Hypoxia-regulated therapeutic gene as a preemptive treatment strategy against ischemia/reperfusion tissue injury

Alok Pachori

Luis Melo

Melanie Hart

Nicholas Noiseux

^{Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115;}^{Department of Physiology, Queen's University, Kingston, ON, Canada K7L 3N6; and}^{Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, MA 02115}

^{To whom correspondence should be sent at the present address: Office of the Chancellor, Duke University Medical Center 3701, Durham, NC 27710. E-mail:}ude.ekud@uazd.rotciv.

Communicated by Eugene Braunwald, Partners HealthCare System, Inc., Boston, MA, June 29, 2004

Received 2004 Apr 16

Abstract

Ischemia and reperfusion represent major mechanisms of tissue injury and organ failure. The timing of administration and the duration of action limit current treatment approaches using pharmacological agents. In this study, we have successfully developed a preemptive strategy for tissue protection using an adenoassociated vector system containing erythropoietin hypoxia response elements for ischemia-regulated expression of the therapeutic gene human heme-oxygenase-1 (hHO-1). We demonstrate that a single administration of this vector several weeks in advance of ischemia/reperfusion injury to multiple tissues such as heart, liver, and skeletal muscle yields rapid and timely induction of hHO-1 during ischemia that resulted in dramatic reduction in tissue damage. In addition, overexpression of therapeutic transgene prevented long-term pathological tissue remodeling and normalized tissue function. Application of this regulatable system using an endogenous physiological stimulus for expression of a therapeutic gene may be a feasible strategy for protecting tissues at risk of ischemia/reperfusion injury.

Abstract

Ischemia and reperfusion (I/R)-induced tissue injury are major causes of mortality and morbidity in the civilized world (1). I/R injury can develop as a consequence of hypotension, shock, or bypass surgery leading to end-organ failure such as acute renal tubular necrosis, liver failure, and bowel infarct. I/R injury can also develop as a result of complications of vascular disease such as stroke and myocardial infarction. In addition, multiple subclinical I/R incidents can induce cumulative tissue injury leading to chronic degenerative diseases such as vascular dementia, ischemic cardiomyopathy, and renal insufficiency. Cytoprotective strategies using pharmacological agents have yielded limited success in the prevention of I/R injury (2–6). Practical difficulties with timing of administration of the therapy, achieving adequate tissue levels of therapeutic product, and unregulated transgene expression pose significant clinical challenges. Several drug-inducible gene expression systems have been developed (7–10). However, these systems require the exogenous administration of a ligand to induce expression of the therapeutic product and usually yield high basal levels of gene expression, thus reducing the efficacy of regulation.

One of the major mechanisms by which the cells control gene expression during low oxygen tension involves the activation of transcription factor hypoxia-inducible factor 1α (HIF1α), which is quickly degraded during normoxic conditions by ubiquitination mechanisms (11, 12) that include proline hydroxylation and acetylation (13, 14). Activation of HIF1α leads to transcription of several target genes such as vascular endothelial growth factor (15), erythropoietin, nitric oxide synthase, and several antioxidant enzyme systems such as superoxide dismutase, and heme-oxygenase-1 (HO-1), which may provide protection against I/R injury. We have previously shown marked reduction of postinfarction myocardial injury with constitutive HO-1 overexpression (16). However, constitutive HO-1 expression could potentially lead to long-term tissue toxicity and cellular damage (17, 18).

Therefore, an ideal strategy for tissue protection against I/R injury would be a single administration of a therapeutic gene using a vector that will provide regulated transgene expression in response to an endogenous pathophysiological stimulus such as hypoxia. Accordingly, in this study, we have developed an adenoassociated vector (AAV) containing tandem repeats of erythropoietin hypoxia response elements (HREs) for hypoxic/ischemic regulation of human HO-1 (hHO-1) expression. Our data demonstrate that a single administration of this vector before injury into rat skeletal muscle, liver, and heart yields low basal expression during normoxic conditions but is readily induced to express high levels of therapeutic gene in response to acute I/R in vivo, providing protection against such injury.

SWD, septal wall thickness (diastole); PWD, posterior wall thickness (diastole); LAVD, long axis volume (diastole); LAVS, long axis volume (systole); LAAD, long axis area (diastole); LAAS, Long axis area (systole); EF, ejection fraction; LALD, long axis length (diastole).

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Acknowledgments

We thank Mikhail Bourgoun for expert technical assistance with echocardiography. This work was supported in part by National Institutes of Health Grants HL 72010, HL73219, HL 58516, and HL 35610 and gifts from the Edna Mandel Foundation (to V.J.D.) and National Institutes of Health Grants HL 52886, HL 56086, and DE 13499 (to G.L.S.). A.S.P. is supported by National Institutes of Health Postdoctoral Training Grant HL 69601-01. L.G.M. is Canada Research Chair in Molecular Cardiology and a New Investigator of the Heart and Stroke Foundation of Canada, and is supported by grants from the Canadian Institutes of Health Research.

Acknowledgments

Notes

Abbreviations: I/R, ischemia and reperfusion; HO-1, heme-oxygenase-1; hHO-1, human HO-1; AAV, adenoassociated vector; HRE, hypoxia response element.

Notes

Abbreviations: I/R, ischemia and reperfusion; HO-1, heme-oxygenase-1; hHO-1, human HO-1; AAV, adenoassociated vector; HRE, hypoxia response element.

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