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

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 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

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 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 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 Model of GAG/MIP-2/CXCR2 Interfaces and Its Functional Effects

MIP-2/CXCL2 is a murine chemokine related to human chemokines that possesses the Glu-Leu-Arg (ELR) activation motif and activates CXCR2 for neutrophil chemotaxis. We determined the structure of MIP-2 to 1.9 Å resolution and created a model with its murine receptor CXCR2 based on the coordinates of human CXCR4. Chemokine-induced migration of cells through specific G-protein coupled receptors is regulated by glycosaminoglycans (GAGs) that oligomerize chemokines. MIP-2 GAG-binding residues were identified that interact with heparin disaccharide I–S by NMR spectroscopy. A model GAG/MIP-2/CXCR2 complex that supports a 2:2 complex between chemokine and receptor was created. Mutants of these disaccharide-binding residues were made and tested for heparin binding, in vitro neutrophil chemotaxis, and in vivo neutrophil recruitment to the mouse peritoneum and lung. The mutants have a 10-fold decrease in neutrophil chemotaxis in vitro. There is no difference in neutrophil recruitment between wild-type MIP-2 and mutants in the peritoneum, but all activity of the mutants is lost in the lung, supporting the concept that GAG regulation of chemokines is tissue-dependent