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Inducible nitric oxide synthase iNOS can be coexpressed with acute phase reactants in hepatocytes however, it is unknown if NO can regulate the acute phase response. We tested the hypothesis that iNOSderived nitric oxide NO attenuates the acute phase response by inhibiting IL6enhanced Stat3 DNAbinding activity and type II acute phase mRNA expression. iNOS was overexpressed in cultured rat hepatocytes via transduction with a replication defective adenovirus containing cDNA for human iNOS AdiNOS, and Stat3 DNAbinding activity was determined by electrophoretic mobility shift assay EMSA. EMSAs demonstrated that AdiNOS inhibits IL6induced Stat3 activation and that this inhibition is reversible in the presence of the NOS inhibitor NGmonomethylLarginine LNMA. The induction of fibrinogen mRNA by IL6, a Stat3 dependent process, is attenuated in AdiNOStransduced cells and partially reversed by LNMA. Thus, iNOS overexpression suppresses IL6induced Stat3 activation and type II acute phase mRNA expression in cultured hepatocytes. This suppression may represent a mechanism by which NO downregulates the acute phase response.

ABSTRACTMetallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotection during metal exposure and oxidative stress. The roles of MT in copper (Cu) binding and release and modulation of redox cycling are unresolved. We hypothesized that Cu-binding to MT renders Cu redox inactive, but that oxidation of free thiols critical for metal binding can reduce MT/Cu interactions and potentiate Cu redox cycling. Overexpression of MT in cells by cadmium pretreatment or ectopic overexpression by gene transfer confers protection from Cu-dependent lipid oxidation and cytotoxicity. Using a chemically defined model system (Cu/ascorbate/H2O2) to study Cu/MT interactions, we observed that MT inhibited Cu-dependent oxidation of luminol. In the absence of H2O2, MT blocked Cu-dependent ascorbyl radical production with a stoichiometry corresponding to Cu/MT ratios 12. In the presence of H2O2, Cu-dependent hydroxyl radical formation was inhibited only up to Cu/MT ratios 6. Using low-temperature EPR of free Cu2to assess Cu/MT physical interactions, we observed that the maximal amount of Cu1bound to MT corresponded to 12 molar equivalents of Cu/MT with Cu and ascorbate alone and was reduced in the presence of H2O2. 2,2-Dithiodipyridine titration of MT SH-groups revealed a 50% decrease after H2O2, which could be regenerated by dihydrolipoic acid (DHLA). DHLA regeneration of thiols in MT was accompanied by restoration of MT's ability to inhibit Cu-dependent oxidation of ascorbate. Thus, optimum ability of MT to inhibit Cu-redox cycling directly correlates with its ability to bind Cu. Some of this Cu, however, appears releasable following oxidation of the thiolate metal-binding clusters. We speculate that redox-dependent release of Cu from MT serves both as a mechanism for physiological delivery of Cu to specific target proteins, as well as potentiation of cellular damage during oxidative stress.

Transforming growth factor-α (TGF-α) is a structural homologue of epidermal growth factor and competes for binding on a common transmembrane protein receptor/kinase. Although TGF-α appears to be more important than epidermal growth factor in embryogenesis and mammalian organogenesis, there is little information regarding its expression in developing lung. Accordingly, we measured levels of immunoreactive TGF-α and its gene expression in late-term fetal rat lung during the transition from the canalicular (19–20 d) to the saccular (21 d) stage. We report that at 19 d gestation intrapulmonary levels of TGF-a were 1.4 ± 0.3 pmol/mg protein as determined by RIA, but had decreased by 50 at 21 d. To determine if the TGF-α gene is expressed in lung, RNA isolated from fetal rat lung was reverse transcribed, and a 302-bp fragment corresponding to a portion of the TGF-α gene was amplified by polymerase chain reaction. Southern blot hybridization with a 32P-labeled 2.3-kb EcoRI fragment of rat TGF-α cDNA clone showed a pattern of declining expression during late gestation. Therefore, fetal rat lung expresses TGF-α, as is evidenced by the synthesis of both the message and the protein. Because levels of protein were highest in the period of canalicular lung development when the respiratory acinus is formed and vascularized, a potential role for this intrapulmonary growth factor in pulmonary remodeling is suggested.

Platelet-activating factor causes pulmonary hypertension, shock, hypoxemia, neutropenia, and increased pulmonary vascular permeability; some of its effects are due to thromboxane A2release. Evidence for possible role of these mediators in the genesis of group B Streptococcus (GBS)-induced pulmonary hypertension was sought using specific receptor antagonists for PAF and thromboxane A2(TxA2) in anesthetized, ventilated piglets (< 12 d of age; n = 22). Infusion of 1 x 108 GBS/kg/min for one hour resulted in a sustained and significant increase in pulmonary artery pressure (PPA) from 17 ± 1 to 35 ± 3 torr. Pretreatment with the TxA2 antagonist SQ 29548 (0.75 mg/kg intravenous), completely inhibited the effect of GBS on PPA. Pretreatment with either platelet-activating factor antagonists SRI 63072 (3 mg/kg intravenous) or SRI 63441 (1 mg/kg) did not affect the pulmonary hypertension due to GBS infusion. GBS-induced pulmonary hypertension could be reversed by SQ 29548; SRI 63072 did not affect PpA when administered to pigs with GBS-induced elevation in PPA. Inasmuch as prevention and reversal of GBS-induced pulmonary hypertension are accomplished with the TxA2antagonist but not with PAF antagonists, these data suggest that TxA2, rather than PAF, is responsible for the early pulmonary hypertension in this model of neonatal GBS sepsis. Therefore, TxA2antagonists may be clinically useful in treating pulmonary hypertension related to GBS sepsis.

ABSTRACT: Transforming growth factor-a (TGF-a) is a structural homologue of epidermal growth factor and competes for binding on a common transmembrane protein receptor/kinase. Although TGF-a appears to be more important than epidermal growth factor in embryogenesis and mammalian organogenesis, there is little information regarding its expression in developing lung. Accordingly, we measured levels of immunoreactive TGF-a and its gene expression in late-term fetal rat lung during the transition from the canalicular (19–20 d) to the saccular (21 d) stage. We report that at 19 d gestation intrapulmonary levels of TGF-a were 1.4 ± 0.3 pmol/mg protein as determined by RIA, but had decreased by 50% at 21 d. To determine if the TGF-a gene is expressed in lung, RNA isolated from fetal rat lung was reverse transcribed, and a 302-bp fragment corresponding to a portion of the TGF-a gene was amplified by polymerase chain reaction. Southern blot hybridization with a 32P-labeled 2.3-kb EcoRI fragment of rat TGF-a cDNA clone showed a pattern of declining expression during late gestation. Therefore, fetal rat lung expresses TGF-a, as is evidenced by the synthesis of both the message and the protein. Because levels of protein were highest in the period of canalicular lung development when the respiratory acinus is formed and vascularized, a potential role for this intrapulmonary growth factor in pulmonary remodeling is suggested.

The preceding discussion focused on some of the unique aspects of cardiopulmonary interaction in the infant and child with congenital cardiac anomalies. We have extracted liberally from our previous reviews and have tried to select information from the literature that helps to elucidate mechanisms for many of the observed phenomena. Where data were not available, we attempted to draw inference from analogous conditions or speculated about the pathophysiology.

Background: Nitric oxide (NO) has been shown to decrease myointimal hyperplasia in injured blood vessels. We hypothesize inducible NO synthase (iNOS) gene transfer even at low efficiency will provide adequate local NO production to achieve this goal. Materials and Methods: A retroviral vector containing the human iNOS cDNA (DFGiNOS) was used to transfer the iNOS gene into vascular cells and isolated blood vessels to answer the following questions: can vascular endothelial and smooth muscle cells support iNOS activity and will low efficiency iNOS gene transfer suppress myointimal hyperplasia in injured porcine arteries? Results: DFGiNOS-infected sheep pulmonary artery endothelial cells (SPAEC) expressed significant iNOS mRNA and protein, releasing nitrite levels of 155.0 ± 10.7 nmol/mg protein/24 h vs. 5.5 ± 1.1 by control cells. Transduced rat smooth muscle cells (RSMC) also expressed abundant iNOS mRNA and protein, but, in contrast to SPAEC, NO synthesis was dependent on exogenous tetrahydrobiopterin (BH4) (291.8 ± 10.4 nmol nitrite/mg protein/24 hr with BH4, 37.7 ± 2.6 without BH4). Only porcine arteries infected with DFGiNOS following balloon injury exhibited a 3-fold increase in total NO synthesis and a 15-fold increase in cGMP levels over control vessels in a BH4dependent fashion, despite only a 1% gene transfer efficiency. Transfer of iNOS completely prevented the 53% increase in myointimal thickness induced by balloon catheter injury; the administration of a NOS inhibitor reversed this effect. Conclusions: These in vitro findings suggest that vascular iNOS gene transfer may be feasible. Furthermore, a low gene transfer efficiency may be sufficient to inhibit myointimal hyperplasia following arterial balloon injury, although a source of BH4may be required.

Quantitative assessment of pulmonary metabolic function has been shown to be a novel and important approach to obtaining specific and subtle biochemical information regarding endothelial cell function in the normal and diseased lung. A primary consideration in this measurement of whole organ metabolism, whether it be enzyme, transport, or binding functions, it is to be able to separate convective from kinetic aspects of endothelial cell physiology. Significant progress has been made in both our understanding of the influence of convective factors, as well as of possible solutions relying on mixed order kinetics.