Endothelial cell apoptosis in chronically obstructed and reperfused pulmonary artery.

BACKGROUND: Endothelial dysfunction is a major complication of pulmonary endarterectomy (PTE) that can lead to pulmonary edema and persistent pulmonary hypertension. We hypothesized that endothelial dysfunction is related to increased endothelial-cell (EC) death. METHODS: In piglets, the left pulmonary artery (PA) was ligated to induce lung ischemia then reimplanted into the main PA to reperfuse the lung. Animals sacrificed 5 weeks after ligation (n = 5), 2 days after reperfusion (n = 5), or 5 weeks after reperfusion (n = 5) were compared to a sham-operated group (n = 5). PA vasoreactivity was studied and eNOS assayed. EC apoptosis was assessed by TUNEL in the proximal and distal PA and by caspase-3 activity assay in the proximal PA. Gene expression of pro-apoptotic factors (thrombospondin-1 (Thsp-1) and plasminogen activator inhibitor 1 (PAI-1)) and anti-apoptotic factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) was investigated by QRT-PCR. RESULTS: Endothelium-dependent relaxation was altered 5 weeks after ligation (p = 0.04). The alterations were exacerbated 2 days after reperfusion (p = 0.002) but recovered within 5 weeks after reperfusion. EC apoptosis was increased 5 weeks after PA ligation (p = 0.02), increased further within 2 days after reperfusion (p < 0.0001), and returned to normal within 5 weeks after reperfusion. Whereas VEGF and bFGF expressions remained unchanged, TSP and PAI-1 expressions peaked 5 weeks after ligation (p = 0.001) and returned to normal within 2 days after reperfusion. CONCLUSION: Chronic lung ischemia induces over-expression of pro-apoptotic factors. Lung reperfusion is followed by a dramatic transient increase in EC death that may explain the development of endothelial dysfunction after PE. Anti-apoptotic agents may hold considerable potential for preventing postoperative complications.

Transgenic mice overexpressing the 5-hydroxytryptamine transporter gene in smooth muscle develop pulmonary hypertension.

One intrinsic abnormality of pulmonary artery smooth muscle cells (PA-SMCs) in human idiopathic pulmonary hypertension (iPH) is an exaggerated proliferative response to internalized serotonin (5-HT) caused by increased expression of the 5-HT transporter (5-HTT). To investigate whether 5-HTT overexpression in PA-SMCs is sufficient to produce PH, we generated transgenic mice overexpressing 5-HTT under the control of the SM22 promoter. Studies in SM22-LacZ(+) mice showed that the transgene was expressed predominantly in SMCs of pulmonary and systemic vessels. Compared with wild-type mice, SM22-5-HTT(+) mice exhibited a 3- to 4-fold increase in lung 5-HTT mRNA and protein, together with increased lung 5-HT uptake activity, but no changes in platelet 5-HTT activity or blood 5-HT levels. At 8 weeks of age, SM22-5-HTT(+) mice exhibited PH, with marked increases in right ventricular systolic pressure (RVSP), right ventricle/left ventricle+septum ratio, and muscularization of distal pulmonary vessels, but no changes in systemic arterial pressure. PH worsened with age. Except a marked decrease in Kv channels, no changes in the lung expression of mediators of pulmonary vascular remodeling were observed in SM22-5-HTT(+) mice. Compared with wild-type mice, SM22-5-HTT(+) mice showed depressed hypoxic pulmonary vasoconstriction contrasting with greater severity of hypoxia- or monocrotaline-induced PH. These results show that increased 5-HTT expression in PA-SMCs, to a level close to that found in human iPH, lead to PH in mice. They further support a central role for 5-HTT in the pathogenesis of PH, making 5-HTT a potential therapeutic target.

Cross talk between endothelial and smooth muscle cells in pulmonary hypertension: critical role for serotonin-induced smooth muscle hyperplasia.

BACKGROUND: The mechanism of pulmonary artery smooth muscle cell (PA-SMC) hyperplasia in idiopathic pulmonary artery hypertension (iPH) may involve both an inherent characteristic of PA-SMCs and abnormal control by external stimuli. We investigated the role of pulmonary microvascular endothelial cells (P-ECs) in controlling PA-SMC growth. METHODS AND RESULTS: Serum-free medium of quiescent P-ECs elicited marked PA-SMC proliferation, and this effect was greater with P-ECs from patients with iPH than from control subjects and greater with PA-SMCs from these patients than from control subjects. Fluoxetine, which inhibits serotonin-induced mitogenesis by blocking the serotonin transporter, and p-chlorophenylalanine, which inhibits serotonin synthesis by blocking tryptophan hydroxylase (TPH), caused a similar 60% reduction in the growth-promoting effect of P-EC media, whereas endothelin receptor blockers had no effect. Assays of TPH activity in P-EC medium based on p-chlorophenylalanine-sensitive 5-hydroxytryptophan accumulation or serotonin determination indicated serotonin synthesis by P-ECs and an increase in this TPH-dependent process in iPH. Expression of the tph1 gene encoding the peripheral form of the TPH enzyme was increased in lungs and P-ECs from patients with iPH. Lung TPH1 immunostaining was confined to the pulmonary vessel intima. CONCLUSIONS: P-ECs produce paracrine factors governing PA-SMC growth. Serotonin, the main P-EC-derived growth factor, is overproduced in iPH and contributes to PA-SMC hyperplasia.

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.

Decreased lung fibroblast growth factor 18 and elastin in human congenital diaphragmatic hernia and animal models.

RATIONALE: Lung hypoplasia in congenital diaphragmatic hernia (CDH) seems to involve impaired alveolar septation. We hypothesized that disturbed deposition of elastin and expression of fibroblast growth factor 18 (FGF18), an elastogenesis stimulus, occurs in CDH. OBJECTIVES: To document FGF18 and elastin in human CDH and ovine surgical and rat nitrofen models and to use models to evaluate the benefit of treatments. METHODS: Human CDH and control lungs were collected post mortem. Diaphragmatic hernia was created in sheep at 85 days; fetal lungs were collected at 139 days (term = 145 days). Pregnant rats received nitrofen at 12 days; fetal lungs were collected at 21 days (term = 22 days). Some of the sheep fetuses with hernia underwent tracheal occlusion (TO); some of the nitrofen-treated pregnant rats received vitamin A. Both treatments are known to promote lung growth. MEASUREMENTS AND MAIN RESULTS: Coincidental with the onset of secondary septation, FGF18 protein increased threefold in control human lungs, which failed to occur in CDH. FGF18 labeling was found in interstitial cells of septa. Elastin staining demonstrated poor septation and markedly decreased elastin density in CDH lungs. Consistently, lung FGF18 transcripts were diminished 60 and 83% by CDH in sheep and rats, respectively, and elastin density and expression were diminished. TO and vitamin A restored FGF18 and elastin expression in sheep and rats, respectively. TO restored elastin density. CONCLUSIONS: Impaired septation in CDH is associated with decreased FGF18 expression and elastic fiber deposition. Simultaneous correction of FGF18 and elastin defects by TO and vitamin A suggests that defective elastogenesis may result, at least partly, from FGF18 deficiency.

Angiopoietin/Tie2 pathway influences smooth muscle hyperplasia in idiopathic pulmonary hypertension.

RATIONALE: Angiopoietins are involved in blood vessel maturation and remodeling. OBJECTIVES: One consequence of endothelium-specific tyrosine kinase-2 (Tie2) receptor activation by angiopoietin-1 (Ang1) is the release of endothelium-derived growth factors that recruit vascular wall cells. We investigated this process in idiopathic pulmonary arterial hypertension (iPAH). METHODS: Ang1, Ang2, and total and phosphorylated Tie2 expression (mRNA and protein) was evaluated in human lung specimens and in cultured pulmonary artery smooth muscle cells (PA-SMCs) and pulmonary endothelial cells (P-ECs) isolated from patients with iPAH and control subjects. Media collected from Ang1-treated P-ECs were assessed for their PA-SMC growth-promoting effect. MEASUREMENTS AND MAIN RESULTS: Tie2 receptor was fourfold higher in lungs and P-ECs from patients with iPAH than in those from control subjects, with a parallel increase in phosphorylated lung Tie2 receptor. In contrast, Ang1 and Ang2 expression in lungs, P-ECs, and PA-SMCs did not differ. Incubation of PA-SMCs with medium collected from P-EC cultures induced marked proliferation, and this effect was stronger when using P-ECs from patients with iPAH than from control subjects. Ang1 pretreatment of P-ECs from either patients or control subjects induced a further increase in PA-SMC proliferation. Fluoxetine, an inhibitor of the mitogenic action of serotonin, reduced the growth-promoting effect of P-EC media. Ang1 added to P-ECs from patients with iPAH increased the production of endothelin-1 (ET-1) and serotonin, but not of platelet-derived growth factor-BB or epidermal growth factor, and increased the amount of mRNA encoding tryptophan hydroxylase-1 (the rate-limiting enzyme of serotonin synthesis), preproET-1, and ET-1-converting enzyme. CONCLUSIONS: The Ang1/Tie2 pathway is potentiated in iPAH, contributing to PA-SMC hyperplasia via increased stimulation of endothelium-derived growth factors synthesis by P-ECs.

Surfactant phospholipids and proteins are increased in fetal sheep with pulmonary hypertension secondary to fetal systemic arteriovenous fistula.

To determine whether prenatal surfactant storage was altered in a model of systemic arteriovenous fistula (SAVF) with pulmonary hypertension, a fistula was created between the internal jugular vein and the carotid artery in 120-day fetal lambs, and surfactant material was explored at 134 days. Total phospholipids (TPL) and disaturated phosphatidylcholine (DSPC) were increased in whole lung tissue. Phospholipid analysis of isolated lamellar body fraction evidenced a specific increase of surfactant pool size: TPL and DSPC in this fraction were enhanced 1.9 and 2.9 times, respectively, when referred to DNA. Although the steady-state level of transcripts of surfactant protein (SP)-A and SP-B was not found to be changed at the time of death, semiquantitative Western blot analysis revealed elevated SP-A and SP-B protein contents three- and twofold, respectively. These findings indicate markedly enhanced accumulation of surfactant material in the presence of surgically induced prenatal pulmonary hypertension. Although total lung cell number was increased by 26%, SP-B immunolabeling indicated that increased surfactant amount did not result from an increased alveolar type II cell proportion, but rather from an increased rate of storage. Whether similar changes in surfactant are encountered in human neonates with persistent pulmonary hypertension is worthy of investigation.

FGF-18 is upregulated in the postnatal rat lung and enhances elastogenesis in myofibroblasts.

The fibroblast growth factors (FGFs) are key players in fetal lung development, but little is known about their status in postnatal lung. Here, we investigated the expression pattern of FGF-18 transcripts through the perinatal period and evidenced a sevenfold increase after birth that paralleled changes in elastin expression. In vitro, recombinant human (rh)FGF-18 had a mitogenic activity on day 21 fetal rat lung fibroblasts and stimulated its own expression in the latter, whereas FGF-2 inhibited it. At 50 or 100 ng/ml, rhFGF-18 increased the expression of alpha-smooth muscle actin (alpha-SMA; 2.5-fold), a characteristic marker of myofibroblasts, of tropoelastin (6.5-fold), of lysyl oxidase (2-fold), and of fibulins 1 and 5 (8- and 2.2-fold) in confluent fibroblasts isolated from fetal day 21 lung; similar results were obtained with fibroblasts from day 3 postnatal lungs. Elastin protein expression was also slightly increased in fetal fibroblasts. Lung analysis on day 4 in rat pups that had received rhFGF-18 (3 microg) on days 0 and 1 showed a 1.7-fold increase of tropoelastin transcripts, whereas alpha-SMA transcripts were unchanged. In contrast, rhFGF-2 markedly decreased expression of elastin in vitro and in vivo and of fibulin 5 in vitro. In addition, vitamin A, which is known to enhance alveolar development, elevated FGF-18 and elastin expressions in day 2 lungs, thus advancing the biological increase. We postulate that FGF-18 is involved in postnatal lung development through stimulating myofibroblast proliferation and differentiation.

Expression of surfactant proteins and thyroid transcription factor 1 in an ovine model of congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: The question of delayed lung maturation in congenital diaphragmatic hernia (CDH) is pending. Data about surfactant proteins (SPs) are sparse in human fetuses and discrepant in the ovine CDH model. The purpose of this study was to investigate, in the ovine surgically created CDH model, the expression of SPs and of thyroid transcription factor 1 (TTF-1), a key regulator of lung development that also controls the expression of surfactant proteins. METHODS: Diaphragmatic hernia (DH) was created surgically in lamb fetuses on day 85 of gestation. On day 139, 5 DH and 6 control fetuses were retrieved by cesarean section. The mRNA levels for SPs and TTF-1 were determined by Northern blot analysis; SP-A and SP-B protein levels were assessed by Western blot analysis. RESULTS: In DH lungs, SP-A, SP-B, and SP-C messenger RNAs were diminished by 82%, 67%, and 32%, respectively, compared with control level. SP-A and SP-B protein amounts were decreased consistently. TTF-1 expression was not altered in the surgical model. CONCLUSIONS: SP's deficiency appears to be a common feature of the various CDH models. By contrast with the nitrofen model, TTF-1 expression was not altered in the surgical model indicating different underlying molecular mechanisms in both models.

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.

Maturational factors modulate transcription factors CCAAT/enhancer-binding proteins alpha, beta, delta, and peroxisome proliferator-activated receptor-gamma in fetal rat lung epithelial cells.

Previous investigations have evidenced the importance of CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor (PPAR)gamma for lung development, especially for alveolar type II cells (ATII). This prompted us to explore whether ATII maturation-promoting mediators controlled their expression in isolated ATII. In whole rat lung, C/EBPalpha, beta, delta, and PPARgamma mRNAs increased 3-5 times between gestational day 18 and term (Day 22), dropped around birth, then reincreased. C/EBPbeta and delta, but not PPARgamma, displayed similar profile in isolated ATII; C/EBPalpha transcript disappeared and the protein became hardly detectable in isolated cells. In cultured ATII, dexamethasone increased C/EBPbeta and PPARgamma mRNAs 2-4 times, and cyclic AMP increased C/EBPbeta and delta mRNAs approximately 1.5 times. Whereas retinoic acid increased C/EBPbeta and PPARgamma mRNAs 1.5 times in ATII in vitro, vitamin-A deficiency strongly decreased fetal lung C/EBPalpha, beta, and PPARgamma transcripts in vivo. C/EBPbeta, delta, and PPARgamma mRNAs were also increased in vitro by epidermal growth factor and keratinocyte growth factor, whereas they were unchanged by the maturation inhibitor transforming growth factor-beta. C/EBPalpha expression was not reinduced by any mediator. Changes in transcripts were reflected in protein levels analyzed through Western blotting. These results argue for a role of these factors in ATII functional maturation, and indicate a multifactorial control of their ontogeny.

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.