Essential role of nitric oxide in VEGF-induced, asthma-like angiogenic, inflammatory, mucus, and physiologic responses in the lung

Abstract

VEGF, nitric oxide (NO), inflammation, and vascular- and extravascular remodeling coexist in asthma and other disorders. In these responses, VEGF regulates angiogenesis. VEGF also induces inflammation and remodeling. The mechanisms of the latter responses have not been defined, however. We hypothesized that VEGF-induces extravascular tissue responses via NO-dependent mechanisms. To evaluate this hypothesis, we compared the effects of transgenic VEGF165 in lungs from normal mice, mice treated with pan-NO synthase (NOS) or endothelial NOS (eNOS) inhibitors, and mice with null mutations of inducible NOS (iNOS) or eNOS. These studies demonstrate that VEGF selectively stimulates eNOS and iNOS. They also demonstrate that VEGF induces pulmonary alterations via NO-dependent and -independent mechanisms with angiogenesis, edema, mucus metaplasia, airway hyperresponsiveness, lymphocyte accumulation, dendritic cell hyperplasia and S-nitrosoglutathione reductase stimulation being NO-dependent and dendritic cell activation being NO-independent. Furthermore, they demonstrate that eNOS and iNOS both contribute to these responses. NO/NOS-based interventions may be therapeutic in VEGF-driven inflammation and remodeling.

Fig. 1.

VEGF regulation of NOS and vascular effects of NO. Tg− and Tg+ mice received dox water for 2 weeks in the presence and absence of l-NAME. The levels of mRNA encoding eNOS and iNOS were assessed (A), pulmonary angiogenesis was evaluated with CD31 staining of the trachea (B), and pulmonary edema was quantitated with wet/dry lung weight ratio(s) (C). The noted values represent assessments in a minimum of four animals. **, P ≤ 0.01; #, P ≤ 0.02; ##, P ≤ 0.05.

Fig. 2.

NO in VEGF-induced inflammatory and immune alterations. Tg− and Tg+ mice received normal water or dox water in the presence or absence of l-NAME for 2 weeks. BAL total cell (A) and differential cell [macrophages (B); lymphocytes (C); and eosinophils (D)] recovery were then evaluated. CD4⁺ and CD8+ T cell accumulation was also evaluated with FACS (E). NO inhibition (with l-NAME) abrogated the VEGF induction of the number of T cells (E). The noted values represent assessments in a minimum of four animals. *, P ≤ 0.0001; **, P ≤ 0.01; #, P ≤ 0.02.

Fig. 3.

NO in VEGF-induced DC alterations. Tg− and Tg+ mice received dox water in the presence or absence of l-NAME for 2 weeks. DC number (A) and DC activation (B) were evaluated. The noted values represent assessments in a minimum of four animals.

Fig. 4.

NO in VEGF-induced mucus metaplasia, AHR, and GSNOR expression. Tg− and Tg+ mice received dox water in the presence and absence of l-NAME for 2 weeks, and mucus metaplasia (periodic acid/Schiff stain) (A), the histologic mucin index (B), mucin-related gene expression (C), methacholine responsiveness (D), and GSNOR mRNA levels (E) were evaluated. The noted values represent assessments in a minimum of four animals. *, P ≤ 0.0001; #, P ≤ 0.02; ##, P ≤ 0.05 vs. others.

Fig. 5.

Relative contributions of iNOS and eNOS and levels of BAL VEGF. Tg− and Tg+ mice with WT (+) or null (−) iNOS or eNOS loci and mice treated with cavtratin or its vehicle control (AP) were incubated with dox water for 2 weeks. Mucus metaplasia (A), the histologic mucin index (B), and the regulation of mucin-related genes and GSNOR (C) and BAL VEGF levels (D) were evaluated. A and C are representative of four similar experiments. The noted values reflect assessments in at least four animals. *, P ≤ 0.0001; **, P ≤ 0.01.