Clusterin protects the lung from leukocyte-induced injury

Clusterin (CLU) is a multifunctional 75- to 80-kDa glycoprotein that is upregulated during cellular stress and might represent a defense mechanism during local cellular damage. Mechanisms discussed are antiapoptotic, antioxidative, and anticomplement properties as well as chaperone-like features protecting stressed proteins. The aim of this study was to investigate potential protective effects of CLU on pulmonary vasculature after in situ PMN activation in isolated rabbit lungs. The experiments were performed on 24 isolated and ventilated rabbit lungs that were perfused with 200 mL of Krebs-Henseleit-10% blood buffer with a constant flow of 150 mL/min in a recirculating system. It was tested whether pretreatment with CLU (2.5 microg/ml; n = 8) or catalase (CAT, 5000 U/ml; n = 8) before N-formyl-Met-Leu-Phe (fMLP; 10(-8) M) injection influenced pulmonary artery pressure (PAP) peak airway pressures (PAW) and edema formation as compared with controls (n = 8). Baseline values of PAP were 9-11 mmHg and PAW 11-13 cm H2O. Application of fMLP resulted in an acute significant (P < 0.01) increase of PAP (48 +/- 29 mmHg) within 2 min in the control group and PAW increased to 35 +/- 7 cm H2O within 30 min. Pretreatment with CLU completely suppressed the PAP and PAW response as a result of the fMLP challenge (P < 0.001), whereas a transient PAW increase up to 27 +/- 15 mmHg was observed after CAT. Complement factor C3a release was suppressed by CAT, whereas CLU blocked the complement cascade at the level of C5b-9 formation. Moreover, generation of thromboxane A(2) was reduced after CLU and CAT. Lung edema occurred in the fMLP group but was absent (P < 0.001) after CLU and CAT treatment. Both CLU and CAT prevented fMLP-induced lung injury. Stabilizing effects of CLU, point towards complement regulating features at the level of the terminal complement sequence. Elevated levels of CLU during inflammation could reflect a compensatory organ protective mechanism. Further studies are required to elucidate the clinical impact of the observed organ-protective properties of CLU.     

Interaction of acetylcholine and endothelin-1 in the modulation of pulmonary arterial pressure

OBJECTIVE: The study was designed to investigate the effects of acetylcholine (ACh) on pulmonary circulation with special regard to mediators that could be involved in the mediation of ACh-induced effects. ACh has been reported to induce either vasodilation or vasoconstriction in the pulmonary circulation of different species. DESIGN: Prospective experimental study in rabbits. SETTING: Experimental laboratory in a university teaching hospital. SUBJECTS: Sixty-six adult rabbits of either sex. INTERVENTIONS: The experiments were performed on 66 isolated and ventilated rabbit lungs that were perfused with a cell- and plasma-free buffer solution. ACh was injected in various concentrations after pulmonary artery preconstriction and in untreated lungs. MEASUREMENTS AND MAIN RESULTS: Pulmonary arterial pressure (PAP) and lung weight gain were monitored continuously. Perfusate samples were taken intermittently to determine endothelin-1 (ET-1), thromboxane A2 (TXA2), and prostacyclin (PGI2) concentrations. ACh in final dosages from 10(-5) to 10(-2) M (n = 6 each) was injected into the pulmonary artery of lungs treated with U46619 to induce pulmonary arterial hypertension or was injected into untreated lungs. To analyze the potential mechanisms of action, ACh (10(-5) M) was administered in additional experiments after pretreatment with either ETA receptor antagonist BQ123 (10(-6) M; n = 6) or the cyclooxygenase inhibitor diclofenac (10 microg/mL; n = 6). In preconstricted pulmonary vessels, ACh (10(-3) and 10(-2) M) initially induced a PAP rise for 10 mins followed by a sustained decrease. In untreated lungs, ACh induced an immediate dose-dependent increase in PAP, requiring as long as 30 mins to return to predrug levels. Simultaneously, significantly elevated TXA2 and PGI2 levels were observed. Furthermore, ET-1 was detected in the perfusate, which was free from ET-1 before ACh administration. Pretreatment with BQ123 reduced substantially the ACh (10(-5) M)-induced PAP increase and the release of TXA2 and PGI2. At 5 mins, the PAP maximum was reduced from 18.5 +/- 3.2 mm Hg to 9.9 +/- 0.65 mm Hg by BQ123 pretreatment (p < .01). An inhibition of PAP increase was also observed after diclofenac pretreatment (11.6 +/- 0.4 mm Hg at 5 mins; p < .05). Inhibitory effects at 5 mins were significantly more pronounced in the BQ123 group compared with the diclofenac group. CONCLUSIONS: The effects of ACh on the pulmonary circulation of isolated rabbit lungs depend on ACh concentration and the basal tone of the arterial vasculature. In lungs with a normal pulmonary vascular resistance, ACh administration causes vasoconstriction via the release of ET-1 and TXA2, whereas vasodilation is induced in preconstricted pulmonary vessels.     

Primed stimulation of isolated perfused rabbit lung by endotoxin and platelet activating factor induces enhanced production of thromboxane and lung injury.

Bacterial sepsis often precedes the development of the adult respiratory distress syndrome (ARDS) and bacterial endotoxin (LPS) produces a syndrome similar to ARDS when infused into experimental animals. We determined in isolated, buffer-perfused rabbit lungs, free of plasma and circulating blood cells that LPS synergized with platelet activating factor (PAF) to injure the lung. In lungs perfused for 2 h with LPS-free buffer (less than 100 pg/ml), stimulation with 1, 10, or 100 nM PAF produced transient pulmonary hypertension and minimal edema. Lungs perfused for 2 h with buffer containing 100 ng/ml of Escherichia coli 0111:B4 LPS had slight elevation of pulmonary artery pressure (PAP) and did not develop edema. In contrast, lungs exposed to 100 ng/ml of LPS for 2 h had marked increases in PAP and developed significant edema when stimulated with PAF. LPS treatment increased capillary filtration coefficient, suggesting that capillary leak contributed to pulmonary edema. LPS-primed, PAF-stimulated lungs had enhanced production of thromboxane B2 (TXB) and 6-keto-prostaglandin F1 alpha (6KPF). Indomethacin completely inhibited PAF-stimulated production of TXB and 6KPF in control and LPS-primed preparations, did not inhibit the rise in PAP produced by PAF in control lungs, but blocked the exaggerated rise in PAP and edema seen in LPS-primed, PAF-stimulated lungs. The thromboxane synthetase inhibitor dazoxiben, and the thromboxane receptor antagonist, SQ 29,548, similarly inhibited LPS-primed pulmonary hypertension and edema after PAF-stimulation. These studies indicate that LPS primes the lung for enhanced injury in response to the physiologic mediator PAF by amplifying the synthesis and release of thromboxane in lung tissue.     

Adenoviral-mediated transfer of the human endothelial nitric oxide synthase gene reduces acute hypoxic pulmonary vasoconstriction in rats.

Nitric oxide (NO), a vasodilator involved in the regulation of pulmonary vascular tone, is synthesized by a family of enzymes, nitric oxide synthases (NOS). To investigate whether adenoviral-mediated overexpression of constitutive endothelial NOS (ceNOS) would attenuate hypoxic pulmonary vasoconstriction, we aerosolized 3 X 10(9) plaque forming units of a recombinant adenovirus containing the ceNOS gene (AdCMVceNOS) into rat lungs. Four days after infection, transgene expression was confirmed using immunoblot techniques. Diffuse ceNOS immunostaining was detected in alveoli and medium-sized and small pulmonary vessels of AdCMVceNOS-transduced lungs. AdCMVceNOS-transduction was associated with an 86% increase in [3H]arginine to [3H]citrulline conversion and a rise in pulmonary cGMP levels from 7 +/- 1 to 59 +/- 9 pmol/mg protein in lungs from AdCMVceNOS versus control rats, (P < 0.05). During acute hypoxia (FIO2 = 0.10) for 25 min, mean pulmonary artery pressure (PAP) increased significantly from 17 +/- 1 to 27 +/- 1 mmHg in rats aerosolized with saline (n = 4) and from 18 +/- 1 to 28 +/- 1 mmHg in rats given an adenoviral vector expressing a nuclear-targeted beta-galactosidase gene (AdCMV beta gal, n = 8). In contrast, in AdCMVceNOS-transduced rats (n = 8) the hypoxia-induced increase in PAP was significantly attenuated (18 +/- 1 to 23 +/- 2 mmHg). Systemic blood pressure was not affected by aerosol gene transfer. Thus, adenoviral-mediated ceNOS gene transfer to rat lungs increases ceNOS expression and activity, and reduces acute hypoxic pulmonary vasoconstriction. Aerosolized recombinant adenovirus overexpressing vasodilatory proteins can act as a selective pulmonary vasodilator and may hold promise as a future therapeutic strategy for pulmonary hypertension.     

Plasma levels of C1- inhibitor complexes and cleaved C1- inhibitor in patients with hereditary angioneurotic edema.

C1- inhibitor (C1(-)-Inh) catabolism in plasma of patients with hereditary angioneurotic edema (HANE) was assessed by measuring the complexes formed by C1(-)-Inh with its target proteases (C1-s, Factor XIIa, and kallikrein) and a modified (cleaved) inactive form of C1(-)-Inh (iC1(-)-Inh). This study was performed in plasma from 18 healthy subjects and 30 patients with HANE in remission: 20 with low antigen concentration (type I) and 10 (from 5 different kindreds) with dysfunctional protein (type II). Both type-I and type-II patients had increased C1(-)-C1(-)-Inh complexes (P less than 0.0001), which in type I inversely correlated with the levels of C1(-)-Inh (P less than 0.001). iC1(-)-Inh was normal in all type-I patients and in type-II patients from three families with increased C1(-)-Inh antigen, whereas iC1(-)-Inh was higher than 20 times the normal values in patients from the remaining two families with C1(-)-Inh antigen in the normal range. None of the subjects had an increase of either Factor XIIa-C1(-)-Inh or kallikrein-C1(-)-Inh complexes. This study shows that the hypercatabolism of C1(-)-Inh in HANE patients at least in part occurs via the formation of complexes with C1- and that genetically determined differences in catabolism of dysfunctional C1(-)-Inh proteins are present in type-II patients.     

Impact of endothelin-1 in endotoxin-induced pulmonary vascular reactions

OBJECTIVES: Elevated endothelin-1 (ET-1) levels have been detected during sepsis. The aim of the study was to examine the role of thromboxane A2 (TXA2) and ET-1 in pulmonary vascular reactions after endotoxin (LPS) challenge. DESIGN: Prospective experimental study in rabbits. SETTING: Experimental laboratory in a university teaching hospital. SUBJECTS: Twenty-four adult rabbits of either sex. INTERVENTIONS: Experiments were performed on 30 isolated and ventilated rabbit lungs, which were perfused with a saline solution containing 10% autologous blood. MEASUREMENTS AND MAIN RESULTS: Pulmonary arterial pressure and lung weight gain were continuously registered. Perfusate samples were drawn intermittently to determine ET-1, TXA2, and prostacyclin (PGI2) concentrations. LPS isolated from Escherichia coli (0.5 mg/mL; n = 6) was added to the perfusate. A marked pulmonary arterial pressure increase followed by massive edema formation after 60 mins was observed after LPS injection. At the same time, elevated TXA2 and PGI2 levels in the perfusate were measured. ET-1 was detected 30 mins after LPS infusion (13.4+/-2.6 fmol/L). Pretreatment with the ET(A) receptor antagonist LU135252 (10(-6) M; n = 6) almost completely suppressed the pressure reaction after endotoxin injection (p < .01 at 50 and 60 mins) and reduced edema formation (p < .05). The cyclooxygenase inhibitor diclofenac (10 microg/mL; n = 6) was as effective as LU135252 in preventing vascular reactions after LPS injection. CONCLUSIONS: Pretreatment with the ET(A) receptor antagonist LU135252 and the cyclooxygenase inhibitor diclofenac reduced pulmonary vascular reactions after LPS challenge. Based on the current data, we conclude that the pulmonary arterial pressure increase and edema formation after LPS injection are related to an ET-1- and TXA2-dependent mechanism.     

Autoantibody facilitated cleavage of C1-inhibitor in autoimmune angioedema.

C1-inhibitor (C1-Inh) is an important inhibitor of the inflammatory response and deficiency of this inhibitor, which may be hereditary or acquired, is associated with recurrent episodes of edema. Recently, an autoimmune form of angioedema has been described that is associated with functional deficiency of C1-Inh and an autoantibody that impedes C1-Inh function. In this report we describe the isolation of C1-Inh from the monocytes and plasma of a patient with autoimmune angioedema and demonstrate that the patient's monocytes secrete structurally and functionally normal C1-Inh, but show that this protein circulates in the patient's plasma in an inactive, structurally altered form. Furthermore, using analytic gel electrophoresis techniques it is demonstrated that the patient's autoantibody facilitates cleavage of normal C1-Inh, by its target proteases, to the same species of C1-Inh that is found circulating in the patient's plasma. This autoantibody facilitated cleavage of normal C1-Inh is apparently a consequence of destabilization of protease/inhibitor complexes. These findings contribute to our understanding of protease/C1-Inh interactions and document important observations on pathogenic mechanisms in autoimmune disease.     

Inhibition of lung phosphodiesterase improves responsiveness to inhaled nitric oxide in isolated-perfused lungs from rats challenged with endotoxin

OBJECTIVES: To investigate the ability of phosphodiesterase (PDE) selective inhibitors to improve responsiveness to inhaled nitric oxide (NO) in isolated-perfused lungs of rats pretreated with endotoxin/lipopolysaccharide (LPS). DESIGN AND SETTING: Prospective, controlled animal study in the animal research facility of a university hospital. INTERVENTIONS: Sixteen hours after adult Sprague-Dawley rats were injected intraperitoneally with 0.4 mg/ kg E. coli 0111:B4 LPS administration, lungs were isolated and perfused, and the thromboxane mimetic U46619 was employed to increase the mean pulmonary artery pressure by 5-7 mmHg. The lungs were then ventilated with or without 0.4 ppm NO, and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA; PDE type 2 inhibitor), milrinone (PDE type 3 inhibitor), or zaprinast (inhibitor of PDE types 5 and 9) were added to the perfusate. MEASUREMENTS AND RESULTS: In the presence of EHNA (12.5, 25, 50 microM) the vasodilator response to inhaled NO was not greater than in its absence (0.25 +/- 0.25, 0.5 +/- 0.25, 0.75 +/- 0.25 mmHg vs. 0.25 +/- 0.25, 0.5 +/- 0.25, 0.75 +/- 0.25 mmHg, respectively). In the presence of milrinone (125, 250, 500 nM), the vasodilator response to inhaled NO was also not improved. In contrast, zaprinast (3.7, 7.4, 14.8 microM) augmented the pulmonary vasodilatory effect of inhaled NO in lungs from LPS-pretreated rats from 0.25 +/- 0.25, 0.5 +/- 0.25, 0.75 +/- 0.25 mmHg to 0.75 +/- 0.25, 1.5 +/- 0.5, 1.75 +/- 0.75 mmHg, respectively (p < 0.05). CONCLUSIONS: Our results demonstrate that inhibition of pulmonary PDE enzyme activity with zaprinast increases vasodilator responsiveness to inhaled NO in lungs obtained from rats 16 h after LPS challenge.     

Dose response of intravenous sildenafil on systemic and regional hemodynamics in hypoxic neonatal piglets

In neonates with acute pulmonary hypertension (PHT), the dose-response effect of sildenafil citrate, a selective phosphodiesterase-5 inhibitor that can alleviate PHT, has not been detailedly examined. We tested the hypothesis that the treatment of hypoxia-induced acute PHT with sildenafil would dose-dependently reduce the elevated pulmonary and systemic arterial pressures (PAP and SAP, respectively) with no effect on the oxygenation in newborn animals. We also examined the regional hemodynamic responses. Using a randomized controlled design, piglets (age range, 1-3 days; weight range, 1.5-2.1 kg) were anesthetized and acutely instrumented to measure cardiac index, left common carotid, superior mesenteric and left renal arterial flow indexes, SAP, and PAP. After stabilization, hypoxia was induced with fractional inspired oxygen concentration at 0.15 and, subsequently, piglets were randomized to receive i.v. sildenafil at 0.06, 0.2, or 2.0 mg/kg per hour or normal saline (controls) for 90 min (n = 6 each). Within 30 min of hypoxia (PaO2, 31 +/- 5 mmHg), the piglets developed PHT (PAP, 33 +/- 5 vs. 26 +/- 4 mmHg at baseline; P < 0.05. Sildenafil dose-dependently reduced the hypoxia-induced PHT (PAP at 90 min: 33 +/- 6, 29 +/- 6, and 26 +/- 6 mmHg of 0.06, 0.2, and 2.0 mg/kg per hour, respectively, vs. 44 +/- 8 mmHg of controls; P < 0.05. Sildenafil at 2.0 mg/kg per hour had the greatest decrease in SAP (P < 0.05) with no significant change at 0.06 and 0.2 mg/kg per hour. Pulmonary selectivity (PAP:SAP ratio) was best in the group treated with 0.2 mg/kg per hour dosage of sildenafil (P < 0.05). There were no differences in cardiac index and regional flow indexes between groups. Although hypoxia decreased oxygen delivery and increased oxygen extraction with no significant effect on oxygen consumption, the administration of sildenafil did not affect the oxygen metabolism (vs. controls). In neonatal piglets, i.v. sildenafil dose-dependently alleviates the hypoxia-induced acute PHT, with the best pulmonary selectivity at 0.2 mg/kg per hour, and shows no significant effect on regional circulation and oxygen metabolism.     

Lung fluid transport in aquaporin-1 and aquaporin-4 knockout mice

The mammalian lung expresses water channel aquaporin-1 (AQP1) in microvascular endothelia and aquaporin-4 (AQP4) in airway epithelia. To test whether these water channels facilitate fluid movement between airspace, interstitial, and capillary compartments, we measured passive and active fluid transport in AQP1 and AQP4 knockout mice. Airspace-capillary osmotic water permeability (Pf) was measured in isolated perfused lungs by a pleural surface fluorescence method. Pf was remarkably reduced in AQP1 (-/-) mice (measured in cm/s x 0.001, SE, n = 5-10: 17 +/- 2 [+/+]; 6.6 +/- 0.6 AQP1 [+/-]; 1.7 +/- 0.3 AQP1 [-/-]; 12 +/- 1 AQP4 [-/-]). Microvascular endothelial water permeability, measured by a related pleural surface fluorescence method in which the airspace was filled with inert perfluorocarbon, was reduced more than 10-fold in AQP1 (-/-) vs. (+/+) mice. Hydrostatically induced lung interstitial and alveolar edema was measured by a gravimetric method and by direct measurement of extravascular lung water. Both approaches indicated a more than twofold reduction in lung water accumulation in AQP1 (-/-) vs. (+/+) mice in response to a 5- to 10-cm H2O increase in pulmonary artery pressure for five minutes. Active, near-isosmolar alveolar fluid absorption (Jv) was measured in in situ perfused lungs using 125I-albumin as an airspace fluid volume marker. Jv (measured in percent fluid uptake at 30 min, n = 5) in (+/+) mice was 6.0 +/- 0.6 (37 degrees C), increased to 16 +/- 1 by beta-agonists, and inhibited to less than 2.0 by amiloride, ouabain, or cooling to 23 degrees C. Jv (with isoproterenol) was not affected by aquaporin deletion (18.9 +/- 2.2 [+/+]; 16.4 +/- 1.5 AQP1 [-/-]; 16.3 +/- 1.7 AQP4 [-/-]). These results indicate that osmotically driven water transport across microvessels in adult lung occurs by a transcellular route through AQP1 water channels and that the microvascular endothelium is a significant barrier for airspace-capillary osmotic water transport. AQP1 facilitates hydrostatically driven lung edema but is not required for active near-isosmolar absorption of alveolar fluid.     

A sensitive method to assay blood complement C1- inhibitor activity

Hereditary angioneurotic edema results from deficiency of complement protein C1- inhibitor. Using a new spectrophotometric assay for C1-s esterase activity on the N-alpha-benzoyl-L-arginine ethyl ester, we describe a routinely available method for quantifying low C1- Inhibitor functional activities in EDTA-treated plasma of hereditary angioneurotic edema patients. C1- Inhibitor activity is deduced from the residual esterase activity of C1-s incubated with 20-80 microliters plasma samples. Arbitrary units (volume of sample inhibiting 50% of C1-s activity) were used to express C1- Inhibitor normal activity which was estimated as 22,500 +/- 5,000 (SD) U/l in 45 healthy individuals. The correlation with C1- Inhibitor antigen in these healthy individuals and 89 patients with varying concentrations of C1 Inhibitor ranging from 0.05-1.05 g/l was r = 0.91. Levels down to 2,000 U/l could be estimated. Specific inhibitory activity is an absolute requirement to distinguish between type I and type II hereditary angioneurotic edema.     

Oxygen free radical-induced injury in isolated rat hearts: effects of ibuprofen and BW 755c

Inhibitors of the arachidonic acid metabolism (AA) as well as scavengers of oxygen free radicals (OFR) have been shown to reduce myocardial infarct size. We investigated the effects of two inhibitors of AA metabolism on the cardiac effects of OFR. Isolated rat hearts were retrogradely perfused for 10 min with buffer containing hypoxanthine (HX, 1 mmol l-1) and xanthine oxidase (XOD: 24 U l-1) alone (n = 11), or with the addition of ibuprofen (IBU) (n = 6) (a cyclooxygenase inhibitor) or BW 755C (n = 6) (a dual inhibitor of cyclo-oxygenase and lipoxygenase). The hearts were observed for 30 min thereafter (total observation time 40 min). Left-ventricular pressures were measured by a balloon in the left ventricle. HX + XOD significantly reduced left-ventricular developed pressure (LVDP) and coronary flow (CF), but not heart rate. The reduction of LVDP and CF was not significantly ameliorated by the addition of IBU (7.6 x 10(-4) mol l-1) or BW 755C (2 x 10(-3) mmol l-1). OFR increased left-ventricular end-diastolic pressure (LVEDP) from (mean +/- SEM) 0 to 22 +/- 4 mmHg (10 min) and 30 +/- 5 mmHg (40 min). Upon addition of IBU, LVEDP increased from 0 to 24 +/- 8 mmHg and 17 +/- 6 mmHg at 10 and 40 min respectively. The addition of BW 755C almost completely inhibited the increase in LVEDP (1 +/- 1 mmHg and 3 +/- 3 mmHg at 10 min and 40 min). In conclusion, except for inhibition of OFR-induced diastolic dysfunction by BW 755C, neither BW 755C nor IBU appear to inhibit cardiac injury induced by OFR.     

Impact of low pulmonary vascular pressure on ventilator-induced lung injury

OBJECTIVE: To study the impact of low pulmonary vascular pressure on ventilator-induced lung injury. DESIGN: Randomized prospective animal study. SUBJECTS: Isolated perfused rabbit heart-lung preparation. SETTINGS: Animal research laboratory in a university hospital. INTERVENTIONS: Twenty isolated sets of normal lungs were perfused (constant flow, 0.3 L/min; left atrial pressure, 6 mm Hg), ventilated for 20 min (pressure control ventilation, 15 cm H2O; baseline period), and then randomized into three groups. Group A (control, n = 7) was perfused and ventilated as previously described during three consecutive 20-min periods. In group B (high airway pressure/normal left atrial pressure, n = 7), pressure control ventilation was 20, 25, and 30 cm H2O during each period. Group C (high airway pressure/low left atrial pressure, n = 6) was ventilated as group B but, in contrast to groups A and B, left atrial pressure was reduced to 1 mm Hg. MEASUREMENTS AND MAIN RESULTS: The rate of edema formation (WGR, weight gain per minute normalized for initial lung weight) and the ultrafiltration coefficient (Kf) were measured during and after each period and their changes from baseline [DeltaWGR (edema formation index) and DeltaKf (vascular permeability index)] calculated to compare groups. The incidence and timing of vascular failure were compared. Vascular failure was considered to be present if all the following conditions were met: pulmonary hypertension, accelerated weight gain, and occurrence of fluid leak from the lungs. At the end of the study, DeltaWGR (g.g.min(-1)) was higher in group C (0.54 +/- 0.17) than in groups B (0.08 +/- 0.04) and A (0.00 +/- 0.01; p<.05), as well as in group B compared with A (p <.05). Similar differences between groups (p <.05) were found for DeltaK (g x min(-1) x cm H2O(-1) x 100 g(-1)): C, 7.24 +/- 2.36; B, 1.40 +/- 0.49; A, 0.01 +/- 0.03. Vascular failure was not observed in groups A and B but occurred in all but one preparation in group C (p <.05; C vs. A and B). CONCLUSION: Reducing left atrial pressure results in more severe ventilator-induced lung injury. These results suggest that lung blood volume modulates cyclic tidal lung stress.     

Neutralization of Knops system antibodies using soluble complement receptor 1

BACKGROUND: Antibodies of the Knops system have been referred to as nonneutralizable because they cannot be inhibited with serum, saliva, or urine. Because the Knops system antigens have been located on complement receptor 1 (CR1), the question of whether the antibodies could be neutralized with soluble CR1 (sCR1) produced by recombinant DNA techniques was studied. STUDY DESIGN AND METHODS: First, radiolabeled immunoprecipitation techniques were used to test sCR1 for the expression of the high-incidence Knops system antigens. Then, a total of 45 antibodies were neutralized with sCR1, including the following: one each of anti-Cr(a), -Dr(a), -Do(b), -Hy, -Ge, -Jr(a), - Sc1, -Jk(a), -Cs(a), and -Kp(b); two each of anti-Lu(b), -Yt(a), and - JMH; three each of anti-McC(a), -Rg, and -Sl(a); and four each of anti- Ch, -Kn(a), -Yk(a), -Kn/McC. In addition, two examples of anti-Kn(a) + K, one example of anti-Sl(a) + K + Fy(a), and one example of anti-Yk(a) + E were tested. The sCR1 was added to each test serum and 6-percent albumin was added to the control; this was followed by neutralization incubation for 5 minutes at 25 degrees C. The antibody samples were then tested by a low-ionic-strength solution, anti-human globulin technique. RESULTS: The sCR1 expressed Kn(a), McC(a), Sl,a and Yk(a). All Knops system antibodies (n = 22) were neutralized by the sCR1, but none of the other 23 alloantibodies decreased in reactivity. The samples containing antibodies of two specificities showed inhibition of the Knops system antibody but not of the second antibody. CONCLUSION: This neutralization method, in which recombinant protein is used, provides an expedient and definitive method of identifying Knops system antibodies.     

Proteolytic inactivation of plasma C1- inhibitor in sepsis.

Activation of both the complement system and the contact system of intrinsic coagulation is implicated in the pathophysiology of sepsis. Because C1 inhibitor (C1-Inh) regulates the activation of both cascade systems, we studied the characteristics of plasma C1-Inh in 48 patients with severe sepsis on admission to the Intensive Care Unit at the Free University of Amsterdam. The ratio between the level of functional and antigenic C1-Inh (functional index) was significantly reduced in the patients with sepsis compared with healthy volunteers (P = 0.004). The assessment of modified (cleaved), inactive C1-Inh (iC1-Inh), and complexed forms of C1-Inh (nonfunctional C1-Inh species) revealed that the reduced functional index was mainly due to the presence of iC1-Inh. On SDS-PAGE, iC1-Inh species migrated with a lower apparent molecular weight (Mr 98,000, 91,000, and 86,000) than native C1-Inh (Mr 110,000). Elevated iC1-Inh levels (greater than or equal to 0.13 microM) were found in 81% of all patients, sometimes up to 1.6 microM. Levels of iC1-Inh on admission appeared to be of prognostic value: iC1-Inh was higher in 27 patients who died than in 21 patients who survived (P = 0.003). The mortality in 15 patients with iC1-Inh levels up to 0.2 microM was 27%, but in 12 patients with plasma iC1-Inh exceeding 0.44 microM, the mortality was 83%. The overall mortality in the patients with sepsis was 56%. We propose that the cleavage of C1-Inh in patients with sepsis reflects processes that play a major role in the development of fatal complications during sepsis.     

Haemodynamic and renal effects of urodilatin in healthy volunteers

Urodilatin (ANF(95-126)), an analogue of the atrial natriuretic factor (ANF(99-126)), has recently been isolated from human urine. To study haemodynamic and renal effects of synthetic urodilatin, 18 healthy male volunteers (age 26.1 +/- 0.8 years; X +/- SEM) received i.v. bolus injections of urodilatin at doses of 1, 2 or 4 micrograms kg-1 body weight (bw) (n = 6 per dosage group). Urodilatin dose-dependently increased heart rate and cardiac index. A dose-dependent increase in plasma cyclic GMP levels was also observed. Urinary cyclic GMP excretion, urine flow and natriuresis increased 7-fold, 5-fold and 4-fold, respectively. Renal effects were not different between dosage groups. Compared with ANF(99-126), after urodilatin the reduction in mean pulmonary arterial pressure (PAP) was more pronounced (2 micrograms kg-1, n = 6; ANF -1.8 +/- 0.5, URO: -5.5 +/- 1.1 mmHg, P less than 0.05). Furthermore, after urodilatin the reduction of PAP lasted continuously from 2 up to 90 min after injection, while ANF(99-126) produced only a transient decrease of PAP. Similarly the reduction of pulmonary capillary wedge pressure (PCWP) by urodilatin from 9.3 +/- 1.2 to 3.8 +/- 0.9 mmHg (P less than 0.05) was also sustained up to 90 min post administration. These data in healthy volunteers suggest that, due to prolonged reduction of PAP and PCWP with increases of cardiac index and reduction of systemic vascular resistance, urodilatin might exhibit beneficial effects in cardiovascular disease.     

Salutary effects of prostaglandin E1 in perfused rat lungs injured with hydrogen peroxide

Studies were conducted in isolated, buffer-perfused rat lungs to determine if prostaglandin (PG) E1 attenuated pulmonary edema provoked by hydrogen peroxide (H2O2). When lungs were challenged by 60 min of perfusion with H2O2 (generated by the reaction between glucose and glucose oxidase) the wet weight-to-dry weight ratio increased from control by 54%, indicating development of pulmonary edema. In contrast, lungs treated simultaneously with H2O2 plus PGE1 (1 microgram/min) failed to exhibit an elevated wet-to-dry weight ratio. H2O2-injured lungs demonstrated a modest 2 torr increase in pulmonary arterial perfusion pressure that was not influenced by simultaneous treatment with PGE1. Both radioimmunoassay (RIA) and high-performance liquid chromatographic (HPLC) analysis detected increased amounts of (5S)-5-hydroxy-6,8,11,14 eicosatetraenoic acid in the perfusion medium of H2O2-injured lungs (RIA, 48.0 +/- 14.7; HPLC, 54.8 +/- 13.5) relative to controls (RIA, 6.6 +/- 1.6; HPLC, 6.8 +/- 1.9), and simultaneous treatment with PGE1 tended to blunt this increase (RIA, 29.2 +/- 8.3; HPLC, 29.8 +/- 7.6). PGE1 abolished the increase in wet weight-to-dry weight ratio induced by exogenous leukotriene C4. Production of H2O2 by the glucose-glucose oxidase reaction was not influenced by PGE1. Taken together, these observations indicate that PGE1 attenuates H2O2-induced pulmonary edema formation in buffer-perfused rat lungs by mechanisms that may relate to inhibition of lung 5'-lipoxygenase activation and/or to inhibition of the injurious effects of endogenously produced lipoxygenase products.     

Effects of erythrapheresis on pulmonary haemodynamics and oxygen transport in patients with secondary polycythaemia and cor pulmonale

Reducing packed cell volume has been advocated as a therapeutic procedure in patients with polycythaemia secondary to hypoxic cor pulmonale. The aim of this investigation was to evaluate the effects of this manoeuvre on resting pulmonary haemodynamics and tissue oxygenation in 12 such patients. The subjects were studied whilst they were breathing air (n = 12), after breathing 35% oxygen for 30 min (n = 11) and breathing air 30 min after isovolaemic reduction in packed cell volume, from 0.61 +/- 0.02 to 0.50 +/- 0.02 (mean +/- SEM), by erythrapheresis (n = 12). Initial values for the group were: PaO2 6.5 +/- 0.3 kPa; red cell mass 152 +/- 12% predicted; mean pulmonary artery pressure (PAP) 41 +/- 3 mmHg; cardiac index 3.1 +/- 0.31 min-1 m-2. Breathing 35% oxygen reduced PAP by 3.1 +/- 1.0 mmHg (P less than 0.02), cardiac index by 0.28 +/- 0.121 min-1 m-2 (P less than 0.05) and right ventricular stroke work by 0.05 +/- 0.01 J (P less than 0.01). Systemic vascular resistance was unchanged. Systemic oxygen transport increased and peripheral oxygen consumption was unaltered. Erythrapheresis reduced blood viscosity at shear rates 23 S-1 and 230 S-1. PAP fell by 2.4 +/- 1.1 mmHg (P less than 0.05) and cardiac index increased by 0.32 +/- 0.091 min-1 m-2 (P less than 0.01), but right ventricular stroke work was unchanged. Systemic vascular resistance was reduced by 25 +/- 7 kPa S l-1 (P less than 0.01). Systemic oxygen transport decreased but peripheral oxygen consumption was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and distribution of endothelin receptors in the pulmonary circulation: investigation of isolated, perfused, and ventilated rabbit lungs.

The aim of the study was to investigate the distribution of 2 subtypes of endothelin-receptors, mediating the effects of endothelin-1 (ET-1) in the pulmonary circulation. Until now, it is still unclear, whether ET(A) receptors or ET(B) receptors or even both are localized in pulmonary vessels. The experiments were performed on 72 isolated and ventilated rabbit lungs that were perfused with a cell- and plasma-free buffer solution. The arterial pressure and the lung weight gain were continuously registered. Intermittently perfusate samples were taken for determination of thromboxane A2 (TXA2) and prostacyclin (PGI2). The injection of ET-1 (10(-8) M, n = 6) resulted in a biphasic increase in pulmonary arterial pressure (PAP) that was accompanied by the generation of TXA2 and PGI2. Pretreatment with the ET(A)-receptor antagonist LU135252 (10(-6) M, n = 6) suppressed the pressure response after ET-1 application (P < 0.01 at 120 min) and reduced the generation of TXA2 (P < 0.05 at 120 min) and PGI2 (P < 0.05 at 120 min). Pretreatment with the cyclooxygenase inhibitor diclofenac (10 microg/mL; n = 6) also reduced the PAP increase after ET-1 injection. In contrast to this, the pulmonary vascular pressure reaction after ET-1 application was elevated, when ET(B)-receptor antagonist BQ788 (10(-6) M; n = 6) was given. Furthermore, the PGI2 to TXA2 ratio was shifted from 2.3 to 0.9, reflecting a predominance of vasoconstrictive TXA2. The simultaneous application of LU135252 and BQ788 significantly reduced the PAP increase after ET-1 application, but no beneficial effects were observed compared with the application of LU135252 solely. The injection of the ET(B)-receptor agonist sarafotoxin S6c (S6c; 10(-8) M, n = 6) also induced an increase in PAP that was not attenuated by pretreatment with the ET(B)-receptor antagonist BQ788 (10(-6) M, n = 6). LU135252 (n = 6) as well as the application of LU135252 in combination with BQ788 (n = 6) failed to suppress the pressure response after S6c, whereas the cyclooxygenase inhibitor diclofenac (10 microg/mL, n = 6) alone and in combination with LU135252 and BQ788 (n = 6) was able to prevent the PAP increase after S6c injection (P < 0.001). The results demonstrate that the ET-1-induced increase in pulmonary vascular resistance is mainly mediated via ET(A) receptors, whereas ET(B) receptors seem to mediate vasodilation, which was shown by an imbalance of TXA2 and PGI2 generation. On the other hand, the ET(B)-receptor agonist S6c induced vasoconstriction, which was only attenuated by the cyclooxygenase inhibitor diclofenac. From the current results we conclude that, apart from vasoconstrictor ET(A) receptors, at least 2 ET(B)-receptor subtypes are expressed in the pulmonary circulation, one mediating vasoconstriction, which was not blocked by BQ788, and one mediating vasodilation, which was influenced by BQ788.     

Uptake of N-[1 (S)-carboxy-(4-OH-3-125I-phenyl)ethyl]-L-Ala-L-Pro, an inhibitor of angiotensin-converting enzyme by rabbit lungs in situ

Single pass extraction of a new iodinated inhibitor of angiotensin-converting enzyme (ACE) was measured by means of indicator-dilution techniques applied to rabbit lungs, perfused in situ at 20 ml/min with Krebs bicarbonate solution containing 3% bovine serum albumin. A bolus containing the inhibitor, N-[1(S)-carboxy-(4-OH-3-125I-phenyl)ethyl]-L-Ala-L-Pro (CPAP), and an intravascular marker, [14C]dextran, was injected and extraction calculated at the peak of the resulting venous outflow-time curve. In 13 of 21 lungs used, a synthetic substrate for ACE, [3H]benzoyl-phenylalanyl-alanyl-proline (BPAP), was added to the bolus and appearance of its hydrolysis product, [3H]benzoyl-Phe, measured in effluent samples. When low amounts (0.15 nmol) of [125I]CPAP were injected, pulmonary extraction (E) of CPAP was 67 +/- 14% (X +/- S.D; n = 21) and metabolism (M) of BPAP was 56 +/- 9% (n = 13). Addition of unlabeled CPAP (3, 34 or 340 nmol) to the Addition of unlabeled CPAP (3, 34 or 340 nmol) to the injected bolus caused dose-dependent reduction of E(CPAP) and M(BPAP) that was no longer evident 10 min after the largest dose of CPAP. Coadministration of the ACE inhibitor, captopril (3, 6, 8 and 28 nmol), also caused dose-dependent, reversible depression of both E(CPAP) and M(BPAP). Accordingly, extraction of CPAP by perfused rabbit lung is saturable. Inasmuch as CPAP inhibits ACE activity (as reflected by BPAP metabolism) and CPAP uptake is inhibited by captopril (which also inhibits BPAP hydrolysis), it appears that a large portion of this saturable process probably reflects binding to vascular ACE.(ABSTRACT TRUNCATED AT 250 WORDS)