Raymond Foust

Immunohistochemical Localization of Protein 3-Nitrotyrosine and S -nitrosocysteine in a Murine Model of Inhaled Nitric Oxide Therapy

Inhaled nitric oxide (INO) therapy is currently used clinically to selectively dilate the pulmonary vasculature and to help treat persistent pulmonary hypertension and bronchopulmonary dysplasia in the neonate. However, in the presence of oxygen or superoxide, nitric oxide forms potentially harmful reactive nitrogen species. Using an experimental mice model, we examined the effects of concurrent hyperoxia and INO on protein tyrosine nitration and cysteine S-nitrosylation in pulmonary tissue. Data showed enhanced 3-nitrotyrosine staining within the airway epithelium and alveolar interstitium of mice lungs treated with hyperoxia, which did not increase significantly with INO administration. Within the alveolar interstitium, 3-nitrotyrosine staining was localized to macrophages. S-Nitrosocysteine staining in airway epithelium was significantly enhanced with INO administration regardless of oxygen content. These data suggest that the formation of protein S-nitrosocysteine is the major protein modification during administration of INO.

COMBINED ECMO AND PARTIAL LIQUID VENTILATION (PLV) IN HUMAN NEONATES: LIQUIVENT® PERFLUOROCHEMICAL (PFC) ELIMINATION. † 1368

COMBINED ECMO AND PARTIAL LIQUID VENTILATION (PLV) IN HUMAN NEONATES: LIQUIVENT® PERFLUOROCHEMICAL (PFC) ELIMINATION. † 1368

Prolonged Total Liquid Ventilation in Premature Lambs. ♦ 1064

Liquid ventilation with continuous tidal movement of oxygenated perfluorochemical (PFC) liquid (total liquid ventilation: TLV) has been shown to improve lung mechanics and provide effective gas exchange in animal models with respiratory failure. We hypothesize that premature lambs can be safely and adequately ventilated for a prolonged period of time with TLV. To test this hypothesis, we evaluated the physiological, histological, and biochemical profile in 9 preterm lambs (132 day gestation; 2.7-4.7 kg) who were supported up to 72 hours with a time-cycled, pressure-limited liquid ventilator (PFC: LiquiVent®Alliance Pharm. Corp. Temp. = 36°C, FiO2 = 0.48-1.0, rate = 4/min, I:E = 1:3, TV = 19-26 ml/kg). Lambs were anesthetized and instrumented with tracheal. carotid, and venous cannulae. Arterial blood samples were obtained for PFC uptake and blood gas measurements. PFC analysis was done by electron capture and flame ionization gas chromatography and expressed in mcg of PFC/gm of blood or PFC gm/tissue. Compliance was calculated using stop-flow alveolar pressure determinations. Tissues were obtained for histologic and biochemical analysis. Animals were time killed at 4, 24, and 72 hours. By 15 min of TLV, PFC levels were 5.1 ± 0.93 and stayed within an average of 7.5 ± 0.30 over 24 hours. The gas exchange and cardiovascular profiles (harmonic mean ± SE) were; pH = 7.34± 0.004, pCO2 =44.3±0.45, pO2 = 177 ± 5.8, compliance = 1.75± 0.04 ml/cmH2O/kg, mean blood pressure = 51± 0.62, heart rate = 189 ± 1.78. Histology demonstrated well-expanded gas exchange spaces without evidence of edema, exudate, or cellular debris. Tissue and blood biochemical analyses are ongoing. These data demonstrate that prolonged TLV can support gas exchange, and provide cardiovascular stability for up to 72 hours with minimal accumulation of PFC in blood in premature lambs.

Detection of Peroxynitrite in Biological Fluids

Peroxynitrite (ONOO(-)) is both an oxidant and a nitrating agent (1-3). However, unlike other strong oxidants, peroxynitrite reacts selectively with biological targets. This selectivity is derived in part from the different second-order rate constants (vary from 10(3)-10(6) M (-1) s(-1)) by which ONOO(-) reacts with biological targets. Competing for peroxynitrite-mediated oxidation of biological targets are two pathways. One is the protonation to form peroxynitrous acid and the second is the reaction with CO(2). Peroxynitrous acid is also an oxidant but it readily isomerizes to nitrate. The reaction with CO(2) results in the formation of the ONO(O)CO(2) (-) adduct that is a more potent nitrating agent but a weaker oxidant than peroxynitrite. Peroxynitrite can diffuse through biological membranes before it encounters and reacts with biological targets. This observation implies that diffusion can effectively compete with the isomerization to nitrate or the reaction with CO(2).

BIOCHEMICAL AND HISTOLOGIC INDICES OF REDUCED PULMONARY TRAUMA DURING PERFLUOROCHEMICAL (PFC) LIQUID VENTILATION |[dagger]| 2119

Urinary excretion of desmosine (DES), an index of proteolytic destruction of lung elastin, has been shown to be greater in premature infants who ultimately developed bronchopulmonary dysplasia (ARRD 131:568, 1985). Tidal liquid ventilation (TLV) in immature animals has been shown to improve pulmonary gas exchange at lower ventilatory pressures while preserving lung architecture as compared to conventional gas ventilation (CMV) (J Appl Physiol 72:1024, 1992). These improvements have been related to removal of the gas-liquid interface, reduction of interfacial alveolar surface tension, improved pulmonary compliance and lung volume recruitment. Larger tidal volumes (VT) and lower breathing frequencies are used during TLV as compared to CMV to minimize resistive pressures and diffusional factors. To compare these two forms of ventilation with respect to biochemical and histologic indices of lung trauma, 17 immature lambs (117 ± 1.4 SE dys gest.) were delivered by cesarean section and supported with CMV (n = 6) or TLV with PFC liquid (LiquiVent)(n = 11) for 4 hrs. Arterial blood chemistry and lung mechanics were serially assessed; urine was continually collected and assayed for DES as an index of elastin turnover. Lung samples were prepared for light microscopy and morphometric analysis including area of gas exchange spaces (GES). Data is expressed as mean ± SE over the 4 hr protocol (*p < 0.05). In addition, there was an increase in DESM over time in both groups which was less in the TLV (+25%) as compared to GV (+49%) animals. TLV lungs were intact and expanded homogeneously and GV lungs demonstrated patchy expansion and disruption of alveolar-capillary membranes across all regions. These data demonstrate that TLV supports improved oxygenation and distribution of ventilation at lower ventilatory pressures with greater gas exchange area and reduced elastin turnover as compared to GV. The results indicate that TLV minimizes pulmonary trauma associated with positive pressure ventilation. As such, this study suggests that TLV provides a gentle form of ventilatory support which has the potential to foster lung development and reduce the risk of chronic pulmonary sequelae in the immature infant. (Supp by NIH R29HD26341; Alliance Pharmaceutical Corp.)Table

The Longitudinal Course of Perfluorochemical (PFC) Intrapulmonary Distribution, Uptake, and Elimination in a Chronic Rabbit Model. † 1547

The Longitudinal Course of Perfluorochemical (PFC) Intrapulmonary Distribution, Uptake, and Elimination in a Chronic Rabbit Model. † 1547

DISTINCT PATTERNS OF APOPTOSIS DURING TOTAL LIQUID VENTILATION (TLV) VS. CONVENTIONAL VENTILATION (CV). |[dagger]| 1516

Mechanical ventilation with high pressures and supraphysiologic concentrations of oxygen is often needed to treat critically ill infants, but it can be quite toxic to the lung. This toxicity can lead to cell death by two distinct modes: apoptosis or necrosis. Apoptosis is often a scheduled and physiologically regulated event, while necrosis is the result of unscheduled, acute injury (often accompanied by inflammation). We have previously shown that in vivo, hyperoxia can cause significant cell death in the lung by apoptosis. TLV is a technique that may be associated with less lung injury and better physiological outcomes compared to CV. To examine whether TLV causes less lung injury and apoptosis compared to CV, we studied 2 groups of premature lambs delivered at 110 and 120 days gestation. Animals were ventilated with 100% O2 and TLV (Liquivent, Alliance Pharm., Hoechst-Marion-Roussel) or CV from birth for up to 4h. Unventilated, gestational matched controls were also studied. Standard physiologic and histologic parameters were analyzed. We also utilized the in situ TUNEL assay, which labels 3′-OH ends of DNA cut by endonucleases that are activated during apoptosis, to evaluate the amount of apoptosis occurring in the lung. Similar to previous reports, the TLV group had better gas exchange and less histologic evidence of lung injury compared to animals receiving CV. Approximately 5% of lung cells were apoptotic in both experimental groups, while gestational matched controls showed virtually no apoptosis. However, the pattern of apoptosis seen with both modes of ventilation was distinctly different. CV was associated with the presence of apoptotic cells within the lumen of immature air spaces, often with these cells completely occluding the lumen. In contrast, during TLV apoptosis was limited to a small number of epithelial cells lining the airspaces, with only a few cells found within the lumen. Although the specific cell types have not yet been identified, these observations suggest that the severity of lung injury might be related to a variety of factors including cell specific apoptosis, rather than apoptosis per se. Supported by a research grant from the March of Dimes(1-FY96-0752).

Pathophysiological Reactivities of Nitric Oxide

Nitric oxide (·NO) is a free radical signal transducing agent that mediates a variety of physiological processes. The cellular reactivity of ·NO may be regulated by reaction with heme proteins, metal complexes, thiols, O2 and superoxide (O 2 − ). To evaluate the pathophysiological role of ·NO in biological systems, its reactivity with thiols, which regulates physiological responses, and with O 2 − , which mediates pathological actions, was examined. Experimental evidence supporting a mechanism for the formation of S-nitrosothiols under physiological conditions indicates that ·NO reacts directly with reduced thiol to form an intermediate that is converted to S-nitrosothiol by the reduction of an electron acceptor. This novel mechanism for the formation of S-nitrosothiols can explain the presence of S-nitrosylated proteins in vivo. The nearly diffusion limited reaction of ·NO with O 2 − forms peroxynitrite (ONOO−), a relatively long-lived, highly reactive species. Peroxynitrite exhibits selective reactivity with proteins to form bioactive nitrotyrosine residues that have been detected in atherosclerosis, sepsis, inflammation, and neurodegenerative diseases. Tyrosine nitration results in inactivation of protein function and interferes with tyrosine phosphorylation, a key event in cellular signal transduction. These data indicate that the reactivity of ·NO with target molecules is critical in regulating its biological function.

Antioxidant Enzyme (AOE) Activities in Liquid Ventilated Premature Lambs.|[dagger]| 1500

Total liquid ventilation (TLV) has been established as a ventilatory strategy for experimental respiratory distress that minimizes lung barotrauma and markedly improves physiologic outcome. The biochemical mechanisms that underlie these improvements are not understood. To evaluate the effect of age and ventilation strategy on pulmonary AOE activity, four groups of premature lambs [n = 27] (110 ± 3 days and 120 ± 2 days gestation) were supported for 4 hrs with either gas ventilation (GV) or TLV with LiquiVent® (FIO2 = 1.0). AOE activity of superoxide dismutase (tSOD), glutathione peroxidase (GPX), and catalase were normalized to tissue protein and compared to their age-matched fetal controls. Histological analysis was performed. (X ± SE; units/ mg protein).Table

Biomarkers of Oxidative Stress in Adult Respiratory Distress Syndrome

Oxidative stress is considered as a significant component in the pathogenesis of the adult respiratory distress syndrome (ARDS) (1-3). Oxidative stress can be defined as the pathogenic outcome created by the oxidation of critical tissue targets by reactive species which are generated at rates that exceed tissue antioxidant capacity. Evidence for the presence of oxidative stress in ARDS patients is scarce. A major limitation for measuring reactive species in biological systems is their short half life. Since reactive species modify biological molecules such as proteins, lipids and DNA measurement of the modified targets provide the experimental tools for their detection and quantification. Previously we identified plasma proteins as a suitable target for quantification of oxidative stress in humans. Therefore the purpose of this study was to measure modified plasma proteins in ARDS patients as well as patients with sepsis. Two protein modifications were measured; protein carbonyl adducts and 3-nitrotyrosine. Protein carbonyls are derived by the direct oxidation of amino acid residues or conjugation of aldehydes that are formed by the oxidation of unsaturated lipids or sugars. Overall, plasma protein carbonyls indicate the formation of oxidants. Nitration of protein tyrosine residues by nitrating species results in the formation of 3-nitrotyrosine. The data collected indicated that elevation in protein carbonyls is associated with ARDS but not sepsis whereas plasma protein 3-nitrotyrosine was found to be elevated in both ARDS and septic patients.