Cell-Free DNA to Detect Heart Allograft Acute Rejection.

BACKGROUND: After heart transplantation, endomyocardial biopsy (EMBx) is used to monitor for acute rejection (AR). Unfortunately, EMBx is invasive, and its conventional histological interpretation has limitations. This is a validation study to assess the performance of a sensitive blood biomarker-percent donor-derived cell-free DNA (%ddcfDNA)-for detection of AR in cardiac transplant recipients. METHODS: This multicenter, prospective cohort study recruited heart transplant subjects and collected plasma samples contemporaneously with EMBx for %ddcfDNA measurement by shotgun sequencing. Histopathology data were collected to define AR, its 2 phenotypes (acute cellular rejection [ACR] and antibody-mediated rejection [AMR]), and controls without rejection. The primary analysis was to compare %ddcfDNA levels (median and interquartile range [IQR]) for AR, AMR, and ACR with controls and to determine %ddcfDNA test characteristics using receiver-operator characteristics analysis. RESULTS: The study included 171 subjects with median posttransplant follow-up of 17.7 months (IQR, 12.1-23.6), with 1392 EMBx, and 1834 %ddcfDNA measures available for analysis. Median %ddcfDNA levels decayed after surgery to 0.13% (IQR, 0.03%-0.21%) by 28 days. Also, %ddcfDNA increased again with AR compared with control values (0.38% [IQR, 0.31-0.83%], versus 0.03% [IQR, 0.01-0.14%]; P<0.001). The rise was detected 0.5 and 3.2 months before histopathologic diagnosis of ACR and AMR. The area under the receiver operator characteristic curve for AR was 0.92. A 0.25%ddcfDNA threshold had a negative predictive value for AR of 99% and would have safely eliminated 81% of EMBx. In addition, %ddcfDNA showed distinctive characteristics comparing AMR with ACR, including 5-fold higher levels (AMR ≥2, 1.68% [IQR, 0.49-2.79%] versus ACR grade ≥2R, 0.34% [IQR, 0.28-0.72%]), higher area under the receiver operator characteristic curve (0.95 versus 0.85), higher guanosine-cytosine content, and higher percentage of short ddcfDNA fragments. CONCLUSIONS: We found that %ddcfDNA detected AR with a high area under the receiver operator characteristic curve and negative predictive value. Monitoring with ddcfDNA demonstrated excellent performance characteristics for both ACR and AMR and led to earlier detection than the EMBx-based monitoring. This study supports the use of %ddcfDNA to monitor for AR in patients with heart transplant and paves the way for a clinical utility study. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02423070.

Nox2 as a potential target of mitochondrial superoxide and its role in endothelial oxidative stress.

Superoxide (O2(·-)) production by the NADPH oxidases is implicated in the pathogenesis of many cardiovascular diseases, including hypertension. We have previously shown that activation of NADPH oxidases increases mitochondrial O2(·-) which is inhibited by the ATP-sensitive K(+) channel (mitoKATP) inhibitor 5-hydroxydecanoic acid and that scavenging of mitochondrial or cytoplasmic O2(·-) inhibits hypertension. We hypothesized that mitoKATP-mediated mitochondrial O2(·-) potentiates cytoplasmic O2(·-) by stimulation of NADPH oxidases. In this work we studied Nox isoforms as a potential target of mitochondrial O2(·-). We tested contribution of reverse electron transfer (RET) from complex II to complex I in mitochondrial O2(·-) production and NADPH oxidase activation in human aortic endothelial cells. Activation of mitoKATP with low dose of diazoxide (100 nM) decreased mitochondrial membrane potential (tetramethylrhodamine methyl ester probe) and increased production of mitochondrial and cytoplasmic O2(·-) measured by site-specific probes and mitoSOX. Inhibition of RET with complex II inhibitor (malonate) or complex I inhibitor (rotenone) attenuated the production of mitochondrial and cytoplasmic O2(·-). Supplementation with a mitochondria-targeted SOD mimetic (mitoTEMPO) or a mitochondria-targeted glutathione peroxidase mimetic (mitoEbselen) inhibited production of mitochondrial and cytoplasmic O2(·-). Inhibition of Nox2 (gp91ds) or Nox2 depletion with small interfering RNA but not Nox1, Nox4, or Nox5 abolished diazoxide-induced O2(·-) production in the cytoplasm. Treatment of angiotensin II-infused mice with RET inhibitor dihydroethidium (malate) significantly reduced blood pressure. Our study suggests that mitoKATP-mediated mitochondrial O2(·-) stimulates cytoplasmic Nox2, contributing to the development of endothelial oxidative stress and hypertension.

Therapeutic targeting of mitochondrial superoxide in hypertension.

RATIONALE: Superoxide (O2(-) ) has been implicated in the pathogenesis of many human diseases including hypertension; however, commonly used antioxidants have proven ineffective in clinical trials. It is possible that these agents are not adequately delivered to the subcellular sites of superoxide production. OBJECTIVE: Because the mitochondria are important sources of reactive oxygen species, we postulated that mitochondrial targeting of superoxide scavenging would have therapeutic benefit. METHODS AND RESULTS: In this study, we found that the hormone angiotensin (Ang II) increased endothelial mitochondrial superoxide production. Treatment with the mitochondria-targeted antioxidant mitoTEMPO decreased mitochondrial O2(-), inhibited the total cellular O2(-), reduced cellular NADPH oxidase activity, and restored the level of bioavailable NO. These effects were mimicked by overexpressing the mitochondrial MnSOD (SOD2), whereas SOD2 depletion with small interfering RNA increased both basal and Ang II-stimulated cellular O2(-). Treatment of mice in vivo with mitoTEMPO attenuated hypertension when given at the onset of Ang II infusion and decreased blood pressure by 30 mm Hg following establishment of both Ang II-induced and DOCA salt hypertension, whereas a similar dose of nontargeted TEMPOL was not effective. In vivo, mitoTEMPO decreased vascular O2(-), increased vascular NO production and improved endothelial-dependent relaxation. Interestingly, transgenic mice overexpressing mitochondrial SOD2 demonstrated attenuated Ang II-induced hypertension and vascular oxidative stress similar to mice treated with mitoTEMPO. CONCLUSIONS: These studies show that mitochondrial O2(-) is important for the development of hypertension and that antioxidant strategies specifically targeting this organelle could have therapeutic benefit in this and possibly other diseases.

Age-accelerated atherosclerosis correlates with failure to upregulate antioxidant genes.

Excess food intake leads to obesity and diabetes, both of which are well-known independent risk factors for atherosclerosis, and both of which are growing epidemics in an aging population. We hypothesized that aging enhances the metabolic and vascular effects of high fat diet (HFD) and therefore examined the effect of age on atherosclerosis and insulin resistance in lipoprotein receptor knockout (LDLR(-/-)) mice. We found that 12-month-old (middle-aged) LDLR(-/-) mice developed substantially worse metabolic syndrome, diabetes, and atherosclerosis than 3-month-old (young) LDLR(-/-) mice when both were fed HFD for 3 months, despite similar elevations in total cholesterol levels. Microarray analyses were performed to analyze the mechanism responsible for the marked acceleration of atherosclerosis in middle-aged mice. Chow-fed middle-aged mice had greater aortic expression of multiple antioxidant genes than chow-fed young mice, including glutathione peroxidase-1 and -4, catalase, superoxide dismutase-2, and uncoupling protein-2. Aortic expression of these enzymes markedly increased in young mice fed HFD but decreased or only modestly increased in middle-aged mice fed HFD, despite the fact that systemic oxidative stress and vascular reactive oxygen species generation, measured by plasma F2alpha isoprostane concentration (systemic) and dihydroethidium conversion and p47phox expression (vascular), were greater in middle-aged mice fed HFD. Thus, the mechanism for the accelerated vascular injury in older LDLR(-/-) mice was likely the profound inability to mount an antioxidant response. This effect was related to a decrease in vascular expression of 2 key transcriptional pathways regulating the antioxidant response, DJ-1 and forkhead box, subgroup O family (FOXOs). Treatment of middle-aged mice fed HFD with the antioxidant apocynin attenuated atherosclerosis, whereas treatment with the insulin sensitizer rosiglitazone attenuated both metabolic syndrome and atherosclerosis. Both treatments decreased oxidative stress. A novel effect of rosiglitazone was to increase expression of Nrf2 (nuclear factor [erythroid-derived 2]-like 2), a downstream target of DJ-1 contributing to enhanced expression of vascular antioxidant enzymes. This investigation underscores the role of oxidative stress when multiple atherosclerotic risk factors, particularly aging, converge on the vessel wall and emphasizes the need to develop effective strategies to inhibit oxidative stress to protect aging vasculature.

Anti-inflammatory activity of Chios mastic gum is associated with inhibition of TNF-alpha induced oxidative stress.

BACKGROUND: Gum of Chios mastic (Pistacia lentiscus var. chia) is a natural antimicrobial agent that has found extensive use in pharmaceutical products and as a nutritional supplement. The molecular mechanisms of its anti-inflammatory activity, however, are not clear. In this work, the potential role of antioxidant activity of Chios mastic gum has been evaluated. METHODS: Scavenging of superoxide radical was investigated by electron spin resonance and spin trapping technique using EMPO spin trap in xanthine oxidase system. Superoxide production in endothelial and smooth muscle cells stimulated with TNF-α or angiotensin II and treated with vehicle (DMSO) or mastic gum (0.1-10 µg/ml) was measured by DHE and HPLC. Cellular H2O2 was measured by Amplex Red. Inhibition of protein kinase C (PKC) with mastic gum was determined by the decrease of purified PKC activity, by inhibition of PKC activity in cellular homogenate and by attenuation of superoxide production in cells treated with PKC activator phorbol 12-myristate 13-acetate (PMA). RESULTS: Spin trapping study did not show significant scavenging of superoxide by mastic gum itself. However, mastic gum inhibited cellular production of superoxide and H2O2 in dose dependent manner in TNF-α treated rat aortic smooth muscle cells but did not affect unstimulated cells. TNF-α significantly increased the cellular superoxide production by NADPH oxidase, while mastic gum completely abolished this stimulation. Mastic gum inhibited the activity of purified PKC, decreased PKC activity in cell homogenate, and attenuated superoxide production in cells stimulated with PKC activator PMA and PKC-dependent angiotensin II in endothelial cells. CONCLUSION: We suggest that mastic gum inhibits PKC which attenuates production of superoxide and H2O2 by NADPH oxidases. This antioxidant property may have direct implication to the anti-inflammatory activity of the Chios mastic gum.

Distinct roles of Nox1 and Nox4 in basal and angiotensin II-stimulated superoxide and hydrogen peroxide production.

NADPH oxidases are major sources of superoxide (O2*-) and hydrogen peroxide (H2O2) in vascular cells. Production of these reactive oxygen species (ROS) is essential for cell proliferation and differentiation, while ROS overproduction has been implicated in hypertension and atherosclerosis. It is known that the heme-containing catalytic subunits Nox1 and Nox4 are responsible for oxygen reduction in vascular smooth muscle cells from large arteries. However, the exact mechanism of ROS production by NADPH oxidases is not completely understood. We hypothesized that Nox1 and Nox4 play distinct roles in basal and angiotensin II (AngII)-stimulated production of O2*- and H2O2. Nox1 and Nox4 expression in rat aortic smooth muscle cells (RASMCs) was selectively reduced by treatment with siNox4 or antisense Nox1 adenovirus. Production of O2*- and H2O2 in intact RASMCs was analyzed by dihydroethidium and Amplex Red assay. Activity of NADPH oxidases was measured by NADPH-dependent O2*- and H2O2 production using electron spin resonance (ESR) and 1-hydroxy-3-carboxypyrrolidine (CPH) in the membrane fraction in the absence of cytosolic superoxide dismutase. It was found that production of O2*- by quiescent RASMC NADPH oxidases was five times less than H2O2 production. Stimulation of cells with AngII led to a 2-fold increase of O2*- production by NADPH oxidases, with a small 15 to 30% increase in H2O2 formation. Depletion of Nox4 in RASMCs led to diminished basal H2O2 production, but did not affect O2*- or H2O2 production stimulated by AngII. In contrast, depletion of Nox1 in RASMCs inhibited production of O2*- and AngII-stimulated H2O2 in the membrane fraction and intact cells. Our data suggest that Nox4 produces mainly H2O2, while Nox1 generates mostly O2*- that is later converted to H2O2. Therefore, Nox4 is responsible for basal H2O2 production, while O2*- production in nonstimulated and AngII-stimulated cells depends on Nox1. The difference in the products generated by Nox1 and Nox4 may help to explain the distinct roles of these NADPH oxidases in cell signaling. These findings also provide important insight into the origin of H2O2 in vascular cells, and may partially account for the limited pharmacological effect of antioxidant treatments with O2*- scavengers that do not affect H2O2.

Cardiolipin fatty acid remodeling regulates mitochondrial function by modifying the electron entry point in the respiratory chain.

Modifications of cardiolipin (CL) levels or compositions are associated with changes in mitochondrial function in a wide range of pathologies. We have made the discovery that acetaminophen remodels CL fatty acids composition from tetralinoleoyl to linoleoyltrioleoyl-CL, a remodeling that is associated with decreased mitochondrial respiration. Our data show that CL remodeling causes a shift in electron entry from complex II to the ß-oxidation electron transfer flavoprotein quinone oxidoreductase (ETF/QOR) pathway. These data demonstrate that electron entry in the respiratory chain is regulated by CL fatty acid composition and provide proof-of-concept that pharmacological intervention can be used to modify CL composition.

Mitochondrial Cyclophilin D in Vascular Oxidative Stress and Hypertension.

Vascular superoxide (O˙2 (-)) and inflammation contribute to hypertension. The mitochondria are an important source of O˙2 (-); however, the regulation of mitochondrial O˙2 (-) and the antihypertensive potential of targeting the mitochondria remain poorly defined. Angiotensin II and inflammatory cytokines, such as interleukin 17A and tumor necrosis factor-α (TNFα) significantly contribute to hypertension. We hypothesized that angiotensin II and cytokines co-operatively induce cyclophilin D (CypD)-dependent mitochondrial O˙2 (-) production in hypertension. We tested whether CypD inhibition attenuates endothelial oxidative stress and reduces hypertension. CypD depletion in CypD(-/-) mice prevents overproduction of mitochondrial O˙2 (-) in angiotensin II-infused mice, attenuates hypertension by 20 mm Hg, and improves vascular relaxation compared with wild-type C57Bl/6J mice. Treatment of hypertensive mice with the specific CypD inhibitor Sanglifehrin A reduces blood pressure by 28 mm Hg, inhibits production of mitochondrial O˙2 (-) by 40%, and improves vascular relaxation. Angiotensin II-induced hypertension was associated with CypD redox activation by S-glutathionylation, and expression of the mitochondria-targeted H2O2 scavenger, catalase, abolished CypD S-glutathionylation, prevented stimulation mitochondrial O˙2 (-), and attenuated hypertension. The functional role of cytokine-angiotensin II interplay was confirmed by co-operative stimulation of mitochondrial O˙2 (-) by 3-fold in cultured endothelial cells and impairment of aortic relaxation incubated with combination of angiotensin II, interleukin 17A, and tumor necrosis factor-α which was prevented by CypD depletion or expression of mitochondria-targeted SOD2 and catalase. These data support a novel role of CypD in hypertension and demonstrate that targeting CypD decreases mitochondrial O˙2 (-), improves vascular relaxation, and reduces hypertension.

Nox2-induced production of mitochondrial superoxide in angiotensin II-mediated endothelial oxidative stress and hypertension.

AIMS: Angiotensin II (AngII)-induced superoxide (O2(•-)) production by the NADPH oxidases and mitochondria has been implicated in the pathogenesis of endothelial dysfunction and hypertension. In this work, we investigated the specific molecular mechanisms responsible for the stimulation of mitochondrial O2(•-) and its downstream targets using cultured human aortic endothelial cells and a mouse model of AngII-induced hypertension. RESULTS: Western blot analysis showed that Nox2 and Nox4 were present in the cytoplasm but not in the mitochondria. Depletion of Nox2, but not Nox1, Nox4, or Nox5, using siRNA inhibits AngII-induced O2(•-) production in both mitochondria and cytoplasm. Nox2 depletion in gp91phox knockout mice inhibited AngII-induced cellular and mitochondrial O2(•-) and attenuated hypertension. Inhibition of mitochondrial reverse electron transfer with malonate, malate, or rotenone attenuated AngII-induced cytoplasmic and mitochondrial O2(•-) production. Inhibition of the mitochondrial ATP-sensitive potassium channel (mitoK(+)ATP) with 5-hydroxydecanoic acid or specific PKCɛ peptide antagonist (EAVSLKPT) reduced AngII-induced H2O2 in isolated mitochondria and diminished cytoplasmic O2(•-). The mitoK(+)ATP agonist diazoxide increased mitochondrial O2(•-), cytoplasmic c-Src phosphorylation and cytoplasmic O2(•-) suggesting feed-forward regulation of cellular O2(•-) by mitochondrial reactive oxygen species (ROS). Treatment of AngII-infused mice with malate reduced blood pressure and enhanced the antihypertensive effect of mitoTEMPO. Mitochondria-targeted H2O2 scavenger mitoEbselen attenuated redox-dependent c-Src and inhibited AngII-induced cellular O2(•-), diminished aortic H2O2, and reduced blood pressure in hypertensive mice. INNOVATION AND CONCLUSIONS: These studies show that Nox2 stimulates mitochondrial ROS by activating reverse electron transfer and both mitochondrial O2(•-) and reverse electron transfer may represent new pharmacological targets for the treatment of hypertension.

DC isoketal-modified proteins activate T cells and promote hypertension.

Oxidative damage and inflammation are both implicated in the genesis of hypertension; however, the mechanisms by which these stimuli promote hypertension are not fully understood. Here, we have described a pathway in which hypertensive stimuli promote dendritic cell (DC) activation of T cells, ultimately leading to hypertension. Using multiple murine models of hypertension, we determined that proteins oxidatively modified by highly reactive γ-ketoaldehydes (isoketals) are formed in hypertension and accumulate in DCs. Isoketal accumulation was associated with DC production of IL-6, IL-1ß, and IL-23 and an increase in costimulatory proteins CD80 and CD86. These activated DCs promoted T cell, particularly CD8+ T cell, proliferation; production of IFN-γ and IL-17A; and hypertension. Moreover, isoketal scavengers prevented these hypertension-associated events. Plasma F2-isoprostanes, which are formed in concert with isoketals, were found to be elevated in humans with treated hypertension and were markedly elevated in patients with resistant hypertension. Isoketal-modified proteins were also markedly elevated in circulating monocytes and DCs from humans with hypertension. Our data reveal that hypertension activates DCs, in large part by promoting the formation of isoketals, and suggest that reducing isoketals has potential as a treatment strategy for this disease.

Rapid and specific measurements of superoxide using fluorescence spectroscopy.

Superoxide plays a key role in many pathological processes; however, detection of superoxide by one of the most common methods using dihydroethidium (DHE) may be unspecific because of overlapping fluorescence of the superoxide-specific product, 2-OH-ethidium (2OH-E), and the unspecific oxidation product, ethidium. Here, we show a new optimized fluorescence spectroscopy protocol that allows rapid and specific detection of superoxide in cell-free systems and intact cells using DHE. We defined new optimized fluorescent settings to measure the superoxide-specific product and minimize the interference of unspecific DHE oxidation products. Using this protocol, we studied real-time superoxide production by xanthine oxidase- and menadione-treated cultured cells. Specificity of the plate reader-based superoxide measurements was confirmed by the inhibition of fluorescence with superoxide dismutase and high-performance liquid chromatography (HPLC) analysis. We show that limitations of the HPLC-based analysis can be overcome by the optimized fluorescence spectroscopy.

Does scavenging of mitochondrial superoxide attenuate cancer prosurvival signaling pathways?

It has been previously suggested that overexpression of mitochondrial superoxide dismutase (SOD) attenuates cancer development; however, the exact mechanism remains unclear. In this work, we have studied the direct effect of the mitochondria-targeted superoxide scavenger, (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO), on B16-F0 mouse melanoma cells and tumor growth in a nude mouse model of human melanoma. We show that scavenging of mitochondrial superoxide inhibited cell growth, reduced viability, and induced apoptosis in melanoma cells, but did not affect nonmalignant skin fibroblasts. Diminished mitochondrial superoxide inhibited redox-dependent Akt, restored activity of mitochondrial pyruvate dehydrogenase, and reduced HIF1-α and lactate dehydrogenase expression in cancer cells. Suppression of glycolysis in mitoTEMPO-treated melanoma cells resulted in a significant drop of cellular adenosine-5'-triphosphate and induced cell death. In vivo mitoTEMPO treatment effectively suppressed growth of established tumor in the mouse model of human melanoma. Therefore, our data lead to the hypothesis that scavenging of mitochondrial superoxide selectively inhibits redox-sensitive survival and metabolic pathways, resulting in cancer cell death. In contrast to existing anticancer therapies, inhibition of mitochondrial superoxide may represent a novel specific anticancer treatment with reduced cytotoxic side effects.