Developmental regulation of NO-mediated VEGF-induced effects in the lung

Abstract

Vascular endothelial growth factor (VEGF) is known to have a pivotal role in lung development and in a variety of pathologic conditions in the adult lung. Our earlier studies have shown that NO is a critical mediator of VEGF-induced vascular and extravascular effects in the adult murine lung. As significant differences have been reported in the cytokine responses in the adult versus the neonatal lung, we hypothesized that there may be significant differences in VEGF-induced alterations in the developing as opposed to the mature lung. Furthermore, nitric oxide (NO) mediation of these VEGF-induced effects may be developmentally regulated. Using a novel externally regulatable lung-targeted transgenic murine model, we found that VEGF-induced pulmonary hemorrhage was mediated by NO-dependent mechanisms in adults and newborns. VEGF enhanced surfactant production in adults as well as increased surfactant and lung development in newborns, via an NO-independent mechanism. While the enhanced survival in hyperoxia in the adult was partly NO-dependent, there was enhanced hyperoxia-induced lung injury in the newborn. In addition, human amniotic fluid VEGF levels correlated positively with surfactant phospholipids. Tracheal aspirate VEGF levels had an initial spike, followed by a decline, and then a subsequent rise, in human neonates with an outcome of bronchopulmonary dysplasia or death. Our data show that VEGF can have injurious as well as potentially beneficial developmental effects, of which some are NO dependent, others NO independent. This opens up the possibility of selective manipulation of any VEGF-based intervention using NO inhibitors for maximal potential clinical benefit.

Figure 1.

Effect of vascular endothelial growth factor (VEGF) on nitric oxide (NO) synthase (NOS) isoforms in the newborn (NB) lung. Expression of mRNA for NOS enzymes (iNOS, eNOS, nNOS) in NB VEGF transgenic (TG) mice lungs (A). These were confirmed with real-time RT-PCR (B–D). Mice were on dox water for 2 days. The figure is representative of assessments in a minimum of four mice. Open bars, NB WT; solid bars, NB VEGF. ^#P = 0.02; ^##P < 0.05.

Figure 2.

Role of NO in VEGF-induced pulmonary hemosiderosis in adult and NB lungs. NO-inhibition (with l-NAME) led to decreased pulmonary hemosiderosis (arrows point to hemosiderin-laden macrophages stained blue) in the adult (A and B) and NB (PN7) (C and D) lungs. The noted values represent assessments in a minimum of four mice. Mice were on dox water and/or l-NAME as described in Materials and Methods. *P ≤ 0.0001, **P ≤ 0.01.

Figure 3.

Role of NO in VEGF-induced pulmonary endothelial permeability in NB mice and survival in hyperoxia in adult mice. (A) NO inhibition (with l-NAME) abrogated the increased VEGF-induced lung wet/dry weight ratios in NB VEGF TG mice. (B) NO inhibition (with l-NAME) abrogated the increased VEGF-induced survival in hyperoxia in adult VEGF TG mice. NO-inhibition (with l-NAME) was associated with abrogation of VEGF-induced A1 mRNA expression (C). NB mice were exposed to dox water and/or l-NAME for 1 week via the transmammary route. Adult mice were on dox water and/or l-NAME for 2 weeks. The data are representative of experiments conducted in a minimum of four mice. Open squares, WT + DOX; solid squares, WT + DOX + l-NAME; open triangles, VEGF + DOX; solid triangles, VEGF + DOX + l-NAME. ^#P < 0.03, ^##P < 0.05, **P ≤ 0.01.

Figure 4.

Effect of VEGF on survival in hyperoxia and hyperoxia-induced acute lung injury (HALI) in NB mice. In contrast to the adult, the NB had no benefit to survival in hyperoxia (A), but had evidence of increased HALI by histopathology (B), 8-OhDG (C), and TUNEL staining (D and E) compared with littermate controls (all at PN7). Mice were on dox water for 1 week. The data are representative of experiments conducted in a minimum of four mice. **P ≤ 0.01.

Figure 5.

Role of NO in VEGF-induced surfactant proteins in the adult lung. VEGF-induced increased expression of surfactant protein (SP)-B and SP-C mRNA (A) or proteins (B) (arrows point to positive staining in the Type II cells; upper panel: SP-B, lower panel: SP-C) were not altered with NO inhibition (with l-NAME) (mRNA for [C] SP-B and -C and [D] SP-B protein). Mice were on dox water and/or l-NAME for 2 weeks. The data are representative of experiments conducted in a minimum of four mice.

Figure 6.

Role of NO in VEGF-induced maturation in the NB lung (PN7). VEGF induced the increased expression of SP-B and SP-C (A) mRNA and (B) protein. (C) VEGF-induced lungs had thinner alveolar walls, increased alveolar space, and decreased intervening mesenchyme. The maturational effects on SP-B and SP-C mRNA expression (D) were not altered with l-NAME treatment in the presence of VEGF, and these were confirmed with real-time RT-PCR (E and F). Similarly, the effects on the lung parenchyma (G) with l-NAME treatment in the presence of VEGF were unchanged. Mice were on dox water and/or l-NAME for 1 week. The data are representative of experiments conducted in a minimum of four mice. *P < 0.0001; **P < 0.01, ^#P < 0.02.

Figure 6.

Role of NO in VEGF-induced maturation in the NB lung (PN7). VEGF induced the increased expression of SP-B and SP-C (A) mRNA and (B) protein. (C) VEGF-induced lungs had thinner alveolar walls, increased alveolar space, and decreased intervening mesenchyme. The maturational effects on SP-B and SP-C mRNA expression (D) were not altered with l-NAME treatment in the presence of VEGF, and these were confirmed with real-time RT-PCR (E and F). Similarly, the effects on the lung parenchyma (G) with l-NAME treatment in the presence of VEGF were unchanged. Mice were on dox water and/or l-NAME for 1 week. The data are representative of experiments conducted in a minimum of four mice. *P < 0.0001; **P < 0.01, ^#P < 0.02.

Figure 6.

Role of NO in VEGF-induced maturation in the NB lung (PN7). VEGF induced the increased expression of SP-B and SP-C (A) mRNA and (B) protein. (C) VEGF-induced lungs had thinner alveolar walls, increased alveolar space, and decreased intervening mesenchyme. The maturational effects on SP-B and SP-C mRNA expression (D) were not altered with l-NAME treatment in the presence of VEGF, and these were confirmed with real-time RT-PCR (E and F). Similarly, the effects on the lung parenchyma (G) with l-NAME treatment in the presence of VEGF were unchanged. Mice were on dox water and/or l-NAME for 1 week. The data are representative of experiments conducted in a minimum of four mice. *P < 0.0001; **P < 0.01, ^#P < 0.02.

Figure 7.

Effect of NOS inhibition on VEGF production. l-NAME treatment of VEGFTG+ mice in the adult (A) or newborn (B) lung did not alter BAL hVEGF levels. Mice were on dox water and/or l-NAME as described in Materials and Methods. The data are representative of experiments conducted in a minimum of four mice. *P ≤ 0.0001; **P ≤ 0.01.

Figure 8.

VEGF levels in human amniotic fluid (AF) and tracheal aspirate (TA). AF samples from mothers with infants in late gestation (≥ 34 wk) with a lecithin/sphingomyelin (L/S) ratio of less than 3.5 (n = 58). Presence of PG (“0” absent, “1” present) correlated strongly with L/S ratios (R = 0.55, P < 0.0001) (A). There was a significant positive correlation of L/S ratios with AF VEGF levels (R = 0.26, P < 0.03) (B). TA VEGF levels (C) from premature babies with RDS with (n = 9; solid bars) and without (n = 7; open bars) an adverse outcome (BPD /Death). The noted values represent assessments in a minimum of four infants at each time point. **P ≤ 0.01; ^#P < 0.02; ^##P ≤ 0.05.