Effect of intratracheal adenoviral vector administration on lung development in newborn rats.

Local overexpression of genes that promote lung defense or repair may be helpful in protecting the immature neonatal lung from injuries, but whether the vectors used to administer these genes affect physiological postnatal lung growth has not been investigated. We explored the effect on alveolarization of E1-deleted Adnull vector (Ad5-LMP-null) given intratracheally to 3-day-old rats. Three Adnull doses were evaluated 10(8), 5 x 10(8), and 10(9) TCID(50). Lung morphometry on day 21 showed significant growth disorders with the two higher doses. With 5 x 10(8) TCID(50), absolute lung volume increased significantly (+16%), as did absolute (+20%) and specific (+32%) alveolar airspace volumes, whereas alveolar surface density decreased by 13% (p < 0.009 for all parameters). Lung inflammation was mild, nonsignificant, and occurred mainly with the highest Adnull dose, indicating that it was unlikely to contribute to our results. Adnull instillation induced a significant#10; decrease in terminal bronchiolar cell proliferation as evaluated by proliferating cell nuclear antigen immunostaining (p = 0.02), as well as a 23% decrease in absolute parenchyma elastic fiber length (p = 0.02). Furthermore, lung tropoelastin mRNA content decreased by 25% (p < 0.02). In conclusion, E1-deleted adenoviral vectors can induce lung growth disorders when instilled into the airways of neonatal rats. Interactions with lung matrix turnover may be the main explanation to these deleterious effects.

Negative impact of DEP exposure on human airway epithelial cell adhesion, stiffness, and repair.

Epidemiological and experimental studies suggest that diesel exhaust particles (DEPs) may be associated with increased respiratory mortality and morbidity. Several recent studies have also shown that DEPs increase the production of inflammatory cytokines by human bronchial epithelium (HBE) cells in vitro. The present study investigates the effects of DEPs on the interaction of l-HBE cells (16HBE14o-) with the cell and matrix microenvironment based on evaluation of integrin-type cell/matrix ligand expression, cytoskeleton (CSK) stiffness, and matrix remodeling via matrix metalloproteinase (MMP)-1, MMP-2, and MMP-9 expression. The results showed that DEP exposure induced: 1) a net dose-dependent decrease in CSK stiffness through actin fibers, 2) a concomitant specific reduction of both alpha(3)- and beta(1)-integrin subunits extensively expressed on the HBE cell surface, 3) a decrease in the level of CD44, which is a major HBE cell-cell and HBE cell-matrix adhesion molecule; and 4) an isolated decrease in MMP-1 expression without any change in tissue inhibitor of matrix metalloproteinase (TIMP)-1 or TIMP-2 tissue inhibitors. Restrictive modulation of cell-matrix interaction, cell-cell connection, CSK stiffness, and fibrillary collagen remodeling results in a decreased wound closure capacity and an increased deadhesion capacity. In conclusion, on the basis of these results, we can propose that, in addition to their ability to increase the production of inflammatory cytokines, DEPs could also alter the links between actin CSK and the extracellular matrix, suggesting that they might facilitate HBE cell detachment in vivo.

Time course of actin cytoskeleton stiffness and matrix adhesion molecules in human bronchial epithelial cell cultures.

Human bronchial epithelial (HBE) cells adhere to underlying extracellular matrix (ECM) via integrin-type transmembrane receptors. Integrins link the ECM to the cytoskeleton (CSK), establishing a mechanical continuum by which forces are transmitted between the outside and the inside of the cells. The present study investigates the time course of global and actin CSK stiffness of HBE cells (16HBE14o-) growing on various matrix substrates as a function of culture time until confluence, and the concomitant time course of F-actin and adhesion molecule distribution. Our results showed a progressive increase in actin CSK stiffness from cell seeding to confluence, related to acquisition of highly polymerized cortical and cytosolic F-actin organization and up-regulation of certain matrix ligands, such as beta 1-, alpha 5-, and alpha v-integrin subunit expression. Moreover, compared to fibrillar type I collagen, reticular type IV collagen used as matrix substrate, appeared to amplify actin CSK stiffness of HBE confluent cells probably in relation to up-regulation of alpha 3-integrin subunit. Taken together, these results support the concept of a close interaction among actin CSK stiffness, structural actin organization, specific integrin molecule involvement, cell spreading, and extracellular matrix.

Protection against acute lung injury by intravenous or intratracheal pretreatment with EPI-HNE-4, a new potent neutrophil elastase inhibitor.

Excessive accumulation of active neutrophil elastase (NE) in pulmonary fluids and tissues of patients with cystic fibrosis (CF) is thought to act on the lungs, compromising their structure and function. The aim of this study was to investigate the in vitro and in vivo protective effect of a new, rapidly acting, potent (Ki = 5.45 x 10(-12) M and Kon = 8 x 10(6) M(-1) s(-1)) and specific human NE inhibitor, EPI-HNE-4, engineered from the Kunitz domain. The results demonstrated that this inhibitor was able to (i) effectively inhibit in vitro the high levels of active NE present in a medium as complex as sputum from children with CF, with a measured IC(50) equal or close to the calculated IC(50) in 60% of cases, and (ii) almost completely block (91%) the N-formyl-methionine-leucine-phenylalanine-induced migration of purified human neutrophils across a Matrigel basement membrane. Intratracheal administration (250, 175, or 100 microg per rat) of the inhibitor 5 min before instillation of pure human NE (HNE) (150 microg per rat) to rats induced effective, dose-dependent protection of the lungs, 4 h later, from hemorrhage, serum albumin leakage, residual active NE, and discrete neutrophil influx in air spaces induced by instillation of pure HNE. Intravenous administration (3 mg per rat) of EPI-HNE-4, 15 min before instillation of the soluble fraction of pooled sputum (delivering 120 microg of active NE per rat) from children with CF, effectively reduced (64%), 4 h later, the massive neutrophil influx induced by sputum instillation. Overall, these data strongly suggest that associated aerosol and systemic administration of EPI-HNE-4 would be beneficial in the treatment of CF.

LPS-induced lung injury in neonatal rats: changes in gelatinase activities and consequences on lung growth.

Postnatal lung growth disorders may involve imbalance between metalloproteinases and their inhibitors. Inflammatory cell 92-kDa gelatinase overactivity has been reported in adults with lung injury but has not been looked for in neonates. We compared gelatinase activity in neonatal and adult rats and evaluated postnatal lung growth after lipopolysaccharide (LPS)-induced lung injury. Significant intra-alveolar inflammatory cell recruitment occurred in adults and neonates; cell counts increased 16-fold in adults and 2.7-fold in neonates. Total 92-kDa gelatinase activity was increased in neonates and adults and was significantly correlated to inflammatory cell counts. For a given cell count, 92-kDa gelatinase increased more in neonates than in adults. Morphometric neonatal lung analysis showed that LPS-injured lungs had decreases in absolute values of lung volume (P < 0.03), alveolar surface (P < 0.004), and air space volume (P < 0.03). Doxycycline, a nonspecific metalloproteinase inhibitor, partly inhibited LPS-induced 92-kDa gelatinase overactivity but did not improve LPS-induced alveolar growth disorders. LPS-mediated lung injury in neonatal rats induced both gelatinase B overactivity and alveolar growth disorders, although no causal link between these two effects was demonstrated.

Gelatinase activities in the airways of premature infants and development of bronchopulmonary dysplasia.

Matrix-degrading metalloproteinases may play a role in the pathophysiology of bronchopulmonary dysplasia (BDP). We, therefore, evaluated correlations between gelatinase activities [metalloproteinase (MMP)-2 and MMP-9] or tissue inhibitor of metalloproteinase (TIMP)-1 levels present in the airways during the initial phase of hyaline membrane disease and the onset of BPD. Tracheal aspirates were obtained within 6 h of birth (day 0) from 64 intubated neonates with a gestational age < or =30 wk. Forty-five neonates were resampled on day 3 or 5. Total MMP-2 level measured by zymography fell with time, whereas total MMP-9 level and TIMP-1 levels, assayed by ELISA, increased; the MMP-9 increase correlated with the increase in airway inflammatory cell numbers. Among the parameters measured on day 0, 3, or 5, lower total MMP-2 level, lower birth weight, and higher fraction of inspired oxygen on day 0 were significantly and independently associated with the development of BPD. In conclusion, MMP-9 level and TIMP-1 levels increased after birth but are not linked to BPD outcome. In contrast, low MMP-2 level at birth is strongly associated with the development of BPD.

Neutrophil proteinases in hydrochloric acid- and endotoxin-induced acute lung injury: evaluation of interstitial protease activity by in situ zymography.

We investigated the role of polymorphonuclear neutrophil (PMN) proteinases, elastase, and gelatinase B in rat models of acute lung injury. Three groups of rats were studied 6 hours after unilateral instillation of hydrochloric acid (HCl; 0.1 N), lipopolysaccharide (LPS) (4 microg), or saline. The results demonstrated that HCl-induced lung injury, as compared with LPS-induced lung injury, was associated with an increase in permeability (wet/dry weight ratio and proteins in bronchoalveolar lavage fluid). In contrast, there was similar PMN recruitment (in bronchoalveolar lavage fluid and myeloperoxidase activity in lung homogenates) and similar proteinase exocytosis (residual alveolar PMN content of elastase and gelatinase B) in both types of lung injury. In situ zymography, evaluating interstitial protease/inhibitor balance, demonstrated a decrease in gelatinolytic activity in both HCl- and LPS-injured lungs compared with normal lung. The increase in interleukin 6 concentration in lung homogenates, which is observed after both injuries compared with saline-instilled animals, could be involved in up-regulation of tissue inhibitor of matrix metalloproteinase-1, shown by immunocytochemistry to participate in antiproteinase excess. Neither inhibition of alveolar neutrophil influx using a leukocyte elastase inhibitor (EPI-hNE-4) nor inhibition of gelatinase activities by recombinant adenovirus for the human tissue inhibitor of matrix metalloproteinase 1 gene transfer decreased lung edema in HCl-induced injury. These data suggest that PMN proteinases do not contribute to HCl-induced acute lung injury in rats.