TRIP-1 via AKT modulation drives lung fibroblast/myofibroblast trans-differentiation.

BACKGROUND: Myofibroblasts are the critical effector cells in the pathogenesis of pulmonary fibrosis which carries a high degree of morbidity and mortality. We have previously identified Type II TGFß receptor interacting protein 1 (TRIP-1), through proteomic analysis, as a key regulator of collagen contraction in primary human lung fibroblasts--a functional characteristic of myofibroblasts, and the last, but critical step in the process of fibrosis. However, whether or not TRIP-1 modulates fibroblast trans-differentiation to myofibroblasts is not known. METHODS: TRIP-1 expression was altered in primary human lung fibroblasts by siRNA and plasmid transfection. Transfected fibroblasts were then analyzed for myofibroblast features and function such as α-SMA expression, collagen contraction ability, and resistance to apoptosis. RESULTS: The down-regulation of TRIP-1 expression in primary human lung fibroblasts induces α-SMA expression and enhances resistance to apoptosis and collagen contraction ability. In contrast, TRIP-1 over-expression inhibits α-SMA expression. Remarkably, the effects of the loss of TRIP-1 are not abrogated by blockage of TGFß ligand activation of the Smad3 pathway or by Smad3 knockdown. Rather, a TRIP-1 mediated enhancement of AKT phosphorylation is the implicated pathway. In TRIP-1 knockdown fibroblasts, AKT inhibition prevents α-SMA induction, and transfection with a constitutively active AKT construct drives collagen contraction and decreases apoptosis. CONCLUSIONS: TRIP-1 regulates fibroblast acquisition of phenotype and function associated with myofibroblasts. The importance of this finding is it suggests TRIP-1 expression could be a potential target in therapeutic strategy aimed against pathological fibrosis.

TRIP-1 regulates TGF-ß1-induced epithelial-mesenchymal transition of human lung epithelial cell line A549.

Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells undergo conversion to a mesenchymal phenotype contributing to wound repair by fibrosis and to cancer cell acquisition of invasive ability. Recently, we showed that type II TGF-ß receptor interacting protein-1 (TRIP-1), a protein identified as a phosphorylation target of the TGF-ß type II receptor kinase and as a functional component of eukaryotic translation initiator factor 3 (eiF3) multiprotein complex, is a novel modulator of fibroblast collagen contraction, an important step in wound repair stimulated by TGF-ß1 action. TGF-ß1 drives EMT, but it is not known whether TRIP-1 expression influences EMT induction. To investigate whether TRIP-1 plays a role in EMT induction we studied the effect of downregulating TRIP-1 expression in the well-characterized A549 model of TGF-ß1 induction of EMT. Here we report that short hairpin RNA (shRNA)-mediated depletion of TRIP-1 gene transcripts in A549 cells promotes EMT as assessed by changes in phenotypic markers, morphology, and migrative ability. Knockdown of TRIP-1 dramatically increased A549 responsiveness to TGF-ß1 induction of EMT. Mechanistically, a pathway involving increased TGF-ß type II receptor level, enhanced Smad3 phosphorylation, and the transcription factor SLUG is implicated. Altogether, the findings point to regulation of endogenous TRIP-1 protein expression as a potential strategy to target EMT, and related invasive behavior, in cancer cells.

Epidermal growth factor-like domain 7 protects endothelial cells from hyperoxia-induced cell death.

Hyperoxia is one of the major contributors to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants. Emerging evidence suggests that the arrested lung development of BPD is associated with pulmonary endothelial cell death and vascular dysfunction resulting from hyperoxia-induced lung injury. A better understanding of the mechanism of hyperoxia-induced endothelial cell death will provide critical information for the pathogenesis and therapeutic development of BPD. Epidermal growth factor-like domain 7 (EGFL7) is a protein secreted from endothelial cells. It plays an important role in vascular tubulogenesis. In the present study, we found that Egfl7 gene expression was significantly decreased in the neonatal rat lungs after hyperoxic exposure. The Egfl7 expression was returned to near normal level 2 wk after discounting oxygen exposure during recovery period. In cultured human endothelial cells, hyperoxia also significantly reduced Egfl7 expression. These observations suggest that diminished levels of Egfl7 expression might be associated with hyperoxia-induced endothelial cell death and lung injury. When we overexpressed human Egfl7 (hEgfl7) in EA.hy926 human endothelial cell line, we found that hEgfl7 overexpression could partially block cytochrome c release from mitochondria and decrease caspase-3 activation. Further Western blotting analyses showed that hEgfl7 overexpression could reduce expression of a proapoptotic protein, Bax, and increase expression of an antiapoptotic protein, Bcl-xL. Theses findings indicate that hEGFL7 may protect endothelial cell from hyperoxia-induced apoptosis by inhibition of mitochondria-dependent apoptosis pathway.

Hyperoxia and tidal volume: Independent and combined effects on neonatal pulmonary inflammation.

BACKGROUND: Hyperoxia and tidal volume mechanical ventilation are independent factors in the genesis of lung injury, but it remains unclear the extent to which each is responsible or contributes to this process in newborns. OBJECTIVES: To study the independent and combined effects of hyperoxia and tidal volume mechanical ventilation on the induction of lung inflammation in a newborn piglet model of ventilator-induced lung injury. METHODS: Following exposure to either ambient air or F(I)O2 = 1.0 for a period of 3 days, newborn piglets were randomized to receive mechanical ventilation with either high tidal volume (20 ml/kg) or low tidal volume (6 ml/kg) for 4 h while controlling for pH. RESULTS: Monocyte chemoattractant protein-1 level in the lungs of animals randomized to hyperoxia with high tidal volume ventilation was significantly elevated, compared to all other groups (p < 0.05). Myeloperoxidase assayed in lung homogenate was found to be significantly higher in nonventilated animals exposed to hyperoxia (p < 0.01). Only in animals previously exposed to hyperoxia did the addition of high tidal volume ventilation further increase the level of myeloperoxidase present (p < 0.05). Pulmonary vascular resistance was significantly elevated after 4 h of mechanical ventilation compared to 1 h (p < 0.001). CONCLUSIONS: We conclude that in neonatal piglets undergoing hyperoxic stress, superimposition of high tidal volume ventilation exacerbates the lung inflammation as assessed by lung monocyte chemoattractant protein-1 and level of myeloperoxidase.

Responses of pulmonary platelet-derived growth factor peptides and receptors to hyperoxia and nitric oxide in piglet lungs.

The peptides platelet-derived growth factor-A (PDGF-A) and especially -B have important roles in lung development. The effect of hyperoxic exposure with and without inhaled nitric oxide (iNO) on lung expression of PDGF and its receptors is unknown. We hypothesized that hyperoxia exposure would suppress mRNA expression and protein production of these ligands and their receptors. The addition of iNO to hyperoxia may further aggravate the effects of hyperoxia. Thirteen-day-old piglets were randomized to breathe 1) room air (RA); 2) 0.96 fraction of inspired oxygen (O2), or 3) 0.96 fraction of inspired oxygen plus 50 ppm of NO (O2+NO), for 5 d. Lungs were preserved for mRNA, Western immunoblot, and immunohistochemical analyses for PDGF-A and -B and their receptors PDGFR-alpha and -beta. PDGF-B mRNA expression was greater than that of PDGF-A or PDGFR-alpha and -beta in RA piglet lungs (p<0.05). Hyperoxia with or without iNO reduced lung PDGF-B mRNA and protein expression relative to the RA group lungs (p<0.01). PDGF-B immunostain intensity was significantly increased in the alveolar macrophages, which were present in greater numbers in the hyperoxia-exposed piglet lungs, with or without NO (p<0.01). PDGFR-beta immunostaining was significantly increased in airway epithelial cells in O2- and O2+NO-exposed piglets. PDGF-A and PDGFR-alpha immunostain intensity and distribution pattern were unchanged relative to the RA group. Sublethal hyperoxia decreases PDGF-B mRNA and protein expression but not PDGF-A or their receptors in piglet lungs. iNO neither aggravates nor ameliorates this effect.

Low levels of tissue inhibitors of metalloproteinases with a high matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio are present in tracheal aspirate fluids of infants who develop chronic lung disease.

OBJECTIVE: The pathogenesis of chronic lung disease (CLD) involves inflammation with proteolytic damage to lung extracellular matrix. Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that, acting in concert with their tissue inhibitors, tightly orchestrate extracellular matrix morphogenesis and repair after injury. Imbalances in their levels relative to that of their inhibitors have been implicated in diseases characterized by matrix disruption and remodeling. We investigated the possibility that imbalances in MMP-9 and MMP-2 relative to their tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2, respectively, in tracheal aspirates of preterm infants may be involved in the development of CLD. METHODS: Serial tracheal aspirates collected from birth until extubation in 49 ventilated preterm infants (24-32 weeks' gestations) were analyzed for MMP-2, MMP-9, TIMP-1, and TIMP-2. Data normalized by TA values of free secretory component of immunoglobulin A were compared for CLD (n = 22) versus no CLD (n = 27). Also, known clinical predictors of CLD (gestational age, birth weight, and sex) were assessed for both groups. Association of predictors with the outcome CLD was assessed by logistic regression. RESULTS: Mean gestational age was lower in CLD infants, but birth weight and gender were comparable for both groups. CLD infants had significantly lower TIMP-1 level with higher MMP-9/TIMP-1 ratio during the first 2 weeks of life and low TIMP-2 and MMP-2 levels during the first 3 days of life compared with no-CLD infants. Logistic regression analysis indicated that the findings are predictive of CLD. CONCLUSIONS: We conclude that low tracheal aspirate levels of TIMPs, with a high MMP-9/TIMP-1 ratio early in life, are associated with subsequent development of CLD.

Acinar arterial changes with chronic lung disease of prematurity in the surfactant era.

Because echocardiographic studies on infants with chronic lung disease (CLD) suggest that pulmonary hypertension (PH) may contribute to its severity, we studied acinar arterial walls in the following surfactant-era infants: controls (n=38): 22-41 weeks of gestational age (GA), exposed briefly to oxygen and positive pressure ventilation, died within 48 hr of birth; prolonged rupture of fetal membranes (PROM) and persistent pulmonary hypertension (PPHN) (n=17); and SCORE (integrated area under curve of average daily FiO2 x average daily MAP) groups (<20, 20-69, and 70-500; mild, moderate, and severe clinical lung disease, respectively, n=35): 23-30 weeks GA, lived 7-79 days. Lungs were stained for elastic tissue and smooth muscle actin. Vessels were assessed for percent of vessel circumference with smooth muscle, extent of elastic laminae in the walls, and percent arterial wall thickness (%AWT) at three levels: terminal to respiratory bronchiole transition (TRB), alveolar duct, and saccule. At the alveolar ductal and saccular levels, percent arterial wall thickness (%AWT) in mild CLD (SCORE < 20) was less than controls (P < 0.05) and those with more severe CLD (SCORE 70-500), indicating that normal postnatal arterial wall thinning may be delayed, or there is remodeling associated with increased %AWT. Severe CLD infants also had a significantly higher percent of circumferential actin than those with milder disease (SCORE < or = 69) and controls. In moderate and severe CLD, there was an increase in extent of the elastic laminae compared to controls and mild CLD. These changes were also significantly greater in PROM and PPHN infants compared to even severe CLD. We conclude that PH is a real possibility in severe CLD infants after discharge at 36 weeks. Grading the severity of CLD at discharge, and echocardiographic studies, may guide subsequent oxygen therapy.

Collagen scaffolding during development and its deformation with chronic lung disease.

OBJECTIVE: Infants with chronic lung disease (CLD) have an arrest of primary and secondary septation. We hypothesized that this may be related to damage or abnormal development of lung collagen secondary to positive pressure ventilation. Our aims were to identify the sites and quantity of collagen in control infants 22 to 72 weeks' postconceptional age and compare these with infants with various degrees of severity of CLD. METHODS: The controls were 22 to 42 weeks' gestation (n = 30), received minimal ventilator care, and died within 48 hours of birth, plus 5 term infants who died at 43 to 72 weeks' postconceptional age from nonpulmonary causes. Infants who were 23 to 30 weeks' gestation, were at risk for CLD, and lived 5 to 94 days (n = 33) were separated into 3 groups on the basis of respiratory score (score group; the integrated area under the curve of the average daily fraction of inspired oxygen x mean airway pressure [cm H2O] over the number of days lived). The score groups, <20, 20 to 69, and 70 to 500, related clinically to mild to moderate and severe lung disease. The lungs were tracheally perfused and formalin fixed. Total lung volume was determined by water displacement. The paraffin-embedded lung blocks were sectioned 5 micro m thick, stained with Gomori's reticulum stain, hematoxylin and eosin, and immunohistochemically for collagen IV. The parenchyma was point-counted, and the volume density of collagen was measured. The chord diameter of the peripheral airway saccules and alveoli was measured. Descriptive collagen data were assessed on en face 40- micro m-thick sections through the alveolar or saccular walls on all infants at risk for CLD and in selected controls. RESULTS: In the controls, the volume density of collagen decreased from a maximum of 9% at 22 weeks to 5% at term and 72 weeks. With Scores < or =69, the fraction of collagen was similar to controls, but in infants with scores 70 to 500, it was increased relative to controls. However, when collagen was expressed as the volume density of interstitial tissue, ie, excluding parenchymal air space, it increased from a low of 5% at 22 weeks to 25% at 72 weeks. In infants with scores 70 to 500, 79% of infants had collagens greater than controls. Saccular and alveolar diameter increased from 40 micro m at 23 weeks to 100 microm at 72 weeks. Most infants with severe CLD (scores > or =70) had diameters more than twice that of controls at the same age. The total lung parenchymal collagen had a similar pattern as the volume density of collagen in interstitial tissue, increasing from 0.4 cm3 at 23 weeks to 9.7 cm3 at 72 weeks in the controls. Eighty-five percent of infants with scores 70 to 500 had total parenchymal collagen greater than the 95% confidence interval of the controls. With en face sections, a fine collagen mesh was seen at 23 weeks, which progressively increased in fiber size and quantity until 72 weeks. With severe CLD, the secondary collagen fibers in the saccular wall were thickened, tortuous, and disorganized relative to same-aged controls. Under 30 weeks, in the controls, the interstitium contained a wide, delicate network of interconnected collagen fibers. After positive pressure ventilation, some saccules markedly increased their diameter, which compressed and obliterated the interstitial network. In contrast with severe CLD, the interstitium was wide, with coarse wavy collagen fibers. CONCLUSIONS: Parenchymal collagen increases throughout development. Before 30 weeks, there is a delicate complex interstitial collagen network, which may be important for primary septation and subsequent normal development. Positive pressure ventilation, if excessive, and depending on lung maturity and disease state, over a short time can severely compress the interstitium and damage this collagen network and prevent normal primary septation and arrest or distort future lung development. With severe CLD, distal air space diameter increases. There is a failure of primary and secondary septation, arrested lung development and remodeling, with thickened cnt and remodeling, with thickened collagenous saccular walls, and a wide interstitium with increased quantity and size of collagen fibers that can affect the mechanics of ventilation. We conclude that normal lung development is dependent on a normal interstitium and, perhaps, collagen architecture and that origins of CLD begin early in the course of positive pressure ventilation.

Endostatin and vascular endothelial cell growth factor (VEGF) in piglet lungs: effect of inhaled nitric oxide and hyperoxia.

Pulmonary hyperoxic injury manifests as widespread alveolar-epithelial and microvascular endothelial cell necrosis, resolution of which requires angiogenesis. We investigated the hypothesis that inhaled nitric oxide (iNO) and hyperoxia each decreases lung vascular endothelial growth factor (VEGF) expression but increases endostatin and that concurrent administration of both gases will show a greater effect. Piglets were randomized to breathe for 5 d room air (RA); RA + NO (RA + 50 ppm NO), O(2) (hyperoxia, F(I)O(2) >0.96), O(2) + NO, or O(2) + NO + REC (O(2) + NO plus recovery in 50% O(2) for 72 h. After the piglets were killed, we measured lung capillary leak, VEGF mRNA, VEGF, and endostatin protein in homogenates, plasma, and lavage. VEGF mRNA decreased significantly with O(2) and O(2) + NO compared with breathing RA (p < or = 0.05). VEGF protein declined in the experimental groups with a significant reduction in the recovery group compared with the RA group (p < or = 0.05). Similar but more dramatic, endostatin declined in all groups relative to the RA group (p < 0.001). Lavage fluid VEGF protein and lung capillary leak rose significantly with O(2) and O(2) + NO compared with RA, but endostatin was unchanged. At 72 h of recovery from hyperoxia, VEGF mRNA and lavage fluid VEGF but not lung VEGF protein had normalized. Hyperoxia and iNO suppresses lung endostatin expression, but iNO unlike hyperoxia alone does not alter lung VEGF production. Hyperoxia paradoxically raises lavageable VEGF levels. This latter effect and that on VEGF mRNA level but not protein is abrogated by recovery in reduced F(I)O(2) for 72 h.

Interaction of endogenous endothelin-1 and inhaled nitric oxide in term and preterm infants.

The peptide endothelin-1 (ET-1) plays an unknown role in the pathogenesis and progression of two important neonatal pulmonary disorders, chronic lung disease (CLD) of prematurity and persistent pulmonary hypertension of the newborn (PPHN). Inhaled nitric oxide (INO) is a proven vasodilator therapy in PPHN and is an experimental therapy in CLD. We sought to determine the effects, if any, of the interaction of inhaled INO with ET-1 in these two separate disorders. Infants (n=21) with PPHN (mean gestation age, 39.4 weeks; mean birth weight, 3470 g) were treated with INO. All infants were <72 h of age at baseline. Plasma obtained at baseline and after 24 h of INO therapy was assessed for ET-1. The change in ET-1 levels with INO was inversely correlated with change in arterial partial pressure of O(2) (r=-0.71, P=0.0003). A separate group of 33 patients with CLD (mean gestational age, 27 weeks; mean birth weight, 740 g; mean age, 19 days) had tracheal aspirate levels of ET-1 obtained before, during, and after 7 days' administration of INO. Values were normalized by soluble secretory component of IgA. Tracheal aspirate ET-1 levels were detectable before INO therapy. There was no significant change during or after treatment with INO. There was not a significant correlation between baseline fractional inspired O(2) and ET-1 levels. There was a non-significant trend in the correlation between the change in ET-1 and the change in interleukin-8 levels in tracheal aspirate. This report confirms the presence of ET-1 in tracheal aspirate of premature infants who are developing CLD and reaffirms the presence of ET-1 in plasma of infants with PPHN. Short-term INO therapy was associated with a decrease in plasma ET-1 levels in PPHN, but did not affect tracheal aspirate ET-1 in CLD. Given the vasconstrictive, profibrotic, and proinflammatory properties of ET-1, specific ET-1 receptor antagonists could be considered as candidates for trials as adjunct therapy in either or both of these disorders.