James McCoy

Nox1 Overexpression Potentiates Angiotensin II-Induced Hypertension and Vascular Smooth Muscle Hypertrophy in Transgenic Mice

Background— Reactive oxygen species (ROS) have been implicated in the development of cardiovascular pathologies. NAD(P)H oxidases (Noxes) are major sources of reactive oxygen species in the vessel wall, but the importance of individual Nox homologues in specific layers of the vascular wall is unclear. Nox1 upregulation has been implicated in cardiovascular pathologies such as hypertension and restenosis. Methods and Results— To investigate the pathological role of Nox1 upregulation in vascular smooth muscle, transgenic mice overexpressing Nox1 in smooth muscle cells (TgSMCnox1) were created, and the impact of Nox1 upregulation on the medial hypertrophic response during angiotensin II (Ang II)–induced hypertension was studied. These mice have increased expression of Nox1 protein in the vasculature, which is accompanied by increased superoxide production. Infusion of Ang II (0.7 mg/kg per day) into these mice for 2 weeks led to a potentiation of superoxide production compared with similarly treated negative littermate controls. Systolic blood pressure and aortic hypertrophy were also markedly greater in TgSMCnox1 mice than in their littermate controls. To confirm that this potentiation of vascular hypertrophy and hypertension was due to increased ROS formation, additional groups of mice were coinfused with the antioxidant Tempol. Tempol decreased the level of Ang II-induced aortic superoxide production and partially reversed the hypertrophic and hypertensive responses in these animals. Conclusions— These data indicate that smooth muscle-specific Nox1 overexpression augments the oxidative, pressor, and hypertrophic responses to Ang II, supporting the concept that medial Nox1 participates in the development of cardiovascular pathologies.

Annexin A1, formyl peptide receptor, and NOX1 orchestrate epithelial repair

N-formyl peptide receptors (FPRs) are critical regulators of host defense in phagocytes and are also expressed in epithelia. FPR signaling and function have been extensively studied in phagocytes, yet their functional biology in epithelia is poorly understood. We describe a novel intestinal epithelial FPR signaling pathway that is activated by an endogenous FPR ligand, annexin A1 (ANXA1), and its cleavage product Ac2-26, which mediate activation of ROS by an epithelial NADPH oxidase, NOX1. We show that epithelial cell migration was regulated by this signaling cascade through oxidative inactivation of the regulatory phosphatases PTEN and PTP-PEST, with consequent activation of focal adhesion kinase (FAK) and paxillin. In vivo studies using intestinal epithelial specific Nox1(-/-IEC) and AnxA1(-/-) mice demonstrated defects in intestinal mucosal wound repair, while systemic administration of ANXA1 promoted wound recovery in a NOX1-dependent fashion. Additionally, increased ANXA1 expression was observed in the intestinal epithelium and infiltrating leukocytes in the mucosa of ulcerative colitis patients compared with normal intestinal mucosa. Our findings delineate a novel epithelial FPR1/NOX1-dependent redox signaling pathway that promotes mucosal wound repair.

Nox1 is over‐expressed in human colon cancers and correlates with activating mutations in K‐Ras

The NADPH‐oxidase 1 (Nox1) is a homolog of gp91phox, the catalytic subunit of the phagocyte superoxide‐generating NADPH‐oxidase. Nox1 is expressed in normal colon epithelial cells and in colon tumor cell lines, and overexpression in model cells has been implicated in stimulation of mitogenesis and angiogenesis and inhibition of apoptosis. This suggests that aberrant expression of Nox1 could contribute to the development of colorectal cancer. Herein, we examine the expression of Nox1 mRNA in 24 colon tumors of various stages compared with paired adjacent normal tissue from the same patient, and correlate expression with some common mutations associated with colon cancer. Nox1 was overexpressed compared with paired normal tissue in 57% of tumors as early as the adenoma stage, with no correlation of expression level with tumor stage. Overexpression of Nox1 mRNA correlated with Nox1 protein levels assessed by immunofluorescence and immunohistochemistry with an antibody specific for Nox1. There was a strong correlation between Nox1 mRNA level and activating mutations in codons 12 and 13 of K‐Ras. Eighty percent (8/10) of tumors with codons 12 and 13 mutations had a 2‐fold or more increase in Nox1 mRNA, and 70% (7/10) had a 5‐fold or greater increase. Transgenic mice expressing K‐RasG12V in the intestinal epithelium also expressed markedly elevated Nox1 in both small and large intestine. There was no correlation between inactivating mutations in the tumor suppressor p53 and Nox1 expression. We conclude that Nox1 mRNA and protein are overexpressed in colon cancer and are strongly correlated with activating mutations in K‐Ras.

Ebselen and Congeners Inhibit NADPH Oxidase 2-Dependent Superoxide Generation by Interrupting the Binding of Regulatory Subunits

NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS), which function in normal physiology and, when overproduced, in pathophysiology. Recent studies using mice deficient in Nox2 identify this isoform as a novel target against Nox2-implicated inflammatory diseases. Nox2 activation depends on the binding of the proline-rich domain of its heterodimeric partner p22phox to p47phox. A high-throughput screen that monitored this interaction via fluorescence polarization identified ebselen and several of its analogs as inhibitors. Medicinal chemistry was performed to explore structure-activity relationships and to optimize potency. Ebselen and analogs potently inhibited Nox1 and Nox2 activity but were less effective against other isoforms. Ebselen also blocked translocation of p47phox to neutrophil membranes. Thus, ebselen and its analogs represent a class of compounds that inhibit ROS generation by interrupting the assembly of Nox2-activating regulatory subunits.

Thioxo-dihydroquinazolin-one Compounds as Novel Inhibitors of Myeloperoxidase

Myeloperoxidase (MPO) is a key antimicrobial enzyme, playing a normal role in host defense, but also contributing to inflammatory conditions including neuroinflammatory diseases such as Parkinsons and Alzheimers. We synthesized and characterized more than 50 quinazolin-4(1H)-one derivatives and showed that this class of compounds inhibits MPO with IC50 values as low as 100 nM. Representative compounds showed partially reversible inhibition that was competitive with respect to Amplex Red substrate and did not result in the accumulation of MPO Compound II. Members of this group show promise for therapeutic development for the treatment of diseases in which inflammation plays a pathogenic role.

NADPH Oxidase 1 Is Associated with Altered Host Survival and T Cell Phenotypes after Influenza A Virus Infection in Mice

The role of the reactive oxygen species-producing NADPH oxidase family of enzymes in the pathology of influenza A virus infection remains enigmatic. Previous reports implicated NADPH oxidase 2 in influenza A virus-induced inflammation. In contrast, NADPH oxidase 1 (Nox1) was reported to decrease inflammation in mice within 7 days post-influenza A virus infection. However, the effect of NADPH oxidase 1 on lethality and adaptive immunity after influenza A virus challenge has not been explored. Here we report improved survival and decreased morbidity in mice with catalytically inactive NADPH oxidase 1 (Nox1*/Y) compared with controls after challenge with A/PR/8/34 influenza A virus. While changes in lung inflammation were not obvious between Nox1*/Y and control mice, we observed alterations in the T cell response to influenza A virus by day 15 post-infection, including increased interleukin-7 receptor-expressing virus-specific CD8+ T cells in lungs and draining lymph nodes of Nox1*/Y, and increased cytokine-producing T cells in lungs and spleen. Furthermore, a greater percentage of conventional and interstitial dendritic cells from Nox1*/Y draining lymph nodes expressed the co-stimulatory ligand CD40 within 6 days post-infection. Results indicate that NADPH oxidase 1 modulates the innate and adaptive cellular immune response to influenza virus infection, while also playing a role in host survival. Results suggest that NADPH oxidase 1 inhibitors may be beneficial as adjunct therapeutics during acute influenza infection.

Nasal delivery of H5N1 avian influenza vaccine formulated with GenJet™ or in vivo-jetPEI® induces enhanced serological, cellular and protective immune responses

Abstract Avian influenza virus infection is a serious public health threat and preventive vaccination is the most cost-effective public health intervention strategy. Unfortunately, currently available unadjuvanted avian influenza vaccines are poorly immunogenic and alternative vaccine formulations and delivery strategies are in urgent need to reduce the high risk of avian influenza pandemics. Cationic polymers have been widely used as vectors for gene delivery in vitro and in vivo. In this study, we formulated H5N1 influenza vaccines with GenJet™ or in vivo-jetPEI®, and showed that these formulations significantly enhanced the immunogenicity of H5N1 vaccines and conferred protective immunity in a mouse model. Detailed analyses of adaptive immune responses revealed that both formulations induced mixed TH1/TH2 antigen-specific CD4 T-cell responses, antigen-specific cytotoxic CD8 T-cell and memory B-cell responses. Our findings suggest that cationic polymers merit future development as potential adjuvants for mucosal delivery of poorly immunogenic vaccines.