Vascular endothelial growth factor (VEGF) is a potent stimulator of vascular angiogenesis, permeability, and remodeling that also plays important roles in wound healing and tissue cytoprotection. To begin to define the roles of VEGF in diseases like asthma and COPD, we characterized the effects of lung-targeted transgenic VEGF165 and defined the innate immune pathways that regulate VEGF tissue responses. The former studies demonstrated that VEGF plays an important role in Th2 inflammation because, in addition to stimulating angiogenesis and edema, VEGF induced eosinophilic inflammation, mucus metaplasia, subepithelial fibrosis, myocyte hyperplasia, dendritic cell activation, and airways hyperresponsiveness via IL-13–dependent and -independent mechanisms. VEGF was also produced at sites of aeroallergen-induced Th2 inflammation, and VEGF receptor blockade ameliorated adaptive Th2 inflammation and Th2 cytokine elaboration. The latter studies demonstrated that activation of the RIG-like helicase (RLH) innate immune pathway using viral pathogen–associated molecular patterns such as Poly(I:C) or viruses ameliorated VEGF-induced tissue responses. In accord with these findings, Poly(I:C)-induced RLH activation also abrogated aeroallergen-induced Th2 inflammation. When viewed in combination, these studies suggest that VEGF excess can contribute to the pathogenesis of Th2 inflammatory disorders such as asthma and that abrogation of VEGF signaling via RLH activation can contribute to the pathogenesis of viral disorders such as virus-induced COPD exacerbations. They also suggest that RLH activation may be a useful therapeutic strategy in asthma and related disorders.

Rationale: Vascular endothelial growth factor (VEGF) regulates vascular, inflammatory, remodeling, and cell death responses. It plays a critical role in normal pulmonary physiology, and VEGF excess and deficiency have been implicated in the pathogenesis of asthma and chronic obstructive pulmonary disease, respectively. Although viruses are an important cause of chronic obstructive pulmonary disease exacerbations and innate responses play an important role in these exacerbations, the effects of antiviral responses on VEGF homeostasis have not been evaluated.

Objectives: We hypothesized that antiviral innate immunity regulates VEGF tissue responses.

Methods: We compared the effects of transgenic VEGF165 in mice treated with viral pathogen–associated molecular pattern polyinosinic:polycytidylic acid [poly(I:C)], mice treated with live virus, and control mice.

Measurements and Main Results: Transgenic VEGF stimulated angiogenesis, edema, inflammation, and mucin accumulation. Each of these was abrogated by poly(I:C). These inhibitory effects were dose dependent, noted when poly(I:C) was administered before and after transgene activation, and mediated by a Toll-like receptor-3–independent and RIG-like helicase (RLH)– and type I IFN receptor–dependent pathway. VEGF stimulated the expression of VEGF receptor-1 and poly(I:C) inhibited this stimulation. Poly(I:C) also inhibited the ability of VEGF to activate extracellular signal–regulated kinase-1, Akt, focal adhesion kinase, and endothelial nitric oxide synthase, and aeroallergen-induced adaptive helper T-cell type 2 inflammation. Influenza and respiratory syncytial virus also inhibited VEGF-induced angiogenesis.

Conclusions: These studies demonstrate that poly(I:C) and respiratory viruses inhibit VEGF-induced tissue responses and adaptive helper T-cell type 2 inflammation and highlight the importance of a RLH- and type I IFN receptor–dependent pathway(s) in these regulatory events. They define a novel link between VEGF and antiviral and RLH innate immune responses and a novel pathway that regulates pulmonary VEGF activity.

To define the factors that control the tissue effects of IL-4, we compared the effects of Tg IL-4 in Balb/c and C57BL/6 mice. In the former, IL-4 caused modest eosinophilic inflammation and mild airway fibrosis and did not shorten survival. In C57BL/6 mice, IL-4 caused profound eosinophilic inflammation, airway fibrosis, emphysematous alveolar destruction, and premature death. These differences could not be accounted for by changes in Th2 or Th1 cytokines, receptor components, STAT6 activation, MMPs, or cathepsins. In contrast, in C57BL/6 mice, alveolar remodeling was associated with decreased levels of tissue inhibitors of metalloproteinase 2, -3, and -4 and α1-antitrypsin, and fibrosis was associated with increased levels of total and bioactive TGF-β1. Impressive differences in adenosine metabolism were also appreciated, with increased tissue adenosine levels and A1, A2B, and A3 adenosine receptor expression and decreased adenosine deaminase (ADA) activity in C57BL/6 animals. Treatment with ADA also reduced the inflammation, fibrosis, and emphysematous destruction and improved the survival of C57BL/6 Tg animals. These studies demonstrate that genetic influences control IL-4 effector pathways in the murine lung. They also demonstrate that IL-4 has different effects on adenosine metabolism in Balb/c and C57BL/6 mice and that these differences contribute to the different responses that IL-4 induces in these inbred animals.

IL-13 dysregulation plays a critical role in the pathogenesis of a variety of inflammatory and remodeling diseases. In these settings, STAT6 is believed to be the canonical signaling molecule mediating the tissue effects of IL-13. Signaling cascades involving MAPKs have been linked to inflammation and remodeling. We hypothesized that MAPKs play critical roles in effector responses induced by IL-13 in the lung. We found that Tg IL-13 expression in the lung led to potent activation of ERK1/2 but not JNK1/2 or p38. ERK1/2 activation also occurred in mice with null mutations of STAT6. Systemic administration of the MAPK/ERK kinase 1 (MEK1) inhibitor PD98059 or use of Tg mice in which a dominant-negative MEK1 construct was expressed inhibited IL-13–induced inflammation and alveolar remodeling. There were associated decreases in IL-13–induced chemokines (MIP-1α/CCL-3, MIP-1β/CCL-4, MIP-2/CXCL-1, RANTES/CCL-5), MMP-2, -9, -12, and -14, and cathepsin B and increased levels of α1-antitrypsin. IL-13–induced tissue and molecular responses were noted that were equally and differentially dependent on ERK1/2 and STAT6 signaling. Thus, ERK1/2 is activated by IL-13 in the lung in a STAT6-independent manner where it contributes to IL-13–induced inflammation and remodeling and is required for optimal IL-13 stimulation of specific chemokines and proteases as well as the inhibition of specific antiproteases. ERK1/2 regulators may be useful in the treatment of IL-13–induced diseases and disorders.

Th1 inflammation and remodeling characterized by tissue destruction frequently coexist in human diseases. To further understand the mechanisms of these responses, we defined the role(s) of CCR5 in the pathogenesis of IFN-γ–induced inflammation and remodeling in a murine emphysema model. IFN-γ was a potent stimulator of the CCR5 ligands macrophage inflammatory protein–1α/CCL-3 (MIP-1α/CCL-3), MIP-1β/CCL-4, and RANTES/CCL-5, among others. Antibody neutralization or null mutation of CCR5 decreased IFN-γ–induced inflammation, DNA injury, apoptosis, and alveolar remodeling. These interventions decreased the expression of select chemokines, including CCR5 ligands and MMP-9, and increased levels of secretory leukocyte protease inhibitor. They also decreased the expression and/or activation of Fas, FasL, TNF, caspase-3, -8, and -9, Bid, and Bax. In accordance with these findings, cigarette smoke induced pulmonary inflammation, DNA injury, apoptosis, and emphysema via an IFN-γ–dependent pathway(s), and a null mutation of CCR5 decreased these responses. These studies demonstrate that IFN-γ is a potent stimulator of CC and CXC chemokines and highlight the importance of CCR5 in the pathogenesis of IFN-γ–induced and cigarette smoke–induced inflammation, tissue remodeling, and emphysema. They also demonstrate that CCR5 is required for optimal IFN-γ stimulation of its own ligands, other chemokines, MMPs, caspases, and cell death regulators and the inhibition of antiproteases.

Hyperoxic acute lung injury (HALI) is characterized by a cell death response with features of apoptosis and necrosis that is inhibited by IL-11 and other interventions. We hypothesized that Bfl-1/A1, an antiapoptotic Bcl-2 protein, is a critical regulator of HALI and a mediator of IL-11–induced cytoprotection. To test this, we characterized the expression of A1 and the oxygen susceptibility of WT and IL-11 Tg(+) mice with normal and null A1 loci. In WT mice, 100% O2 caused TUNEL+ cell death, induction and activation of intrinsic and mitochondrial-death pathways, and alveolar protein leak. Bcl-2 and Bcl-xl were also induced as an apparent protective response. A1 was induced in hyperoxia, and in A1-null mice, the toxic effects of hyperoxia were exaggerated, Bcl-2 and Bcl-xl were not induced, and premature death was seen. In contrast, IL-11 stimulated A1, diminished the toxic effects of hyperoxia, stimulated Bcl-2 and Bcl-xl, and enhanced murine survival in 100% O2. In A1-null mice, IL-11–induced protection, survival advantage, and Bcl-2 and Bcl-xl induction were significantly decreased. VEGF also conferred protection via an A1-dependent mechanism. In vitro hyperoxia also stimulated A1, and A1 overexpression inhibited oxidant-induced epithelial cell apoptosis and necrosis. A1 is an important regulator of oxidant-induced lung injury, apoptosis, necrosis, and Bcl-2 and Bcl-xl gene expression and a critical mediator of IL-11– and VEGF-induced cytoprotection.

IL-13 potently stimulates eosinophilic and lymphocytic inflammation and alveolar remodeling in the lung, effects that depend on the induction of various matrix metalloproteinases (MMPs). Here, we compared the remodeling and inflammatory effects of an IL-13 transgene in lungs of wild-type, MMP-9–deficient, or MMP-12–deficient mice. IL-13–induced alveolar enlargement, lung enlargement, compliance alterations, and respiratory failure and death were markedly decreased in the absence of MMP-9 or MMP-12. Moreover, IL-13 potently induced MMPs-2, -12, -13, and -14 in the absence of MMP-9, while induction of MMPs-2, -9, -13, and -14 by IL-13 was diminished in the absence of MMP-12. A deficiency in MMP-9 did not alter eosinophil, macrophage, or lymphocyte recovery, but increased the recovery of total leukocytes and neutrophils in bronchoalveolar lavage (BAL) fluids from IL-13 transgenic mice. In contrast, a deficiency in MMP-12 decreased the recovery of leukocytes, eosinophils, and macrophages, but not lymphocytes or neutrophils. These studies demonstrate that IL-13 acts via MMPs-9 and -12 to induce alveolar remodeling, respiratory failure, and death and that IL-13 induction of MMPs-2, -9, -13, and -14 is mediated at least partially by an MMP-12–dependent pathway. The also demonstrate that MMPs-9 and -12 play different roles in the generation of IL-13–induced inflammation, with MMP-9 inhibiting neutrophil accumulation and MMP-12 contributing to the accumulation of eosinophils and macrophages.