Molecular Impact of Conventional and Electronic Cigarettes on Pulmonary Surfactant.

The alveolar epithelium is covered by a non-cellular layer consisting of an aqueous hypophase topped by pulmonary surfactant, a lipo-protein mixture with surface-active properties. Exposure to cigarette smoke (CS) affects lung physiology and is linked to the development of several diseases. The macroscopic effects of CS are determined by several types of cell and molecular dysfunction, which, among other consequences, lead to surfactant alterations. The purpose of this review is to summarize the published studies aimed at uncovering the effects of CS on both the lipid and protein constituents of surfactant, discussing the molecular mechanisms involved in surfactant homeostasis that are altered by CS. Although surfactant homeostasis has been the topic of several studies and some molecular pathways can be deduced from an analysis of the literature, it remains evident that many aspects of the mechanisms of action of CS on surfactant homeostasis deserve further investigation.

Optimization of Fibrin Scaffolds to Study Friction in Cultured Mesothelial Cells.

To study the friction of cell monolayers avoiding damage due to stress concentration, cells can be cultured on fibrin gels, which have a structure and viscoelasticity similar to that of the extracellular matrix. In the present research, we studied different gel compositions and surface coatings in order to identify the best conditions to measure friction in vitro. We examined the adhesion and growth behavior of mesothelial cell line MET-5A on fibrin gels with different fibrinogen concentrations (15, 20, and 25 mg/mL) and with different adhesion coatings (5 µg/mL fibronectin, 10 µg/mL fibronectin, or 10 µg/mL fibronectin + 10 µg/mL collagen). We also investigated whether different substrates influenced the coefficient of friction and the ability of cells to stick to the gel during sliding. Finally, we studied the degradation rates of gels with and without cells. All substrates tested provided a suitable environment for the adherence and proliferation of mesothelial cells, and friction measurements did not cause significant cell damage or detachment. However, in gels with a lower fibrinogen concentration, cell viability was higher and cell detachment after friction measurement was lower. Fibrinolysis was negligible in all the substrates tested.

Acute and chronic acidosis influence on antioxidant equipment and transport proteins of rat jejunal enterocyte.

Acidosis elicits the formation of oxidants and, in turn, ROS (reactive oxygen species)-induced intestinal diseases cause acidosis. This research investigated whether both acute and chronic acidosis influence the antioxidant enzymatic equipment of rat jejunocyte, including γ-GT activity, involved in GSH (glutathione) homoeostasis. Lipid peroxidation level and the expressions of (Na+, K+)-ATPase and GLUT2 were also investigated. The possible influence of acidosis on ROS action was tested. Isolated apical membranes, everted sac preparations and homogenates from acidotic rats were used. γ-GT activity is inhibited after incubation of isolated membranes at acidic pH, but using the whole intestinal tract this inhibition disappears, while SOD (superoxide dismutase) and GR (glutathione reductase) activities are enhanced. Also, in conditions of chronic acidosis, γ-GT activity is unaffected, but no variations of antioxidant activities are apparent. (Na+, K+)-ATPase expression increases, while GLUT2 decreases in acidotic animals. Lipid peroxidation level is unaffected by acidosis. H2O2 inhibits γ-GT activity only in isolated membranes; in the whole tissue, it enhances CAT (catalase) and SOD activities and reduces GLUT2 expression. The pattern of responses to oxidant agents is unaffected by acidosis. Although jejunum seems quite resistant to acidosis, results, suggesting specific responses to this condition, may direct further research on antioxidant supplementation.

Role of MUC1 in lubrication of pleural mesothelial cells cultured on fibrine gel.

To elucidate the role of sialomucin in friction reduction, we investigated the sliding friction of pleural mesothelial cells monolayers cultured on fibrine gel. These measurements were performed on normal (4/4 RM-4) and on tumor (CARM-L1 TG3) cell lines. The effect of treatment with neuraminidase, which removes sialic acid from sialomucin, and of dexamethasone, which has shown to increase sialomucin expression, were also assessed. Furthermore, the expression of the main form of cell-surface-associated mucin (MUC1) present in the mesothelium, was assessed by western blot and immunofluorescence, under different experimental conditions. Expression of MUC1 was not significantly different in the two cell lines. Moreover, dexamethasone did not increase the expression of MUC1. Coefficient of kinetic friction (µ) was significantly higher in tumor cells than in normal cells. Neuraminidase increased µ in both cell lines. These results suggest that sialomucin may play a role in reducing the friction of pleural mesothelial cells.

Evidence for Na+-glucose cotransporter in type I alveolar epithelium.

Functional evidence of Na(+)-glucose cotransport in rat lung has been provided by Basset et al. (J. Physiol. 384:325-345, 1987). By autoradiography [(3)H]phloridzin binding has been found confined to alveolar epithelial type II cells in mouse and rabbit lungs (Boyd, J. Physiol. 422: 44P, 1990). In this research we checked by immunofluorescence whether Na(+)-glucose cotransporter (SGLT1) is also expressed in alveolar type I cells. Lungs of anesthetized rats and lambs were fixed by paraformaldehyde, perfused in pulmonary artery, or instilled into a bronchus, respectively. Tissue blocks embedded in paraffin or frozen were sectioned. Two specific anti-SGLT1 antibodies for rat recognizing aminoacid sequence 402-420, and 546-596 were used in both species. Bound primary antibody was detected by secondary antibody conjugated to fluorescein isothiocianate or Texas red, respectively. In some sections cellular nuclei were also stained. In rats alveolar type I cells were identified by fluorescent Erythrina cristagalli lectin. Sections were examined by confocal laser-scanning microscope. Both in rats and lambs alveolar epithelium was stained by either antibody; no labeling occurred in negative controls. Hence, SGLT1 appears to be also expressed in alveolar type I cells. This is functionally relevant because type I cells provide 95-97% of alveolar surface, and SGLT1, besides contributing to removal of lung liquid under some circumstances, keeps low glucose concentration in lining liquid, which is useful to prevent lung infection.

Effects of Creatine Treatment on Jejunal Phenotypes in a Rat Model of Acidosis.

We investigated the effects of creatine treatment on jejunal phenotypes in a rat model of oxidative stress induced by acidosis. In particular, the activities of some antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase, and glutathione reductase), the level of lipid peroxidation, the expression of heat shock proteins (HSP70), and the expression of the major carriers of the cells (Na+/K+-ATPase, sodium-glucose Transporter 1-SGLT1, and glucose transporter 2-GLUT2) were measured under control and chronic acidosis conditions. Creatine did not affect the activity of antioxidant enzymes in either the control or acidosis groups, except for catalase, for which the activity was reduced in both conditions. Creatine did not change the lipid peroxidation level or HSP70 expression. Finally, creatine stimulated (Na+/K+)-ATPase expression under both control and chronic acidosis conditions. Chronic acidosis caused reductions in the expression levels of GLUT2 and SGLT1. GLUT2 reduction was abolished by creatine, while the presence of creatine did not induce any strengthening effect on the expression of SGLT1 in either the control or chronic acidosis groups. These results indicate that creatine has antioxidant properties that are realized through direct interaction of the molecule with reactive oxygen species. Moreover, the administration of creatine seems to determine a functional strengthening of the tissue, making it more resistant to acidosis.

Na+-glucose cotransporter is also expressed in mesothelium of species with thick visceral pleura.

Molecular evidence for Na+-glucose cotransporter (SGLT1) in rabbit pleural mesothelium has been recently provided, confirming earlier functional findings on solute-coupled liquid absorption from rabbit pleural space. In this research we checked whether SGLT1 is also expressed in pleural mesothelium of species with thick visceral pleura, which receives blood from systemic circulation, but drains it into pulmonary veins. To this end immunoblot assays were performed on total protein extract of scraped visceral and parietal mesothelium of lambs and adult sheep, and of a human mesothelial cell line. All of them showed SGLT1 specific bands. Moreover, confocal immunofluorescence images of lamb pleural mesothelium showed that SGLT1 is located in apical membrane. Therefore, a solute-coupled liquid absorption should also occur from pleural space of species with thick visceral pleura. Because of this protein-free liquid entering interstitium between visceral mesothelium and capillaries, inherent Starling forces should be different than hitherto considered, and visceral pleura capillaries could absorb liquid even in these species.

The Fate of Lateral Ridge Augmentation: A Systematic Review and Meta-Analysis.

PURPOSE: Owing to volumetric changes after tooth extraction, lateral ridge augmentation has become a common procedure prior or simultaneous to implant placement. Nonetheless, little is known with regard to the dimensional remodeling after healing of these lateral ridge augmentation procedures. Hence, the purpose of this systematic review was to assess the stability of bone grafting material between augmentation procedures and final healing, in terms of resorption rate. MATERIALS AND METHODS: An electronic and hand literature search was conducted in several databases, such as the Cochrane Oral Health Group Trials Register, Embase, and Cochrane Central Register of Controlled Trials, up until February 2017. Only randomized controlled trials (RCTs) with a mean follow-up of at least 6 months after implant placement aiming to evaluate the stability of grafting material for lateral ridge augmentation were included and quantitatively analyzed. RESULTS: A total of 35 articles were evaluated; however, only 17 RCTs met the inclusion criteria. A total of 15 studies reported information on bone resorption, leading to a total sample of 304 implants. The estimated overall mean horizontal bone gain at the time of regeneration was 3.71 ± 0.24 mm, with 4.18 ± 0.56 mm for the block graft technique and 3.61 ± 0.27 mm for guided bone regeneration (GBR). The estimated overall net bone gain at final re-evaluation (11.9 ± 7.8) was 2.86 ± 0.23 mm. The estimated mean (± SD) resorption after 6 months was 1.13 ± 0.25 mm, with 0.75 ± 0.59 mm for the block graft technique and 1.22 ± 0.28 mm for GBR. The implant survival rate was 97% to 100%. CONCLUSION: Regardless of the material used for regeneration, different degrees of graft resorption should be expected. Given the sample of investigations analyzed in this review, block grafts seemed to maintain the volume of the initial augmentation site more than GBR techniques. During the initial stages of healing, the GBR technique experienced more changes compared with block grafts. The resorption of the xenograft group was inferior compared with the combination of xenograft and autologous bone groups. Consequently, overcorrection of the horizontal defects should be performed to compensate for the resorption of the grafting materials.

Pleural liquid during hemorrhagic hypotension.

The effect of approximately 25% or 35% blood loss (b.l.) on volume, pressure, and protein concentration of pleural liquid has been determined in anesthetized rabbits in lateral or supine posture. Volume and pressure of pleural liquid did not change with 25% b.l. 30 and 60 min after beginning of hemorrhage, and with 35% b.l. at 30 min (bleeding time approximately 10 and 12 min, respectively). With 35% b.l. protein concentration of pleural liquid was 85% greater (P<0.01) than control; moreover, percent albumin was smaller (P<0.05), and percent globulin greater (P<0.05) than control. Decrease in arterial plasma protein concentration, hematocrit, and pH after hemorrhage fit literature data. Ventilation at 15 and 30 min increased (P<0.01) by 16% and 23%, respectively, with 25% b.l., but it did not change with 35% b.l., a condition borderline to survival in anesthetized rabbits without ad hoc treatment. Pleural liquid seems protected against derangements from hemorrhage up to 25% b.l. for periods shorter than 1 h.

Expression of Na+-glucose cotransporter (SGLT1) in visceral and parietal mesothelium of rabbit pleura.

Indirect evidence for a solute-coupled liquid absorption from rabbit pleural space indicated that it should be caused by a Na(+)/H(+)-Cl(-)/HCO(3)(-) double exchanger and a Na(+)-glucose cotransporter [Agostoni, E., Zocchi, L., 1998. Mechanical coupling and liquid exchanges in the pleural space. In: Antony, V.B. (Ed.), Clinics in Chest Medicine: Diseases of the Pleura, vol. 19. Saunders, Philadelphia, pp. 241-260]. In this research we tried to obtain molecular evidence for Na(+)-glucose cotransporter (SGLT1) in visceral and parietal mesothelium of rabbit pleura. To this end we performed immunoblot assays on total protein extracts of scraped visceral or parietal mesothelium of rabbits. These showed two bands: one at 72kDa (m.w. of SGLT1), and one at 55kDa (which should also provide Na(+)-glucose cotransport). Both bands disappeared in assays in which SGLT1 antibody was preadsorbed with specific antigen. Molecular evidence for Na(+)/K(+) ATPase (alpha1 subunit) was also provided. Immunoblot assays for SGLT1 on cultured mesothelial cells of rabbit pleura showed a band at 72kDa, and in some cases also at 55kDa, irrespectively of treatment with a differentiating agent. Solute-coupled liquid absorption hinders liquid filtration through parietal mesothelium caused by Starling forces, and favours liquid absorption through visceral mesothelium caused by these forces.

Distribution and mixing of a liquid bolus in pleural space.

Distribution and mixing time of boluses with labeled albumin in pleural space of anesthetized, supine rabbits were determined by sampling pleural liquid at different times in various intercostal spaces (ics), and in cranial and caudal mediastinum. During sampling, lung and chest wall were kept apposed by lung inflation. This was not necessary in costo-phrenic sinus. Here, 10 min after injection, lung inflation increased concentration of labeled albumin by 50%. Lung inflation probably displaces some pleural liquid cranio-caudally, increasing labeled albumin concentration caudally to injection point (6th ics), and decreasing it cranially. Boluses of 0.1-1 ml did not preferentially reach mediastinal regions, as maintained by others. Time for an approximate mixing was approximately 1 h for 0.1 ml, and approximately 30 min for 1 ml. This relatively long mixing time does not substantially affect determination of contribution of lymphatic drainage through stomata to overall removal of labeled albumin from 0.3 ml hydrothoraces lasting 3 h [Bodega, F., Agostoni, E., 2004. Contribution of lymphatic drainage through stomata to albumin removal from pleural space. Respir. Physiol. Neurobiol. 142, 251-263].

Pleural liquid and kinetic friction coefficient of mesothelium after mechanical ventilation.

Volume and protein concentration of pleural liquid in anesthetized rabbits after 1 or 3h of mechanical ventilation, with alveolar pressure equal to atmospheric at end expiration, were compared to those occurring after spontaneous breathing. Moreover, coefficient of kinetic friction between samples of visceral and parietal pleura, obtained after spontaneous or mechanical ventilation, sliding in vitro at physiological velocity under physiological load, was determined. Volume of pleural liquid after mechanical ventilation was similar to that previously found during spontaneous ventilation. This finding is contrary to expectation of Moriondo et al. (2005), based on measurement of lymphatic and interstitial pressure. Protein concentration of pleural liquid after mechanical ventilation was also similar to that occurring after spontaneous ventilation. Coefficient of kinetic friction after mechanical ventilation was 0.023±0.001, similar to that obtained after spontaneous breathing.

Lubricating recovery of damaged pleural mesothelium: effect of time and of phosphatidylcholines.

Effect of time and phosphatidylcholines (PCs) on lubrication of damaged mesothelium has been investigated. Marked increase in coefficient of kinetic friction (µ) of pleural specimens after mesothelial blotting and rewetting decreased by 23.4±3.5%, 41.8±3.8%, and 40.5±2.7% after 30min, 1h, and 2h. Hence, damaged mesothelium is able to partially reset lubricating molecules on its surface. Increase in µ of post-blotting Ringer 2h after addition of unsaturated PCs (3mg/ml) decreased a little more than after 2h Ringer. Effects of unsaturated and saturated PCs were similar, contrary to expectation raised by their different percentage in pleural and alveolar lavage. Effect of PCs did not increase at 6mg/ml, and was nil at 0.4mg/ml. Increase of µ after short phospholipase treatment decreased by 45.9±2.0% after 2h Ringer, and a little more after addition of unsaturated or saturated PCs. Hence, PCs, as other phospholipids, have a small effect, likely because of difficulty in resetting their relationships with main lubricating molecules.

Pleural mesothelium lubrication after phospholipase treatment.

Coefficient of kinetic friction (µ) of rabbit pleural mesothelium increased after short treatment of specimens with phospholipase C. This increase was removed by addition of a solution with hyaluronan or sialomucin, as previously shown in post-blotting Ringer or after short pronase treatment. After phospholipase µ decreased with increase in sliding velocity, but at highest velocity it was still greater than control; this difference was removed by addition of hyaluronan or sialomucin, as in post-blotting Ringer or after short pronase treatment. Hyaluronan placed on specimen before phospholipase treatment reduced increase in µ by protecting phospholipids from enzyme, as shown by others for alveolar and synovial phospholipids. Samples of parietal pleura stained with silver nitrate showed that mesothelial cells were not disrupted by short phospholipase treatment. Instead, they were disrupted if this treatment was preceded by a short pronase treatment; but even after this disruption addition of hyaluronan or sialomucin brought µ back to control.

Pleural mesothelium lubrication after hyaluronidase, neuraminidase or pronase treatment.

Coefficient of kinetic friction (µ) of pleural mesothelium has been found to increase markedly after mesothelial blotting and rewetting. This increase disappeared after addition of a solution with hyaluronan or sialomucin, though previous morphological studies showed that only sialomucin occurs in mesothelial glycocalyx. In this research we investigated whether µ of rabbit pleural mesothelium increased after hyaluronidase, neuraminidase or pronase treatment. Hyaluronidase and neuraminidase did not increase µ, though neuraminidase cleaved sialic acid from mesothelial glycocalyx of diaphragm specimens, and removed hystochemical stain of sialic acid from glycocalyx. Sialomucin treated with neuraminidase lowered µ of blotted mesothelium, though less than untreated sialomucin; this feature plus lubrication provided by other molecules could explain why µ did not increase after neuraminidase. Short pronase treatment (in order to affect only glycocalyx proteins) increased µ; this increase was removed by hyaluronan or sialomucin. After pronase treatment µ decreased with increase in sliding velocity, indicating a regime of mixed lubrication, as in blotted mesothelium.

Mixed lubrication after rewetting of blotted pleural mesothelium.

Coefficient of kinetic friction (µ) of pleural mesothelium blotted with filter paper, and rewetted with Ringer solution markedly increases; this increase is removed if a sufficient amount of sialomucin or hyaluronan is added to Ringer (Bodega et al., 2012. Respiratory Physiology and Neurobiology 180, 34-39). In this research we found that µ of pleural mesothelium blotted, rewetted, and sliding at physiological velocities and loads, decreased with increase of velocity, mainly at low velocities. Despite this decrease, µ at highest velocity was still double that before blotting. With small concentration of sialomucin or hyaluronan µ was markedly smaller at each velocity, decreased less with increase of velocity, and at highest velocity approached preblotting value. These findings indicate a regime of mixed lubrication in post-blotting Ringer, at variance with boundary lubrication occurring before blotting or postblotting with sufficient macromolecule addition. Greater roughness of mesothelial surface, caused by blotting, likely induces zones of elastohydrodynamic lubrication, which increase with velocity, while contact area decreases.

Lubricating effect of sialomucin and hyaluronan on pleural mesothelium.

Coefficient of kinetic friction (µ) between rabbit visceral and parietal pleura, sliding in vitro at physiological velocities and load, increases markedly after blotting mesothelial surface with filter paper; this increase is only partially reduced by wetting blotted mesothelium with Ringer solution. Given that mesothelial surface is covered by a thick coat with sialomucin and hyaluronan, we tested whether addition of sialomucin or hyaluronan solution after blotting lowers µ more than Ringer alone. Actually, these macromolecules lowered µ more than Ringer, so that µ was no longer significantly higher than its preblotting value. Moreover, Ringer addition, after washout of macromolecule solution, increased µ, in line with their dilution. These findings indicate that mesothelial blotting removes part of these molecules from the coat covering mesothelial surface, and their relevance for pleural lubrication. Transmission electron micrographs of pleural specimens after mesothelial blotting showed that microvilli were partially or largely removed from mesothelium, consistent with a substantial loss of macromolecules normally entrapped among them.

ß2-Adrenergic receptors and G-protein-coupled receptor kinase 2 in rabbit pleural mesothelium.

Former studies on net rate of liquid absorption from small Ringer or 1% albumin-Ringer hydrothoraces in rabbits indicated that Na+ transport and solute-coupled liquid absorption by mesothelium is increased by pleural liquid dilution, and stimulation of ß2-adrenoreceptors (ß2AR). In this research we tried to provide molecular evidence for ß2AR in visceral and parietal mesothelium of rabbit pleura. Moreover, because prolonged stimulation of ß2AR may lead to desensitization mediated by G-protein-coupled receptor kinase 2 (GRK2), we also checked whether GRK2 is expressed in pleural mesothelium. To this end we performed immunoblot assays on total protein extracts from scraped visceral and parietal mesothelium, and from cultured pleural mesothelial cells of rabbits. All three samples showed ß2AR and GRK2 specific bands.

Oxidative stress reduces transintestinal transports and (Na+, K+) -ATPase activity in rat jejunum.

Because oxidative stress is a component of gastrointestinal injury, we investigated the effect of H(2)O(2) on transintestinal transport using isolated rat jejunum incubated in vitro. Millimolar concentrations of H(2)O(2) inhibited all the tested parameters without inducing any cytotoxic effect. Electrophysiological experiments indicated that H(2)O(2) decreases significantly both short circuit current and transepithelial electrical potential difference without affecting transepithelial resistance. The possibility that H(2)O(2) could influence (Na+, K+) -ATPase activity was explored using isolated basolateral membranes. Besides H(2)O(2), free radicals (O(2)(*-), HO*) were generated using different iron-dependent and independent systems; (Na+, K+) -ATPase activity was inhibited after membrane exposure to all ROS tested. The inhibition was prevented by allopurinol, superoxide dismutase or desferrioxamine. Western blot analysis showed a decreased expression of the alpha(1)-subunit of (Na+, K+) -ATPase. We conclude that H(2)O(2) may be a modulator of jejunal ion and water transport by multiple mechanisms, among which a significant inhibition of the basolateral (Na+, K+) -ATPase.