Claudin-18 deficiency results in alveolar barrier dysfunction and impaired alveologenesis in mice.

Claudins are a family of transmembrane proteins that are required for tight junction formation. Claudin (CLDN)-18.1, the only known lung-specific tight junction protein, is the most abundant claudin in alveolar epithelial type (AT) 1 cells, and is regulated by lung maturational agonists and inflammatory mediators. To determine the function of CLDN18 in the alveolar epithelium, CLDN18 knockout (KO) mice were generated and studied by histological, biochemical, and physiological approaches, in addition to whole-genome microarray. Alveolar epithelial barrier function was assessed after knockdown of CLDN18 in isolated lung cells. CLDN18 levels were measured by quantitative PCR in lung samples from fetal and postnatal human infants. We found that CLDN18 deficiency impaired alveolar epithelial barrier function in vivo and in vitro, with evidence of increased paracellular permeability and architectural distortion at AT1-AT1 cell junctions. Although CLDN18 KO mice were born without evidence of a lung abnormality, histological and gene expression analysis at Postnatal Day 3 and Week 4 identified impaired alveolarization. CLDN18 KO mice also had evidence of postnatal lung injury, including acquired AT1 cell damage. Human fetal lungs at 23-24 weeks gestational age, the highest-risk period for developing bronchopulmonary dysplasia, a disease of impaired alveolarization, had significantly lower CLDN18 expression relative to postnatal lungs. Thus, CLDN18 deficiency results in epithelial barrier dysfunction, injury, and impaired alveolarization in mice. Low expression of CLDN18 in human fetal lungs supports further investigation into a role for this tight junction protein in bronchopulmonary dysplasia.

Activated alveolar epithelial cells initiate fibrosis through secretion of mesenchymal proteins.

Fibrosis is characterized by accumulation of activated fibroblasts and pathological deposition of fibrillar collagens. Activated fibroblasts overexpress matrix proteins and release factors that promote further recruitment of activated fibroblasts, leading to progressive fibrosis. The contribution of epithelial cells to this process remains unknown. Epithelium-directed injury may lead to activation of epithelial cells with phenotypes and functions similar to activated fibroblasts. Prior reports that used a reporter gene fate-mapping strategy are limited in their ability to investigate the functional significance of epithelial cell-derived mesenchymal proteins during fibrogenesis. We found that lung epithelial cell-derived collagen I activates fibroblast collagen receptor discoidin domain receptor-2, contributes significantly to fibrogenesis, and promotes resolution of lung inflammation. Alveolar epithelial cells undergoing transforming growth factor-ß-mediated mesenchymal transition express several other secreted profibrotic factors and are capable of activating lung fibroblasts. These studies provide direct evidence that activated epithelial cells produce mesenchymal proteins that initiate a cycle of fibrogenic effector cell activation, leading to progressive fibrosis. Therapy targeted at epithelial cell production of type I collagen offers a novel pathway for abrogating this progressive cycle and for limiting tissue fibrosis but may lead to sustained lung injury/inflammation.

Claudin-4 levels are associated with intact alveolar fluid clearance in human lungs.

The removal of edema from the air spaces is a critical function of the alveolar barrier requiring intact tight junctions. Alveolar fluid clearance contributes to graft function after transplantation and is associated with survival in patients with acute lung injury. Claudin-4 concentrations are known to increase during lung injury and the loss of claudin-4 decreases alveolar fluid clearance in mice. This study was therefore undertaken to evaluate whether differences in lung expression of the tight junction protein claudin-4 are associated with alveolar fluid clearance or clinical measures of lung function. Alveolar fluid clearance rates were measured in ex vivo perfused human lungs not used for transplantation and were compared with histological lung injury and clinical measures of lung injury in the donors. Claudin-4 staining demonstrated a positive correlation with alveolar fluid clearance (Spearman rank correlation [r(s)] = 0.71; P < 0.003); however, claudin-4 staining was not strongly associated with histological measures of lung injury. The expression of other tight junction proteins (including ZO-1) was not associated with alveolar fluid clearance or claudin-4 levels. Claudin-4 staining was lower in lungs from donors with greater impairment in respiratory physiology. These data suggest that claudin-4 may promote alveolar fluid clearance and demonstrate that the amount of claudin-4 expressed may provide specific information regarding alveolar epithelial barrier function that strengthens the link between histological changes and physiological impairment.

Keratinocyte growth factor enhances barrier function without altering claudin expression in primary alveolar epithelial cells.

Keratinocyte growth factor (KGF) has efficacy in several experimental models of lung injury; however, the mechanisms underlying KGF's protective effect remain incompletely understood. This study was undertaken to determine whether KGF augments barrier function in primary rat alveolar epithelial cells grown in culture, specifically whether KGF alters tight junction function via claudin expression. KGF significantly increased alveolar epithelial barrier function in culture as assessed by transepithelial electrical resistance (TER) and paracellular permeability. Fluorescence-activated cell sorting of freshly isolated type 1 (AT1) and type 2 (AT2) cells followed by quantitative real-time RT-PCR revealed that more than 97% of claudin mRNA transcripts in these cells were for claudins-3, -4, and -18. Using cultured AT2 cells, we then examined the effect of KGF on the protein levels of the claudins with the highest mRNA levels: -3, -4, -5, -7, -12, -15, and -18. KGF did not alter the levels of any of the claudins tested, nor of zona occludens-1 (ZO-1) or occludin. Moreover, localization of claudins-3, -4, -18, and ZO-1 was unchanged. KGF did induce a marked increase in the apical perijunctional F-actin ring. Actin depolymerization with cytochalasin D blocked the KGF-mediated increase in TER without significantly changing TER in control cells. Together, these data support a novel mechanism by which KGF enhances alveolar barrier function, modulation of the actin cytoskeleton. In addition, these data demonstrate the complete claudin expression profile for AT1 and AT2 cells and indicate that claudins-3, -4, and -18 are the primary claudins expressed in these cell types.

Acute hypoxia-ischemia results in hydrogen peroxide accumulation in neonatal but not adult mouse brain.

The neonatal brain responds differently to hypoxic-ischemic injury and may be more vulnerable than the mature brain due to a greater susceptibility to oxidative stress. As a measure of oxidative stress, the immature brain should accumulate more hydrogen peroxide (H2O2) than the mature brain after a similar hypoxic-ischemic insult. To test this hypothesis, H2O2 accumulation was measured in postnatal day 7 (P7, neonatal) and P42 (adult) CD1 mouse brain regionally after inducing HI by carotid ligation followed by systemic hypoxia. H2O2 accumulation was quantified at 2, 12, 24, and 120 h after HI using the aminotriazole (AT)-mediated inhibition of catalase spectrophotometric method. Histologic injury was determined by an established scoring system, and infarction volume was determined. P7 and P42 animals were subjected to different durations of hypoxia to create a similar degree of brain injury. Despite similar injury, significantly less H2O2 accumulated in P42 mouse cortex compared with P7 at 2, 12, and 24 h after HI. In addition, less H2O2 accumulated in P42 mouse hippocampus compared with P7 hippocampus at 2 h. Since immature neurons are more vulnerable to the toxic effects of H2O2 than mature neurons, this increased accumulation in the immature brain may explain why the neonatal brain may be more devastated, even after a milder degree of acute hypoxic-ischemic injury.