Membrane Of Alveolar Epithelial Cells Research Articles

Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting

PurposeThe incidence of acute lung injury (ALI) in severe trauma patients is 48% and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%. Alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s) are the key cells in the repair of injured lungs as well as fetal lung development. Therefore, the purification and culture of AEC1s and AEC2s play an important role in the research of repair and regeneration of lung tissue. MethodsSprague-Dawley rats (3–4 weeks, 120–150 g) were purchased for experiment. Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells, and then magnetic bead cell sorting was performed to isolate T1α positive cells as AEC1s from the single-cell suspension by using polyclonal rabbit anti-T1a (a specific AEC1s membrane protein) antibodies combined with anti-rabbit IgG microbeads. Afterwards, alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining T1α-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads. Cell purity was identified by immunofluorescence staining and flow cytometry. ResultsThe purity of AEC1s and AEC2s was 88.3% ± 3.8% and 92.6% ± 2.7%, respectively. The cell growth was observed as follows: AEC1s stretched within the 12–16 h, but the cells proliferated slowly; while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days. ConclusionAEC1s and AEC2s sorted by this method have high purity and good viability. Therefore, our method provides a new approach for the isolation and culture of AEC1s and AEC2s as well as a new strategy for the research of lung repair and regeneration.

Characteristics of pulmonary microvascular structure in postnatal yaks

Yaks are typical plateau-adapted animals, however the microvascular changes and characteristics in their lungs after birth are still unclear. Pulmonary microvasculature characteristics and changes across age groups were analysed using morphological observation and molecular biology detection in yaks aged 1, 30 and 180 days old in addition to adults. Results: Our experiments demonstrated that yaks have fully developed pulmonary alveolar at birth but that interalveolar thickness increased with age. Immunofluorescence observations showed that microvessel density within the interalveolar septum in the yak gradually increased with age. In addition, transmission electron microscopy (TEM) results showed that the blood–air barrier of 1-day old and 30-days old yaks was significantly thicker than that observed at 180-days old and in adults (P < 0.05), which was caused by the thinning of the membrane of alveolar epithelial cells. Furthermore, Vegfa and Epas1 expression levels in 30-day old yaks were the highest in comparison to the other age groups (P < 0.05), whilst levels in adult yaks were the lowest (P < 0.05). The gradual increase in lung microvessel density can effectively satisfy the oxygen requirements of ageing yaks. In addition, these results suggest that the key period of yak lung development is from 30 to 180 days.

Dysregulation of ion transport in the lung epithelium infected with SARS-CoV-2.

Dysregulation of ion transport in the lung epithelium infected with SARS-CoV-2.

Effects and mechanism of fecal transplantation on acute lung injury induced by lipopolysaccharide in rats

Objective: To investigate the effect of fecal microbiota transplantation (FMT) on acute lung injury (ALI) induced by lipopolysaccharide (LPS) and its regulatory mechanism. Methods: Fifteen rats were divided into control group, LPS group and LPS+FMT group by random number table method. LPS group and LPS+FMT group were intraperitoneally injected with LPS to generate rat ALI model. After 24 h of modeling, feces (10 ml/kg) were given to the LPS+FMT group twice a day, and the control group and LPS group were given the same amount of normal saline. The intervention lasted for 2 days. After 24 h of the last fecal microbiota transplantation, arterial blood gas analysis was performed in each group. Then rats were sacrificed and enzyme-linked immunosorbent (ELISA) method was used to detect intercellular adhesion molecule 1 (ICAM-1) content in the serum and bronchoalveolar lavage fluid (BALF). The lung wet-dry weight ratio (W/D) was evaluated; HE staining and lung tissue pathology scoring, immunohistochemical detection of nuclear factor-kappa B (NF-κB) predominate nuclear expression and expression of ICAM-1 of alveolar epithelial cells were conducted; Western blot was used to detect the expression of proteins related to the intracellular phosphatidylinositol kinase (PI3K)/protein kinase (AKT) signaling pathway. Samples of rat feces were collected and DNA was extracted. Polymerase chain reaction (PCR) products of the V3 and V4 regions of the 16S ribosomal RNA gene (16SrDNA) were sequenced at high throughput, and bioinformatics analysis was conducted on the microbial community based on the operational classification unit. Results: The lung W/D and lung histopathological score of the LPS group were significantly higher than those of the control group, while the arterial partial oxygen pressure (PaO(2)) of the LPS group was significantly lower than that of the control group [(79.2±5.89 vs 95.2±2.77) mmHg, 1 mmHg=0.133 kPa](all P<0.05). The results of intestinal flora sequencing revealed that the diversity index of LPS group was significantly higher than that of the control group, while the lactobacillus of LPS group rats was significantly lower than that of the control group. The content of ICAM-1 in serum, BALF and its relative expression on the cell membrane in the LPS group was significantly higher than that in the control group [(8.64±0.87) vs (7.40±0.32) ng/L; (0.941±0.035) vs (0.739±0.079) ng/L; (0.250±0.010) vs (0.076±0.010)] (all P<0.05). Moreover, the relative expression levels of phosphorylated P65 (p-P65), p-PI3K and p-AKT nucleoprotein in the LPS group were significantly higher than those in the control group (4.89±0.27 vs 3.28±0.13, 0.265±0.030 vs 0.036±0.013 and 0.444±0.040 vs 0.109±0.016) (all P<0.05). The above injury effect was reduced after fecal fungus transplantation. The lung W/D and lung pathological score of LPS+FMT group were significantly lower than those of LPS group, and PaO(2) of LPS+FMT group was significantly higher than that of LPS group [(88.0±3.53) mmHg]. The results of intestinal flora sequencing revealed that the diversity index of LPS+FMT group was significantly lower than that of LPS group, and the lactobacillus genus of LPS+FMT group was significantly higher than that of LPS group. ICAM-1 in the blood serum ((7.44±0.46) ng/L), BALF (0.834±0.040) ng/L) and its relative expression on alveolar epithelial cell membrane (0.173±0.030), the relative expression of p-P65, p-PI3K and p-AKT protein of NF-κB in alveolar epithelial cells was down-regulated ((2.99±0.28, 0.090±0.013 and 0.206±0.018) in LPS+FMT group than those of LPS group, the differences were statistically significant (all P<0.05). Conclusion: Fecal transplantation can alleviate lipopolysaccharide-induced acute lung injury in rats, and its regulatory effect may be related to inhibiting the activation of PI3K/AKT/NF-κB signaling pathway and reducing the expression of inflammatory factor ICAM-1.

Sialidase Attenuates Epidermal Growth Factor Response and Abolishes Antiproliferative Effects of Erlotinib in A549Alveolar Epithelial Cells.

Erlotinib is a widely used, reversible tyrosine kinase inhibitor (TKI), targeting pro-proliferative signaling of epidermal growth factor receptor (EGFR). The drug is approved for the first-line treatment of patients with metastatic non-small cell lung cancer with EGFR mutations. Extracellular glycans can affect EGFR expression, dimerization, phosphorylation, and EGF binding. In this study we investigated the effects of EGF and erlotinib on the cell cycle of naive and sialidase (alpha-neuraminidase)-pretreated human A549 alveolar epithelial cells. A549 cells were labeled with propidium iodide, and fractions of cells in different phases of cycle were quantified by flow cytometry. We found that neither did desialilation nor EGF, as well as erlotinib treatment, increase the number of damaged cells (subG0/G1 cell fraction), while erlotinib did significantly increase the number of G0/G1 cells and decrease S+G2/M cell fractions. In naive cells, EGF increased proliferating cell numbers by more than 40%, and this effect was blocked by erlotinib. In desialylated cells, however, proliferation was significantly decreased by about 29%, and EGF and erlotinib did not exert significant effects. We conclude that changes in alveolar epithelial cell membrane glycosylation may affect function of growth-promoting receptors and erlotinib effectiveness.

The effects of L-type amino acid transporter 1 on milk protein synthesis in mammary glands of dairy cows

The mammary gland requires the uptake of AA for milk protein synthesis during lactation. The L-type amino acid transporter 1 (LAT1, encoded by SLC7A5), found in many different types of mammalian cells, is indispensable as a transporter of essential AA to maintain cell growth and protein synthesis. However, the function of LAT1 in regulating milk protein synthesis in the mammary gland of the dairy cow remains largely unknown. For the current study, we characterized the relationship between LAT1 expression and milk protein synthesis in lactating dairy cows and investigated whether the mammalian target of rapamycin complex 1 (mTORC1) signaling controls the expression of LAT1 in their mammary glands. We found that LAT1 and the heavy chain of its chaperone, 4F2, were expressed in mammary tissues of lactating cows, with the expression levels of LAT1 and the 4F2 heavy chain being significantly greater in lactating mammary tissues with high-milk protein content (milk yield, 33.8 ± 2.1 kg/d; milk protein concentration >3%, wt/vol,; n = 3) than in tissues from cows with low-milk protein content (milk yield, 33.7 ± 0.5 kg/d; milk protein concentration <3%, wt/vol; n = 3). Immunofluorescence staining of sectioned mammary tissues from cows with high and low milk protein content showed that LAT1 was located on the whole plasma membrane of alveolar epithelial cells, suggesting that LAT1 provides essential AA to the mammary gland. In cultured mammary epithelial cells from the dairy cows with high-milk protein content, knockdown of LAT1 expression decreased cell viability and β-casein expression; in contrast, overexpression of LAT1 had the opposite effect. Inhibition of mTORC1 by rapamycin attenuated the phosphorylation of molecules related to mTORC1 signaling and caused a marked decrease in LAT1 expression in the cultured cells; expression of β-casein also decreased significantly. These results suggest that LAT1 is involved in milk protein synthesis in the mammary glands of lactating dairy cows and that the mTORC1 signaling pathway might be a control point for regulation of LAT1 expression, which could ultimately be used to alter milk protein synthesis.

Protective effects of imipramine on alveolar epithelial barrier function against acute lung injury in mice

Objective To investigate the protective effects of imipramine on alveolar epithelial barrier function in mice against LPS-induced acute lung injury (ALI), and explore the possible mechanisms. Methods Total of 32 SPF male Balb/c mice were randomly (random number) divided into four groups: control group, Imipramine group, LPS group, LPS + Imipramine group. To establish an animal model of ALI, mice were administered intraperitoneally with LPS in 20 mg/kg. Mice were treated with imipramine in 25 mg/kg 30 min prior to LPS administration. FITC-FD4 was administered in mice via the tail vein with FITC-FD4 10 min before mice sacrificed under anesthesia at 12 hours after LPS administration, then bronchoalveolar lavage fluid (BALF) and lung tissue were obtained. HE staining was used to observe histopathological changes, and pathology scores; lung tissue wet-to-dry weight ratio and BALF/serum FD4 ratio were used to assess pulmonary edema and alveolar epithelial permeability. Real-time PCR, western blot and immunochemistry were employed to detect the mRNA expressions and protein levels of Occludin, Claudin-4 and ZO-1. Data were analyzed with SPSS 16.0 software, one way analysis of variance (ANOVA) was used to compare multiple sets of variables, the intergroup comparisons were analyzed by the least-significant-difference (LSD) tests with P<0.05 for the statistically significant difference. Results Compared with LPS group, LPS+ Imipramine group had a statistically significant decrease in pathological score [(9.22±0.21) vs. (11.23±0.55), P<0.05); the wet-to-dry weight ratio in LPS + Imipramine group was less than that in LPS group and the difference was statistically significant (P<0.05); compared with LPS group, the ratio of BALF/serum FD4 in LPS+ Imipramine was less and the difference was statistically significant (P<0.05); compared with LPS group, the mRNA expressions and protein levels of Occludin, Claudin-4 and ZO-1 in LPS + Imipramine group were significantly increased (mRNA: Occludin: P<0.05; Claudin-4: P<0.05; ZO-1: P<0.05. western blot: Occludin: P<0.05; Claudin-4: P<0.05; ZO-1: P<0.05). Immunochemistry showed that Occludin and Claudin-4 were present mainly in alveolar epithelial cell membrane, Z0-1 was found mainly in cytoplasm of alveolar epithelial cell. In control group and Imipramine group, tight junction proteins were obviously expressed. Compared with control group, protein levels in LPS group were significantly decreased (Occludin: P<0.05; Claudin-4: P<0.05; ZO-1: t=6.59, P<0.05); compared with LPS group, the tight junction proteins in LPS+ Imipramine group were significantly increased (Occludin: P<0.05; Claudin-4: P<0.05; ZO-1: P<0.05). Conclusion The protective effects of imipramine on alveolar epithelial barrier function by up-regulating tight junction proteins expression in murine LPS-induced ALI. Key words: Imipramine; Alveolar epithelial barrier function; Tight junction proteins; LPS; Mice; Acute lung injury; Mechanisms

CYR61 (CCN1) overexpression induces lung injury in mice.

Cysteine-rich protein-61 (CYR61), also known as connective tissue growth factor, CYR61, and nephroblastoma overexpressed gene 1 (CCN1), is a heparin-binding protein member of the CCN family of matricellular proteins. Gene expression profiles showed that Cyr61 is upregulated in human acute lung injury (ALI), but its functional role is unclear. We hypothesized that CYR61 contributes to ALI in mice. First, we demonstrated that CYR61 expression increases after bleomycin-induced lung injury. We then used adenovirus-mediated gene transfer to determine whether CYR61 overexpression in the lungs was sufficient to cause ALI. Mice instilled with CYR61 adenovirus showed greater weight loss, increased bronchoalveolar lavage total neutrophil counts, increased protein concentrations, and increased mortality compared with mice instilled with empty-vector adenovirus. Immunohistochemical studies in lungs from humans with idiopathic pulmonary fibrosis revealed CYR61 expression on the luminal membrane of alveolar epithelial cells in areas of injury. We conclude that CYR61 is upregulated in ALI and that CYR61 overexpression exacerbates ALI in mice.

Calcium release-activated calcium (CRAC) channels mediate the β2-adrenergic regulation of Na,K-ATPase

β2-Adrenergic agonists have been shown to regulate Na,K-ATPase in the alveolar epithelium by recruiting Na,K-ATPase-containing vesicles to the plasma membrane of alveolar epithelial cells (AEC). Here, we provide evidence that β2-agonists induce store-operated calcium entry (SOCE) in AECs. This calcium entry is necessary for β2-agonist-induced recruitment of Na,K-ATPase to the plasma membrane of AECs. Specifically, we show that β2-agonists induce SOCE via stromal interaction molecule 1 (STIM1)-associated calcium release-activated calcium (CRAC) channels. We also demonstrate that the magnitude of SOCE affects the abundance of Na,K-ATPase at the plasma membrane of AECs.

Hypoxia-Induced Inhibition of Epithelial Na+ Channels in the Lung. Role of Nedd4-2 and the Ubiquitin-Proteasome Pathway

Transepithelial sodium transport via alveolar epithelial Na(+) channels (ENaC) and Na(+),K(+)-ATPase constitutes the driving force for removal of alveolar edema fluid. Alveolar hypoxia associated with pulmonary edema may impair ENaC activity and alveolar Na(+) absorption through a decrease of ENaC subunit expression at the apical membrane of alveolar epithelial cells (AECs). Here, we investigated the mechanism(s) involved in this process in vivo in the β-Liddle mouse strain mice carrying a truncation of β-ENaC C-terminus abolishing the interaction between β-ENaC and the ubiquitin protein-ligase Nedd4-2 that targets the channel for endocytosis and degradation and in vitro in rat AECs. Hypoxia (8% O2 for 24 h) reduced amiloride-sensitive alveolar fluid clearance by 69% in wild-type mice but had no effect in homozygous mutated β-Liddle littermates. In vitro, acute exposure of AECs to hypoxia (0.5-3% O2 for 1-6 h) rapidly decreased transepithelial Na(+) transport as assessed by equivalent short-circuit current Ieq and the amiloride-sensitive component of Na(+) current across the apical membrane, reflecting ENaC activity. Hypoxia induced a decrease of ENaC subunit expression in the apical membrane of AECs with no change in intracellular expression and induced a 2-fold increase in α-ENaC polyubiquitination. Hypoxic inhibition of amiloride-sensitive Ieq was fully prevented by preincubation with the proteasome inhibitors MG132 and lactacystin or with the antioxidant N-acetyl-cysteine. Our data strongly suggest that Nedd4-2-mediated ubiquitination of ENaC leading to endocytosis and degradation of apical Na(+) channels is a key feature of hypoxia-induced inhibition of transepithelial alveolar Na(+) transport.

Treatment of Pulmonary Edema by ENaC Activators/Stimulators

Lungs contain a particular amount of fluid that is crucial for proper lung function. This fluid content is tightly controlled within certain limits. Fluid accumulation in the alveolar airspace impairs gas exchange and represents a lifethreatening condition referred to as pulmonary edema. Ion transport processes by pulmonary epithelia represent a mechanism, responsible for fluid absorption from the airspace. Thus, it is obvious to consider ion transport processes as target for therapeutic interventions in pulmonary edema. The principle mechanism responsible for fluid absorption from the airspace is: Na(+) diffuses through luminal Na(+) channels into epithelial cells and is extruded by Na(+)/K(+)-ATPases at the basolateral side. This process generates an osmotic gradient that represents the driving force for fluid absorption. The rate of Na(+) absorption is limited by the number/activity of Na+ channels in the luminal membrane of alveolar epithelial cells. Although different Na+ channels have been identified, the epithelial Na+ channel (ENaC) is a major player that participates in Na(+)-driven fluid absorption and thus a suitable target for the treatment of pulmonary edema. This article reviews cellular mechanisms by which ENaC activity can be increased in alveolar epithelia (lectins, proteases, β-adrenoceptors, mineralo-/glucocorticoid-receptors). These mechanisms are involved in regulating ENaC-dependent fluid absorption under physiological conditions. Additionally, pre-clinical as well as some preliminary clinical studies revealed that "ENaC-activators/stimulators" (β2-adrenoceptor agonists and mineralo-/glucocorticoid-receptor agonists) could be beneficial for therapeutic interventions in patients with pulmonary edema. However, the outcome of subsequently performed multicenter clinical trials with "ENaC-activators/stimulators" for treatment of patients with pulmonary edema was disappointing.

Effect of surfactants on polystyrene nanoparticle (PNP) interactions with primary rat alveolar epithelial cell monolayers (RAECM)

We investigated effects of apical 0.1% (w/v) Pluronic (P) or 1 mg/mL Infasurf (I) on flux of 20 nm carboxyl‐modified (negatively charged) PNP (20‐PNP) or 20 nm amidine‐modified (positively charged) PNP (20+ PNP) across RAECM, across which the bioelectric properties (transepithelial resistance (Rt) and equivalent short circuit current (Ieq)) were unchanged in the presence of these two surfactants. Results of flux measurements, along with hydrodynamic diameter (HD) measured by dynamic light scattering and zeta potential (ZP) measured in the presence and absence of P or I, are summarized below. 20+ PNP 20‐ PNP Flux (pg/cm2/sec) PNP alone 38±7 1.6±0.1 PNP with P 35±7 2.5±0.2 PNP with I 42±6 0.9±0.2 HD (nm) PNP alone 543±28 156±7 PNP with P 374±13 80±3 PNP with I 316±9 278±9 ZP (mV) PNP alone 37±10 −61±6 PNP with P 33±2 −29±2 PNP with I −44±1 −82±4 These data indicate that there is remarkably little effect of P or I on PNP flux despite significant changes in measured HD and ZP. We conclude that PNP (and RAECM) exposure to either surfactant P or I surprisingly does not cause meaningful alterations in PNP translocation across RAECM, which may be explainable by the behavior of PNP in the extracellular microdomain adjacent to the apical alveolar epithelial cell membrane.(Funded by NIH and Hastings &amp; Whittier Fdns.)

Determination of Functional ABCG2 Activity and Assessment of Drug–ABCG2 Interactions in Dairy Animals Using a Novel MDCKII In Vitro Model

The ATP-binding cassette subfamily G member 2 (ABCG2) transporter is a member of the ATP-binding cassette (ABC) family of efflux carriers that mediates cellular extrusion of various drugs and toxins. In the mammary gland, ABCG2 is expressed at the apical membrane of alveolar epithelial cells and is induced during lactation. It is well established that ABCG2 plays the main role in active secretion of xenobiotics into milk of humans and mice. In contrast, no detailed information is as yet available about functional activity and substrate spectrum of ABCG2 in dairy animals. Therefore, we cloned full-length ABCG2 from bovine, ovine and caprine lactating mammary gland tissues using rapid amplification of complementary DNA (cDNA) ends polymerase chain reaction. The generated full-length ABCG2 cDNA constructs were stably transduced in MDCKII cells. Functional ABCG2 efflux activity was demonstrated with the Hoechst H33342 accumulation assay using the specific ABCG2 inhibitor Ko143. The established ruminant MDCKII-ABCG2 cell culture models in conjunction with the H33342 transport assay showed interaction of various drugs such as cefalexin and albendazole with bABCG2, oABCG2 or cABCG2. Moreover, the flavonoids equol and quercetin exhibited interaction with all ruminant ABCG2 clones. Altogether, our generated cell culture models allowed rapid and high-throughput screening of potential ruminant ABCG2 substrates and thus increase the understanding of carrier-associated secretion of xenobiotics into milk.

The Role of Lipid Raft Aggregation in the Infection of Type II Pneumocytes by Mycobacterium tuberculosis

Dynamic, cholesterol-dense regions of the plasma membrane, known as lipid rafts (LR), have been observed to develop during and may be directly involved in infection of host cells by various pathogens. This study focuses on LR aggregation induced in alveolar epithelial cells during infection with Mycobacterium tuberculosis (Mtb) bacilli. We report dose- and time-dependent increases in LR aggregation after infection with three different strains at multiplicities of infection of 1, 10 and 100 from 2–24 hr post infection (hpi). Specific strain-dependent variations were noted among H37Rv, HN878 and CDC1551 with H37Rv producing the most significant increase from 15 aggregates per cell (APC) to 27 APC at MOI 100 during the 24 hour infection period. Treatment of epithelial cells with Culture Filtrate Protein, Total Lipids and gamma-irradiated whole cells from each strain failed to induce the level of LR aggregation observed during infection with any of the live strains. However, filtered supernatants from infected epithelial cells did produce comparable LR aggregation, suggesting a secreted mycobacterial product produced during infection of host cells is responsible for LR aggregation. Disruption of lipid raft formation prior to infection indicates that Mtb bacilli utilize LR aggregates for internalization and survival in epithelial cells. Treatment of host cells with the LR-disruption agent Filipin III produced a nearly 22% reduction in viable bacteria for strains H37Rv and HN878, and a 7% reduction for strain CDC1551 after 6 hpi. This study provides evidence for significant mycobacterial-induced changes in the plasma membrane of alveolar epithelial cells and that Mtb strains vary in their ability to facilitate aggregation and utilization of LR.

Lung Cell Hypoxia: Role of Mitochondrial Reactive Oxygen Species Signaling in Triggering Responses

Lung cells experience hypoxia during development, during travel to high altitude, and in acute and chronic lung diseases. The functional responses evoked by hypoxia are diverse and generally act to protect the cells from hypoxic injury, although some lung cell responses are counterproductive because they degrade normal function of the organ. The cellular O(2) sensor responsible for many of these responses involves the mitochondrial electron transport chain. Under hypoxic conditions, increased release of reactive oxygen species from the inner mitochondrial membrane to the intermembrane space leads to the activation of transcription factors, including hypoxia-inducible factor, activation of hypoxic pulmonary vasoconstriction, activation of AMP-dependent protein kinase, and internalization of the membrane Na,K-ATPase from the basolateral membrane of alveolar epithelial cells. Although the specific targets of reactive oxygen species signals are not fully understood, this signaling pathway is critical for development and for normal lung responses in the newborn and the mature lung.

An experimental study on the safety of intratracheal drug administration

To explore whether injury and repair occur in the trachea and the lung after intra-tracheal administration of different drugs. Wistar rats were randomly divided into 5 groups, a normal group, a blank control (BC) group, a normal saline (NS) group, a lidocaine (LD) group and an amikacin (AK) group. For the latter 3 groups, normal saline, lidocaine and amikacin were injected into trachea by needle puncture. Scanning electron microscope was used to observe the ultra-structural changes of the epithelium, and the percentage of the area of damage (PAD) in tracheal mucosa was calculated. Moreover, pathological changes of the mucous membrane of bronchioles and alveolar epithelial cells were also examined, and the degree of lung pathology was semi-quantified. Two hours after the injection of the 3 drugs, derangement and edema of the cilia were evident by scanning electron microscopy. The PAD of the NS group, the LD group and the AK group were (94.2 ± 3.2)%, (93.1 ± 3.0)% and (95.5 ± 1.8)%, respectively; all being significantly higher than that of the BC group (1.3 ± 0.3)%. For the NS group and the LD group, the PAD decreased significantly after 24 h, which were (73.7 ± 7.8)% and (81.0 ± 4.6)% respectively, and returned to normal at 48 h and 96 h. While for the AK group, the damage began to improve at 72 h [PAD (62.1 ± 5.2)%], and recovered at 96 h. Airway epithelial derangement and cell edema in the alveoli and the bronchioles also occurred 2 h after drug injection, and inflammatory cell infiltration became evident at 24 h. At this time, the score of pathology was 1.80 ± 0.84, 2.60 ± 0.55 and 2.80 ± 0.45 for the NS group, the LD group and the AK group, respectively; all being higher than that of the BC group (0). These pathological changes recovered totally after 72 h for the NS and the LD groups, and 96 h for the AK group. Intra-tracheal administration of normal saline, lidocaine and amikacin in rats led to reversible airway mucosal and lung tissue damages.

Sepsis and Therapeutic Hypercapnia

Sepsis and Therapeutic Hypercapnia

Role of kinesin light chain‐2 of kinesin‐1 in the traffic of Na,K‐ATPase‐containing vesicles in alveolar epithelial cells

Recruitment of the Na,K-ATPase to the plasma membrane of alveolar epithelial cells results in increased active Na(+) transport and fluid clearance in a process that requires an intact microtubule network. However, the microtubule motors involved in this process have not been identified. In the present report, we studied the role of kinesin-1, a plus-end microtubule molecular motor that has been implicated in the movement of organelles in the Na,K-ATPase traffic. We determined by confocal microscopy and biochemical assays that kinesin-1 and the Na,K-ATPase are present in the same membranous cellular compartment. Knockdown of kinesin-1 heavy chain (KHC) or the light chain-2 (KLC2), but not of the light chain-1 (KLC1), decreased the movement of Na,K-ATPase-containing vesicles when compared to sham siRNA-transfected cells (control group). Thus, a specific isoform of kinesin-1 is required for microtubule-dependent recruitment of Na,K-ATPase to the plasma membrane, which is of physiological significance.

Syndecan-1 mediates the coupling of positively charged submicrometer amorphous silica particles with actin filaments across the alveolar epithelial cell membrane

The cellular interactions and pathways of engineered submicro- and nano-scale particles dictate the cellular response and ultimately determine the level of toxicity or biocompatibility of the particles. Positive surface charge can increase particle internalization, and in some cases can also increase particle toxicity, but the underlying mechanisms are largely unknown. Here we identify the cellular interaction and pathway of positively charged submicrometer synthetic amorphous silica particles, which are used extensively in a wide range of industrial applications, and are explored for drug delivery and medical imaging and sensing. Using time lapse fluorescence imaging in living cells and other quantitative imaging approaches, it is found that heparan sulfate proteoglycans play a critical role in the attachment and internalization of the particles in alveolar type II epithelial cell line (C10), a potential target cell type bearing apical microvilli. Specifically, the transmembrane heparan sulfate proteoglycan, syndecan-1, is found to mediate the initial interactions of the particles at the cell surface, their coupling with actin filaments across the cell membrane, and their subsequent internalization via macropinocytosis. The observed interaction of syndecan molecules with the particle prior to their engagement with actin filaments suggests that the particles initiate their own internalization by facilitating the clustering of the molecules, which is required for the actin coupling and subsequent internalization of syndecan. Our observations identify a new role for syndecan-1 in mediating the cellular interactions and fate of positively charged submicrometer amorphous silica particles in the alveolar type II epithelial cell, a target cell for inhaled particles.

Inactivation of Channel-Activating Protease 1 in mouse distal lung impairs ENaC-mediated lung fluid clearance

Transepithelial sodium (Na + ) transport via amiloride-sensitive epithelial Na + channels (ENaC) expressed in the apical membrane of alveolar epithelial cells provides the driving force for removal of fluid from the alveolar space. In vitro , the membrane-anchored channel-activating serine protease 1 (CAP1) strongly activates ENaC channels at the cell surface by increasing their open probability through proteolytic cleavage of γ-ENaC subunit, but the physiological relevance of this process in vivo is unknown. We investigated whether inactivation of CAP1 gene in mouse distal lung epithelium would affect in vivo transepithelial alveolar sodium transport and alveolar fluid clearance. Mice lacking CAP1 in the alveolar epithelium were generated by CreLoxP-recombination inducible in utero by doxycycline (Tet-ON system). Specificity and efficiency of Cre-recombination as well as expression of ENaC subunits were examined by PCR, real-time RT-PCR and Western-blotting in freshly isolated alveolar epithelial cells. Alveolar fluid clearance was measured in vivo in an in situ lung model under basal and β 2 -agonist-stimulated conditions using radiolabeled albumin as a tracer of alveolar fluid volume. Cre-recombination was restricted to alveolar epithelial cells by PCR and led to a 95% decrease in CAP1 mRNA expression by quantitative RT-PCR, without affecting ENaC mRNA and protein levels. CAP1-deficient mice had normal growth and survival rate, and normal lung histology. Basal alveolar fluid clearance was reduced by 30% in CAP1-deficient mice as compared with control littermates (p<0.01), due to a 50%-decrease in amiloride-sensitive clearance reflecting ENaC activity (p<0.001). β 2 -agonists failed to stimulate alveolar fluid clearance in CAP1-deficient mice. Basal alveolar fluid clearance and stimulation by β 2 -agonists were fully restored by addition in the alveolar instillate of human neutrophil elastase, a serine protease that reproduces the effect of CAP1 on ENaC. Conditional inactivation of CAP1 in mouse alveolar epithelium impairs ENaC-mediated alveolar fluid clearance, and abolishes the response to β 2 -agonists. These data provide in vivo evidence that epithelium-derived CAP1 is a potent activator of ENaC in mouse distal lung. Funded by Inserm, SPLF, Fondation du Legs Poix and Swiss National Foundation