Angiotensin II (ANG II) plays an important role in the central regulation of systemic cardiovascular function. ANG II-mediated intraneuronal signaling has been shown to be predicated by an increase in mitochondrial superoxide (O₂˙⁻), yet the source of this reactive oxygen species (ROS) production remains unclear. NADPH oxidase 4 (Nox4), a member of the NADPH oxidase family, has been reported to be localized in mitochondria of various cell types and has been implicated in brain angiotensinergic signaling. However, the subcellular localization and function of Nox4 in neurons has not been fully elucidated. In this study, we hypothesized that Nox4 is expressed in neuron mitochondria and is involved in ANG II-dependent O₂˙⁻-mediated intraneuronal signaling. To query this, Nox4 immunofluorescent staining and mitochondrial enrichment were performed in a mouse catecholaminergic neuronal cell model (CATH.a). Nox4 was shown to be present in neuron mitochondria as evidenced by colocalization with both the mitochondrial-localized protein manganese superoxide dismutase (MnSOD) and dye MitoTracker Red. Moreover, Nox4 expression was significantly increased in enriched mitochondrial fractions compared with whole cell lysates. Additionally, adenoviral-encoded small interfering RNA for Nox4 (AdsiNox4) caused a robust knockdown in Nox4 mRNA and protein levels, which led to the attenuation of ANG II-induced mitochondrial O₂˙⁻ production. Finally, in the subfornical organ (SFO) of the brain, Nox4 not only demonstrated mitochondrial localization but was induced by chronic, peripheral infusion of ANG II. Collectively, these data suggest that Nox4 is a source of O₂˙⁻ in neuron mitochondria that contributes to ANG II intraneuronal signaling.

Cardiovascular diseases, including hypertension and heart failure, are associated with activation of the renin-angiotensin system (RAS) and increased circulating and tissue levels of ANG II, a primary effector peptide of the RAS. Through its actions on various cell types and organ systems, ANG II contributes to the pathogenesis of cardiovascular diseases by inducing cardiac and vascular hypertrophy, vasoconstriction, sodium and water reabsorption in kidneys, sympathoexcitation, and activation of the immune system. Cardiovascular research over the past 15–20 years has clearly implicated an important role for elevated levels of reactive oxygen species (ROS) in mediating these pathophysiological actions of ANG II. As such, the use of antioxidants, to reduce the elevated levels of ROS, as potential therapies for various ANG II-associated cardiovascular diseases has been intensely investigated. Although some antioxidant-based therapies have shown therapeutic impact in animal models of cardiovascular disease and in human patients, others have failed. In this review, we discuss the benefits and limitations of recent strategies, including gene therapy, dietary sources, low-molecular-weight free radical scavengers, polyethylene glycol conjugation, and nanomedicine-based technologies, which are designed to deliver antioxidants for the improved treatment of cardiovascular diseases. Although much work has been completed, additional research focusing on developing specific antioxidant molecules or proteins and identifying the ideal in vivo delivery system for such antioxidants is necessary before the use of antioxidant-based therapies for cardiovascular diseases become a clinical reality.

Reactive oxygen species (ROS), particularly superoxide (O₂^·−), have been identified as key signaling intermediates in ANG II-induced neuronal activation and sympathoexcitation associated with cardiovascular diseases, such as hypertension and heart failure. Studies of the central nervous system have identified NADPH oxidase as a primary source of O₂^·− in ANG II-stimulated neurons; however, additional sources of O₂^·−, including mitochondria, have been mostly overlooked. Here, we tested the hypothesis that ANG II increases mitochondria-produced O₂^·− in neurons and that increased scavenging of mitochondria-produced O₂^·− attenuates ANG II-dependent intraneuronal signaling. Stimulation of catecholaminergic (CATH.a) neurons with ANG II (100 nM) increased mitochondria-localized O₂^·− levels, as measured by MitoSOX Red fluorescence. This response was significantly attenuated in neurons overexpressing the mitochondria-targeted O₂^·−-scavenging enzyme Mn-SOD. To examine the biological significance of the ANG II-mediated increase in mitochondria-produced O₂^·−, we used the whole cell configuration of the patch-clamp technique to record the well-characterized ANG II-induced inhibition of voltage-gated K⁺ current ( I_Kv) in neurons. Adenovirus-mediated Mn-SOD overexpression or pretreatment with the cell-permeable antioxidant tempol (1 mM) significantly attenuated ANG II-induced inhibition of I_Kv. In contrast, pretreatment with extracellular SOD protein (400 U/ml) had no effect. Mn-SOD overexpression also inhibited ANG II-induced activation of Ca²⁺/calmodulin kinase II, a redox-sensitive protein known to modulate I_Kv. These data indicate that ANG II increases mitochondrial O₂^·−, which mediates, at least in part, ANG II-induced activation of Ca²⁺/calmodulin kinase II and inhibition of I_Kv in neurons.

Obesity-induced insulin resistance is a risk factor for diabetes and cardiovascular disease. However, the mechanisms underlying endothelial senescence in obesity, and how it impacts obesity-induced insulin resistance remain incompletely understood. In this study, transcriptome analysis revealed that the long non-coding RNA (lncRNA) Maternally expressed gene 3 (Meg3) is one of the top differentially expressed lncRNAs in the vascular endothelium in diet-induced obese mice. Meg3 knockdown induces cellular senescence of endothelial cells characterized by increased senescence-associated β-galactosidase activity, increased levels of endogenous superoxide, impaired mitochondrial structure and function, and impaired autophagy. Moreover, Meg3 knockdown causes cellular senescence of hepatic endothelium in diet-induced obese mice. Furthermore, Meg3 expression is elevated in human nonalcoholic fatty livers and nonalcoholic steatohepatitis livers, which positively correlates with the expression of CDKN2A encoding p16, an important hallmark of cellular senescence. Meg3 knockdown potentiates obesity-induced insulin resistance and impairs glucose homeostasis. Insulin signaling is reduced by Meg3 knockdown in the liver and, to a lesser extent, in the skeletal muscle, but not in the visceral fat of obese mice. We found that the attenuation of cellular senescence of hepatic endothelium by ablating p53 expression in vascular endothelium can restore impaired glucose homeostasis and insulin signaling in obesity. In conclusion, our data demonstrate that cellular senescence of hepatic endothelium promotes obesity-induced insulin resistance, which is tightly regulated by the expression of Meg3. Our results suggest that manipulation of Meg3 expression may represent a novel approach to managing obesity-associated hepatic endothelial senescence and insulin resistance.

Doxycycline (DOX), a derivative of tetracycline, is a broad-spectrum antibiotic that exhibits a number of therapeutic activities in addition to its antibacterial properties. For example, DOX has been used in the management of a number of diseases characterized by chronic inflammation. One potential mechanism by which DOX inhibits the progression of these diseases is by reducing oxidative stress, thereby inhibiting subsequent lipid peroxidation and inflammatory responses. Herein, we tested the hypothesis that DOX directly scavenges reactive oxygen species (ROS) and inhibits the formation of redox-mediated malondialdehyde-acetaldehyde (MAA) protein adducts. Using a cell-free system, we demonstrated that DOX scavenged reactive oxygen species (ROS) produced during the formation of MAA-adducts and inhibits the formation of MAA-protein adducts. To determine whether DOX scavenges specific ROS, we examined the ability of DOX to directly scavenge superoxide and hydrogen peroxide. Using electron paramagnetic resonance (EPR) spectroscopy, we found that DOX directly scavenged superoxide, but not hydrogen peroxide. Additionally, we found that DOX inhibits MAA-induced activation of Nrf2, a redox-sensitive transcription factor. Together, these findings demonstrate the under-recognized direct antioxidant property of DOX that may help to explain its therapeutic potential in the treatment of conditions characterized by chronic inflammation and increased oxidative stress.