Simvastatin attenuates vascular leak and inflammation in murine inflammatory lung injury

Therapies to limit the life-threatening vascular leak observed in patients with acute lung injury (ALI) are currently lacking. We explored the effect of simvastatin, a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor that mediates endothelial cell barrier protection in vitro, in a murine inflammatory model of ALI. C57BL/6J mice were treated with simvastatin (5 or 20 mg/kg body wt via intraperitoneal injection) 24 h before and again concomitantly with intratracheally administered LPS (2 microg/g body wt). Inflammatory indexes [bronchoalveolar lavage (BAL) myeloperoxidase activity and total neutrophil counts assessed at 24 h with histological confirmation] were markedly increased after LPS alone but significantly reduced in mice that also received simvastatin (20 mg/kg; approximately 35-60% reduction). Simvastatin also decreased BAL albumin (approximately 50% reduction) and Evans blue albumin dye extravasation into lung tissue (100%) consistent with barrier protection. Finally, the sustained nature of simvastatin-mediated lung protection was assessed by analysis of simvastatin-induced gene expression (Affymetrix platform). LPS-mediated lung gene expression was significantly modulated by simvastatin within a number of gene ontologies (e.g., inflammation and immune response, NF-kappaB regulation) and with respect to individual genes implicated in the development or severity of ALI (e.g., IL-6, Toll-like receptor 4). Together, these findings confirm significant protection by simvastatin on LPS-induced lung vascular leak and inflammation and implicate a potential role for statins in the management of ALI.     

Microsatellite genotyping of post-PCR fluorescently labeled markers

We show that a post-PCR multicolor fluorescence-labeling technique is applicable to multiplex microsatellite genotyping. Forty-three dinucleotide microsatellite markers, which are located on 11q13-23, a candidate region for dominant familial exudative vitreoretinopathy (FEVR), were used to evaluate the quality of the marker profile produced by this technique. Thirty-eight people from six families with this disease were subjects for genotyping. The samples revealed clearly separated fragment peaks with signal intensities sufficient for analysis. All genotypes were consistent with Mendelian inheritance within each family. This method is cost effective because it does not use expensive fluorescently labeled primers. It also has the advantage of avoiding ambiguity in the analysis, which may arise from the addition of non-template nucleotides by Taq DNA polymerase.     

Interaction of chemical and high vascular pressure injury in isolated canine lung

Because both chemical and mechanical insults to the lung may occur concomitantly with trauma, we hypothesized that the pressure threshold for vascular pressure-induced (mechanical) injury would be decreased after a chemical insult to the lung. Normal isolated canine lung lobes (N, n = 14) and those injured with either airway acid instillation (AAI, n = 18) or intravascular oleic acid (OA, n = 25) were exposed to short (5-min) periods of elevated venous pressure (HiPv) ranging from 19 to 130 cmH2O. Before the HiPv stress, the capillary filtration coefficient (Kf,c) was 0.12 +/- 0.01, 0.27 +/- 0.03, and 0.31 +/- 0.02 ml.min-1.cmH2O-1 x 100 g-1 and the isogravimetric capillary pressure (Pc,i) was 9.2 +/- 0.3, 6.8 +/- 0.5, and 6.5 +/- 0.3 cmH2O in N, AAI, and OA lungs, respectively. However, the pattern of response to HiPv was similar in all groups: Kf,c was no different from the pre-HiPv value when the peak venous pressure (Pv) remained less than 55 cmH2O, but it increased reversibly when peak Pv exceeded 55 cmH2O (P less than 0.05). The reflection coefficient (sigma) for total proteins measured after pressure exposure averaged 0.60 +/- 0.03, 0.32 +/- 0.04, and 0.37 +/- 0.09 for N, AAI, and OA lobes respectively. However, in contrast to the result expected if pore stretching had occurred at high pressure, in all groups the sigma measured during the HiPv stress when Pv exceeded 55 cmH2O was significantly larger than that measured during the recovery period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and preliminary biological evaluation of the 99mTc labeled nitrobenzoimidazole and nitrotriazole as tumor hypoxia markers

1-(3-1,2,4-Nitrotriazole-1-yl)-propanhydroxyiminoamide and 1-(6-nitrobenzoimidazole-1-yl)-propanhydroxyiminoamide were synthesized and radiolabeled with (99m)Tc. The (99m)Tc labeled complexes continuously accumulated in hypoxic murine sarcoma S180 cells in vitro but not in aerobic cells. Biodistribution results in mice bearing S180 tumor indicated that the tracers could localize in tumor and eliminate from it slowly. These results suggested that the (99m)Tc labeled nitrobenzoimidazole and nitrotriazole might be the novel tumor hypoxia markers.     

Role of neutrophils in lung vascular injury and edema after premature birth in lambs

Carlton, David P., Kurt H. Albertine, Soo Chul Cho, MennoLont, and Richard D. Bland. Role of neutrophils in lung vascular injury and edema after premature birth in lambs. J. Appl. Physiol. 83(4): 1307-1317, 1997.Toinvestigate the role of neutrophils in the pathogenesis of respiratorydistress after premature birth, we assessed the relationship betweencirculating neutrophil concentration and neutrophil accumulation in thelung, lung lymph and pleural liquid flow, and extravascular lungwater in 10 chronically catheterized preterm lambs (127 ± 1 days gestation) that were mechanically ventilated for 8 h afterbirth. Circulating neutrophil concentration transiently decreasedwithin 2 h after birth and then returned to prenatal values by 6-8h. The decrease in circulating neutrophil concentration was relateddirectly to the accumulation of neutrophils in the air spaces, drainageof liquid and protein from the lung 6-8 h after delivery, andpostmortem extravascular lung water. In additional studies, weintravenously administered mechlorethamine to 5 fetal lambs to reducecirculating neutrophils before delivery (neutrophil concentrationbefore birth: 9 ± 11 cells/µl). Compared with control lambs,neutrophil-depleted lambs had significantly less drainage of liquid(7.8 ± 5.9 vs. 2.6 ± 1.9 ml/h, respectively) and protein (116 ± 74 vs. 42 ± 27 mg/h, respectively) from the lung 6-8 hafter birth and significantly less extravascular lung water atpostmortem (6.5 ± 0.8 vs. 4.8 0.6 g/g dry lung,respectively). Thus neutrophils contribute to thepathogenesis of respiratory distress after premature birth byincreasing lung vascular protein permeability and promoting lung edema.

     

Arginase and ornithine, as markers in human non-small cell lung carcinoma

The arginase activity and ornithine level were determined in tissue obtained from patients with non-small cell lung carcinoma (NSLC). The arginase activity and ornithine level in tumor tissues were 1.89 +/- 1.28 U/mg protein and 42.32 +/- 25.82 nmol/mg protein, respectively versus 0.67 +/- 0.19 U/mg protein and 10.12 +/- 3.69 nmol/mg protein for normal tissues (p < 0.01).     

Comparison of the efficiency of urea,aqueous ammonia and ammonium sulphate as nitrogen fertilizers

Summary Nitrogen-15 labelled urea, aqueous NH₃ and (NH₄)₂SO₄ were applied to soils contained in pots. The fertilizers were injected in 5 cm³ of solution, 3.5 cm below the soil surface, to simulate a fertilizer band in the field. Ryegrass (Lolium perenne) was planted, and several cuttings and roots were harvested. Efficiency was determined as the recovery of fertilizer-N in the plant tissues and soil.Total recovery varied from 94 to 100%. There was no significant difference between the total recovery of the 3 fertilizer forms, although recovery in the soil component was lower for (NH₄)₂SO₄ than for urea or NH₃. There was a significant difference in total recovery between soils due to the soil component. Only small amounts of¹⁵N were not recovered, whereas laboratory experiments reported elsewhere had demonstrated that substantial gaseous losses of N as N₂, N₂O and NO +NO₂ occurred in these soils during nitrification of added NH₃ fertilizer.     

Crystallization and phase behavior of fatty acid esters of 1,3 propanediol III: 1,3 propanediol dicaprylate/1,3 propanediol distearate (CC/SS) and 1,3 propanediol dicaprylate/1,3 propanediol dipalmitate (CC/PP) binary systems

The phase behavior of the 1,3 propanediol dicaprylate/1,3 propanediol distearate (CC/SS) and the 1,3 propanediol dicaprylate/1,3 propanediol dipalmitate (CC/PP) binary systems were investigated using different techniques. The two systems presented essentially the same overall features. XRD measurements detected CC-CC, PP-PP and SS-SS bilayers which crystallized in beta forms but no mixed bilayers for all mixtures. The phase diagrams of both systems were comparable and displayed a monotectic behavior. As strongly evidenced by XRD data, both phase diagrams suggested that CC, PP and SS formed largely separate phases but were probably not completely immiscible. Avrami analysis of SFC vs. time indicated heterogeneous nucleation and spherulitic crystal development from sporadic nuclei. However, noticeable differences in the manifestation of the molecular interactions have been detected at all levels of structure and confirmed by the interchange coupling determined by the enthalpy of melt, the final SFC and the hardness data. This was obviously related to the difference in chain size between SS and PP molecules. The effect on texture was highlighted by drastic microstructural differences between the two systems. Furthermore, the differences in nucleation and crystal growth, the more pronounced tendency for phase separation in the CC/SS system compared to the CC/PP system, and the relatively better crystallization of the CC/PP mixtures, particularly visibly for x(CC)< or =0.3 compared to the CC/SS mixtures were associated with the chain length difference.     

TAK1 is essential for endothelial barrier maintenance and repair after lung vascular injury

TGF–β-activated kinase 1 (TAK1) plays crucial roles in innate and adaptive immune responses and is required for embryonic vascular development. However, TAK1’s role in regulating vascular barrier integrity is not well defined. Here we show that endothelial TAK1 kinase function is required to maintain and repair the injured lung endothelial barrier. We observed that inhibition of TAK1 with 5Z-7-oxozeaenol markedly reduced expression of β-catenin (β-cat) and VE-cadherin at endothelial adherens junctions and augmented protease-activated receptor-1 (PAR-1)- or toll-like receptor-4 (TLR-4)-induced increases in lung vascular permeability. In inducible endothelial cell (EC)-restricted TAK1 knockout (TAK1^i∆EC) mice, we observed that the lung endothelial barrier was compromised and in addition, TAK1^i∆EC mice exhibited heightened sensitivity to septic shock. Consistent with these findings, we observed dramatically reduced β-cat expression in lung ECs of TAK1^i∆EC mice. Further, either inhibition or knockdown of TAK1 blocked PAR-1- or TLR-4-induced inactivation of glycogen synthase kinase 3β (GSK3β), which in turn increased phosphorylation, ubiquitylation, and degradation of β-cat in ECs to destabilize the endothelial barrier. Importantly, we showed that TAK1 inactivates GSK3β through AKT activation in ECs. Thus our findings in this study point to the potential of targeting the TAK1-AKT-GSK3β axis as a therapeutic approach to treat uncontrolled lung vascular leak during sepsis.     

Immature lung and acute lung injury

Acute lung injury occurs mostly in the very low birth weight and extremely low birth weight infants. The pathological process leading to acute lung injury includes immature and/or diseased lung that experienced oxidative stress, inflammation and mechanical insult with the bronchial, alveolar and capillary injuries and cell death. It may be the first step to the subsequent development of chronic lung disease of prematurity or bronchopulmonary dysplasia. The mechanisms of lung injury are extensively investigated in the experimental models and clinical studies, mostly performed on the adult patients. At present, the explanations of the mechanism(s) leading to lung tissue injury in tiny premature babies are just derived from these studies. Acute lung injury seems to be rather a syndrome than a well-defined nosological unit and is of multifactorial etiology. The purpose of this review is to discuss the main factors contributing to the development of acute lung injury in the very low or extremely low birth weight infants--lung immaturity, mechanical injury, oxidative stress and inflammation. Nevertheless, numerous other factors may influence the status of immature lung after delivery.     

Urinary desmosine as a biomarker in acute lung injury

Acute lung injury (ALI) is a complex disorder associated with an acute inflammatory response thought to contribute to tissue injury. Desmosine, a cross-linking amino acid present in elastin, is released during matrix degradation and cleared by the kidney. Results from animal models and human disease studies have suggested that ALI is associated with the release of desmosine, resulting in increased urinary desmosine. A radioimmunoassay was used to monitor urinary desmosine levels over 10 days in ten patients with ALI. The concentration of desmosine was measured with and without acid hydrolysis. Baseline urinary desmosine was increased in two of ten patients. The concentration of desmosine at baseline did not appear to be related to age, gender, neutrophil elastase (NE)/α₁-antiprotease complex concentration or P_aO₂/F_iO₂ ratio. No meaningful changes in desmosine levels were noted after removal from mechanical ventilation. Baseline desmosine concentrations did not appear to correlate with the risk of death. The limited sensitivity, predictive correlations and dynamic modulation would suggest that urine desmosine has a limited role as a biomarker for ALI. Hydrolysis of urine samples appears necessary for optimal measurement of urine desmosine.     

Considering TWEAK as a target for therapy in renal and vascular injury

TWEAK is a cytokine of the TNF superfamily that activates the Fn14 receptor. TWEAK may regulate cell proliferation, cell death, cell differentiation, angiogenesis and inflammation. The expression of TWEAK and Fn14 is increased during vascular and renal injury. Inflammatory cytokines increase Fn14 receptor expression in tubular and vascular smooth muscle cells. Moreover, TWEAK induces tubular cell apoptosis under proinflammatory conditions. TWEAK itself contributes to renal and vascular inflammation by promoting chemokine and inflammatory cytokine secretion. Confirmation of its role in acute kidney injury and atherosclerotic lesions formation came from functional studies in experimental animal models. The available evidence suggests that TWEAK might be a target for therapeutic intervention in renal and vascular injury and its role in different forms of tissue damage should be further explored.     

Protective role of vascular endothelial growth factor in endotoxin-induced acute lung injury in mice

Background

Vascular endothelial growth factor (VEGF), a substance that stimulates new blood vessel formation, is an important survival factor for endothelial cells. Although overexpressed VEGF in the lung induces pulmonary edema with increased lung vascular permeability, the role of VEGF in the development of acute lung injury remains to be determined.

Methods

To evaluate the role of VEGF in the pathogenesis of acute lung injury, we first evaluated the effects of exogenous VEGF and VEGF blockade using monoclonal antibody on LPS-induced lung injury in mice. Using the lung specimens, we performed TUNEL staining to detect apoptotic cells and immunostaining to evaluate the expression of apoptosis-associated molecules, including caspase-3, Bax, apoptosis inducing factor (AIF), and cytochrome C. As a parameter of endothelial permeability, we measured the albumin transferred across human pulmonary artery endothelial cell (HPAEC) monolayers cultured on porous filters with various concentrations of VEGF. The effect of VEGF on apoptosis HPAECs was also examined by TUNEL staining and active caspase-3 immunoassay.

Results

Exogenous VEGF significantly decreased LPS-induced extravascular albumin leakage and edema formation. Treatment with anti-VEGF antibody significantly enhanced lung edema formation and neutrophil emigration after intratracheal LPS administration, whereas extravascular albumin leakage was not significantly changed by VEGF blockade. In lung pathology, pretreatment with VEGF significantly decreased the numbers of TUNEL positive cells and those with positive immunostaining of the pro-apoptotic molecules examined. VEGF attenuated the increases in the permeability of the HPAEC monolayer and the apoptosis of HPAECs induced by TNF-α and LPS. In addition, VEGF significantly reduced the levels of TNF-α- and LPS-induced active caspase-3 in HPAEC lysates.

Conclusion

These results suggest that VEGF suppresses the apoptosis induced by inflammatory stimuli and functions as a protective factor against acute lung injury.     

Time-dependent reversal of sepsis-induced PMN uptake and lung vascular injury by expression of CD18 antagonist

We determined the time-dependent effects of conditional expression of neutrophil inhibitory factor (NIF), a specific 41-kDa CD18 integrin antagonist, on the time course of NIF expression and lung PMN (polymorphonuclear leukocyte) infiltration and vascular injury in a model of Escherichia coli-induced sepsis in mice. Studies were made in mice transduced with the E-selectin (ES) promoter-NIF construct (using liposomes) in which the NIF cDNA was driven by the inflammation- and endothelial cell-specific ES promoter. We observed time-dependent expression of NIF in pulmonary vascular endothelium that paralleled the ES expression. Expression of both was evident at 1 h after E. coli challenge, peaked at 3-6 h, and returned to basal level within 48 h. We observed that increases in PMN uptake and transalveolar PMN migration induced by E. coli challenge were reversed in a time-dependent manner following NIF expression in mice. NIF expression also prevented the progression of lung vascular injury and edema formation following E. coli challenge. Thus the conditional expression of NIF using the ES promoter can reverse, in a time-dependent manner, lung PMN infiltration and vascular injury induced by gram-negative sepsis. The results support the model that initial engagement of CD18 integrins enables the further recruitment of additional PMN into lung tissues such that PMN continue to sequester and migrate after E. coli challenge.     

Comparison of three tracers for detecting lung epithelial injury in anesthetized sheep

We compared the ability of three aerosolized tracers to discriminate among control, lung inflation with a positive end expired pressure of 10 cmH2O, lung vascular hypertension and edema without lung injury, and lung edema with lung injury due to intravenous oleic acid. The tracers were 99mTc-diethylenetriaminepentaacetate (99mTc-DTPA, mol wt 492), 99mTc-human serum albumin (99mTc-ALB, mol wt 69,000), and 99mTc-aggregated albumin (99mTc-AGG ALB, mol wt 383,000). 99mTc-DTPA clearance measurements were not able to discriminate lung injury from lung inflation. The 99mTc-AGG ALB clearance rate was unchanged by lung inflation and increased slightly with lung injury. The 99mTc-ALB clearance rate (0.06 +/- 0.02%/min) was unchanged by lung inflation (0.09 +/- 0.02%/min, P greater than 0.05) or 4 h of hypertension without injury (0.09 +/- 0.04%/min, P greater than 0.05). Deposition of 99mTc-ALB within 15 min of the administration of the oleic acid increased the clearance rate to 0.19 +/- 0.06%/min, which correlated well with the postmortem lung water volume (r = 0.92, P less than 0.01). This did not occur when there was a 60-min delay in the deposition of 99mTc-ALB. We conclude that 99mTc-ALB is the best indicator for studying the effects of lung epithelial injury on protein and fluid transport into and out of the air spaces of the lungs in a minimally invasive manner.     

Comparison of cellular and medium insulin and GABA content as markers for living beta-cells

Experimental and therapeutic use of islet cell preparations could benefit from assays that measure variations in the mass of living beta-cells. Because processes of cell death can be followed by depletion and/or discharge of cell-specific substances, we examined whether in vitro conditions of beta-cell death resulted in changes in tissue and medium content of insulin and of gamma-aminobutyric acid (GABA), two beta-cell-specific compounds with different cellular localization and turnover. Exposure of rat purified beta-cells to streptozotocin (5 mM, 120 min) or to the nitric oxide donor GEA-3162 (GEA; 50 microM, 120 min) caused 80% necrosis within 24 h; at the end of this period, cellular insulin content was not significantly decreased, but cellular GABA content was reduced by 70%; when cultured at basal glucose (6 mM), the toxin-exposed cells did not discharge less insulin but released 80% less GABA in the period 8-24 h. As in rat beta-cell purification, GABA comigrated with insulin during human islet cell isolation. Twenty-four hours after GEA (500 microM, 120 min), human islet cell preparations exhibited 90% dead cells and a 45 and 90% reduction, respectively, in tissue insulin and GABA content; in the period 9-24 h, insulin discharge in the medium was not reduced, but GABA release was decreased by 90%. When rat beta-cells were cultured for 24 h with nontoxic interleukin (IL)-1beta concentrations that suppressed glucose-induced insulin release, cellular GABA content was not decreased and GABA release increased by 90% in the period 8-24 h. These data indicate that a reduction in cellular and medium GABA levels is more sensitive than insulin as a marker for the presence of dead beta-cells in isolated preparations. Pancreatic GABA content also rapidly decreased after streptozotocin injection and remained unaffected by 12 h of hyperglycemia. At further variance with insulin, GABA release from living beta-cells depends little on its cellular content but increases with IL-1beta-induced alterations in beta-cell phenotype.     

Comparison of self-reported and measured BMI as correlates of disease markers in US adults

Objective: The purpose of this study is to evaluate the validity of BMI based on self‐reported data by comparison with technician‐measured BMI and biomarkers of adiposity. Research Methods and Procedures: We analyzed data from 10,639 National Health and Nutrition Education Study III participants ≥20 years of age to compare BMI calculated from self‐reported weight and height with BMI from technician‐measured values and body fatness estimated from bioelectrical impedance analysis in relation to systolic blood pressure, fasting blood levels of glucose, high‐density lipoprotein‐cholesterol, triglycerides, C‐reactive protein, and leptin. Results: BMI based on self‐reported data (25.07 kg/m²) was lower than BMI based on technician measurements (25.52 kg/m²) because of underreporting weight (?0.56 kg; 95% confidence interval, ?0.71, ?0.41) and overreporting height (0.76 cm; 95% confidence interval, 0.64, 0.88). However, the correlations between self‐reported and measured BMI values were very high (0.95 for whites, 0.93 for blacks, and 0.90 for Mexican Americans). In terms of biomarkers, self‐reported and measured BMI values were equally correlated with fasting blood glucose (r = 0.43), high‐density lipoprotein‐cholesterol (r = ?0.53), and systolic blood pressure (r = 0.54). Similar correlations were observed for both measures of BMI with plasma concentrations of triglycerides and leptin. These correlations did not differ appreciably by age, sex, ethnicity, or obesity status. Correlations for percentage body fat estimated through bioelectrical impedance analysis with these biomarkers were similar to those for BMI. Discussion: The accuracy of self‐reported BMI is sufficient for epidemiological studies using disease biomarkers, although inappropriate for precise measures of obesity prevalence.