The NOX toolbox: validating the role of NADPH oxidases in physiology and disease

Reactive oxygen species (ROS) are cellular signals but also disease triggers; their relative excess (oxidative stress) or shortage (reductive stress) compared to reducing equivalents are potentially deleterious. This may explain why antioxidants fail to combat diseases that correlate with oxidative stress. Instead, targeting of disease-relevant enzymatic ROS sources that leaves physiological ROS signaling unaffected may be more beneficial. NADPH oxidases are the only known enzyme family with the sole function to produce ROS. Of the catalytic NADPH oxidase subunits (NOX), NOX4 is the most widely distributed isoform. We provide here a critical review of the currently available experimental tools to assess the role of NOX and especially NOX4, i.e. knock-out mice, siRNAs, antibodies, and pharmacological inhibitors. We then focus on the characterization of the small molecule NADPH oxidase inhibitor, VAS2870, in vitro and in vivo, its specificity, selectivity, and possible mechanism of action. Finally, we discuss the validation of NOX4 as a potential therapeutic target for indications including stroke, heart failure, and fibrosis.     

Direct evidence that the N-terminal extensions of the TAP complex act as autonomous interaction scaffolds for the assembly of the MHC I peptide-loading complex

The loading of antigenic peptides onto major histocompatibility complex class I (MHC I) molecules is an essential step in the adaptive immune response against virally or malignantly transformed cells. The ER-resident peptide-loading complex (PLC) consists of the transporter associated with antigen processing (TAP1 and TAP2), assembled with the auxiliary factors tapasin and MHC I. Here, we demonstrated that the N-terminal extension of each TAP subunit represents an autonomous domain, named TMD(0), which is correctly targeted to and inserted into the ER membrane. In the absence of coreTAP, each TMD(0) recruits tapasin in a 1:1 stoichiometry. Although the TMD(0)s lack known ER retention/retrieval signals, they are localized to the ER membrane even in tapasin-deficient cells. We conclude that the TMD(0)s of TAP form autonomous interaction hubs linking antigen translocation into the ER with peptide loading onto MHC I, hence ensuring a major function in the integrity of the antigen-processing machinery.     

Nanoparticle-based drug delivery: case studies for cancer and cardiovascular applications

Nanoparticles (NPs) comprised of nanoengineered complexes are providing new opportunities for enabling targeted delivery of a range of therapeutics and combinations. A range of functionalities can be included within a nanoparticle complex, including surface chemistry that allows attachment of cell-specific ligands for targeted delivery, surface coatings to increase circulation times for enhanced bioavailability, specific materials on the surface or in the nanoparticle core that enable storage of a therapeutic cargo until the target site is reached, and materials sensitive to local or remote actuation cues that allow controlled delivery of therapeutics to the target cells. However, despite the potential benefits of NPs as smart drug delivery and diagnostic systems, much research is still required to evaluate potential toxicity issues related to the chemical properties of NP materials, as well as their size and shape. The need to validate each NP for safety and efficacy with each therapeutic compound or combination of therapeutics is an enormous challenge, which forces industry to focus mainly on those nanoparticle materials where data on safety and efficacy already exists, i.e., predominantly polymer NPs. However, the enhanced functionality affordable by inclusion of metallic materials as part of nanoengineered particles provides a wealth of new opportunity for innovation and new, more effective, and safer therapeutics for applications such as cancer and cardiovascular diseases, which require selective targeting of the therapeutic to maximize effectiveness while avoiding adverse effects on non-target tissues.     

Regenerating functional heart tissue for myocardial repair

Heart disease is one of the leading causes of death worldwide and the number of patients with the disease is likely to grow with the continual decline in health for most of the developed world. Heart transplantation is one of the only treatment options for heart failure due to an acute myocardial infarction, but limited donor supply and organ rejection limit its widespread use. Cellular cardiomyoplasty, or cellular implantation, combined with various tissue-engineering methods aims to regenerate functional heart tissue. This review highlights the numerous cell sources that have been used to regenerate the heart as well as cover the wide range of tissue-engineering strategies that have been devised to optimize the delivery of these cells. It will probably be a long time before an effective regenerative therapy can make a serious impact at the bedside.     

Multispecies-compatible antitumor effects of a cross-species small-interfering RNA against mammalian target of rapamycin

Successful development of sequence-specific siRNA (small interfering RNA)-based drugs requires an siRNA design that functions consistently in different organisms. Utilizing the CAPSID program previously developed by our group, we here designed siRNAs against mammalian target of rapamycin (mTOR) that are entirely complementary among various species and investigated their multispecies-compatible gene-silencing properties. The mTOR siRNAs markedly reduced mTOR expression at both the mRNA and protein levels in human, mouse, and monkey cell lines. The reduction in mTOR expression resulted in inactivation of both mTOR complex I and II signaling pathways, as confirmed by reduced phosphorylation of p70S6K (70-kDa ribosomal protein S6 kinase), 4EBP1 (eIF4E-binding protein 1), and AKT, and nuclear accumulation of FOXO1 (forkhead box O1), with consequent cell-cycle arrest, proliferation inhibition, and autophagy activation. Moreover, interfering with mTOR activity in vivo using mTOR small-hairpin RNA-expressing recombinant adeno-associated virus led to significant antitumor effects in xenograft and allograft models. Thus, the present study demonstrates that cross-species siRNA successfully silences its target and readily produces multispecies-compatible phenotypic alterations-antitumor effects in the case of mTOR siRNA. Application of cross-species siRNA should greatly facilitate the development of siRNA-based therapeutic agents.