The theme of this renewal is that strategies involving overexpression of pulmonary endothelial cell metallothionein (MT) will provide a novel approach to augmenting the antioxidant system of the neonatal lung. Metallothioneins, the major intracellular thiol-containing (cysteine: 30 mol%) heavy metal (Cd,Zn,Cu) binding proteins, are inducible by a variety of pharmacological and environmental conditions and participate in cellular defense against partially reduced oxygen species (PROS). We propose to study the effects of overexpression of MT in the sensitivity to hyperoxia of cultured ovine pulmonary artery endothelial cells (OPAEC) as well as intact newborn lambs. In preliminary experiments, we have noted that induction (with cadmium pre-treatment or direct gene transfer) of MT expression in OPAEC is associated with decreased sensitivity to oxidant injury (tert butyl hydroperoxide or 95% O2). The late term fetal lamb is ideal to evaluate a protective role for MT against pulmonary vascular oxidant injury since it can be safely manipulated in utero and unlike many other experimental animals, it remains sensitive to hyperoxia as a neonate. Accordingly, we will determine if overexpression of MT in OPAEC with cadmium pretreatment (AIM I) or direct transfer of mouse MT-I gene (AIM II) affects the sensitivity of these cells to hyperoxia (95% O2; 24-72 hrs). We will then determine the mechanism (AIM III) by which overexpression may protect these cells including assessing whether MT: a) interferes with hyperoxic induced increases in PROS; b) acts as a direct interceptor of these radicals; or c) indirectly affects other antioxidants (CuZn SOD; glutathione peroxidase). We will then (AIM IV) use immunoliposomes targeted to the pulmonary vascular endothelium of 143d fetal lamb as a vehicle for transferring mouse MT-I gene and determine the sensitivity of the full term (147d) neonate to hyperoxia (100%; 1-4d postnatal). Overexpression of MT will be studied at mRNA (Northern blotting and in situ hybridization) and protein (ELISA and immunocytochemistry) levels in OPAEC and lung tissue. Functional determinations of oxidant injury in OPAEC will include 5-hydroxytrypta- mine transport and radioactive chromium release. Structural (capillary stereology) and functional (lung water balance via lymph fistula) studies will be performed in the intact neonate exposed to hyperoxia with or without MT gene transfer. These studies will provide new information regarding the function of MT in vascular endothelium. Furthermore a novel strategy regarding augmentation of antioxidant defense mechanisms of the newborn lung via in utero gene transfer in the late term fetus will be evaluated.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL032154-11
Application #
2216962
Study Section
Lung Biology and Pathology Study Section (LBPA)
Project Start
1987-09-01
Project End
1997-12-31
Budget Start
1994-01-01
Budget End
1994-12-31
Support Year
11
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Pharmacology
Type
Schools of Dentistry
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Stitt, Molly S; Wasserloos, K J; Tang, X et al. (2006) Nitric oxide-induced nuclear translocation of the metal responsive transcription factor, MTF-1 is mediated by zinc release from metallothionein. Vascul Pharmacol 44:149-55
Wilson, Annette; He, Fengtian; Li, Jiang et al. (2005) Targeted delivery of therapeutic oligonucleotides to pulmonary circulation. Adv Genet 54:21-41
Wilson, Annette; Zhou, Wen; Champion, Hunter C et al. (2005) Targeted delivery of oligodeoxynucleotides to mouse lung endothelial cells in vitro and in vivo. Mol Ther 12:510-8
Li, Jiang; Ma, Zheng; Tang, Zhi-Lue et al. (2004) CpG DNA-mediated immune response in pulmonary endothelial cells. Am J Physiol Lung Cell Mol Physiol 287:L552-8
Ma, Zheng; Li, Jiang; Yang, Lijuan et al. (2004) Inhibition of LPS- and CpG DNA-induced TNF-alpha response by oxidized phospholipids. Am J Physiol Lung Cell Mol Physiol 286:L808-16
St Croix, Claudette M; Stitt, Molly S; Leelavanichkul, Karanee et al. (2004) Nitric oxide-induced modification of protein thiolate clusters as determined by spectral fluorescence resonance energy transfer in live endothelial cells. Free Radic Biol Med 37:785-92
Collins, Joy L; Vodovotz, Yoram; Hierholzer, Christian et al. (2003) Characterization of the expression of inducible nitric oxide synthase in rat and human liver during hemorrhagic shock. Shock 19:117-22
Zhang, J; Wilson, A; Alber, S et al. (2003) Prolonged gene expression in mouse lung endothelial cells following transfection with Epstein-Barr virus-based episomal plasmid. Gene Ther 10:822-6
Wilson, Annette; Pitt, Bruce; Li, Song (2002) Complex roles of CpG in liposomal delivery of DNA and oligonucleotides. Biosci Rep 22:309-22
Jiang, Jianfei; St Croix, Claudette M; Sussman, Nancy et al. (2002) Contribution of glutathione and metallothioneins to protection against copper toxicity and redox cycling: quantitative analysis using MT+/+ and MT-/- mouse lung fibroblast cells. Chem Res Toxicol 15:1080-7

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