HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5668fjev1 20/12/2156    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, X. Articles by Lee, P. J. Search for Related Content PubMed PubMed Citation Articles by Zhang, X. Articles by Lee, P. J.

Endothelial STAT3 is essential for the protective effects of HO-1 in oxidant-induced lung injury

Xuchen Zhang^*, Peiying Shan^*, Ge Jiang^*, Samuel S-M. Zhang, Leo E. Otterbein, Xin-Yuan Fu and Patty J. Lee^*,1

^* Section of Pulmonary and Critical Care Medicine;

Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut USA;

Department of Surgery, Beth Israel Deaconess Medical Center, Harvard University Medical School, Boston, Massachusetts, USA, and the

Microbiology and Immunology Department, Indiana University, Indianapolis, Indiana, USA

¹Correspondence: Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, P.O. Box 208057, New Haven, CT 06520-8057, USA. E-mail: patty.lee{at}yale.edu

SPECIFIC AIMS

High levels of oxygen therapy (hyperoxia) is a life-saving component in the treatment of critically ill patients, but prolonged oxygen therapy has been shown to exacerbate respiratory failure and lead to increased mortality. Hyperoxia leads to the accumulation of reactive oxidants that initiate endothelial and epithelial cell injury, causing pulmonary capillary permeability, inflammation, and respiratory failure. Heme oxygenase (HO)-1 (HO-1) and STAT3 have been shown to confer protection against hyperoxia-induced lung injury. However, nothing is known about the role of lung endothelial cells and the functional link, if any, between STAT3 and HO-1 during hyperoxia-induced lung injury. We sought to address the role of endothelial STAT3 in hyperoxia and in mediating the cytoprotective effects of the stress response protein HO-1.

PRINCIPAL FINDINGS

1. Silencing of HO-1 induction enhances hyperoxia-induced lung injury in vitro and in vivo
Hyperoxia significantly increased HO-1 expression in mouse lung and murine lung endothelial cells (MLEC) exposed to hyperoxia. HO-1 siRNA knocked down hyperoxia-induced HO-1 protein expression in both mouse lung and MLEC. Administration of HO-1 siRNA prior to hyperoxia significantly increased mouse mortality (Fig. 1 A), bronchoalveolar lavage (BAL) protein (Fig. 1B ), BAL leukocytes (Fig. 1C ), TNF- and interleukin (IL)-1ßbeta; mRNA expression (Fig. 1D ), and lung apoptosis (Fig. 1F ) compared with mice given nonspecific siRNA (NS siRNA). Similarly, HO-1 siRNA enhanced hyperoxia-induced MLEC cell injury and death.

View larger version (14K): [in this window] [in a new window]   Figure 1. HO-1 siRNA enhances hyperoxia-induced mortality and lung injury in vivo. A) Percent survival of mice given nonspecific (NS) siRNA (n=11) or HO-1 siRNA (n=11), then exposed to 100 h of continuous hyperoxia (O2) (*P<0.05, NS siRNA vs. HO-1 siRNA). B) Lung permeability was assessed by bronchoalveolar lavage (BAL) protein content in naive mice, mice administered NS siRNA or HO-1 siRNA, then exposed to 72 h O2 (n=3) C) Lung inflammation was detected by BAL cell counts in naive mice, mice administered NS siRNA or HO-1 siRNA, then exposed to 72 h O2 (n=3). BAL protein and cell counts are shown as mean ± SE. *P < 0.05 compared with NS siRNA mice. D) TNF- and IL-1ßbeta; mRNA expression were assayed by RT-PCR in lung lysates after mice were given NS siRNA or HO-1 siRNA and exposed to 72 h O2. ßbeta;-actin was used as a loading control. Results are representative of 3 independent experiments. E) Graphical quantitation of TUNEL-positive cells expressed as % of total cells in lung sections (n=3). Data are shown as mean ± SE. *P < 0.05 compared with O2/NS siRNA.

2. HO-1 overexpression abrogates hyperoxia-induced lung injury in vitro and in vivo
Mice administered adenoviral heme oxygenase-1 (Ad-HO-1) to overexpress HO-1 in lung displayed lower BAL protein, BAL leukocytes, lung apoptosis, and TNF- and IL-1ßbeta; mRNA expression compared with mice administered empty adenoviral vector (Ad-null) during hyperoxia. MLEC overexpressing HO-1 by retroviral HO-1 expression (LSN/HO-1) showed markedly less cell injury, as assessed by LDH activity, and apoptosis compared with empty vector controls (LXSN).

3. Endothelial STAT3 deficiency enhances hyperoxia-induced lung injury in vitro and in vivo
STAT3 was activated, as assessed by phosphorylated (p)-STAT3 expression, in mouse lung and MLEC during hyperoxia. Mice with targeted disruption of endothelial STAT3^–/– (STAT3^E–/–) had significantly greater lung injury than STAT3 wild-type (WT) mice during hyperoxia. STAT3 siRNA specifically inhibited STAT3 expression in MLEC. Consistent with our in vivo findings, MLEC transfected with STAT3 siRNA were more susceptible to hyperoxia-induced cell injury and death.

4. The protective effect of HO-1 is dependent on endothelial cell STAT3 in vivo and in vitro
STAT3^E–/– mice administered intranasal Ad-HO-1 exhibited high levels of HO-1 expression in lung during normoxia (Fig. 2 A) as well as during hyperoxia (Fig. 2B ), indicating that endogenous HO-1 induction in response to hyperoxia or adenoviral-mediated HO-1 induction was not impaired. Exogenous Ad-HO-1 delivery to WT mice significantly increased survival during hyperoxia, whereas STAT3^E–/– mice showed survival rates indistinguishable from that of control mice given Ad-null vector (Fig. 2C ). Indices of lung injury such as BAL protein (Fig. 2D ), BAL leukocytes (Fig. 2E ), and lung apoptosis (Fig. 2F ) were consistent with the survival data. In addition, MLEC overexpressing (LSN/HO-1) or treated with the HO-1 reaction product carbon monoxide (CO) had increased susceptibility to hyperoxia-induced cell injury and death in the presence of STAT3 siRNA compared with MLEC given nonspecific siRNA. These data indicate that both HO-1 and its reaction product CO depend on endothelial STAT3 to exert their protective effects during hyperoxia.

View larger version (37K): [in this window] [in a new window]   Figure 2. The protective effect of HO-1 during hyperoxia is dependent on endothelial STAT3 in vivo. A) Endothelial-targeted STAT3-deficient (STAT3E–/–) mice were administered intranasal rat HO-1 gene in an adenoviral vector (Ad-HO-1, 5x108 plaque-forming units) and lung lysates were obtained after 48 h. Lung lysates were immunoblotted against HO-1 or ßbeta;-tubulin (loading control). B) STAT3E–/–mice were exposed to 72 h O2 after intranasal administration of Ad-HO-1 (5x108 plaque-forming units). Lung lysates were immunoblotted against HO-1 or ßbeta;-tubulin. C) Percent survival of STAT3 WT and STAT3E–/–mice administered Ad-null or Ad-HO-1, then exposed to continuous O2 (n=8–15). *P < 0.05 compared with STAT3 WT Ad-HO-1 / O2. D) Lung permeability was assessed by BAL protein content in naive STAT3E–/– and STAT3E–/– mice administered Ad-null or Ad-HO-1, then exposed to 72 h O2 (n=3–5). Data are shown as mean ± SE. *P < 0.05 compared with Ad-null/O2 and Ad-HO-1/O2 mice. E) Lung inflammation was detected by BAL cell counts in naive STAT3E–/– and STAT3E–/– mice given Ad-null or Ad-HO-1, then exposed to 72 h O2 (n=3–5). Data are shown as mean ± SE. *P < 0.05 compared with Ad-null/O2 and Ad-HO-1/O2 mice. F) TUNEL quantitation was expressed as % of total cells in lung sections from naive STAT3E–/– and STAT3E–/– mice given Ad-null or Ad-HO-1, then exposed to 72 h O2 (n=3–5). Data are shown as mean ± SE. *P < 0.05 compared with Ad-null/O2 and Ad-HO-1/O2.

5. The protective effect of STAT3 during hyperoxia is dependent in part on HO-1 in endothelial cells
WT MLEC transfected with Ad-STAT3 in order to overexpress STAT3 exhibited significantly less injury and death than control MLEC (Ad-null). In the absence of HO-1 (MLEC were isolated from HO-1^–/– mice), STAT3 overexpression still led to a marked decrease in cell injury, as assessed by LDH activity, and to cell death, albeit not to the low levels achieved by WT cells overexpressing STAT3. MLEC transfected with Ad-STAT3 had increased basal levels of HO-1, p-Akt, Bcl-2, and Bcl-xL proteins. STAT3 siRNA decreased the ability of MLEC to up-regulate HO-1, p-Akt, Bcl-2, and Bcl-xL in response to hyperoxia compared with WT or nonspecific siRNA-transfected MLEC. We postulated that a positive feedback system between STAT3 and HO-1 exists. HO-1 overexpression in mouse lung and MLEC led to increased phospho-STAT3, Bcl-xL, and Bcl-2 expression. However, HO-1-deficient MLEC did not exhibit increased Akt, Bcl-2, or Bcl-xL expression despite STAT3 overexpression. These data indicate that STAT3 can activate HO-1 and vice versa, but Akt, Bcl-2, and Bcl-xL are likely directly downstream of HO-1 and not dependent on STAT3.

CONCLUSIONS AND SIGNIFICANCE

Both HO-1 and CO have been shown to be protective against a variety of injuries, but the precise mechanisms remain poorly defined. Previous studies examining the consequences of HO-1 inhibition or deficiency have been limited by the lack of a specific HO-1 inhibitor and by difficulties in generating extensive numbers of HO-1-deficient mice. Interpreting data from HO-1-deficient mice exposed to hyperoxia during adulthood is also perplexing because HO-1 deficiency likely triggers compensatory responses that are probably present before birth. In the current studies we were able to study the function of postnatal HO-1 induction in a highly specific manner using HO-1 siRNA. In the presence of HO-1 siRNA, the mice exhibited increased mortality, injury, and apoptosis. However, HO-1 overexpression using viral delivery systems successfully rescued WT mice and endothelial cells from hyperoxia-induced injury and apoptosis.

Although endothelial integrity is recognized as important during oxidant injury, studies demonstrating the protective functions of the endothelium during hyperoxia in vivo do not exist. We demonstrated that endothelial STAT3 is important in ameliorating lung injury but is essential for the protective functions of exogenous HO-1 and its reaction product CO during hyperoxia. We earlier described CO-mediated STAT3 induction in lung endothelial cells. Together with our current results, the data point to the presence of a positive feedback system in which STAT3 activates HO-1, leading to CO production, which in turn can potentiate STAT3 activation. STAT3 overexpression still had significant antiapoptotic effects despite the absence of HO-1, indicating the presence of both HO-1-dependent and independent mechanisms of STAT3-mediated protection during hyperoxia. This is likely due to the multiple downstream protective pathways modulated by STAT3, of which HO-1 is only one. STAT3 and HO-1 both appear to act as pleiotropic molecules with the ability to modulate various downstream targets that may or may not overlap, but ultimately result in significant protection against noxious stimuli. We speculate that the presence of a positive feedback system between two protective molecules such as HO-1 and STAT3 optimizes defense against lethal injury. Therefore, these studies serve to delineate a previously unappreciated interdependence between HO-1 and STAT3 in vivo and in vitro and may enhance our understanding of lung protective strategies.

View larger version (9K): [in this window] [in a new window]   Figure 3. Schematic diagram.

This article has been cited by other articles:

				M.-F. Mashreghi, R. Klemz, I. S. Knosalla, B. Gerstmayer, U. Janssen, R. Buelow, A. Jozkowicz, J. Dulak, H.-D. Volk, and K. Kotsch Inhibition of Dendritic Cell Maturation and Function Is Independent of Heme Oxygenase 1 but Requires the Activation of STAT3 J. Immunol., June 15, 2008; 180(12): 7919 - 7930. [Abstract] [Full Text] [PDF]

				L. J. Quinton, M. R. Jones, B. E. Robson, B. T. Simms, J. A. Whitsett, and J. P. Mizgerd Alveolar Epithelial STAT3, IL-6 Family Cytokines, and Host Defense during Escherichia coli Pneumonia Am. J. Respir. Cell Mol. Biol., June 1, 2008; 38(6): 699 - 706. [Abstract] [Full Text] [PDF]

				J. M. Siner, G. Jiang, Z. I. Cohen, P. Shan, X. Zhang, C. G. Lee, J. A. Elias, and P. J. Lee VEGF-induced heme oxygenase-1 confers cytoprotection from lethal hyperoxia in vivo FASEB J, May 1, 2007; 21(7): 1422 - 1432. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5668fjev1 20/12/2156    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, X. Articles by Lee, P. J. Search for Related Content PubMed PubMed Citation Articles by Zhang, X. Articles by Lee, P. J.