Defective angiogenesis in hypoplastic human fetal lungs correlates with nitric oxide synthase deficiency that occurs despite enhanced angiopoietin-2 and VEGF.

Lung hypoplasia (LH) is a life-threatening congenital abnormality with various causes. It involves vascular bed underdevelopment with abnormal arterial muscularization leading to pulmonary hypertension. Because underlying molecular changes are imperfectly known and sometimes controversial, we determined key factors of angiogenesis along intrauterine development, focusing at the angiopoietin (ANG)/Tie-2 system. Lung specimens from medical terminations of pregnancy (9-37 wk) were used, including LH due to congenital diaphragmatic hernia (CDH) or other causes, and nonpulmonary disease samples were used as controls. ELISA determination indicated little ANG-1 change during pregnancy and no effect of LH, whereas Tie-2 declined similarly between 9 and 37 wk in LH and controls. By contrast, ANG-2 markedly increased in LH from 24 wk, whereas it remained stable in controls. Because VEGF increased also, this was interpreted as an attempt to overcome vascular underdevelopment. Hypothesizing that its inefficiency might be due to impaired downstream mechanism, endothelial nitric oxide synthase (eNOS) was determined by semiquantitative Western blot and found to be reduced by approximately 75%, mostly in the instance of CDH. In conclusion, angiogenesis remains defective in hypoplastic lungs despite reactive enhancement of VEGF and ANG-2 production, which could be due, at least in part, to insufficient eNOS expression.

Pulmonary effects of keratinocyte growth factor in newborn rats exposed to hyperoxia.

Acute lung injury and compromised alveolar development characterize bronchopulmonary dysplasia (BPD) of the premature neonate. High levels of keratinocyte growth factor (KGF), a cell-cell mediator with pleiotrophic lung effects, are associated with low BPD risk. KGF decreases mortality in hyperoxia-exposed newborn rodents, a classic model of injury-induced impaired alveolarization, although the pulmonary mechanisms of this protection are poorly defined. These were explored through in vitro and in vivo approaches in the rat. Hyperoxia decreased by 30% the rate of wound closure of a monolayer of fetal alveolar epithelial cells, due to cell death, which was overcome by recombinant human KGF (100 ng/ml). In rat pups exposed to >95% O2 from birth, increased viability induced by intraperitoneal injection of KGF (2 microg/g body wt) every other day was associated with prevention of neutrophil influx in bronchoalveolar lavage (BAL), prevention of decreases in whole lung DNA content and cell proliferation rate, partial prevention of apoptosis increase, and a markedly increased proportion of surfactant protein B-immunoreactive cells in lung parenchyma. Increased lung antioxidant capacity is likely to be due in part to enhanced CAAT/enhancer binding protein alpha expression. By contrast, KGF neither corrected changes induced by hyperoxia in parameters of lung morphometry that clearly indicated impaired alveolarization nor had any significant effect on tissue or BAL surfactant phospholipids. These findings evidence KGF alveolar epithelial cell protection, enhancing effects on alveolar repair capacity, and anti-inflammatory effects in the injured neonatal lung that may account, at least in part, for its ability to reduce mortality. They argue in favor of a therapeutic potential of KGF in the injured neonatal lung.

Bronchopulmonary dysplasia and emphysema: in search of common therapeutic targets.

Bronchopulmonary dysplasia of the premature neonate and emphysema of the adult lung are common diseases that are characterized by increased airspace size and respiratory insufficiency and that presently lack efficient treatment. Although the former leads to impaired alveolar development and the latter to alveolar destruction, they have striking similarities in their pathophysiology, including the precipitating effect of oxidative stress, sustained inflammation, enhanced apoptosis, protease-antiprotease imbalance, elastic fiber deterioration and altered microvascularization. This review aims to comparatively analyze their molecular mechanisms to try identify common therapeutic targets. The recent discovery that alveolar developmental and maintenance programs share the same signal molecules and pathways, together with considerable increase in their understanding, have facilitated the development of common innovative strategies that have started to be tested in experimental models and pilot clinical studies.

Gene expression profiling in lung fibroblasts reveals new players in alveolarization.

Little is known about the molecular basis of lung alveolarization. We used a microarray profiling strategy to identify novel genes that may regulate the secondary septation process. Rat lung fibroblasts were extemporaneously isolated on postnatal days 2, 7, and 21, i.e., before, during, and after septation, respectively. Total RNA was extracted, and cRNAs were hybridized to Affymetrix rat genome 230 2.0 microarrays. Expression levels of a selection of genes were confirmed by real-time PCR. In addition to genes already known to be upregulated during alveolarization including drebrin, midkine, Fgfr3, and Fgfr4, the study allowed us to identify two remarkable groups of genes with opposite profiles, i.e., gathering genes either transiently up- or downregulated on day 7. The former group includes the transcription factors retinoic acid receptor (RXR)-gamma and homeobox (Hox) a2, a4, and a5 and genes involved in Wnt signaling (Wnt5a, Fzd1, and Ndp); the latter group includes the extracellular matrix components Comp and Opn and the signal molecule Slfn4. Profiling in whole lung from fetal life to adulthood confirmed that changes were specific for alveolarization. Two treatments that arrest septation, hyperoxia and dexamethasone, inhibited the expression of genes that are upregulated during alveolarization and conversely enhanced that of genes weakly expressed during alveolarization and upregulated thereafter. The possible roles of these genes in secondary septation are discussed. Gene expression profiling analysis on freshly isolated cells represents a powerful approach to provide new information about differential regulation of genes during alveolarization and pathways potentially involved in the pathogenesis of bronchopulmonary dysplasia.

Differential expression of matrix metalloproteinases and inhibitors in developing rat lung mesenchymal and epithelial cells.

Lung development requires extracellular matrix remodeling. This involves matrix metalloproteinases (MMPs) and their endogenous inhibitors [tissue inhibitors of metalloproteinases (TIMPs)]. Because these have been generally studied only in whole lung, we focused specifically on mesenchymal and epithelial cells freshly isolated at various developmental stages. In fibroblasts, the most striking developmental change was a peak (fourfold the prenatal level) of membrane type 1 (MT1)-MMP transcript during alveolarization, consistent with the known crucial role of MT1-MMP in this process. TIMP-1 and -2 mRNAs transiently increased on postnatal d (pn) 3. In alveolar epithelial cells (AECs), MMP-2 expression was maximal on fetal d (f) 19 when alveolar type II cells (ATII) differentiate and on pn5; by contrast, MT1-MMP expression changed little and TIMP-1 expression decreased with advancing gestation. In cells expressing in vitro the ATI phenotype, TIMP-1 and -2 activities were nine- and fivefold those in cells expressing ATII features, respectively, whereas ATII presented higher MMP-2 activity and were the only cell type to express MMP-9. This indicates higher remodeling potential for ATII. Pulmonary mesenchymal and epithelial cells have therefore quite distinct MMP/TIMP expression patterns. Changes in cell compartments should be specifically documented in developing lung diseases such as bronchopulmonary dysplasia in which changes in MMP activities have been reported.

Nitric oxide donor restores lung growth factor and receptor expression in hyperoxia-exposed rat pups.

Exposure of newborn rats to hyperoxia impairs alveolarization. Nitric oxide (NO) may prevent this evolution. Angiogenesis and factors involved in this process, but also other growth factors (GFs) involved in alveolar development, are likely potential therapeutic targets for NO. We studied the effects of the NO donor, [Z]-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)aminio]diazen-1-ium-1, 2-diolate, also termed DETANONOate (D-NO), on hyperoxia-induced changes in key regulatory factors of alveolar development in neonatal rats, and its possible preventive effect on the physiologic consequences of hyperoxia. Newborn rat pups were randomized at birth to hyperoxia (> 95% O2) or room air exposure for 6 or 10 d, while receiving D-NO or its diluent. On Day 6, several GFs and their receptors were studied at pre- and/or post-translational levels. Elastin transcript determination on Day 6, and elastin deposition in tissue and morphometric analysis of the lungs on Day 10, were also performed. Hyperoxia decreased the expression of vascular endothelial growth factor (VEGF) receptor (VEGFR) 2, fibroblast growth factor (FGF)-18, and FGF receptors (FGFRs) FGFR3 and FGFR4, increased mortality, and impaired alveolarization and capillary growth. D-NO treatment of hyperoxia-exposed pups restored the expression level of FGF18 and FGFR4, induced an increase of both VEGF mRNA and protein, enhanced elastin expression, and partially restored elastin deposition in alveolar walls. Although, under the present conditions, D-NO failed to prevent the physiologic consequences of hyperoxia in terms of survival and lung alveolarization, our findings demonstrate molecular effects of NO on GFs involved in alveolar development that may have contributed to the protective effects previously reported for NO.

Effects of vascular endothelial growth factor on isolated fetal alveolar type II cells.

Previous investigations gained from in vivo or lung explant studies suggested that VEGF is an autocrine proliferation and maturation factor for developing alveolar type II cells. The objective of this work was to determine whether VEGF exerted its growth and maturation effects directly on isolated type II cells. These were isolated from 19-day fetal rat lung and cultured in defined medium. The presence of VEGF receptor-2 was assessed in cultured cells at the pre- and posttranslational levels. Recombinant VEGF(165), formerly found to be active on lung explants, failed to enhance type II cell proliferation estimated by thymidine and 5-bromo-2'-deoxy-uridine incorporation. It increased choline incorporation in saturated phosphatidylcholine by 27% but did not increase phospholipid surfactant pool size. VEGF (100 ng/ml) left unchanged the transcript level of surfactant proteins (SP)-A, SP-C, and SP-D but increased SP-B transcripts to four times the control steady-state level. VEGF slightly retarded, but did not prevent, the in vitro transdifferentiation of type II into type I cells, as assessed by immunolabeling of the type I cell marker T1alpha. We conclude that, with the exception of SP-B expression, which appears to be controlled directly, the previously observed effects of this VEGF isoform on type II cells are likely to be exerted indirectly through reciprocal paracrine interactions involving other lung cell types.

Dexamethasone potentiates keratinocyte growth factor-stimulated SP-A and SP-B gene expression in alveolar epithelial cells.

Keratinocyte growth factor (KGF, or fibroblast growth factor 7) was previously reported to enhance the synthesis of surfactant in alveolar type II cells. We investigated the possible interactions between KGF and a glucocorticoid, dexamethasone (Dex), on surfactant protein (SP) gene expression. In cultured fetal rat type II cells, KGF and Dex induced greater-than-additive stimulating effects on SP-A and SP-B expressions that were enhanced three-fold and 30-fold, respectively, but had only additive effects on SP-C expression. Using murine lung epithelial (MLE) cells, KGF increased SP-A, SP-B (up to two-fold), and SP-C (up to three-fold) mRNA levels in a dose-dependent way. Dex 10(-9) to 10(-7) M increased SP-A and SP-B mRNA 1.5-fold and SP-C mRNA two-fold. Consistent with type II cell findings, simultaneous treatment by KGF and Dex induced a synergistic increase of SP-A and SP-B transcripts (three-fold and 4.5-fold, respectively), but not of SP-C transcripts. SP-A protein was present in MLE-15 and was increased about three-fold by KGF plus Dex. Expression study of a reporter gene placed under either the SP-A or the SP-B gene regulatory sequences and transfected in MLE-15 cells indicated that the Dex-KGF synergy was achieved mainly through a transcriptional effect for SP-A, and both transcriptional and nontranscriptional effects for SP-B. For the latter, increased mRNA stability was evidenced with the aid of actinomycin D. The Dex-KGF synergy may have potential interest for diseases associated with surfactant deficiency.