MicroRNA-126 Attenuates the Effect of Chemokine CXCL8 on Proliferation, Migration, Apoptosis, and MAPK-Dependent Signaling Activity of Vascular Endothelial Cells Cultured in a Medium with High Glucose Concentration.

We studied the mechanisms by which microRNA-126 regulates proliferation and migration of human umbilical vein endothelial cells (HUVEC) cultured in a medium with high glucose concentration and treated with chemokine CXCL8. Cell proliferation, apoptosis, and migration were analyzed by the CCK-8 assay, Annexin V-PI staining, and Transwell assay, respectively. The ratios of p-ERK/ERK, p-P38/P38, p-JNK/JNK were determined by ELISA. HUVEC cells cultured in the presence of high glucose concentration (30 mmol/ml) and treated with CXCL8 (50 ng/ml) demonstrated more intensive proliferation, migration, and p-ERK/ERK, p-P38/P38, and p-JNK/JNK ratios and significantly lower apoptosis rate than control cells (high glucose, no treatment) and cells treated with CXCL8 and transfected with microRNA-126-mimic. Thus, microRNA-126 regulates proliferation and migration of HUVEC cells cultured in the presence of high glucose concentrations and treated with CXCL8 through inhibition of MAPK signaling pathway.

Lung-derived mediators induce cytokine production in downstream organs via an NF-κB-dependent mechanism.

In the setting of acute lung injury, levels of circulating inflammatory mediators have been correlated with adverse outcomes. Previous studies have demonstrated that injured, mechanically ventilated lungs represent the origin of the host inflammatory response; however, mechanisms which perpetuate systemic inflammation remain uncharacterized. We hypothesized that lung-derived mediators generated by mechanical ventilation (MV) are amplified by peripheral organs in a "feed forward" mechanism of systemic inflammation. Herein, lung-derived mediators were collected from 129X1/SVJ mice after 2 hours of MV while connected to the isolated perfused mouse lung model setup. Exposure of liver endothelial cells to lung-derived mediators resulted in a significant increase in G-CSF, IL-6, CXCL-1, CXCL-2, and MCP-1 production compared to noncirculated control perfusate media (P < 0.05). Furthermore, inhibition of the NF-κB pathway significantly mitigated this response. Changes in gene transcription were confirmed using qPCR for IL-6, CXCL-1, and CXCL-2. Additionally, liver tissue obtained from mice subjected to 2 hours of in vivo MV demonstrated significant increases in hepatic gene transcription of IL-6, CXCL-1, and CXCL-2 compared to nonventilated controls. Collectively, this data demonstrates that lung-derived mediators, generated in the setting of MV, are amplified by downstream organs in a feed forward mechanism of systemic inflammation.

The fabrication and characteristics of indium-oxide covered porous InP.

Uniform and vertical indium-oxide nanotube (IONT) arrays embedded well in n-type InP single crystal have been successfully prepared in situ by porous InP-template-assisted chemical vapor deposition (CVD). This IONT/InP nanostructure reveals high sensitivity to humidity at room temperature, which is ascribed to the ultrahigh surface-to-volume ratio of this nanostructure and the large number of oxygen defected states in IONTs. Such a nanostructure of IONT arrays embedded in a III-V semiconductor substrate could be expected to have potential applications, such as superior gas sensors. This work provides a novel approach for fabricating low-melting metal oxide semiconductor nanotubes.

Mechanisms responsible for surfactant changes in sepsis-induced lung injury.

Pulmonary surfactant is altered in sepsis, and these changes contribute to the predisposition of septic lungs to subsequent insults, ultimately leading to acute lung injury. Specifically, the total amount of surfactant is lower in sepsis, mainly due to decreased small aggregate (SA) surfactant pools. The amount of large aggregate (LA) surfactant is not altered. To evaluate the mechanisms responsible for these alterations, trace doses of tritium-labelled dipalmitoylphosphatidylcholine (3H-DPPC)-labelled LA were instilled intratracheally into adult rats 20 hrs after caecal ligation and perforation (CLP) or sham surgery. Animals were sacrificed at 0, 1 and 4 h after instillation and recovery of 3H-DPPC in alveolar macrophages (AM), LA and SA was measured. In separate in vitro experiments, AM isolated from CLP/sham rats were incubated with LA or SA isolated from normal animals to evaluate the uptake of these aggregates into the AM. Results showed increased surfactant radioactivity associated with AM of CLP animals compared with sham animals both in vivo and in vitro. In addition, more 3H-DPPC label remained in LA forms in the CLP animals in vivo compared with sham. These findings indicate that differences in surfactant aggregate uptake and large aggregate conversion occur in septic lungs, resulting in changes in surfactant pools.

Polo-like kinase-1 in porcine oocyte meiotic maturation, fertilization and early embryonic mitosis.

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that regulate multiple stages of mitosis. Expression and distribution of polo-like kinase 1 (Plk1) were characterized during porcine oocyte maturation, fertilization and early embryo development in vitro, as well as after microtubule polymerization modulation. The quantity of Plk1 protein remained stable during meiotic maturation. Plk1 accumulated in the germinal vesicles (GV) in GV stage oocytes. After germinal vesicle breakdown (GVBD), Plk1 was localized to the spindle poles at metaphase I (MI) stage, and then translocated to the middle region of the spindle at anaphase-telophase I. Plk1 was also localized in MII spindle poles and on the spindle fibers and on the middle region of anaphase-telophase II spindles. Plk1 was not found in the spindle region when colchicine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. After fertilization, Plk1 concentrated around the female and male pronuclei. During early embryo development, Plk1 was found to be in association with the mitotic spindle at metaphase, but distributed diffusely in the cytoplasm at interphase. Our results suggest that Plk1 is a pivotal regulator of microtubule organization and cytokinesis during porcine oocyte meiotic maturation, fertilization, and early embryo cleavage in pig oocytes.

Evaluation of alveolar surfactant aggregates in vitro and in vivo.

In acute lung injury, a decrease in surface-active large aggregates and an increase in the less surface-active small surfactant aggregates are observed. The objective of the current study was to determine if the increase in small aggregates interfered with the function of large aggregates, thereby independently contributing to lung dysfunction. Isolated large aggregates, small aggregates, and large aggregate+small aggregate combinations were analysed for in vitro surface activity utilizing a pulsating bubble surfactometer. Subsequently, large aggregates, small aggregates, and large aggregate+ small aggregate combinations were administered to surfactant-deficient, adult Sprague-Dawley rats. Physiological parameters were measured during 1 h of ventilation. After sacrifice, the whole lung lavage was analysed for protein concentration, and surface activity of the recovered large aggregates. The minimum surface tension of the large aggregate+small aggregate preparations (10 mN x m(-1)) was significantly higher than large aggregates alone (1 mN x m(-1)), but lower than small aggregates alone (21 mN x m(-1) ) after 100 pulsations. In vivo, rats receiving large aggregates+small aggregates showed immediate increases in oxygenation, similar to animals given large aggregates, whereas animals given small aggregates and control animals maintained low oxygenation values. In conclusion, small aggregates interfered with large aggregates function in vitro, but this was not observed in vivo in this experimental model.

Surfactant treatment for ventilation-induced lung injury in rats: effects on lung compliance and cytokines.

The objective of this study was to determine if exogenous surfactant therapy could prevent the harmful effects of ventilation at high tidal volumes without positive end-expiratory pressure (PEEP). Rats were randomized to either a nontreated control group (8 mL/kg 4 cm H2O PEEP), a nontreated injuriously ventilated group (20 mL/kg 0 cm H2O PEEP) or a treatment group of either 50 mg/kg, 50 mg/kg + 5% surfactant-associated protein A, 100 mg/kg exogenous surfactant followed by injurious ventilation. Isolated lungs from animals in all 5 groups were ventilated in a humidified box at 37 degrees C for 2 hours. Pressure-volume curves and light microscopy showed that surfactant treatment reduced the ventilation-induced lung injury (VILI). Inflammatory cytokines (tumor necrosis factor-alpha [TNFalpha], interleukin [IL]-1beta, and IL-6) in the lavage were significantly higher in injuriously ventilated lungs compared to the control group. However the 3 treatment groups had cytokine concentrations that were similar to the injuriously ventilated group. We conclude that surfactant treatment is beneficial in preventing VILI; however, it does not prevent the release of inrflammatory cytokines during mechanical ventilation.

Mechanical ventilation of isolated septic rat lungs: effects on surfactant and inflammatory cytokines.

The effects of mechanical ventilation (MV) on the surfactant system and cytokine secretion were studied in isolated septic rat lungs. At 23 h after sham surgery or induction of sepsis by cecal ligation and perforation (CLP), lungs were excised and randomized to one of three groups: 1) a nonventilated group, 2) a group subjected to 1 h of noninjurious MV (tidal volume = 10 ml/kg, positive end-expiratory pressure = 3 cmH(2)O), or 3) a group subjected to 1 h of injurious MV (tidal volume = 20 ml/kg, positive end-expiratory pressure = 0 cmH(2)O). Nonventilated sham and CLP lungs had similar compliance, normal lung morphology, surfactant, and cytokine concentrations. Injurious ventilation decreased compliance, altered surfactant, increased cytokines, and induced morphological changes compared with nonventilation in sham and CLP lungs. In these lungs, the surfactant system was similar in sham and CLP lungs; however, tumor necrosis factor-alpha and interleukin-6 levels were significantly higher in CLP lungs. We conclude that injurious ventilation altered surfactant independent of sepsis and that the CLP lungs were predisposed to the secretion of larger amounts of cytokines because of ventilation.

Evaluation of exogenous surfactant in HCL-induced lung injury.

The efficacy of exogenous surfactant administration is influenced by numerous factors, which has resulted in variable outcomes of clinical trials evaluating this treatment for the acute respiratory distress syndrome (ARDS). We investigated several of these factors in an animal model of acid aspiration including different surfactant preparations, and different delivery methods. In addition, high-frequency oscillation (HFO), a mode of mechanical ventilation known to recruit severely damaged lungs, was utilized. Lung injury was induced in adult rabbits via intratracheal instillation of 0.2 N HCl followed by conventional mechanical ventilation (CMV) until Pa(O2)/FI(O2) values ranged from 220 to 270 mm Hg. Subsequently, animals were given one of three surfactants administered via three different methods and physiological responses were assessed over a 1-h period. Regardless of the surfactant treatment strategy utilized, oxygenation responses were not sustained. In contrast, HFO resulted in a superior response compared with all surfactant treatment strategies involving CMV. The deterioration in physiological parameters after surfactant treatment was likely due to overwhelming protein inhibition of the surfactant. In conclusion, various surfactant treatment strategies were not effective in this model of lung injury, although the lungs of these animals were recruitable with HFO, as reflected by the acute and sustained oxygenation improvements.

Effects of alveolar surfactant aggregates on T-lymphocyte proliferation.

The effects of alveolar large aggregate (LA) and small aggregate (SA) surfactant subfractions isolated from healthy adult rats on mitogen-stimulated proliferative responses of human peripheral blood mononuclear cells (PBMC) was examined. Various concentrations of total surfactant suppressed proliferation of stimulated lymphocytes by up to 95% of mitogen-stimulated cells alone. LA subfractions of total surfactant had no effect on proliferation, whereas SA significantly enhanced the lymphocyte proliferation at lower concentrations (7.8 microg/ml) compared to mitogen-stimulated cells alone. Higher concentrations of SA (62.5 microg/ml) inhibited lymphocyte proliferation. This concentration-dependent effect of SA on proliferation of PBMC was also present when cells were stimulated with various lectins including anti-CD3, concanavalin A and phytohemagglutinin. Analysis of the supernatant of mitogen-stimulated cell cultures treated with inhibitory concentrations of SA showed decreased amounts of interleukin (IL)-2, compared to cells alone, which could be reversed by adding exogenous IL-2 to the cell cultures with the SA. These results suggest that alveolar surfactant subfractions have distinct functions within the alveoli, both biophysically and with respect to their effects on the host's immunomodulatory responses.

[The determination of lorazepam in human urine by gas chromatography/nitrogen-phosphorus detector].

A method to assay lorazepam in human urine has been developed. After addition of hydroxyethylflurazepam (internal standard) and hydrolysis with beta-glucuronidase, the lorazepam and hydroxyethylflurazepam were extracted with ethyl ether at pH 10.8. The analysis was performed on an HP-5 capillary column with nitrogen-phosphorus detector(NPD). The detection limit and recovery of analytes in urine were 5 micrograms/L and (83.4 +/- 3.1)% respectively. The method was successfully applied to urine specimens collected from healthy human volunteers who have ingested 2 mg of lorazepam. The method was sensitive enough to assay urine specimen excreted at 32 h after taking the medicine by volunteers.

Ovine surfactant protein cDNAs: use in studies on fetal lung growth and maturation after prolonged hypoxemia.

cDNAs for ovine surfactant-associated protein (SP) A, SP-B, and SP-C have been cloned and shown to possess strong similarity to cDNAs for surfactant apoproteins in other species. These reagents were employed to examine the effect of fetal hypoxia on the induction of surfactant apoprotein expression in the fetal lamb. Postnatal lung function is dependent on adequate growth and maturation during fetal development. Insulin-like growth factor (IGF) I and IGF-II, which are present in all fetal tissues studied, possess potent mitogenic and proliferative actions, and their effects can be modulated by IGF-specific binding proteins (IGFBPs). Hypoxia can lead to increases in circulating cortisol and catecholamines that can influence lung maturation. Therefore, the effects of mild hypoxia in chronically catheterized fetal lambs at gestational days 126-130 and 134-136 (term 145 days) on the expression of pulmonary surfactant apoproteins and IGFBPs were examined. Mild hypoxia for 48 h resulted in an increase in plasma cortisol that was more pronounced at later gestation, and in these animals, there was a twofold increase in SP-A mRNA. SP-B mRNA levels also increased twofold, but this was not significant. SP-C mRNA was not altered. No significant changes in apoprotein mRNA were observed with the younger fetuses. However, these younger animals selectively exhibited reduced IGFBP-5 mRNA levels. IGF-I mRNA was also reduced at 126-130 days, although this conclusion is tentative due to low abundance. IGF-II levels were not affected at either gestational age. We conclude that these data suggest that mild prolonged fetal hypoxia produces alterations that could affect fetal cellular differentiation early in gestation and can induce changes consistent with lung maturation closer to term.

Alveolar environment influences the metabolic and biophysical properties of exogenous surfactants.

Several factors have been shown to influence the efficacy of exogenous surfactant therapy in the acute respiratory distress syndrome. We investigated the effects of four different alveolar environments (control, saline-lavaged, N-nitroso-N-methylurethane, and hydrochloric acid) on the metabolic and functional properties of two exogenous surfactant preparations: bovine lipid extract surfactant and recombinant surfactant-associated protein (SP) C drug product (rSPC) administered to each of these groups. The main difference between these preparations was the lack of SP-B in the rSPC. Our results demonstrated differences in the large aggregate pool sizes recovered from each of the experimental groups. We also observed differences in SP-A content, surface area cycling characteristics, and biophysical activities of these large aggregate forms after the administration of the two exogenous surfactant preparations. We conclude that the alveolar environment plays a critical role, influencing the overall efficacy of exogenous surfactant therapy. Thus further preclinical studies are warranted to investigate the specific factors within the alveolar environment that lead to the differences observed in this study.

An examination of the different variables affecting surfactant aggregate conversion in vitro.

Pulmonary surfactant exists in 2 major subtypes, the freshly secreted, surface-active large surfactant aggregates (LA) and their metabolic product, the less surface active, small aggregates (SA). Conversion of LA into SA can be studied using an in vitro technique, surface area cycling, which involves the rotation of a suspension of LA end-over-end so that the surface area of the liquid changes twice each cycle. In order to further elucidate the mechanisms involved in aggregate conversion, we have examined the effects of time, temperature, change in surface area, cycling speed, surfactant concentration, and albumin on aggregate conversion in vitro. Three different surfactant preparations were used; rabbit LA, sheep LA, and an exogenous surfactant preparation, bovine lipid extract surfactant (BLES). Based on our results that showed that these variables affected aggregate conversion, we concluded that the adsorption of surfactant at the changing air-liquid interface was an important step in aggregate conversion. However, the results also indicated that aggregate conversion was not solely due to the adsorption characteristics of the surfactant. Other surfactant properties, such as the activity of serine protease or film stability at the air-liquid interface, may also be important in aggregate conversion.

[Research of the extraction method of morphine from biological fluids].

A solid-phase extraction method of morphine from urine and blood has introduced. The effect of 5 SPE columns, 3 eluents and pH on morphine recovery has been investigated systematically. Derivative GC was used as a method of detection. The result showed that the column and the eluent of such as GDX-301, GDX-403 and C18 chloroform:isopropanol (9:1) had good behaviors to extraction of morphine. When GDX-301 was used as a sorbent, the recovery of morphine from urine was above 90% at pH 9, then went down with the increase of pH. While the recovery from blood was growing with the increase of pH, which reached above 90% in strong alkaline. The extraction method is simple, inexpensive, efficient and reproducible, which provides an effective and practical method to extract morphine and similar illicit drugs from biological fluids.

Effects of surfactant distribution and ventilation strategies on efficacy of exogenous surfactant.

The effects of both surfactant distribution patterns and ventilation strategies utilized after surfactant administration were assessed in lung-injured adult rabbits. Animals received 50 mg/kg surfactant via intratracheal instillation in volumes of either 4 or 2 ml/kg. A subset of animals from each treatment group was euthanized for evaluation of the exogenous surfactant distribution. The remaining animals were randomized into one of three ventilatory groups: group 1 [tidal volume (VT) of 10 ml/kg with 5 cmH2O positive end-expiratory pressure (PEEP)]; group 2 (VT of 5 ml/kg with 5 cmH2O PEEP); or group 3 (VT of 5 ml/kg with 9 cmH2O PEEP). Animals were ventilated and monitored for 3 h. Distribution of the surfactant was more uniform when it was delivered in the 4 ml/kg volume. When the distribution of surfactant was less uniform, arterial PO2 values were greater in groups 2 and 3 compared with group 1. Oxygenation differences among the different ventilation strategies were less marked in animals with the more uniform distribution pattern of surfactant (4 ml/kg). In both surfactant treatment groups, a high mortality was observed with the ventilation strategy used for group 3. We conclude that the distribution of exogenous surfactant affects the response to different ventilatory strategies in this model of acute lung injury.

Effects of ventilation strategies on the efficacy of exogenous surfactant therapy in a rabbit model of acute lung injury.

We evaluated the effects of various ventilation strategies on the efficacy of exogenous surfactant therapy in lung-injured adult rabbits. Lung injury was induced by repetitive whole-lung saline lavage followed by mechanical ventilation. Three hours after the final lavage, 100 mg lipid/kg bovine lipid extract surfactant was instilled. After confirmation of similar responses to exogenous surfactant, animals were then randomized to one of four ventilation groups; (1) Normal tidal volume (VT) (5 cm H2O): VT = 10 ml/kg, respiratory rate (RR) = 30/min, positive end-expiratory pressure (PEEP) = 5 cm H2O; (2) Normal VT (9 cm H2O): VT = 10 ml/kg, RR = 30/min, PEEP = 9 cm H2O; (3) Low VT (5 cm H2O): VT = 5 ml/kg, RR = 60/min, PEEP = 5 cm H2O; (4) Low VT (9 cm H2O): VT = 5 ml/kg, RR = 60/min, PEEP = 9 cm H2O. Animals were ventilated for an additional 3 h and then killed, and lung lavage fluid was analyzed. Animals ventilated with the low-VT modes (Low VT [5 cm H2O] and Low VT [9 cm H2O]) had higher PaO2 values (430 +/- 7 mm Hg and 425 +/- 18 mm Hg versus 328 +/- 13 mm Hg) and higher percentages of surfactant in large aggregate forms (83 +/- 2% and 82 +/- 2% versus 67 +/- 4%) at 3 h after treatment than did the Normal VT (5 cm H2O) group (p < 0.05). Increasing the PEEP level was beneficial for a short period after surfactant administration to maintain oxygenation, but did not affect exogenous surfactant aggregate conversion. We speculate that ventilation strategies resulting in low exogenous surfactant aggregate conversion will result in superior physiologic responses to exogenous surfactant.

Alterations of the endogenous surfactant system in septic adult rats.

Sepsis is the most common factor leading to the acute respiratory distress syndrome (ARDS) and is associated with the highest mortality rate. It has been suggested that the pulmonary surfactant system is altered and contributes to the lung dysfunction associated with ARDS. The objective of this study was to characterize the lung injury, specifically the endogenous surfactant system in septic adult rats. Sepsis was induced in male Sprague-Dawley rats by cecal ligation and perforation and resulted in significant increases in heart rates, respiratory rates, and lactate levels along with positive blood cultures in septic animals compared with a sham control group. Two distinct septic groups were developed, a septic group and a sepsis with lung injury (septic+LI) group. The septic group had no significant differences in oxygenation compared with the sham group, whereas the septic+LI group had significantly lower PaO2 and higher A-a gradient values compared to both the sham and septic groups. The total surfactant pool size was significantly lower in the septic+LI group compared with the sham group. The small surfactant aggregate to large surfactant aggregate ratio was significantly lower in the septic group and was further reduced in the septic+LI group. There were also significantly higher levels of surfactant protein A (SP-A) in both septic and septic+LI groups compared to the sham group. These results demonstrated that the endogenous surfactant system was altered in systemic sepsis without lung dysfunction and is further altered when a lung injury is present.

Effects of lung injury on pulmonary surfactant aggregate conversion in vivo and in vitro.

Within the alveolar space pulmonary surfactant is converted from the surface active large aggregates (LA) to the inactive small aggregates (SA). This conversion is affected by a change in surface area, lung injury, breathing pattern, and protease activity. This study examined the effect of N-nitroso-N-methylurethane-induced acute lung injury on aggregate conversion in mechanically ventilated and spontaneously breathing rabbits. Both the in vitro surface area cycling techniques and the in vivo technique of intratracheally injecting radiolabeled LA were used for analyzing aggregate conversion. Mechanical ventilation of injured lungs resulted in increased aggregate conversion and increased surfactant aggregate ratios compared with controls. Spontaneously breathing injured animals had aggregate conversion and aggregate ratios that were not significantly different from controls. In vitro aggregate conversion was slower for LA obtained from injured animals compared with normal animals. We conclude that the mechanical stress of mechanical ventilation results in increased aggregate conversion and aggregate ratios. Furthermore, in vitro conversion of isolated LA does not necessarily reflect the conversion of aggregates within the alveoli.