout reperfusion, mice were exposed to AIR or AIR + CO (250 ppm). Following reperfusion, the distal ileum was exteriorized to assess the intestinal inflammatory response by quantifying leukocyte rolling and adhesion in submucosal postcapillary venules with the use of intravital microscopy. Ileum samples were also analyzed for proinflammatory cytokine expression [tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta] and malondialdehyde (MDA) with the use of enzyme-linked immunosorbent assay and thiobarbituric acid reactive substances assays, respectively. I/R + AIR led to a significant decrease in leukocyte rolling velocity and a sevenfold increase in leukocyte adhesion. This was also accompanied by a significant 1.3-fold increase in ileum MDA and 2.3-fold increase in TNF-alpha expression. Treatment with AIR + CO led to a significant reduction in leukocyte recruitment and TNF-alpha expression elicited by I/R; however, MDA levels remained unchanged. Our data suggest that low-dose inhaled CO selectively attenuates the remote intestinal inflammatory response elicited by hindlimb I/R, yet does not provide protection against intestinal lipid peroxidation. CO may represent a novel anti-inflammatory therapeutic treatment to target remote organs following acute trauma and/or I/R injury.

Carbon monoxide liberated from carbon monoxide-releasing molecule CORM-2 attenuates inflammation in the liver of septic mice.

Recent studies suggest that exogenously administered CO is beneficial for the resolution of acute inflammation. In this study, we assessed the role of CO liberated from a systemically administered tricarbonyldichlororuthenium-(II)-dimer (CORM-2) on modulation of liver inflammation during sepsis. Polymicrobial sepsis in mice was induced by cecal ligation and perforation (CLP). CORM-2 (8 mg/kg iv) was administered immediately after CLP induction, and neutrophil [polymorphonuclear leukocyte (PMN)] tissue accumulation, activation of transcription factor, NF-kappaB, and changes in adhesion molecule ICAM-1 expression (inflammation-relevant markers) were assessed in murine liver 24 h later. In addition, the effects and potential mechanisms of CORM-2-released CO in modulation of vascular endothelial cell proinflammatory responses were assessed in vitro. To this end, human umbilical vein endothelial cells (HUVEC) were stimulated with LPS (1 microg/ml) in the presence or absence of CORM-2 (10-100 microM) and production of intracellular reactive oxygen species (ROS), (DHR123 oxidation) and NO (DAF-FM nitrosation) and subsequent activation of NF-kappaB were assessed 4 h later. In parallel, expression of ICAM-1 and inducible NO synthase (iNOS) proteins along with PMN adhesion to LPS-challenged HUVEC were also assessed. Induction of CLP resulted in increased PMN accumulation, ICAM-1 expression, and activation of NF-kappaB in the liver of septic mice. These effects were significantly attenuated by systemic administration of CORM-2. In in vitro experiments, CORM-2-released CO attenuated LPS-induced production of ROS and NO, activation of NF-kappaB, increase in ICAM-1 and iNOS protein expression and PMN adhesion to LPS-stimulated HUVEC. Taken together, these findings indicate that CO released from systemically administered CORM-2 provides anti-inflammatory effects by interfering with NF-kappaB activation and subsequent downregulation of proadhesive vascular endothelial cell phenotype in the liver of septic mice.

Role of heme oxygenase in the protection afforded skeletal muscle during ischemic tolerance.

OBJECTIVE: Ischemic tolerance (IT) is known to improve resistance to ischemia/reperfusion (I/R)-induced injury; however, the mechanisms remain unknown. The authors hypothesized that induction of heme oxygenase (HO), a heat shock protein, would provide anti-inflammatory benefits during IT, thereby preventing leukocyte-derived I/R injury. METHODS: Male Wistar rats were randomly assigned to sham (n = 4), I/R (n = 9), preconditioning (PC)+I/R (n = 7), chromium mesoporphyrin, to inhibit HO (CrMP; n = 4), or PC+I/R+CrMP (n = 6) groups. PC consisted of 5 cycles of I/R, each lasting 10 min, induced by tightening a tourniquet placed above the greater trochantor of the hindlimb. Twenty-four hours later, the hindlimb underwent 2 h of no-flow ischemia followed by intravital microscopy during 90 min reperfusion to assess capillary perfusion (#/mm), tissue injury (ratio of ethidium bromide to bisbenzimide labeled cells/100 microm2), leukocyte rolling (Lr, #/1000 microm2), and adhesion (La, #/1000 microm2) in postcapillary venules of the extensor digitorum longus (EDL) muscle. RESULTS: In the I/R group, Lr was significantly increased (7.1 +/- 0.4) compared to sham (3.1 +/- 0.4). PC+I/R increased Lr (10.8 +/- 0.72), which was further exacerbated by the removal of HO (14.2 +/- 1.3). La (7.8 +/- 2.0) was significantly increased compared to sham (2.4 +/- 0.9), while PC returned La back to sham levels (1.9 +/- 0.7). Removal of HO activity, via CrMP, had no significant effect on La (3.9 +/- 0.7). However, CrMP removed the protection to microvascular perfusion (I/R = 9.4 +/- 1.1, PC = 16.6 +/- 1.8, sham = 20.5 +/- 2.8, PC+CrMP+I/R = 12.3 +/- 2.3) and prevented protection from ischemia-induced tissue injury. CONCLUSION: The data suggest that HO is an important protective mechanism during IT in skeletal muscle, but such protection was by mechanisms other than altered leukocyte-endothelial cell interaction.

Role of tumor necrosis factor-alpha in myocardial dysfunction and apoptosis during hindlimb ischemia and reperfusion.

OBJECTIVE: Peripheral vascular surgery involving limb ischemia/reperfusion is associated with tumor necrosis factor-alpha production and an increased risk of cardiac complications. The objective of this study was to investigate the role of tumor necrosis factor-alpha in myocardial apoptosis and dysfunction following hindlimb ischemia/reperfusion. DESIGN: Randomized perspective animal study. SETTING: Research laboratory. SUBJECTS: Adults male tumor necrosis factor-alpha(-/-) and littermate wild-type mice. INTERVENTIONS: Bilateral hindlimb ischemia/reperfusion was induced in wild-type and tumor necrosis factor-alpha(-/-) mice using tourniquet occlusion. After 2 hrs of hindlimb ischemia, the tourniquets were released, allowing reperfusion for 0.5-24 hrs. MEASUREMENTS AND MAIN RESULTS: In wild-type mice, hindlimb ischemia/reperfusion resulted in myocardial depression early during the reperfusion period (p < .05). These effects were temporally correlated with enhanced levels of myocardial and plasma tumor necrosis factor-alpha. All variables were restored to baseline levels by 24 hrs of reperfusion. Myocardial apoptosis, assessed by cell death enzyme-linked immunosorbent assay, terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling staining, and caspase-3 activity, was also significantly higher at 6 hrs of reperfusion (p < .05) but returned to baseline levels by 24 hrs. Interestingly, cardiac dysfunction and myocardial apoptosis were abolished in tumor necrosis factor-alpha mice subjected to the same degree of hindlimb ischemia/reperfusion as the wild-type mice. Treatment of etanercept restored cardiac function in wild-type mice. CONCLUSIONS: Tumor necrosis factor-alpha contributes significantly to myocardial dysfunction and apoptosis in hindlimb ischemia/reperfusion. Although a causal link between myocardial apoptosis and cardiac dysfunction is not established, our study does suggest that tumor necrosis factor-alpha may be a potential therapeutic target for cardiac injury in clinical situations involving prolonged remote ischemia/reperfusion.

Heme oxygenase modulates small intestine leukocyte adhesion following hindlimb ischemia/reperfusion by regulating the expression of intercellular adhesion molecule-1.

OBJECTIVE: Heme oxygenase is the rate-limiting enzyme in the degradation of heme into carbon monoxide, iron, and bilirubin. Recent evidence suggests that the induction of heme oxygenase-1 is associated with potent anti-inflammatory properties. The objectives of this study were to determine the temporal, regional, and cellular distribution of heme oxygenase-1 within the small intestine and its role in modulating remote intestinal leukocyte recruitment following trauma induced by hindlimb ischemia/reperfusion. DESIGN: Randomized, controlled, prospective animal study. SETTING: Hospital surgical research laboratory. SUBJECTS: Male C57BL/6 mice. INTERVENTIONS: Mice underwent 1 hr of bilateral hindlimb ischemia, followed by 3, 6, 12, or 24 hrs of reperfusion. MEASUREMENTS AND MAIN RESULTS: Heme oxygenase-1 messenger RNA, heme oxygenase-1 protein, and heme oxygenase activity were measured using reverse transcription polymerase chain reaction, Western blot, immunohistochemistry, and spectrophotometric assay, respectively. The jejunum was also exteriorized to quantify the flux of rolling and adherent leukocytes and R-Phycoerythrin conjugated intercellular adhesion molecule-1 monoclonal antibody fluorescence intensity in submucosal postcapillary venules with the use of intravital microscopy. Ischemia/reperfusion led to a significant increase in heme oxygenase-1 messenger RNA in the jejunum and ileum 3 hrs following limb reperfusion, with a subsequent increase in heme oxygenase-1 protein and heme oxygenase activity at 6 hrs. Ischemia/reperfusion also led to a significant 1.4-fold increase in leukocyte rolling, whereas inhibition of heme oxygenase via injection of tin protoporphyrin IX (20 micromol/kg intraperitoneally) resulted in a three-fold increase in leukocyte adhesion, compared with ischemia/reperfusion alone. This increase in adhesion was significantly reduced to baseline in mice treated with intercellular adhesion molecule-1 monoclonal antibody before heme oxygenase inhibition (40 microg/mouse), whereas inhibition of heme oxygenase activity following ischemia/reperfusion also led to a significant increase in R-Phycoerythrin intercellular adhesion molecule-1 monoclonal antibody fluorescence intensity. CONCLUSIONS: Our data suggest that remote trauma induced by hindlimb ischemia/reperfusion leads to an increase in heme oxygenase activity within the small intestine, which modulates intercellular adhesion molecule-1 dependent intestinal leukocyte adhesion.

Inhalation of carbon monoxide prevents liver injury and inflammation following hind limb ischemia/reperfusion.

The induction of heme oxygenase (HO), the rate limiting enzyme in the conversion of heme into carbon monoxide (CO) and biliverdin, limits liver injury following remote trauma such as hind limb ischemia/reperfusion (I/R). Using intravital video microscopy, we tested the hypothesis that inhaled CO (250 ppm) would mimic HO-derived liver protection. Hind limb I/R significantly decreased sinusoidal diameter and volumetric flow, increased leukocyte accumulation within sinusoids, increased leukocyte rolling and adhesion within postsinusoidal venules, and significantly increased hepatocyte injury compared with naive animals. Inhalation of CO alone did not alter any microcirculatory or inflammatory parameters. Inhalation of CO following I/R restored volumetric flow, decreased stationary leukocytes within sinusoids, decreased leukocyte rolling and adhesion within postsinusoidal venules, and significantly reduced hepatocellular injury following hind limb I/R. HO inhibition did not alter microcirculatory parameters in naive mice, but did increase inflammation, as well as increase hepatocyte injury following hind limb I/R. Inhalation of CO during HO inhibition significantly reduced such microcirculatory deficits, hepatic inflammation, and injury in response to hind limb I/R. In conclusion, these results suggest that HO-derived hepatic protection is mediated by CO, and inhalation of low concentrations of CO may represent a novel therapeutic approach to prevent remote organ injury during systemic inflammatory response syndrome, or SIRS.

Chronic lower extremity ischemia: a human model of ischemic tolerance.

BACKGROUND: Ischemic preconditioning (IPC) has been found in animals to have a protective effect against future ischemic injury to muscle tissue. Such injury is unavoidable during some surgical procedures. To determine whether chronic ischemia in the lower extremities would imitate IPC and reduce ischemic injury during vascular surgery, we designed a controlled clinical study. PATIENTS AND METHODS: Two groups of patients at a university-affiliated medical centre with chronic lower-extremity ischemia served as models of IPC: 6 patients awaiting femoral distal bypass (FDB) and 4 scheduled for aortobifemoral (ABF) bypass grafting for aortoiliac occlusive disease. Seven patients undergoing elective open repair of an infrarenal abdominal aortic aneurysm (AAA) were chosen as non-IPC controls. Three hematologic indicators of skeletal-muscle injury, lactate dehydrogenase (LDH), creatine kinase (CK) and myoglobin, were measured before placement of the proximal clamp, during surgical ischemia, immediately upon reperfusion, 15 minutes after and 1 hour after reperfusion, and during the first, second and third postoperative days. RESULTS: Baseline markers of skeletal-muscle injury were similar in all groups. In postreperfusion samples, concentrations of muscle-injury markers were significantly lower in the 2 PC groups than in the control group. For example, at day 2, LDH levels were increased by about 30% over baseline measures in the elective AAA (control) group, whereas levels in the FDB and ABF groups remained statistically unchanged from baseline. Myoglobin in controls had increased by 977%, but only by 160% in the FDB and 528% in the ABF groups. CK levels, in a similar trend, were 1432% higher in the control group and only 111% (FDB) and 1029% (ABF) in the study groups. Taken together, these data represent a significant level of protection. CONCLUSIONS: Patients with chronic lower-extremity ischemia suffered less severe ischemic injury after a period of acute ischemia than those with acute ischemia alone. Ischemic preconditioning is one proposed mechanism to help explain this protective effect.

Remote liver injury is attenuated by adenovirus-mediated gene transfer of heme oxygenase-1 during the systemic inflammatory response syndrome.

OBJECTIVES: Adenovirus-mediated gene therapy is being investigated with increasing success for future treatment of autoimmune diseases. However, the use of adenoviruses is still limited by inflammatory and immune responses in the target organ. Previous work by the authors' laboratory established that the adenovirus encoding inducible heme oxygenase (Ad-HO-1) does not elicit the acute hepatic inflammation normally caused by adenoviruses, inviting further investigation in models of severe inflammation. Concurrently, there is increasing evidence for an endogenous protective role for heme oxygenase (HO) in the liver during the systemic inflammatory response syndrome (SIRS). Building on our previous results, this study investigated the effect of Ad-HO-1 pretreatment on remote liver injury during normotensive SIRS, induced by bilateral hind limb ischemia and reperfusion. METHODS: Microvascular perfusion and hepatocyte death were quantified using established intravital videomicroscopy techniques. Hepatocellular injury and liver function were assessed using blood-borne indicators. RESULTS: Microvascular perfusion deficits and increased hepatocyte death occurred following limb ischemia and 3 h of reperfusion in vehicle-pretreated animals; however, Ad-HO-1 pretreatment prevented these deficits. In contrast, the increase in serum alanine transaminase levels was unaffected by Ad-HO-1 pretreatment. Serum bilirubin levels were increased during systemic inflammation, predominantly in the conjugated form; and, this increase was prevented by administration of Ad-HO-1. CONCLUSIONS: These data indicate that gene transfer of inducible HO is an effective method to protect the liver during SIRS, providing incentive for further investigation into gene therapy strategies exploiting this anti-inflammatory enzyme.

Endogenous heme oxygenase induction is a critical mechanism attenuating apoptosis and restoring microvascular perfusion following limb ischemia/reperfusion.

BACKGROUND: A protective role for endogenous heme oxygenase (HO) in the initiation of remote liver injury after limb ischemia/reperfusion has been established. This study expands on our previous work by investigating the role of endogenous HO on hepatocellular injury, hepatocyte death (necrotic and apoptotic), and microvascular perfusion at protracted post-reperfusion times. METHODS: Remote liver injury was studied after 1 hour of bilateral hind limb ischemia and 3, 6, or 24 hours of reperfusion in male C57BL6 mice. Inhibition of HO was achieved with the use of chromium mesoporphrin (CrMP). Established intravital videomicroscopy techniques were used to evaluate microvascular perfusion and hepatocyte death. Hepatocellular injury was quantified by serum alanine transaminase. Apoptosis was measured by using DNA laddering, Cell Death ELISA, and caspase-3 activity. RESULTS: Although significant perfusion deficits and hepatocellular injury/death occurred after 3 hours, progression of hepatocellular death beyond 6 hours was not observed. A transient increase in apoptosis was observed at 6 hours. By 24 hours, microvascular perfusion was completely restored. This lack of progression correlated with increased HO activity, observed throughout the protocol. Administration of CrMP reduced HO activity to sham nonstressed levels, and caused increased microvascular perfusion deficits, hepatocellular injury, and hepatocyte death over 24 hours. The transient increase in apoptosis was increased in duration and magnitude in CrMP-treated animals. CONCLUSIONS: These results suggest that endogenous HO activity prevents the progression of remote liver injury after limb ischemia/reperfusion.

Heme oxygenase modulates hepatic leukocyte sequestration via changes in sinusoidal tone in systemic inflammation in mice.

Heme oxygenase (HO) modulates the accumulation of leukocytes within the liver during the early stages of a systemic inflammatory response syndrome (SIRS), but the anti-inflammatory mechanism(s) remain to be tested. The influence of HO on the adhesion molecule expression within the liver and on circulating leukocytes was assessed. In addition, the effect of HO and nitric oxide synthase (NOS) on the liver microcirculation was tested. Mice were subjected to 1 h bilateral hindlimb ischemia followed by 3 h of reperfusion, at which time blood samples and the liver were harvested and adhesion molecule expression determined (ICAM-1, CD49d and CD11b). Direct measures of sinusoidal diameter and estimates of volumetric blood flow were obtained using intravital microscopy. HO was specifically induced and inhibited by hemin and chromium mesoporphyrin (CrMP), respectively, whereas NOS was inhibited by N-nitro-L-arginine methyl ester (L-NAME). ICAM-1 expression was increased following hindlimb ischemia-reperfusion. Hemin caused only a modest, but significant decrease in ICAM-1 expression, whereas inhibition of HO had no effect. However, HO inhibition significantly reduced sinusoidal diameters and volumetric flow and such vessels were correlated with significantly increased numbers of stationary leukocytes. Inhibition of NOS had no effect on sinusoidal diameter or volumetric flow. In conclusion, the anti-inflammatory benefits afforded by HO activity within the liver appear to involve the control of sinusoidal diameter and volumetric blood flow rather than altered adhesion molecule expression during the early stages of SIRS.

Protective mechanisms during ischemic tolerance in skeletal muscle.

The purpose of this study was to test specific mechanisms of protection afforded the rat extensor digitorum longus (EDL) muscle during ischemic tolerance. Two days following five cycles of 10 min ischemia and 10 min reperfusion, heme oxygenase (HO) and calcium-dependent nitric oxide synthase (cNOS) activities were increased 2- and 2.5-fold (p <.05), respectively. Interestingly, calcium-independent NOS (iNOS) activity was completely downregulated (p <.05). The levels of superoxide dismutase (SOD) and catalase were increased 2-fold (p <.05), while glutathione peroxidase activity remained unchanged from non-preconditioned controls. Using intravital microscopy combined with chromium mesoporphyrin (CrMP), a selective HO inhibitor, and l-NAME, a NOS inhibitor, the roles of HO and cNOS were evaluated. Ischemic tolerance in the EDL muscle, 48 h after the preconditioning stimulus, was characterized by complete protection from both microvascular perfusion deficits and tissue injury after a 2-h period of ischemia. Removal of NOS activity completely removed the benefit afforded microvascular perfusion, while inhibition of HO activity prevented the parenchymal protection. These data suggest that ischemic tolerance within skeletal muscle is associated with the upregulation of specific cytoprotective proteins and that the benefits afforded by cNOS and HO activity are spatially discrete to the microvasculature and parenchyma, respectively.