Characterization of a versatile organometallic pro-drug (CORM) for experimental CO based therapeutics.

The complex fac-[Mo(CO)(3)(histidinate)]Na has been reported to be an effective CO-Releasing Molecule in vivo, eliciting therapeutic effects in several animal models of disease. The CO releasing profile of this complex in different settings both in vitro and in vivo reveals that the compound can readily liberate all of its three CO equivalents under biological conditions. The compound has low toxicity and cytotoxicity and is not hemolytic. CO release is accompanied by a decrease in arterial blood pressure following administration in vivo. We studied its behavior in solution and upon the interaction with proteins. Reactive oxygen species (ROS) generation upon exposure to air and polyoxomolybdate formation in soaks with lysozyme crystals were observed as processes ensuing from the decomposition of the complex and the release of CO.

Nitric oxide-dependent bone marrow progenitor mobilization by carbon monoxide enhances endothelial repair after vascular injury.

BACKGROUND: Carbon monoxide (CO) has emerged as a vascular homeostatic molecule that prevents balloon angioplasty-induced stenosis via antiproliferative effects on vascular smooth muscle cells. The effects of CO on reendothelialization have not been evaluated. METHODS AND RESULTS: Exposure to CO has diametrically opposite effects on endothelial cell (EC) and vascular smooth muscle cell proliferation in rodent models of carotid injury. In contrast to its effect of blocking vascular smooth muscle cell growth, CO administered as a gas or as a CO-releasing molecule enhances proliferation and motility of ECs in vitro by >50% versus air controls, and in vivo, it accelerates reendothelialization of the denuded artery by day 4 after injury versus day 6 in air-treated animals. CO enhanced EC proliferation via rapid activation of RhoA (Ras homolog gene family, member A), followed by downstream phosphorylation of Akt, endothelial nitric oxide (NO) synthase phosphorylation, and a 60% increase in NO generation by ECs. CO drives cell cycle progression through phosphorylation of retinoblastoma, which is dependent in part on endothelial NO synthase-generated NO. Similarly, endothelial repair in vivo requires NO-dependent mobilization of bone marrow-derived EC progenitors, and CO yielded a 4-fold increase in the number of mobilized green fluorescent protein-Tie2-positive endothelial progenitor cells versus controls, with a corresponding accelerated deposition of differentiated green fluorescent protein-Tie2-positive ECs at the site of injury. CO was ineffective in augmenting EC repair and the ensuing development of intimal hyperplasia in eNOS(-/-) mice. CONCLUSIONS: Collectively, the present data demonstrate that CO accelerates EC proliferation and vessel repair in a manner dependent on NO generation and enhanced recruitment of bone marrow-derived endothelial progenitor cells.

Tissue hypoxia activates JNK in the liver during hemorrhagic shock.

The earliest signaling pathways responsible for initiating the systemic response to hemorrhagic shock (HS) remain poorly characterized. We have investigated the involvement of the mitogen-activated protein (MAP) kinase C-JUN N-terminal kinase (JNK) and its activation in the liver as an early response to tissue hypoxia soon after the initiation of hemorrhage. In the present studies, hemorrhage of mice to 25 mmHg for 30 min resulted in a significant (2.1-fold) increase in JNK phosphorylation within the liver. Results were similar in rats hemorrhaged to 40 mmHg for 1 h. Hypoxia alone, replicated by warm isolated hepatic ischemia in vivo or hepatocytes cultured under 1% oxygen, also resulted in JNK phosphorylation. Finally, preservation of tissue perfusion and oxygenation by pretreatment with a blood-soluble drag-reducing polymer (DRP) in the rat HS model prevented phosphorylation of JNK in the liver. These results identify tissue hypoxia as a key factor in activating early signaling events in the liver following hemorrhage, as measured by JNK phosphorylation.

A role for angiotensin II in the activation of extracellular signal-regulated kinases in the liver during hemorrhagic shock.

Hemorrhagic shock (HS) is a complex process that initiates a global stress response. However, the earliest signaling pathways responsible for initiating this response remain unidentified. We have investigated the involvement of the extracellular signal-regulated kinases (ERK 1/2; also known as p42/44) and their activation in the liver by angiotensin II in the early signal transduction after HS. Hemorrhage of mice to 25 mmHg for 30 min was associated with the activation of ERK 1/2 in the liver, and this was accompanied by a 6.7-fold elevation of circulating angiotensin II levels. Similar results were obtained in rats. Both the angiotensin II levels and ERK 1/2 phosphorylation were suppressed by administration of an angiotensin-converting enzyme inhibitor peptide. Plasma from shocked rats, but not shocked rats treated with the angiotensin-converting enzyme inhibitor, increased ERK 1/2 phosphorylation in cultured hepatocytes. Together, these data suggest that angiotensin II is an important stimulus for ERK 1/2 activation in the liver during HS.

Effect of hemorrhagic shock on gut barrier function and expression of stress-related genes in normal and gnotobiotic mice.

We sought to determine whether gut-derived microbial factors influence the hepatic or intestinal inflammatory response to hemorrhagic shock and resuscitation (HS/R). Conventional and gnotobiotic mice contaminated with a defined microbiota without gram-negative bacteria were subjected to either a sham procedure or HS/R. Tissue samples were obtained 4 h later for assessing ileal mucosal permeability to FITC dextran and hepatic and ileal mucosal steady-state IL-6, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and TNF mRNA levels. Whereas HS/R significantly increased ileal mucosal permeability in conventional mice, this effect was not apparent in gnotobiotic animals. HS/R markedly increased hepatic mRNA levels for several proinflammatory genes in both conventional and gnotobiotic mice. HS/R increased ileal mucosal IL-6 and COX-2 mRNA expression in conventional but not gnotobiotic mice. If gnotobiotic mice were contaminated with Escherichia coli C25, HS/R increased ileal mucosal permeability and upregulated expression of IL-6 and COX-2. These data support the view that the hepatic inflammatory response to HS/R is largely independent of the presence of potentially pathogenic gram-negative bacteria colonizing the gut, whereas the local mucosal response to HS/R is profoundly influenced by the microbial ecology within the lumen during and shortly after the period of hemorrhage.

Ethyl pyruvate modulates inflammatory gene expression in mice subjected to hemorrhagic shock.

Administration of pyruvate, an effective scavenger of reactive oxygen species, has been shown to be salutary in numerous models of redox-mediated tissue or organ injury. Pyruvate, however, is unstable in solution and, hence, is not attractive for development as a therapeutic agent. Herein, ethyl pyruvate, which is thought to be more stable than the parent compound, was formulated in a calcium-containing balanced salt solution [Ringer ethyl pyruvate solution (REPS)] and evaluated in a murine model of hemorrhagic shock and resuscitation (HS/R). Resuscitation with REPS instead of Ringer lactate solution (RLS) significantly improved survival at 24 h and abrogated bacterial translocation to mesenteric lymph nodes and the development of increased ileal mucosal permeability to FITC-labeled dextran (4,000 Da) at 4 h. Mice treated with REPS instead of RLS also had lower circulating levels of alanine aminotransferase at 4 h. Treatment with REPS instead of RLS decreased activation of nuclear factor-kappaB in liver and colonic mucosa after HS/R and also decreased the expression of inducible nitric oxide synthase, tumor necrosis factor, cyclooxygenase-2, and interleukin-6 mRNA in liver, ileal mucosa, and/or colonic mucosa. These data support the view that resuscitation with REPS modulates the inflammatory response and decreases hepatocellular and gut mucosal injury in mice subjected to HS/R.

Role of toll-like receptors in changes in gene expression and NF-kappa B activation in mouse hepatocytes stimulated with lipopolysaccharide.

The liver is an important site of host-microbe interaction. Although hepatocytes have been reported to be responsive to lipopolysaccharide (LPS), the global gene expression changes by LPS and mechanism(s) by which LPS stimulates cultured hepatocytes remain uncertain. Cultures of primary mouse hepatocytes were incubated with LPS to assess its effects on the global gene expression, hepatic transcription factors, and mitogen-activated protein (MAP) kinase activation. DNA microarray analysis indicated that LPS modulates the selective expression of more than 80 genes and expressed sequence tags. We have shown previously that hepatocytes express CD14, which is required both for uptake and responsiveness to LPS. In other cells, responsiveness to microbial products requires expression of Toll-like receptors (TLR) and their associated accessory molecules. Hepatocytes expressed TLR1 through TLR9 as well as MyD88 and MD-2 transcripts, as shown by reverse transcriptase PCR analysis, indicating that hepatocytes express all known microbe recognition molecules. The MAP kinase extracellular signal-regulated kinase 1/2 was phosphorylated in response to LPS in mouse hepatocytes, and the levels of phosphorylation were lower in hepatocytes from TLR4-null mice. NF-kappa B activation was reduced in TLR4-mutant or -null hepatocytes compared to control hepatocytes, and this defect was partially restored by adenoviral transduction of mouse TLR4. Thus, hepatocytes respond to nanogram concentrations of LPS through a TLR4 response pathway.