In keeping with the pleiotropic effects of VEGF in injury and repair and its roles in normal homeostasis and disease, studies were undertaken to define the mechanisms that control its tissue effector responses. Specifically, we hypothesized that antiviral innate immune responses are powerful regulators of VEGF effector function. The present studies confirm this hypothesis by demonstrating that the viral PAMP, poly(I:C), inhibits VEGF-induced angiogenic responses and tissue edema. The viral relevance of these findings was confirmed in studies that demonstrated that influenza virus and RSV also inhibit VEGF angiogenesis. Innate immune regulation of VEGF responses was not restricted to vascular targets because VEGF-induced inflammation, mucous metaplasia, and Th2 inflammation were also abrogated by poly(I:C). Importantly, these studies also demonstrated that these effects of poly(I:C) are mediated by a TLR3-independent, RLH-dependent pathway that selectively regulates VEGF receptor expression and inhibits VEGF activation of ERK, FAK, Akt, and eNOS. When viewed in combination, these studies define a novel link between VEGF and antiviral and RLH-mediated innate immune responses. In so doing, they provide essential insights into the consequences of viral recognition and innate immune activation. These insights define mechanisms that can mediate the adverse consequences of viral infections and RLH-mediated immune responses. They also define an agonist and pathway that can be used to regulate VEGF-induced tissue responses in health and disease.
Host antiviral responses are initiated via the detection of viral PAMPs by host pattern recognition receptors (PRRs). On recognition, PRR signaling results in the expression of type I IFNs that suppress viral replication and facilitate adaptive immune responses (31
). Double-stranded (ds) RNA, which is produced during the replication of many viruses, is recognized by several innate pathways including TLR3 and the RNA helicases RIG-I and Mda5 (31
). In addition, many singled-stranded RNA viruses including influenza virus have been shown to activate the RIG-I pathway via the generation of 5′-triphosphorylated single-stranded RNA. TLR3 resides in endosomal membranes, where it recognizes dsRNA and poly(I:C). RIG-I and Mda5 detect dsRNA and poly(I:C) in the cytoplasm, where they are linked to downstream signaling molecules via MAVS (38
). When our studies demonstrated that poly(I:C) inhibited VEGF-induced tissue responses we expected that this inhibition would be mediated, to a great extent, by TLR3. Our studies, however, proved that this is not correct because similar poly(I:C)-induced VEGF-inhibitory effects were seen in mice that were sufficient and deficient in this receptor. In contrast, poly(I:C) regulation of VEGF-induced tissue responses appeared to be RLH mediated because it was entirely abrogated by the elimination of MAVS. In accordance with the prominent role that the RLH pathway plays in the induction of type I IFNs, our studies also demonstrated that poly(I:C) stimulated the production of IFN-α and IFN-β and that the inhibitory effects of poly(I:C) are significantly diminished in the absence of a common type I IFN receptor. In contrast, null mutations of PKR did not alter the VEGF-regulatory effects of poly(I:C). These are the first studies to define a relationship between VEGF and the RLH innate immune signaling pathway. They are also the first to demonstrate that type I IFNs inhibit VEGF-induced tissue responses via a PKR-independent mechanism. Additional investigation will be required to define the pathways that type I IFNs use to inhibit VEGF effector responses.
VEGF mediates its tissue effects by recruiting and activating multiple signaling networks that regulate cell and organ function. These ligands bind and signal through VEGF receptors, which are receptor tyrosine kinases, and neuropilin-1 and -2, which are coreceptors. Studies of VEGF signaling in a variety of tissues and modeling systems have demonstrated that phospholipase C-γ, PI3 kinase, Akt, Ras pathways, and MAP kinases play important roles in many VEGF-induced cellular and tissue responses (4
). To further understand the mechanisms that RLH pathway activation might use to control VEGF-induced tissue responses, we compared the expression of VEGF receptors and the activation of these pathways in mice treated with poly(I:C) or vehicle control. Our studies demonstrate that the ability of VEGF to stimulate the expression of VEGFR1 on dendritic cells and endothelial cells and to activate pulmonary FAK, ERK, Akt, and eNOS was significantly diminished after treatment with poly(I:C). These studies also demonstrated that the inhibition of VEGF signaling is mediated via a type I IFN receptor–dependent mechanism. In accordance with the widespread inhibition that was noted, these studies, in composite, demonstrate that poly(I:C) selectively regulates VEGF receptor expression and inhibits a variety of VEGF-induced signaling events.
The lung is among the organs with the highest levels of basal VEGF expression and activity. In this setting VEGF plays a critical role in early lung development. More recently it has been shown to confer protection against injury and oxidative stress. This protection may be particularly important in the respiratory alveolus because pharmacological inhibition of VEGF activity and lung-targeted inactivation of the VEGF gene result in emphysematous phenotypes whereas VEGF expression diminishes emphysema in murine modeling systems (11
). In accordance with this finding, a number of investigators have reported decreased levels of VEGF expression/accumulation in biological samples from patients with emphysema (16
). Our demonstration that VEGF effector function is abrogated by antiviral innate immune responses has important implications regarding the pathogenesis of COPD. First, these studies highlight a mechanism by which the protective effects of VEGF can be abrogated in the setting of viral infection. One can easily see how this inhibition of VEGF effector responses could contribute to the RLH-mediated, exaggerated inflammatory, apoptotic, and emphysematous responses that are seen in mice simultaneously exposed to cigarette smoke and poly(I:C) or influenza virus (31
) and to the pathological effects of virus-induced COPD exacerbations. These studies also suggest that one cannot develop a clear understanding of the integrity of the effects of VEGF in the lung simply by measuring BAL VEGF levels. Because the tissue effects of VEGF can be abrogated without corresponding alterations in the levels of VEGF in a biological fluid the levels of BAL VEGF are a “best case” estimate of the tissue effects of this important cytokine.
A number of lines of evidence have emerged that suggest that VEGF plays an important role in asthma pathogenesis. This includes studies from our laboratory that demonstrated that lung-targeted VEGF transgenic mice have an asthma-like phenotype with eosinophilic inflammation, airway remodeling, and airway hyperresponsiveness and that VEGFR blockade diminishes aeroallergen-induced Th2 inflammation and Th2 cytokine production in vivo
). It also includes human investigations that demonstrated that the levels of expression of VEGF and the VEGFRs are increased in asthma (50
) and studies that noted associations between VEGF polymorphisms and the disease (53
). To add to this literature we characterized the effects of poly(I:C) on OVA-induced adaptive Th2 immune responses. In accordance with reports from others (54
), our studies demonstrated that poly(I:C) inhibits tissue inflammation in this setting. In contrast, others have reported that poly(I:C) can exacerbate pulmonary allergic reactions (55
). These divergent results may be due to differences in the protocols that were employed or the appreciation that different doses of poly(I:C) can activate different immune pathways (56
). However, it is tempting to hypothesize that both reports are correct and that poly(I:C) inhibits when it predominantly activates the RLH pathway (as described in this article) and stimulates when it predominantly activates TLR3 as described by Torres and colleagues (55
). It will be interesting to see whether subsequent studies confirm this speculation.
In summary, our studies demonstrate that RLH activation blocks VEGF tissue responses and ameliorates aeroallergen-induced adaptive Th2 inflammation. These studies suggest that innate immunity agonists such as poly(I:C) can be used to activate the RLH pathway and control VEGF tissue responses in diseases such as asthma. Elevated levels of VEGF are also believed to be involved in the pathogenesis of a variety of other diseases including tumor angiogenesis, rheumatoid arthritis, inflammatory bowel disease, psoriasis, allergic rhinitis, and wet macular degeneration (3
). Thus, it is tempting to speculate that poly(I:C) and/or other RLH agonists can be used to treat these disorders as well. Additional investigations into the agonists that can be employed, the mechanisms of their effect, and the utility of RLH agonists in the regulation of VEGF-induced tissues responses are warranted.