Cathepsin B is activated as an executive protease in fetal rat alveolar type II cells exposed to hyperoxia

Alveolar type II cells are main target of hyperoxia-induced lung injury. The authors investigated whether lysosomal protease, cathepsin B (CB), is activated in fetal alveolar type II cells in the transitional period from the canalicular to saccular stages during 65%-hyperoxia and whether CB is related to fetal alveolar type II cell (FATIIC) death secondary to hyperoxia. FATIICs were isolated from embryonic day 19 rats and exposed to 65%-oxygen for 24 h and 36 h. The cells exposed to room air were used as controls. Cell cytotoxicity was assessed by lactate dehydrogenase-release and flow cytometry, and apoptosis was analyzed by TUNEL assay and flow cytometry. CB activity was assessed by colorimetric assay, qRT-PCR and western blots. 65%-hyperoxia induced FATIIC death via necrosis and apoptosis. Interestingly, caspase-3 activities were not enhanced in FATIICs during 65%-hyperoxia, whereas CB activities were greatly increased during 65%-hyperoxia in a time-dependent manner, and similar findings were observed with qRT-PCR and western blots. In addition, the preincubation of CB inhibitor prior to 65%-hyperoxia reduced FATIIC death significantly. Our studies suggest that CB activation secondary to hyperoxia might have a relevant role in executing the cell death program in FATIICs during the acute stage of 65%-hyperoxia.

Inhibitory effect of receptor for advanced glycation end products (RAGE) on the TGF-beta-induced alveolar epithelial to mesenchymal transition

Idiopathic pulmonary fibrosis (IPF) is a lethal parenchymal lung disease characterized by myofibroblast proliferation. Alveolar epithelial cells (AECs) are thought to produce myofibroblasts through the epithelial to mesenchymal transition (EMT). Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptors whose activation is associated with renal fibrosis during diabetes and liver fibrosis. RAGE is expressed at low basal levels in most adult tissues except the lung. In this study, we evaluated the interaction of ligand advanced glycation end products (AGE) with RAGE during the epithelial to myofibroblast transition in rat AECs. Our results indicate that AGE inhibited the TGF-beta-dependent alveolar EMT by increasing Smad7 expression, and that the effect was abolished by RAGE siRNA treatment. Thus, the induction of Smad7 by the AGE-RAGE interaction limits the development of pulmonary fibrosis by inhibiting TGF-beta-dependent signaling in AECs.

Primary culture of alveolar epithelial type II cells and its bionomic study / 华中科技大学学报（医学）（英德文版）

To establish a better method of primary culture for alveolar epithelial type II cells (AEC II) and to study its bionomics, alveolar epithelial type II cells were isolated by digestion with trypsin and collagenase, which were then purified by plated into culture flask coated with rat immunoglobulin G. The purified AEC II were identified by alkaline phosphatase staining, electron microscopy, immunocytochemical staining of pulmonary surfactant protein A (SPA). The SPA expression and transfection characteristics were compared with those of A549 cell line. The results showed that AEC II could be isolated by digestion with trysin and collagenase and purified by adhesive purification by using IgG, with a yield of about 2-3 x 10(7), and a purity of about 75%-84%. Cells could be quickly identified with AKP staining. AEC II were different from A549 cell line in terms of SPA expression and transfection characteristics. It is concluded that adhesive purification with IgG can improve the purity of AEC II, and AKP staining is simple in cell identification. AEC II can not be completely replaced by A549 cells in some studies because the differences between them, such as SPA expression.

Monocrotaline-induced pulmonary hypertension correlates with upregulation of connective tissue growth factor expression in the lung

Pulmonary hypertension (PH) is characterized by structural and functional changes in the lung including proliferation of vascular smooth muscle cells (VSMCs) and excessive collagen synthesis. Although connective tissue growth factor (CTGF) is known to promote cell proliferation, migration, adhesion, and extracellular matrix production in various tissues, studies on the role of CTGF in pulmonary hypertension have been limited. Here, we examined CTGF expression in the lung tissues of male Sprague Dawley rats treated with monocrotaline (MCT, 60 microgram/kg), a pneumotoxic agent known to induce PH in animals. Establishment of PH was verified by the significantly increased right ventricular systolic pressure and right ventricle/left ventricle weight ratio in the MCT-treated rats. Histological examination of the lung revealed profound muscular hypertrophy in the media of pulmonary artery and arterioles in MCT-treated group. Lung parenchyma, vein, and bronchiole did not appear to be affected. RT-PCR analysis of the lung tissue at 5 weeks indicated significantly increased expression of CTGF in the MCT-treated group. In situ hybridization studies also confirmed abundant CTGF mRNA expression in VSMCs of the arteries and arterioles, clustered pneumocytes, and infiltrated macrophages. Interestingly, CTGF mRNA was not detected in VSMCs of vein or bronchiole. In saline-injected control, basal expression of CTGF was seen in bronchial epithelial cells, alveolar lining cells, and endothelial cells. Taken together, our results suggest that CTGF upregulation in arterial VSMC of the lung might be important in the pathogenesis of pulmonary hypertension. Antagonizing the role of CTGF could thus be one of the potential approaches for the treatment of PH.

Avaliaçäo da permeabilidade epitelial pulmonar através da taxa de depuraçäo pulmonar do 99mTc-DTPA / Evaluation of lung epithelial permeability by the pulmonary clearance of 99mTc-DTPA

A taxa de depuraçäo pulmonar do 99mTc-DTPA constitui um índice da permeabilidade epitelial pulmonar. O interesse crescente nesta técnica diagnóstico decorre de sua natureza näo-invasiva, da facilidade de repetiçöes das medidas, de seu relativo baixo custo e, principalmente, de sua elevada sensibilidade em detectar precocemente a lesäo pulmonar. Este artigo revisa os princípios básicos e as aplicaçöes desta técnica, procurando dar ênfase a seu uso como método diagnóstico na prática clínica

Pulmonary Alveoli and Macrophages of Rats: A Study of Aging Changes by Electron Microscopy

Lung tissues of rats from two different age groups (2-12 and 16-26 months of age) were studied by both light and electron microscopy. Proliferation of granular pneumocytes in pulmonary alveolar lining was a frequent occurrence in older rats. Lungs of older rats showed not only an increase in number of granular pneumocytes, but also a remarkable increase of lamellar bodies and other forms of lipid vacuoles in individual granular pneumocytes. Spontaneously-occurring nodular lesions characterized by the accumulation of macrophages in the alveolar spaces were accompanied by desquamation and proliferation of granular pneumocytes. These lesions developed only in the lungs of rats older than l7 months of age. Such lessions in lungs of old rats were similar in many respects to desquamative interstitial pneumonitis of human lungs. Atrophy of alveolar walls and emphysematous areas seen in senile rats was characterized by irregular cytoplasmic breakdown of Type I alveolar lining epithelial cells. Obliteration of capillaries by spontaneously-occurring thrombus formation or a herniated cytoplasm of the septal cell and collagen fibers was considered to be a cause of atrophy of alveolar walk. Degeneration and actual breakdown of endothe1ial cytoplasm of puImonary capillaries enhanced herniation of the septal tissue. Vacuolar degeneration of epithelial cytoplasm was occasionally observed, but only in rats older than 20 months of age. The basement membrane of pulmonary alveolar walls was often thicker in old rats than in younger rats. Hyperplasia of granular pneumocytes invariably accompanied large septal cells, some of which contained many of the organelles found in granular pneumocytes.