Expression of antioxidant protective proteins in the rat retina during prenatal and postnatal development.
(1/1786)
PURPOSE: In retinopathy of prematurity, capillary growth in the retina is attenuated. Subsequent cyclic elevation of oxygen levels leads to renewed capillary growth that may eventually result in retinal detachment. It is hypothesized that the sensitivity of the premature retina to oxidative shock results from the absence of antioxidant protective proteins. METHODS: The expression of heme oxygenase-1, metallothionein, superoxide dismutase, and catalase mRNAs was measured in retinas of rats from 6 days before birth to 4 days after birth using in situ hybridization and semiquantitative reverse transcription-polymerase chain reaction with Southern blot analysis. RESULTS: Superoxide dismutase mRNA was expressed to a similar extent at all time points. Metallothionein mRNA expression, which was high at embryonic days (E) 16 and 18, decreased to low levels by the time of birth and remained low at least until 4 days after birth. Catalase mRNA expression was low until birth and increased until at least postnatal day 4. Heme oxygenase-1 mRNA showed low expression at E16 and E18, increased before birth, and then diminished. CONCLUSIONS: Four antioxidant protein mRNAs showed very different patterns of expression in the rat retina. Two of these proteins, heme oxygenase-1 and catalase, were expressed at relatively low levels until approximately the time of birth. The former is important in protection against heme-mediated generation of reactive oxygen species, whereas the latter protects against hydrogen peroxide-generated damage. As a result of the low expression of these mRNAs, and presumably the proteins encoded by them, the premature rat (and probably the premature human) is likely to be born without a full complement of antioxidant defenses. (+info)
Protective effects of transient HO-1 overexpression on susceptibility to oxygen toxicity in lung cells.
(2/1786)
Rat fetal lung cells (RFL-6) were transiently transfected with a full-length rat heme oxygenase (HO)-1 cDNA construct and then exposed to hyperoxia (95% O2-5% CO2) for 48 h. Total HO activity and HO-1 protein were measured as well as cell viability, lactate dehydrogenase (LDH) release, protein oxidation, lipid peroxidation, and total glutathione to measure oxidative injury. HO-1 overexpression resulted in increased total HO activity (2-fold), increased HO-1 protein (1.5-fold), and increased cell proliferation. Immunohistochemistry revealed perinuclear HO-1 localization, followed by migration to the nucleus by day 3. Decreased cell death, protein oxidation, and lipid peroxidation but increased LDH release and glutathione depletion were seen with HO-1 overexpression. Reactive iron content could not explain the apparent loss of cell membrane integrity. With the addition of tin mesoporphyrin, total HO activity was decreased and all changes in injury parameters were normalized to control values. We conclude that moderate overexpression of HO-1 is protective against oxidative injury, but we speculate that there is a beneficial threshold of HO-1 expression. (+info)
Crystallization of recombinant human heme oxygenase-1.
(3/1786)
Heme oxygenase catalyzes the NADPH, O2, and cytochrome P450 reductase dependent oxidation of heme to biliverdin and carbon monoxide. One of two primary isozymes, HO-1, is anchored to the endoplasmic reticulum membrane via a stretch of hydrophobic residues at the C-terminus. While full-length human HO-1 consists of 288 residues, a truncated version with residues 1-265 has been expressed as a soluble active enzyme in Escherichia coli. The recombinant enzyme crystallized from ammonium sulfate solutions but the crystals were not of sufficient quality for diffraction studies. SDS gel analysis indicated that the protein had undergone proteolytic degradation. An increase in the use of protease inhibitors during purification eliminated proteolysis, but the intact protein did not crystallize. N-terminal sequencing and mass spectral analysis of dissolved crystals indicated that the protein had degraded to two major species consisting of residues 1-226 and 1-237. Expression of the 1-226 and 1-233 versions of human HO-1 provided active enzyme that crystallizes in a form suitable for diffraction studies. These crystals belong to space group P2(1), with unit cell dimensions a = 79.3 A, b = 56.3 A, c = 112.8 A, and beta = 101.5 degrees. (+info)
Heme and acute inflammation role in vivo of heme in the hepatic expression of positive acute-phase reactants in rats.
(4/1786)
Acute-phase protein synthesis in the liver during inflammation is regulated via cytokines and glucocorticoids. Using quantitative reverse transcription (RT)-PCR analysis and immunoassay, we explored, in the rat, the response of the acute-phase protein, alpha-2 macroglobulin (A2M), after systemic inflammation induced by lipopolysaccharide (LPS) or localized inflammation induced by turpentine oil (TO). The results indicate that synthesis of A2M is higher following TO-induced inflammation than LPS-induced inflammation and is not correlated with interleukin (IL)-6 or glucocorticoid levels. We studied the putative role of heme in this differential A2M expression following localized vs. systemic inflammation; addition of heme during LPS-induced inflammation can boost the expression of A2M, whereas blocking heme synthesis (by succinyl acetone) or enhancing its consumption in parallel biosynthetic pathways (cytochrome P450 induction by phenobarbital) decreases A2M expression. This decrease was abolished by exogenous heme supplementation. Finally, we demonstrate that heme supplementation is also able to increase the A2M response in female rats to a level similar to that in male rats providing a new insight into the puzzling sexual dimorphism observed previously during localized inflammation. We propose that heme should be considered a new regulatory element in controlling liver A2M expression during inflammation. (+info)
Role of nitric oxide in pathogenesis of herpes simplex virus encephalitis in rats.
(5/1786)
The role of nitric oxide (NO) in the pathogenesis of viral encephalitis was investigated by using an experimental model of herpes simplex virus type 1 (HSV-1) encephalitis in Lewis rats. The expression of inducible NO synthase (iNOS) mRNA determined by Northern blotting was observed first in the olfactory bulb and the brain stem on day 5 after intranasal inoculation of HSV-1, and thereafter iNOS mRNA was detected in other brain regions, i.e., cerebrum and cerebellum. In various parts of the brain, excessive NO production was identified by electron spin resonance spectroscopy. The temporal and spatial patterns of iNOS expression coincided with those of viral propagation, as demonstrated by polymerase chain reaction for HSV-1 gene expression as well as by the plaque-forming assay. Immunohistochemical study determined that iNOS was localized mainly in monocyte-derived macrophages. Treatment of virus-infected animals with the NOS inhibitor Nomega-monomethyl-l-arginine (l-NMMA), but not Nomega-monomethyl-d-arginine, significantly ameliorated not only clinical symptoms such as paralysis and seizures but also mortality. Virus yield from brain tissue was not affected by l-NMMA treatment. It is of interest that increased expression of the antioxidant enzyme heme oxygenase-1 was observed in the HSV-1-infected brain; this increased expression was strongly inhibited by l-NMMA treatment. These data suggest that the high level of NO produced by iNOS is a pathogenic factor in HSV-1-induced encephalitis in rats. (+info)
Exogenous administration of heme oxygenase-1 by gene transfer provides protection against hyperoxia-induced lung injury.
(6/1786)
Heme oxygenase-1 (HO-1) confers protection against a variety of oxidant-induced cell and tissue injury. In this study, we examined whether exogenous administration of HO-1 by gene transfer could also confer protection. We first demonstrated the feasibility of overexpressing HO-1 in the lung by gene transfer. A fragment of the rat HO-1 cDNA clone containing the entire coding region was cloned into plasmid pAC-CMVpLpA, and recombinant adenoviruses containing the rat HO-1 cDNA fragment Ad5-HO-1 were generated by homologous recombination. Intratracheal administration of Ad5-HO-1 resulted in a time-dependent increase in expression of HO-1 mRNA and protein in the rat lungs. Increased HO-1 protein expression was detected diffusely in the bronchiolar epithelium of rats receiving Ad5-HO-1, as assessed by immunohistochemical studies. We then examined whether ectopic expression of HO-1 could confer protection against hyperoxia-induced lung injury. Rats receiving Ad5-HO-1, but not AdV-betaGal, a recombinant adenovirus expressing Escherichia coli beta-galactosidase, before exposure to hyperoxia (>99% O2) exhibited marked reduction in lung injury, as assessed by volume of pleural effusion and histological analyses (significant reduction of edema, hemorrhage, and inflammation). In addition, rats receiving Ad5-HO-1 also exhibited increased survivability against hyperoxic stress when compared with rats receiving AdV-betaGal. Expression of the antioxidant enzymes manganese superoxide dismutase (Mn-SOD) and copper-zinc superoxide dismutase (CuZn-SOD) and of L-ferritin and H-ferritin was not affected by Ad5-HO-1 administration. Furthermore, rats treated with Ad5-HO-1 exhibited attenuation of hyperoxia-induced neutrophil inflammation and apoptosis. Taken together, these data suggest the feasibility of high-level HO-1 expression in the rat lung by gene delivery. To our knowledge, we have demonstrated for the first time that HO-1 can provide protection against hyperoxia-induced lung injury in vivo by modulation of neutrophil inflammation and lung apoptosis. (+info)
Carbon monoxide provides protection against hyperoxic lung injury.
(7/1786)
Findings in recent years strongly suggest that the stress-inducible gene heme oxygenase (HO)-1 plays an important role in protection against oxidative stress. Although the mechanism(s) by which this protection occurs is poorly understood, we hypothesized that the gaseous molecule carbon monoxide (CO), a major by-product of heme catalysis by HO-1, may provide protection against oxidative stress. We demonstrate here that animals exposed to a low concentration of CO exhibit a marked tolerance to lethal concentrations of hyperoxia in vivo. This increased survival was associated with highly significant attenuation of hyperoxia-induced lung injury as assessed by the volume of pleural effusion, protein accumulation in the airways, and histological analysis. The lungs were completely devoid of lung airway and parenchymal inflammation, fibrin deposition, and pulmonary edema in rats exposed to hyperoxia in the presence of a low concentration of CO. Furthermore, exogenous CO completely protected against hyperoxia-induced lung injury in rats in which endogenous HO enzyme activity was inhibited with tin protoporphyrin, a selective inhibitor of HO. Rats exposed to CO also exhibited a marked attenuation of hyperoxia-induced neutrophil infiltration into the airways and total lung apoptotic index. Taken together, our data demonstrate, for the first time, that CO can be therapeutic against oxidative stress such as hyperoxia and highlight possible mechanism(s) by which CO may mediate these protective effects. (+info)
Hypoxia induces severe right ventricular dilatation and infarction in heme oxygenase-1 null mice.
(8/1786)
Heme oxygenase (HO) catalyzes the oxidation of heme to generate carbon monoxide (CO) and bilirubin. CO increases cellular levels of cGMP, which regulates vascular tone and smooth muscle development. Bilirubin is a potent antioxidant. Hypoxia increases expression of the inducible HO isoform (HO-1) but not the constitutive isoform (HO-2). To determine whether HO-1 affects cellular adaptation to chronic hypoxia in vivo, we generated HO-1 null (HO-1(-/-)) mice and subjected them to hypoxia (10% oxygen) for five to seven weeks. Hypoxia caused similar increases in right ventricular systolic pressure in wild-type and HO-1(-/-) mice. Although ventricular weight increased in wild-type mice, the increase was greater in HO-1(-/-) mice. Similarly, the right ventricles were more dilated in HO-1(-/-) mice. After seven weeks of hypoxia, only HO-1(-/-) mice developed right ventricular infarcts with organized mural thrombi. No left ventricular infarcts were observed. Lipid peroxidation and oxidative damage occurred in right ventricular cardiomyocytes in HO-1(-/-), but not wild-type, mice. We also detected apoptotic cardiomyocytes surrounding areas of infarcted myocardium by terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) assays. Our data suggest that in the absence of HO-1, cardiomyocytes have a maladaptive response to hypoxia and subsequent pulmonary hypertension. J.Clin. Invest. 103:R23-R29 (1999). (+info)