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.

Abstract-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 ϩ mice showed that the transgene was expressed predominantly in SMCs of pulmonary and systemic vessels. Compared with wild-type mice, SM22-5-HTT ϩ 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 ϩ mice exhibited PH, with marked increases in right ventricular systolic pressure (RVSP), right ventricle/left ventricleϩseptum 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 ϩ mice. Compared with wild-type mice, SM22-5-HTT ϩ 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.

Keratinocyte growth factor (KGF) or fibroblast growth factor (FGF)-7, a peptide produced by stromal cells and in particular by lung mesenchyme, has recently been shown to influence early lung morphogenesis and to be a mitogen for fetal and adult alveolar type II cells. Although contradictory findings have been reported regarding its effects on surfactant protein expression, its effects on surfactant phospholipids have not been studied. We investigated the effects of KGF on the synthesis of surfactant components by cultured fetal rat type II cells isolated during the late gestational period, when surfactant accumulates in preparation for extrauterine life. We show that KGF is a potent stimulus of surfactant phospholipid synthesis, particularly for the major component of surfactant, disaturated phosphatidylcholine (DSPC). KGF increased choline incorporation into DSPC in a dose-dependent manner up to 25 ng/ml (1.3 x 10(-9) M), and this effect was greater for surfactant than for nonsurfactant DSPC. KGF was several times more potent in this respect than acidic FGF at the same molar concentration. KGF, similar to epidermal growth factor, also stimulated acetate incorporation and increased the surfactant phospholipid and DSPC content of cultured cells twofold. These effects correlated with increased choline phosphate cytidylyltransferase activity and increased fatty acid synthase activity and gene expression. KGF also induced a dose-dependent stimulation of surfactant protein-A, -B, and -C gene expression, leading to a 2- to 3-fold increase in their messenger RNAs. KGF therefore stimulates the synthesis of all surfactant components in developing type II cells at the time of surfactant accumulation. Its secretion by lung fibroblasts may thus be an important factor in promoting the maturation of fetal lung epithelium and the synthesis of sufficient surfactant. The results suggest that KGF could provide a new therapeutic agent for the management of the immature or injured lung.

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.

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.

Congenital diaphragmatic hernia (CDH) is a major cause of refractory respiratory failure in the newborn. Besides pulmonary hypoplasia, the pathophysiology of CDH also includes surfactant deficiency. Vitamin A (vit A) is important for various aspects of lung development. We hypothesized that antenatal treatment with vit A would stimulate lung surfactant synthesis in experimental CDH induced in rats by maternal ingestion of the herbicide nitrofen (2,4-dichloro-phenyl-p-nitrophenyl-ether) on Day 12. Fetuses were assigned to six experimental groups: (1) controls from rats that received olive oil, the vehicle; (2) fetuses from rats that received olive oil on Day 12 and vit A orally (15,000 IU) on Day 14; (3) nitrofen (N)-exposed fetuses without diaphragmatic hernia (N/no DH); (4) N/no DH from rats given vit A on Day 14; (5 ) nitrofen-exposed fetuses with DH (N/+DH); (6) N/+DH from rats given vit A on Day 14. Fetuses were delivered by C-section at Day 21. Lung DNA content was lowered in the nitrofen group as compared with the controls group, but increased by subsequent vit A treatment. Lung surfactant disaturated phosphatidylcholine was reduced in the N/+DH group and restored to control level by vit A. The expression level of surfactant proteins (SP) -A and -C was decreased in vit A-treated control rats and in nitrofen-exposed fetuses with or without DH. Vit A restored SP-A and -C mRNA expression to control levels in N/+DH. SP-B expression was lowered in N/no DH and increased by vit A in this group. The proportion of type II cells assessed by SP-B immunolabeling was lowered in N/+DH and restored by vit A treatment. We conclude that antenatal treatment with vit A restores lung maturation in nitrofen-induced hypoplastic lungs with CDH. These findings point out vit A as a potential therapeutical agent for correcting surfactant deficiency in CDH.

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.

Serotonin [5-hydroxytryptamine (5-HT)] biosynthesis depends on two rate-limiting tryptophan hydroxylases (Tph): Tph1, which is expressed in peripheral organs, and Tph2, which is expressed in neurons. Because 5-HT is involved in pulmonary hypertension (PH), we investigated whether genetic variations in Tph1 and/or Tph2 affected PH development in mice. To examine the functional impact of peripheral Tph1 deficiency on hypoxic PH, we used Tph1(-/-) mice characterized by very low 5-HT synthesis rates and contents in the gut and lung and increased 5-HT synthesis in the forebrain. With chronic hypoxia, 5-HT synthesis in the forebrain increased further. Hypoxic PH, right ventricular hypertrophy, and distal pulmonary artery muscularization were less severe (P < 0.001) than in wild-type controls. The Tph inhibitor p-chlorophenylalanine (100 mgxkg(-1)xday(-1)) further improved these parameters. We then investigated whether mouse strains harboring the C1473G polymorphism of the Tph2 gene showed different PH phenotypes during hypoxia. Forebrain Tph activity was greater and hypoxic PH was more severe in C57Bl/6 and 129X1/SvJ mice homozygous for the 1473C allele than in DBA/2 and BALB/cJ mice homozygous for the 1473G allele. p-Chlorophenylalanine reduced PH in all groups and abolished the difference in PH severity across mouse strains. Hypoxia increased 5-hydroxytryptophan accumulation but decreased 5-HT contents in the forebrain and lung, suggesting accelerated 5-HT turnover during hypoxia. These results provide evidence that dysregulation of 5-HT synthesis is closely linked to the hypoxic PH phenotype in mice and that Tph1 and Tph2 may contribute to PH development.