Carbon monoxide (CO), one of the products of heme oxygenase action on heme, prevents arteriosclerotic lesions that occur following aorta transplantation; pre-exposure to 250 parts per million of CO for 1 hour before injury suppresses stenosis after carotid balloon injury in rats as well as in mice. The protective effect of CO is associated with a profound inhibition of graft leukocyte infiltration/activation as well as with inhibition of smooth muscle cell proliferation. The anti-proliferative effect of CO in vitro requires the activation of guanylate cyclase, the generation of cGMP, the activation of p38 mitogen-activated protein kinases and the expression of the cell cycle inhibitor p21Cip1. These findings demonstrate a protective role for CO in vascular injury and support its use as a therapeutic agent.

To better understand the molecular basis of chronic obstructive pulmonary disease (COPD), we used serial analysis of gene expression (SAGE) and microarray analysis to compare the gene expression patterns of lung tissues from COPD and control smokers. A total of 59,343 tags corresponding to 26,502 transcripts were sequenced in SAGE analyses. A total of 327 genes were differentially expressed (1.5-fold up-or down-regulated). Microarray analysis using the same RNA source detected 261 transcripts that were differentially expressed to a significant degree between GOLD-2 and GOLD-0 smokers. We confirmed the altered expression of a select number of genes by using real-time quantitative RT-PCR. These genes encode for transcription factors (EGR1 and FOS), growth factors or related proteins (CTGF, CYR61, CX3CL1, TGFB1, and PDGFRA), and extracellular matrix protein (COL1A1). Immunofluorescence studies on the same lung specimens localized the expression of Egr-1, CTGF, and Cyr61 to alveolar epithelial cells, airway epithelial cells, and stromal and inflammatory cells of GOLD-2 smokers. Cigarette smoke extract induced Egr-1 protein expression and increased Egr-1 DNA-binding activity in human lung fibroblast cells. Cytomix (tumor necrosis factor ␣, IL-1␤, and IFN-␥) treatment showed that the activity of matrix metalloproteinase-2 (MMP-2) was increased in lung fibroblasts from EGR1 control (؉/؉) mice but not detected in that of EGR1 null (؊/؊) mice, whereas MMP-9 was regulated by EGR1 in a reverse manner. Our study represents the first comprehensive analysis of gene expression on GOLD-2 versus GOLD-0 smokers and reveals previously unreported candidate genes that may serve as potential molecular targets in COPD.

Caveolin-1 has been reported to regulate apoptosis, lipid metabolism, and endocytosis in macrophages. In the present study, we demonstrate that caveolin-1 can act as a potent immunomodulatory molecule. We first observed caveolin-1 expression in murine alveolar macrophages by Western blotting and immunofluorescence microscopy. Loss-of-function experiments using small interfering RNA showed that down regulating caveolin-1 expression in murine alveolar and peritoneal macrophages increased LPS-induced proinflammatory cytokine TNF-alpha and IL-6 production but decreased anti-inflammatory cytokine IL-10 production. Gain-of-function experiments demonstrated that overexpression of caveolin-1 in RAW264.7 cells decreased LPS-induced TNF-alpha and IL-6 production and augmented IL-10 production. p38 mitogen-activated protein kinase (MAPK) phosphorylation was increased by overexpressing caveolin-1 in RAW264.7 cells, whereas c-Jun N-terminal kinase, extracellular signal-regulated kinase MAPK, and Akt phosphorylation were inhibited. The antiinflammatory modulation of LPS-induced cytokine production by caveolin-1 was significantly abrogated by the administration of p38 inhibitor SB203580 in RAW264.7 cells. Peritoneal macrophages isolated from MKK3 null mice did not demonstrate any modulation of LPS-induced cytokine production by caveolin-1. LPS-induced activation of NF-kappaB and AP-1 determined by electrophoretic mobility shift assay were significantly reduced by overexpressing caveolin-1 in RAW264.7 cells. The reductions were attenuated by the administration of p38 inhibitor SB203580. Taken together, our data suggest that caveolin-1 acts as a potent immunomodulatory effector molecule in immune cells and that the regulation of LPS-induced cytokine production by caveolin-1 involves the MKK3/p38 MAPK pathway.

Successful lung transplantation has been limited by the high incidence of acute graft rejection. There is mounting evidence that the stress response gene heme oxygenase-1 (HO-1) and/or its catalytic by-product carbon monoxide (CO) confers cytoprotection against tissue and cellular injury. This led us to hypothesize that CO may protect against lung transplant rejection via its anti-inflammatory and antiapoptotic effects. Orthotopic left lung transplantation was performed in Lewis rat recipients from Brown-Norway rat donors. HO-1 mRNA and protein expression were markedly induced in transplanted rat lungs compared to sham-operated control lungs. Transplanted lungs developed severe intraalveolar hemorrhage, marked infiltration of inflammatory cells, and intravascular coagulation. However, in the presence of CO exposure (500 ppm), the gross anatomy and histology of transplanted lungs showed marked preservation. Furthermore, transplanted lungs displayed increased apoptotic cell death compared with the transplanted lungs of CO-exposed recipients, as assessed by TUNEL and caspase-3 immunostaining. CO exposure inhibited the induction of IL-6 mRNA and protein expression in lung and serum, respectively. Gene array analysis revealed that CO also down-regulated other proinflammatory genes, including MIP-1alpha and MIF, and growth factors such as platelet-derived growth factor, which were up-regulated by transplantation. These data suggest that the anti-inflammatory and antiapoptotic properties of CO confer potent cytoprotection in a rat model of lung transplantation.

Human epithelial (A549) cells exposed to hyperoxia die by cellular necrosis. In the current study, we demonstrated the involvement of apoptogenic factors in epithelial cell necrosis in response to hyperoxia, including the formation of the Fas-related death-inducing signaling complex and initiation of mitochondria-dependent apoptotic pathways. We showed increased activation of both Bid and Bax in A549 cells subjected to hyperoxia. Bax activation involved a Bid-assisted conformational change. We discovered that the response to hyperoxia in vivo predominantly involved the activation of the Bid/ caspase-8 pathway without apparent increases in Bax expression. Disruption of the Bid pathway by gene deletion protected against cell death in vivo and in vitro. Likewise, inhibition of caspase-8 by Flip also protected against cell death. Taken together, we have demonstrated the involvement of apoptogenic factors in epithelial cell responses to hyperoxia, despite a final outcome of cellular necrosis. We have, for the first time, identified a predominant role for the caspase-8/Bid pathway in signaling associated with hyperoxic lung injury and cell death in vivo and in vitro.

The gaseous molecule carbon monoxide (CO) is elevated in the breath of individuals with asthma. The physiologic function of CO in asthma is poorly understood. Here we demonstrate that CO (250 ppm) markedly inhibits human airway smooth muscle cell (HASMC) proliferation, arresting cells at the G0/G1 phase. This CO-induced cell growth arrest of HASMC was associated with upregulation of p21 and downregulation of cyclin D1 expression. It is generally believed that the signaling pathway by which CO affects biologic processes is primarily mediated via the guanylyl cyclase/3',5'-Guanylate cyclic monophosphate (cGMP) pathway. To examine whether guanylyl cyclase/cGMP was involved in CO-induced growth arrest of HASMC, Rp-8-Br-cGMP, a selective inhibitor of cGMP-dependent protein kinase and ODQ, a selective inhibitor of soluble guanylate cyclase, were administered to HASMC in the presence of CO. Interestingly, CO-induced cell growth arrest was not reversed by these inhibitors. We next examined whether the extracellular signal-regulated kinase (ERK) 1/ERK2 mitogen-activated protein kinase (MAPK) signaling pathway may regulate the antiproliferative effect of CO. We first showed time-dependent activation of the various MAPKs in HASMC in response to serum, including phosphorylated ERK1/ERK2, p38, and JNK and then demonstrated that CO exerted negligible effect on activated p38 and JNK; however, ERK activation was significantly attenuated in the presence of CO. These data suggest that CO can inhibit HASMC proliferation via the ERK1/ERK2 MAPK pathway, independent of a guanylyl cyclase/cGMP independent pathway. CO may act as an important mediator of remodeling of human airways in asthma via its ability to regulate cell growth of airway smooth muscle cells.

Idiopathic pulmonary fibrosis is an incurable fibrosing disorder that progresses relentlessly to respiratory failure. We hypothesized that a product of heme oxygenase activity, carbon monoxide (CO), may have anti-fibrotic effects. To test this hypothesis, mice treated with intratracheal bleomycin were exposed to low-concentration inhaled CO or ambient air. Lungs of mice treated with CO had significantly lower hydroxyproline accumulation than controls. Fibroblast proliferation, thought to play a central role in the progression of fibrosis, was suppressed by in vitro exposure to CO. CO caused increased cellular levels of p21(Cip1) and decreased levels of cyclins A and D. This effect was independent of the observed suppression of MAPK's phosphorylation by CO but was dependent on increased cGMP levels. Further, CO-exposed cells elaborated significantly less fibronectin and collagen-1 than control cells. This same effect was seen in vivo. Suppression of collagen-1 production did not depend on MAPK or guanylate cyclase signaling pathways but did depend on the transcriptional regulator Id1. Taken together, these data suggest that CO exerts an anti-fibrotic effect in the lung, and this effect may be due to suppression of fibroblast proliferation and/or suppression of matrix deposition by fibroblasts.

T lymphocyte activation and proliferation is involved in many pathological processes. We have recently shown that carbon monoxide (CO), an enzymatic product of heme oxyenase-1 (HO-1), confers potent antiproliferative effects in airway and vascular smooth muscle cells. The purpose of this study was to determine whether CO can inhibit T lymphocyte proliferation and then to determine the mechanism by which CO can modulate T lymphocyte proliferation. In the presence of 250 parts per million CO, CD3-activated T lymphocyte proliferation was, remarkably, inhibited by 80% when compared with controls. We observed that the antiproliferative effect of CO in T lymphocytes was independent of the mitogen-activated protein kinase or cGMP signaling pathways, unlike what we demonstrated previously in smooth muscle cells. We demonstrate that CO inhibited caspase-3 and caspase-8 expression and activity, and caspase inhibition with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK pan-caspase inhibitor) blocked T lymphocyte proliferation. Furthermore, in caspase-8-deficient lymphocytes, the antiproliferative effect of CO was markedly attenuated, further supporting the involvement of caspase-8 in the antiproliferative effects of CO. CO also increased the protein level of p21Cip1, and CO-mediated inhibition of caspase activity is partially regulated by p21Cip1. Taken together, these data suggest that CO confers potent antiproliferative effects in CD3-activated T lymphocytes and that these antiproliferative effects in T lymphocytes are mediated by p21Cip1-dependent caspase activity, in particular caspase-8, independent of cGMP and mitogen-activated protein kinase signaling pathways.