A critical role for fibroblast growth factor-7 during early alveolar formation in the neonatal rat.

Mesenchymal cell-derived FGF-7 (fibroblast growth factor-7) induces proliferation in both epithelial and endothelial cells. We found FGF-7 to be expressed in the lungs of neonatal rats from birth to d 14 of age. A role for FGF-7 in early postnatal lung growth and alveolar formation, by an action on type II pneumocytes, has been excluded by the work of others. However, a role through an action of FGF-7 on other cell types has not been excluded. We used intraperitoneal injections of neutralizing antibodies on d 3, 4, and 5 of life to inhibit binding of FGF-7 to its receptors, and assessed alveolar formation on d 6 of life. This intervention inhibited DNA synthesis in, and number of, alveoli-forming secondary crests, resulting in a significantly reduced alveolar number. This failure of alveolar formation was associated with a reduction in the number of small blood vessels in the lung periphery. We conclude that FGF-7, most likely through its effect on the vascular bed, is required for normal early postnatal alveolar formation from secondary crests.

8-Isoprostane-induced endothelin-1 production by infant rat pulmonary artery smooth muscle cells is mediated by Rho-kinase.

We have reported that 8-isoprostane stimulated the production of endothelin (ET)-1, a potent vasoconstrictor and critical mediator of chronic pulmonary hypertension, by infant rat pulmonary artery smooth muscle cells (PASMCs), through stimulation of the thromboxane A2 receptor. The aim of this study was to examine the contribution of putative downstream intracellular mediators of thromboxane A2 receptor stimulation to this effect. PASMCs from infant rats were treated with calcium ionophore (A23187), 8-isoprostane, or 8-isoprostane together with inhibitors of tyrosine kinase, protein kinase C, phosphatidylinositol 3-kinase, mitogen-activated protein kinases, or Rho-kinases (ROCK). A23187 had no effect on ET-1 production, excluding raised intracellular Ca2+ as a major contributor. Increased ET-1 production induced by 8-isoprostane was significantly attenuated by the ROCK inhibitors Y-27632 and hydroxyfasudil, but not by inhibitors of the other pathways. 8-Isoprostane also increased membrane binding of RhoA, a major determinant of ROCK activity, and ROCK-II expression through the protein kinase C pathway. These data indicate that the RhoA/ROCK pathway mediates increased ET-1 production by PASMCs, which we speculate may at least partly explain the beneficial effects of both antioxidants and ROCK inhibitors in animal models of chronic pulmonary hypertension.

Hepatocyte growth factor is required for alveologenesis in the neonatal rat.

RATIONALE: Our core hypothesis is that growth factors that have dysregulated expression during experimental neonatal lung injury are likely to be involved in normal postnatal lung growth and alveologenesis. OBJECTIVES: To determine if hepatocyte growth factor (HGF) is upregulated in neonatal lung injury and is essential for postnatal alveologenesis. METHODS: A neonatal lung injury, in which there were patchy areas of interstitial thickening with a relative increase in the proportion of epithelial cells, was induced in newborn rats by exposing them to 60% oxygen for 14 days. Air-exposed pups had binding of endogenous HGF to its natural receptor, c-Met, inhibited by the intraperitoneal injection of either neutralizing antibody to HGF, or a truncated soluble c-Met receptor. MEASUREMENTS AND MAIN RESULTS: The 60% oxygen-mediated lung injury was associated with increased lung mRNAs for hepatocyte growth factor and c-Met, relative to air-exposed control lungs, at Day 7 after birth. After exposure to 60% oxygen, immunoreactive HGF was increased at Days 4 and 7, and immunoreactive c-Met was increased at Day 14. In air-exposed pups, intraperitoneal injections of neutralizing antibody to HGF inhibited DNA synthesis in alveoli-forming secondary crests, and reduced the number of alveoli in 6-day-old pups. Intraperitoneal injections of a truncated soluble c-Met receptor inhibited DNA synthesis in secondary crests in 4-day-old air-exposed rat pups. CONCLUSIONS: HGF and its c-Met receptor are required for normal postnatal alveolar formation from secondary crests, and are upregulated during 60% oxygen-induced neonatal lung injury.

Fibroblast growth factor receptor-1 and neonatal compensatory lung growth after exposure to 95% oxygen.

Neonatal rats exposed to 95% oxygen (O2) for 7 days from birth had inhibited lung growth, DNA synthesis, and secondary septation. These parameters were rapidly restored by a period of recovery in air. Northern and Western blot analysis and immunohistochemistry were used to screen for the fibroblast growth factor receptor-1 (FGF-R1) and its high affinity ligand, basic fibroblast growth factor (bFGF), which could have a role in this recovery process. Expression of bFGF in the lung was significantly reduced at the end of the 7-day exposure to 95% O2 and was increased after 3 days of recovery in air. Expression of FGF-R1 was not affected by exposure to 95% O2 or recovery in air. We hypothesized that the increase in bFGF after removal from 95% O2, acting through the FGF-R1, would be critical for compensatory growth. Intraperitoneal injection of soluble truncated FGF-R1 at the onset of the recovery phase arrested compensatory lung DNA synthesis and secondary septation seen in control animals after 3 days of recovery, confirming a role for FGF-R1 in this model of compensatory neonatal lung growth.

Transfection of lung cells in vitro and in vivo: effect of antioxidants and intraliposomal bFGF.

We hypothesized that constitutive formation of reactive oxygen species by respiratory cells is a barrier to gene transfer when liposome-DNA complexes are used, by contributing to rapid degradation of plasmid DNA. When plasmid DNA is complexed to liposomes it is protected against H(2)O(2)-mediated degradation but not that mediated by the hydroxyl radical. Treatment of distal rat fetal lung epithelial cells (RFL(19)Ep) with the vitamin E analog Trolox (50 microM) reduced intracellular plasmid degradation. Both Trolox (50 microM) and an iron chelator, phenanthroline (0.1 microM), significantly increased transgene expression in RFL(19)Ep approximately twofold, consistent with a hydroxyl radical-mediated inhibition of transgene expression. When basic fibroblast growth factor (bFGF; 20 ng/ml), a growth factor with antioxidant properties, was included within liposomes, we observed a significantly greater enhancement of RFL(19)Ep transgene expression (approximately 2-fold) over that seen with Trolox or phenanthroline. Inclusion of bFGF within liposomes also significantly enhanced (approximately 4-fold) transgene expression in mice following intratracheal instillation.

A role for platelet-derived growth factor-BB in rat postpneumonectomy compensatory lung growth.

Unilateral pneumonectomy leads to compensatory growth in the residual lung, the mediators of which are largely unknown. We hypothesized, based on its other known roles in lung cell growth, that platelet-derived growth factor (PDGF)-BB would be an essential mediator of postpneumonectomy compensatory lung growth. Left-sided pneumonectomies were performed on 21-d-old rats, for comparison with sham-operated or unoperated control animals. Body weights were not different between groups. Right lung weights and DNA content were significantly increased (p < 0.05), compared with controls, by 10 d after pneumonectomy. The rate of DNA synthesis was maximal on d 5 postpneumonectomy. Total right lung PDGF-B mRNA and PDGF-BB protein increased after pneumonectomy, but were apparently tightly regulated, relative to total right lung beta-actin mRNA and protein content, respectively. However, PDGF-BB expression after pneumonectomy was apparently not purely constitutive, in that daily i.p. injections of a truncated soluble PDGF beta-receptor both reduced activation of the native PDGF beta-receptor, and attenuated increased lung DNA synthesis on d 3 after pneumonectomy. These findings are consistent with a critical role for PDGF-BB in postpneumonectomy lung growth.