Rational Design and Therapeutic Potential of a Novel Nox1 Inhibitor for the Treatment of Pulmonary Hypertension: In Vitro and In Vivo Effects of Nox1 Inhibition

NADPH oxidases (Noxes) represent a family of enzymes who produce reactive oxygen species. Excessive Nox activity is associated with multiple pathological conditions, including hypertension. Despite Nox1’s association with morbidity, there is a paucity of specific Nox1 inhibitors. The overarching hypothesis of this project was that Nox1 promotes endothelial phenotypes contributing to pulmonary hypertension and associated cardiac dysfunction. Pharmacological Nox1 inhibition testing this hypothesis was performed via designing the first specific peptidic Nox1 inhibitor (NoxA1ds). Our results show that Nox1 is key to endothelial O2•- and VEGF-stimulated migration and that Nox1 contributes to left ventricle cardiac dysfunction. Functional Nox1 is activated in part by association of Nox1 with one of its essential cytosolic subunits NOXA1. NoxA1ds was designed to mimic a putative activation domain in NOXA1 with a single F199A amino acid mutation. NoxA1ds specifically inhibited Nox1 but not Nox2, Nox4, Nox5 in reconstituted cell-free systems. Mechanistically, we found that NoxA1ds binds to Nox1 and disrupts Nox1:NOXA1 association and thus enzyme assembly. To identify the relative roles of Nox1 and Nox2 in human pulmonary artery endothelial cell (HPAEC) physiology, the relative specificity of Nox2ds for Nox2 vs Nox1 required validation. In part, this thesis established Nox2ds as specific for Nox2 over canonical, hybrid, or inducible Nox1 and Nox4. NoxA1ds and Nox2ds were then used to establish that Nox1, but not Nox2, is responsible for hypoxia-induced O2•- in HPAEC and VEGF-stimulated HPAEC migration. Additional data revealed that VEGF stimulates Nox1:NOXA1 association and identified fibroblasts as a source for hypoxic VEGF production. The role of Nox1 in HPAEC O2- and migration suggested that Nox1 may contribute to of the development of pulmonary arterial hypertension. Treatment of pulmonary hypertensive rats with aerosolized NoxA1ds improved left ventricular dilation but displayed minimal benefit in the right ventricle, indicating Nox1 may play a predominant role in the systemic vs pulmonary vasculature. Major contributions of this study include the design and characterization a novel Nox1 inhibitor (NoxA1ds), the identification of pulmonary endothelial phenotypes mediated by Nox1 rather than Nox2, and that the contribution of Nox1 to left ventricular dilation in the context of severe PAH

MEF2C-MYOCD and Leiomodin1 Suppression by miRNA-214 Promotes Smooth Muscle Cell Phenotype Switching in Pulmonary Arterial Hypertension.

BACKGROUND: Vascular hyperproliferative disorders are characterized by excessive smooth muscle cell (SMC) proliferation leading to vessel remodeling and occlusion. In pulmonary arterial hypertension (PAH), SMC phenotype switching from a terminally differentiated contractile to synthetic state is gaining traction as our understanding of the disease progression improves. While maintenance of SMC contractile phenotype is reportedly orchestrated by a MEF2C-myocardin (MYOCD) interplay, little is known regarding molecular control at this nexus. Moreover, the burgeoning interest in microRNAs (miRs) provides the basis for exploring their modulation of MEF2C-MYOCD signaling, and in turn, a pro-proliferative, synthetic SMC phenotype. We hypothesized that suppression of SMC contractile phenotype in pulmonary hypertension is mediated by miR-214 via repression of the MEF2C-MYOCD-leiomodin1 (LMOD1) signaling axis. METHODS AND RESULTS: In SMCs isolated from a PAH patient cohort and commercially obtained hPASMCs exposed to hypoxia, miR-214 expression was monitored by qRT-PCR. miR-214 was upregulated in PAH- vs. control subject hPASMCs as well as in commercially obtained hPASMCs exposed to hypoxia. These increases in miR-214 were paralleled by MEF2C, MYOCD and SMC contractile protein downregulation. Of these, LMOD1 and MEF2C were directly targeted by the miR. Mir-214 overexpression mimicked the PAH profile, downregulating MEF2C and LMOD1. AntagomiR-214 abrogated hypoxia-induced suppression of the contractile phenotype and its attendant proliferation. Anti-miR-214 also restored PAH-PASMCs to a contractile phenotype seen during vascular homeostasis. CONCLUSIONS: Our findings illustrate a key role for miR-214 in modulation of MEF2C-MYOCD-LMOD1 signaling and suggest that an antagonist of miR-214 could mitigate SMC phenotype changes and proliferation in vascular hyperproliferative disorders including PAH

NADPH oxidases: key modulators in aging and age-related cardiovascular diseases?

Reactive oxygen species (ROS) and oxidative stress have long been linked to aging and diseases prominent in the elderly such as hypertension, atherosclerosis, diabetes and atrial fibrillation (AF). NADPH oxidases (Nox) are a major source of ROS in the vasculature and are key players in mediating redox signalling under physiological and pathophysiological conditions. In this review, we focus on the Nox-mediated ROS signalling pathways involved in the regulation of 'longevity genes' and recapitulate their role in age-associated vascular changes and in the development of age-related cardiovascular diseases (CVDs). This review is predicated on burgeoning knowledge that Nox-derived ROS propagate tightly regulated yet varied signalling pathways, which, at the cellular level, may lead to diminished repair, the aging process and predisposition to CVDs. In addition, we briefly describe emerging Nox therapies and their potential in improving the health of the elderly population

Activities of ICP0 Involved in the Reversal of Silencing of Quiescent Herpes Simplex Virus 1 ▿

ICP0 is a transcriptional activating protein required for the efficient replication and reactivation of latent herpes simplex virus 1 (HSV-1). Multiple regions of ICP0 contribute its activity, the most prominent of which appears to be the RING finger, which confers E3 ubiquitin ligase activity. A region in the C terminus of ICP0 has also been implicated in several activities, including the disruption of a cellular repressor complex, REST/CoREST/HDAC1/2/LSD1. We used quiescent infection of MRC-5 cells with a virus that does not express immediate-early proteins, followed by superinfection with various viral mutants to quantify the ability of ICP0 variants to reactivate gene expression and alter chromatin structure. Superinfection with wild-type virus resulted in a 400-fold increase in expression from the previously quiescent d109 genome, the removal of heterochromatin and histones from the viral genome, and an increase in histone marks associated with activated transcription. RING finger mutants were unable to reactivate transcription or remove heterochromatin from d109, while mutants that are unable to bind CoREST activate gene expression from quiescent d109, albeit to a lesser degree than the wild-type virus. One such mutant, R8507, resulted in the partial removal of heterochromatin. Infection with R8507 did not result in the hyperacetylation of H3 and H4. The results demonstrate that (i) consistent with previous findings, the RING finger domain of ICP0 is required for the activation of quiescent genomes, (ii) the RF domain is also crucial for the ultimate removal of repressive chromatin, (iii) activities or interactions specified by the carboxy-terminal region of ICP0 significantly contribute to activation, and (iv) while the effects of the R8507 on chromatin are consistent with a role for REST/CoREST/HDAC1/2/LSD1 in the repression of quiescent genomes, the mutation may also affect other activities involved in derepression

EGF Potentiation of VEGF Production Is Cell Density Dependent in H292 EGFR Wild Type NSCLC Cell Line

Non-small cell lung cancer (NSCLC) affects millions of patients each year worldwide. Existing therapies include epidermal growth factor receptor (EGFR) inhibition using small molecules or antibodies with good efficacy. Unfortunately, intrinsic and acquired resistance to EGFR therapy remains a persistent complication for disease treatment. A greater understanding of the role of EGFR in NSCLC etiology is crucial to improving patient outcomes. In this study, the role of EGFR in tumor angiogenesis was examined in H292 NSCLC cells under the pretense that confluent cells would exhibit a more angiogenic and growth-centered phenotype. Indeed, confluent H292 cells potentiated endothelial cell angiogenesis in co-culture models in an EGFR-dependent manner. While confluent H292 cells did not exhibit any change in EGFR protein expression, EGFR localization to the extracellular membrane was increased. EGFR membrane localization coincided with a comparable potentiation of maximal EGFR phosphorylation and was followed by a 3-fold increase in vascular endothelial growth factor A (VEGF-A) production as compared to subconfluent cells. EGFR-mediated VEGF-A production was determined to be dependent on signal transducer and activator of transcription 3 (STAT3) activation and not phosphoinositide 3-kinase (PI3K) signaling. These results identify unique cell density dependent phenotypes within a monoclonal NSCLC cell line and provide a potential mechanism of resistance to anti-EGFR therapy in metastatic NSCLC

Abstract 195: Nitro-Oleic Acid is a Dual Inhibitor of Nox1 and Nox2

Nitrated fatty acids (NFAs) are α,β-unsaturated keto derivatives of ω-3 fatty acids formed from redox reactions of nitric oxide and nitrite and have recently emerged as anti-inflammatory agents that act as electrophiles. NFAs activate Nrf2 and PPARγ signaling and inhibit NF-κB. Recent data suggest a role in inhibition of reactive oxygen species (ROS) production. This function however, remains largely unexplored. The Nox family of oxidases is a major source of ROS implicated in hypertension and other cardiovascular diseases (CVDs). Here we postulate that the NFA nitro-oleic acid (OA-NO 2 ) specifically inhibits ROS production by the Nox1 and Nox2 oxidases. Treatment of canonical Nox1-expressing COS-7 cells with OA-NO 2 vs its oleic acid (OA) control resulted in a concentration-dependent inhibition of ROS production (101.5, 98.9, 91.7, 94.8, 54.2, and 45.5 % of vehicle treated groups for 0.1, 1, 2, 4, 8, and 10 μM OA-NO 2 , respectively), with an IC 50 of 6.5 μM. Treatment of phorbol ester (PMA)-stimulated Nox2-expressing COS-7 cells yielded similar results (103.6, 106.6, 112.2, 102.1, 51.8 and 35.1 % vehicle treated groups for the same OA-NO 2 concentration range) with an IC 50 of 7.0 μM. Treatment with the same OA-NO 2 concentrations of Nox4- or Nox5-expressing COS-7 cells did not result in any inhibition. The effects of OA-NO 2 on Nox1 and Nox2 were unique to its electrophilic unsaturated keto structure, as OA (lacking NO 2 group), palmitic acid (lacking both double bond and NO 2 group) and linoleic acid (containing two cis double bonds) did not result in any inhibition, demonstrating that fatty acids other than NFAs are incapable of this inhibition. Moreover, experiments using potassium superoxide (O 2 •- ) ruled out any scavenging effects of OA-NO 2 on O 2 •- . Finally, 10 μM OA-NO 2 inhibited both angiotensin II (AngII)- and PMA-stimulated O 2 •- in rat aortic smooth muscle cells (4.4 ± 0.9, 10.8 ± 1.7 and 9.0 ± 2.5 in OA-treated vehicle, AngII and PMA groups vs. 5.8 ± 0.6, 5.7 ± 0.3 and 3.3 ± 1.0 pmol O 2 •- /min/mg protein in OA-NO 2 -treated vehicle, AngII and PMA groups, respectively). Taken together, these results identify NFAs as potent inhibitors of Nox1- and Nox2-derived ROS raising the possibility for their use as therapeutic agents to treat hypertension and other CVDs. </jats:p