Role of Nitric Oxide Isoforms in Vascular and Alveolar Development and Lung Injury in Vascular Endothelial Growth Factor Overexpressing Neonatal Mice Lungs

<div><p>Background</p><p>The role of vascular endothelial growth factor (VEGF)-induced 3 different nitric oxide synthase (NOS) isoforms in lung development and injury in the newborn (NB) lung are not known. We hypothesized that VEGF-induced specific NOS pathways are critical regulators of lung development and injury.</p><p>Methodology</p><p>We studied NB wild type (WT), lung epithelial cell-targeted VEGF165 doxycycline-inducible overexpressing transgenic (VEGFTG), VEGFTG treated with a NOS1 inhibitor (L-NIO), VEGFTG x NOS2^-/- and VEGFTG x NOS3^+/- mice in room air (RA) for 7 postnatal (PN) days. Lung morphometry (chord length), vascular markers (Ang1, Ang2, Notch2, vWF, CD31 and VE-cadherin), cell proliferation (Ki67), vascular permeability, injury and oxidative stress markers (hemosiderin, nitrotyrosine and 8-OHdG) were evaluated.</p><p>Results</p><p>VEGF overexpression in RA led to increased chord length and vascular markers at PN7, which were significantly decreased to control values in VEGFTG x NOS2^−/− and VEGFTG x NOS3^+/- lungs. However, we found no noticeable effect on chord length and vascular markers in the VEGFTG / NOS1 inhibited group. In the NB VEGFTG mouse model, we found VEGF-induced vascular permeability in the NB murine lung was partially dependent on NOS2 and NOS3-signaling pathways. In addition, the inhibition of NOS2 and NOS3 resulted in a significant decrease in VEGF-induced hemosiderin, nitrotyrosine- and 8-OHdG positive cells at PN7. NOS1 inhibition had no significant effect.</p><p>Conclusion</p><p>Our data showed that the complete absence of NOS2 and partial deficiency of NOS3 confers protection against VEGF-induced pathologic lung vascular and alveolar developmental changes, as well as injury markers. Inhibition of NOS1 does not have any modulating role on VEGF-induced changes in the NB lung. Overall, our data suggests that there is a significant differential regulation in the NOS-mediated effects of VEGF overexpression in the developing mouse lung.</p></div

Effect of VEGF overexpression on pulmonary phenotype and caspase 3 mRNA expression in the BPD murine model.

<p>NB WT and VEGF TG+ mice were exposed to room air or hyperoxia to induce the murine model of BPD (hyperoxia from PN1-4; room air from PN5-PN14) and were killed on PN14. All received DOX water from PN day 5 to 14. (<b>A</b>) Representative microphotographs from H&E-stained lung sections showed alveolar size, as confirmed by chord length measurements (<b>B</b>), demonstrated the increased simplification in the NB VEGF TG+ mice as compared to VEGF TG- (WT) in hyperoxia-induced murine model of BPD. (<b>C</b>) The mRNA expression of caspase 3 was also increased in VEGF TG+ lung in NB BPD lungs. Each bar represents the mean ± SEM for a minimum of four animals. Each bar represents the mean ± SEM for a minimum of four animals. *<i>P</i> < 0.05, **<i>P</i> ≤ 0.01 and ***<i>P</i> ≤ 0.001, 2-way ANOVA followed by Tukey test. VEGF: vascular endothelial growth factor; BPD: bronchopulmonary dysplasia; NB: newborn; WT: wild type; NOS: nitric oxide synthase; TG+: transgene positive; TG-: transgene negative; PN: postnatal; DOX: doxycycline.</p

Role of NOS isoforms in VEGF overexpressed lung phenotype.

<p>VEGF TG+ and VEGF TG- mice were received DOX water from PN day1 to 7. (<b>A</b>) NB lungs from a VEGF TG+ showed increased NOS1, NOS2 and NOS3 staining of the endothelial and inflammatory cells was observed in the PN7 lung, compared with control lung samples. The figures are illustrative of a minimum of 5 animals in each group. (<b>B</b> and <b>C</b>) Alveolar size, as measured by chord length, confirmed features noted on lung histology in VEGF TG-, VEGF TG+, VEGF-TG/ NOS2^−/− (<b>D</b> and <b>E</b>) Similarly, chord length of VEGF TG-, VEGF TG+ and VEGF-TG/ NOS3^+/− and (<b>F</b>) chord lengths of VEGF TG+/LNIO groups. Each bar represents the mean ± SEM for a minimum of four animals, *<i>P</i> < 0.05 and **<i>P</i> ≤ 0.01, 2-way ANOVA followed by Tukey test. NOS: nitric oxide synthase; VEGF: vascular endothelial growth factor; TG-: transgene negative; TG+: transgene positive; DOX: doxycycline; NB: newborn; PN: postnatal.</p

Role of NOS isoforms in VEGF overexpressed lung cell proliferation.

<p>(<b>A-C</b>) Representative examples of Ki67 (brown) staining in lungs from 7-day-old VEGF TG-, VEGF TG+, VEGF-TG/ NOS2^−/− and VEGF-TG/ NOS3^+/−. All received DOX water from PN day1 to 7. Decreased percentage of Ki67 staining area in VEGF TG+, which is increased in VEGF-TG/ NOS2^−/− and VEGF-TG/ NOS3^+/− lungs. Each bar represents the mean ± SEM for a minimum of three animals. *<i>P</i> < 0.05, **<i>P</i> ≤ 0.01, and ****<i>P</i> ≤ 0.0001, 2-way ANOVA followed by Tukey test. NOS: nitric oxide synthase; VEGF: vascular endothelial growth factor; TG-: transgene negative; TG+: transgene positive; DOX: doxycycline; PN: postnatal.</p

Effect of VEGF overexpression on cathepsins L and H mRNA expression in the BPD murine model.

<p>NB WT and VEGF TG+ mice were exposed to room air or hyperoxia to induce the murine model of BPD (hyperoxia from PN1-4; room air from PN5-PN14) and were killed on PN14. All received DOX water from PN day 5 to 14. (<b>A</b>) Cathepsin L mRNA expression in lung tissue was decreased in VEGF TG+ mice as compared to VEGF TG- mice, in the BPD mice model. (<b>B</b>) Cathepsin H mRNA expression in lung tissue was increased in VEGF TG+ mice as compared to VEGF TG- mice, in the BPD mice model. Each bar represents the mean ± SEM for a minimum of four animals. **<i>P</i> ≤ 0.01 and ***<i>P</i> ≤ 0.001, 2-way ANOVA followed by Tukey test. VEGF: vascular endothelial growth factor; BPD: bronchopulmonary dysplasia; NB: newborn; WT: wild type; NOS: nitric oxide synthase; TG+: transgene positive; TG-: transgene negative; PN: postnatal; DOX: doxycycline.</p

Role of NOS isoforms in VEGF overexpressed lung on angiogenic markers.

<p>NB VEGF TG-, VEGF TG+ and VEGF-TG/ NOS3^+/− mice were sacrificed at PN7. All received DOX water from PN day1 to 7. (<b>A</b> and <b>B</b>) Ang1 and NOTCH2 proteins, with β-actin as controls, were detected by western blotting and analyzed by densitometry. (<b>C</b>) Ang1 and NOTCH2 proteins, with β-actin were detected by western blotting (and densitometry) in L-NIO (NOS1 inhibitor) treated VEGF TG mice. (<b>D—F</b>) Protein levels (Western blot and densitometry) of Collagen IV, VE-cadherin, Ang2, NOS1, NOS2 and NOS3 are performed in indicated groups of PN7 mouse lungs. The figure is representative of n = 3 mice per group, *<i>P</i> < 0.05, **<i>P</i> ≤ 0.01, ***<i>P</i> ≤ 0.001 and ****<i>P</i> ≤ 0.0001, 2-way ANOVA followed by Tukey test. NOS: nitric oxide synthase; VEGF: vascular endothelial growth factor; NB: newborn; TG-: transgene negative; TG+: transgene positive; PN: postnatal; DOX: doxycycline; Ang: angiopoietin.</p

Role of NOS isoforms in VEGF overexpressed lung on BAL protein levels and endothelial barrier protein (claudin 1) expression.

<p>(<b>A</b>) BAL fluid was isolated from PN7 mouse lungs and protein content was measured. (<b>B</b> and <b>C</b>) Claudin 1 expression was decreased in PN7 VEGF TG+ mice lung as compared to control lungs. Inhibition of NOS2 and NOS3 in the presence of VEGF TG restored the claudin 1 protein expression levels. Each bar represents the mean ± SEM for a minimum of four animals. *<i>P</i> < 0.05, **<i>P</i> ≤ 0.01, ***<i>P</i> ≤ 0.001 and ****<i>P</i> ≤ 0.0001, 2-way ANOVA followed by Tukey test. NOS: nitric oxide synthase; VEGF: vascular endothelial growth factor; BAL: bronchoalveolar; TG+: transgene positive.</p

Role of NOS isoforms in VEGF overexpressed lung on oxidative stress.

<p>(<b>A</b>) PN7 lungs were removed from indicated groups, fixed, stained to allow identification of hemosiderin-laden macrophages. Hemosiderin-laden activated macrophages accumulate in VEGF TG+ PN7 mice lungs, which were significantly reduced in VEGF-TG/ NOS2^−/−, VEGF-TG/ NOS3^+/− and NOS1 inhibited mice lungs. (<b>B</b>) Representative examples of nitrotyrosine staining and quantitation from lung of 7-day-old VEGF TG-, VEGF TG+, VEGF-TG/ NOS2^−/− and VEGF-TG/ NOS3^+/− mice exposed to DOX water from PN1-PN7. (<b>C</b>) Representative examples of staining and quantitation of lung 8-OHdG in VEGF TG+ PN7 newborn mice, which is significantly decreased in VEGF-TG+/ NOS2^−/− and VEGF-TG+/ NOS3^−/− lungs. All immunohistochemistry images were quantified by NIH image J software. Each bar represents the mean ± SEM for a minimum of 3 animals. **<i>P</i> ≤ 0.01 and ****<i>P</i> ≤ 0.0001, 2-way ANOVA followed by Tukey test. NOS: nitric oxide synthase; VEGF: vascular endothelial growth factor; TG-: transgene negative; TG+: transgene positive; PN: postnatal; DOX: doxycycline; 8-OHdG: 8-hydroxy-2'-deoxyguanosine.</p

Role of NOS isoforms in VEGF overexpressed lung on the vascular markers CD31 and vWF.

<p>VEGF TG-, VEGF TG+, VEGF-TG/ NOS2^−/− and VEGF-TG/ NOS3^+/− mice exposed to DOX water from PN1-PN7. (<b>A</b> and <b>B</b>) Increased expression of lung CD31 in VEGF TG+ PN7 newborn mice, which was significantly decreased in VEGF-TG+/ NOS2^−/− and VEGF-TG/ NOS3^+/− (<b>C</b> and <b>D</b>) mice lungs. Similarly another vascular marker vWF showed decreased staining VEGF-TG+/ NOS2^−/− (<b>E</b> and <b>F</b>) and VEGF-TG/ NOS3^+/− (<b>G</b> and <b>H</b>) mice lungs as compared to VEGF TG+ lungs. (<b>I and J</b>) Increased expression of lung vWF and CD31 in VEGF TG+ PN7 newborn mice, with no significant change in L-NIO treated VEGF-TG+ animals. Each bar represents the mean ± SEM for a minimum of four animals. **<i>P</i> ≤ 0.01, ***<i>P</i> ≤ 0.001, ****<i>P</i> ≤ 0.0001, 2-way ANOVA followed by Tukey test. NOS: nitric oxide synthase; VEGF: vascular endothelial growth factor; TG-: transgene negative; TG+: transgene positive; DOX: doxycycline; PN: postnatal; vWF: von Willebrand factor.</p

A Critical Regulatory Role for Macrophage Migration Inhibitory Factor in Hyperoxia-Induced Injury in the Developing Murine Lung

<div><p>Background</p><p>The role and mechanism of action of MIF in hyperoxia-induced acute lung injury (HALI) in the newborn lung are not known. We hypothesized that MIF is a critical regulatory molecule in HALI in the developing lung.</p><p>Methodology</p><p>We studied newborn wild type (WT), MIF knockout (MIFKO), and MIF lung transgenic (MIFTG) mice in room air and hyperoxia exposure for 7 postnatal (PN) days. Lung morphometry was performed and mRNA and protein expression of vascular mediators were analyzed.</p><p>Results</p><p>MIF mRNA and protein expression were significantly increased in WT lungs at PN7 of hyperoxia exposure. The pattern of expression of Angiopoietin 2 protein (in MIFKO>WT>MIFTG) was similar to the mortality pattern (MIFKO>WT>MIFTG) in hyperoxia at PN7. In room air, MIFKO and MIFTG had modest but significant increases in chord length, compared to WT. This was associated with decreased expression of Angiopoietin 1 and Tie 2 proteins in the MIFKO and MIFTG, as compared to the WT control lungs in room air. However, on hyperoxia exposure, while the chord length was increased from their respective room air controls, there were no differences between the 3 genotypes.</p><p>Conclusion</p><p>These data point to the potential roles of Angiopoietins 1, 2 and their receptor Tie2 in the MIF-regulated response in room air and upon hyperoxia exposure in the neonatal lung.</p></div