Inflammation and oxidative stress play important roles in cystic fibrosis (CF) lung disease. Inflammatory/oxidant-mediated induction of heme oxygenase-1 (HO-1) is believed to be a cytoprotective response. This study examined HO-1 expression in lung samples from patients with CF using immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. In addition, we evaluated myeloperoxidase staining as a marker of acute inflammation and potentially an increase in oxidant stress and Prussian blue and ferritin staining to assess iron status of the lung. Macrophage HO-1 staining was increased in diseased lungs as compared with normal control subjects and correlated with myeloperoxidase staining. Quantitative reverse transcription-polymerase chain reaction further supported an increase in HO-1 expression in CF lung disease. Although iron staining was minimal, ferritin staining was increased in diseased lungs in concert with HO-1 staining. To determine whether HO-1 induction was cytoprotective, we evaluated a CF airway epithelial cell line, IB3.1, in response to Pseudomonas aeruginosa-induced injury/apoptosis in cells overexpressing HO-1 by either transient or stable transfection of pcDNA3.1/HO-1 construct. Overexpression of HO-1 resulted in protection against P. aeruginosa-induced injury/apoptosis. This suggests that the induction of HO-1 in patients with CF is a cytoprotective event and that augmenting its expression is a potential therapy against bacterial injury.

Background-Rapamycin is an immunosuppressive agent with antiproliferative properties against not only lymphocytes but also vascular endothelial and smooth muscle cells, and it reduces the fibroproliferative response to vascular injury. Heme oxygenase-1 (HO-1) has also been shown to have graft protective effects and to inhibit vascular remodeling. In this study, we evaluated whether there is an interaction between rapamycin and HO-1. Methods and Results-In human pulmonary artery endothelial or smooth muscle cells, HO-1 expression was evaluated in response to rapamycin or wortmannin, an inhibitor of the upstream modulator of mammalian target of rapamycin (mTOR) PI-3K. We also evaluated whether the inhibitory actions of rapamycin on platelet-derived growth factordependent proliferation was mediated by HO using the chemical inhibitor tin protoporphyrin. Rapamycin induced HO-1 expression in both pulmonary endothelial and smooth muscle cells, whereas no to little increase was seen in response to another immunosuppressive agent, cyclosporin A. HO-1 expression was also increased in response to wortmannin, suggesting that the PI-3K-mTOR pathway is required for this induction. Inhibition of HO activity resulted in a loss of the antiproliferative activity of rapamycin in growth factor-stimulated smooth muscle cells. Conclusions-The induction of HO-1 expression by rapamycin and, more importantly, the effects of tin protoporphyrin, an inhibitor of HO activity, on the antiproliferative actions of rapamycin suggest that the effects of rapamycin may be, at least in part, modulated by its actions on HO-1.

Rapamycin inhibits the development and progression of vascular disease. We previously showed that rapamycin induces the cytoprotective protein, heme oxygenase-1 (HO-1), and more importantly, chemically inhibiting HO-1 blocked the antiproliferative actions of rapamycin. In this study, we evaluated whether HO-1 is required for the vascular protective effects of rapamycin in vivo using a rat monocrotaline-induced pulmonary hypertension model. Rats were exposed to monocrotaline with or without rapamycin and HO activity was altered using the chemical inhibitor, tin protoporphyrin or the inducer, cobalt protoporphyrin. We also evaluated possible mechanisms of rapamycin-dependent induction of HO-1, and how HO-1 mediates growth factor-dependent antiproliferative actions of rapamycin. Proliferation and cell cycle progression were examined in smooth muscle cells derived from both wild-type and HO-1 knockout (HO-1 À/À ) mice in response to growth factors and rapamycin. Similar to our previous findings in vitro, rapamycin induced HO-1 in rat lung. Rapamycin also inhibited the development of monocrotaline-induced pulmonary hypertension, and this protective effect was blocked with the addition of tin protoporphyrin. In addition, treatment with cobalt protoporphyrin resulted in a substantial protection in this model of pulmonary hypertension. Rapamycin induction of HO-1 was dependent upon a transcriptional event; however, it was not mediated through an altered redox state or mammalian targets of rapamycin inhibition. Unlike wild-type cells, the growth of HO-1 À/À mouse aortic smooth muscle cells was not inhibited or cell cycle arrested in G1 in response to rapamycin. This study demonstrates that HO-1 is critical for the antiproliferative and vascular protective effects of rapamycin in vitro and in vivo in monocrotaline-induced pulmonary hypertension. Laboratory Investigation (2006Investigation ( ) 86, 62-71. doi:10.1038 published online 31 October 2005 Keywords: animal model; aorta; heme oxygenase; monocrotaline; pulmonary hypertension; rapamycinThe fibroproliferative response to injury is a pathologic property of vascular diseases, such as pulmonary hypertension.1-3 Pathologic changes observed in pulmonary arterial hypertension (PAH) include medial hypertrophy, adventitial thickening, and abnormal growth of endothelial cells. A limitation of current therapies for PAH is that they are mostly aimed at vasodilatation, rather than against the fibroproliferative response.

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HO-1 protein expression is increased in murine heterotopic airway rejection, and deficiency of HO-1 accelerates the development of the obliterative bronchiolitis-like lesion. IL-10 protein expression parallels expression of HO-1, suggesting that IL-10 may participate in the genesis of HO-1's effects on the inflammatory processes triggered by allotransplantation.