A novel diagnostic tool reveals mitochondrial pathology in human diseases and aging

The inherent complex and pleiotropic phenotype of mitochondrial diseases poses a significant diagnostic challenge for clinicians as well as an analytical barrier for scientists. To overcome these obstacles we compiled a novel database, www.mitodb.com, containing the clinical features of primary mitochondrial diseases. Based on this we developed a number of qualitative and quantitative measures, enabling us to determine whether a disorder can be characterized as mitochondrial. These included a clustering algorithm, a disease network, a mitochondrial barcode and two scoring algorithms. Using these tools we detected mitochondrial involvement in a number of diseases not previously recorded as mitochondrial. As a proof of principle Cockayne syndrome, ataxia with oculomotor apraxia 1 (AOA1), spinocerebellar ataxia with axonal neuropathy 1 (SCAN1) and ataxia-telangiectasia have recently been shown to have mitochondrial dysfunction and those diseases showed strong association with mitochondrial disorders. We next evaluated mitochondrial involvement in aging and detected two distinct categories of accelerated aging disorders, one of them being associated with mitochondrial dysfunction. Normal aging seemed to associate stronger with the mitochondrial diseases than the non-mitochondrial partially supporting a mitochondrial theory of aging

NOXA1-dependent NADPH oxidase regulates redox signaling and phenotype of vascular smooth muscle cell during atherogenesis

Increased reactive oxygen species (ROS) production and inflammation are key factors in the pathogenesis of atherosclerosis. We previously reported that NOX activator 1 (NOXA1) is the critical functional homolog of p67phox for NADPH oxidase activation in mouse vascular smooth muscle cells (VSMC). Here we investigated the effects of systemic and SMC-specific deletion of Noxa1 on VSMC phenotype during atherogenesis in mice.Neointimal hyperplasia following endovascular injury was lower in Noxa1-deficient mice versus the wild-type following endovascular injury. Noxa1 deletion in Apoe-/- or Ldlr-/- mice fed a Western diet showed 50% reduction in vascular ROS and 30% reduction in aortic atherosclerotic lesion area and aortic sinus lesion volume (P < 0.01). SMC-specific deletion of Noxa1 in Apoe-/- mice (Noxa1SMC-/-/Apoe-/-) similarly decreased vascular ROS levels and atherosclerotic lesion size. TNFα-induced ROS generation, proliferation and migration were significantly attenuated in Noxa1-deficient versus wild-type VSMC. Immunofluorescence analysis of atherosclerotic lesions showed a significant decrease in cells positive for CD68 and myosin11 (22% versus 9%) and Mac3 and α-actin (17% versus 5%) in the Noxa1SMC-/-/Apoe-/- versus Apoe-/- mice. The expression of transcription factor KLF4, a modulator of VSMC phenotype, and its downstream targets – VCAM1, CCL2, and MMP2 – were significantly reduced in the lesions of Noxa1SMC-/-/Apoe-/- versus Apoe-/- mice as well as in oxidized phospholipids treated Noxa1SMC-/- versus wild-type VSMC.Our data support an important role for NOXA1-dependent NADPH oxidase activity in VSMC plasticity during restenosis and atherosclerosis, augmenting VSMC proliferation and migration and KLF4-mediated transition to macrophage-like cells, plaque inflammation, and expansion. Keywords: Oxidative stress, NOXA1, Smooth muscle cells, KLF4, Macrophage-like cells, Atherosclerosi

Increased mitochondrial NADPH oxidase 4 (NOX4) expression in aging is a causative factor in aortic stiffening

Aging is characterized by increased aortic stiffness, an early, independent predictor and cause of cardiovascular disease. Oxidative stress from excess reactive oxygen species (ROS) production increases with age. Mitochondria and NADPH oxidases (NOXs) are two major sources of ROS in cardiovascular system. We showed previously that increased mitochondrial ROS levels over a lifetime induce aortic stiffening in a mouse oxidative stress model. Also, NADPH oxidase 4 (NOX4) expression and ROS levels increase with age in aortas, aortic vascular smooth muscle cells (VSMCs) and mitochondria, and are correlated with age-associated aortic stiffness in hypercholesterolemic mice. The present study investigated whether young mice (4 months-old) with increased mitochondrial NOX4 levels recapitulate vascular aging and age-associated aortic stiffness. We generated transgenic mice with low (Nox4TG605; 2.1-fold higher) and high (Nox4TG618; 4.9-fold higher) mitochondrial NOX4 expression. Young Nox4TG618 mice showed significant increase in aortic stiffness and decrease in phenylephrine-induced aortic contraction, but not Nox4TG605 mice. Increased mitochondrial oxidative stress increased intrinsic VSMC stiffness, induced aortic extracellular matrix remodeling and fibrosis, a leftward shift in stress-strain curves, decreased volume compliance and focal adhesion turnover in Nox4TG618 mice. Nox4TG618 VSMCs phenocopied other features of vascular aging such as increased DNA damage, increased premature and replicative senescence and apoptosis, increased proinflammatory protein expression and decreased respiration. Aortic stiffening in young Nox4TG618 mice was significantly blunted with mitochondrial-targeted catalase overexpression. This demonstration of the role of mitochondrial oxidative stress in aortic stiffness will galvanize search for new mitochondrial-targeted therapeutics for treatment of age-associated vascular dysfunction

Transcriptional pausing and stalling causes multiple clustered mutations by human activation-induced deaminase

Transcription of the rearranged immunoglobulin gene and expression of the enzyme activation-induced deaminase (AID) are essential for somatic hypermutations of this gene during antibody maturation. While AID acts as a single-strand DNA-cytosine deaminase creating U · G mispairs that lead to mutations, the role played by transcription in this process is less clear. We have used in vitro transcription of the kan gene by the T7 RNA polymerase (RNAP) in the presence of AID and a genetic reversion assay for kanamycin-resistance to investigate the causes of multiple clustered mutations (MCMs) during somatic hypermutations. We find that, depending on transcription conditions, AID can cause single-base substitutions or MCMs. When wild-type RNAP is used for transcription at physiologically relevant concentrations of ribonucleoside triphosphates (NTPs), few MCMs are found. In contrast, slowing the rate of elongation by reducing the NTP concentration or using a mutant RNAP increases several-fold the percent of revertants containing MCMs. Arresting the elongation complexes by a quick removal of NTPs leads to formation of RNA-DNA hybrids (R-loops). Treatment of these structures with AID results in a high percentage of KanR revertants with MCMs. Furthermore, selecting for transcription elongation complexes stalled near the codon that suffers mutations during acquisition of kanamycin-resistance results in an overwhelming majority of revertants with MCMs. These results show that if RNAP II pauses or stalls during transcription of immunoglobulin gene, AID is likely to promote MCMs. As changes in physiological conditions such as occurrence of certain DNA primary or secondary structures or DNA adducts are known to cause transcriptional pausing and stalling in mammalian cells, this process may cause MCMs during somatic hypermutation.—Canugovi, C., Samaranayake, M., Bhagwat, A. S. Transcriptional pausing and stalling causes multiple clustered mutations by human activation-induced deaminase

Mitochondrial DAMPs-dependent inflammasome activation during aging induces vascular smooth muscle cell dysfunction and aortic stiffness in low aerobic capacity rats

Introduction: Low aerobic exercise capacity is an independent risk factor for cardiovascular disease (CVD) and a predictor of premature death. In combination with aging, low aerobic capacity lowers the threshold for CVD. Aim: Since low aerobic capacity and aging have been linked to mitochondrial oxidative stress and dysfunction, we investigated whether aged Low-Capacity Runner (LCR) rats (27 months) had vascular dysfunction compared to High-Capacity Runner (HCR) rats. Methods and Results: A significant decrease in aortic eNOS levels and vasodilation as well as an increase in aortic collagen and stiffness were observed in aged LCR rats compared to age and sex-matched HCR rats. There was a correlation between age-related vascular dysfunction and increased levels of ROS and DNA damage in aortas of LCR rats. Moreover, mitochondrial oxygen consumption, membrane potential, ATP levels, and mitophagy were lower in VSMCs of aged LCR rats. VSMCs from older LCR rats showed AIM2 inflammasome activation. VSMCs of young (4 months old) LCR rats treated with purified mitochondrial damage-associated molecular patterns (DAMP) recapitulated an inflammasome activation phenotype similar to that seen in aged rat VSMCs. Rapamycin, a potent immunosuppressant, induced mitophagy, stimulated electron transport chain activity, reduced inflammasome activity, mitochondrial ROS and DAMP levels in VSMCs from aged LCR rats. MitoTEMPO, a mitochondrial ROS scavenger, was similarly effective on VSMCs from aged rats.Conclusion: The findings suggest that impaired mitophagy and inflammasome activation in the vasculature under conditions of low aerobic exercise capacity during aging results in arterial dysfunction and aortic stiffness. In older adults with reduced aerobic capacity, mitochondrial antioxidants, mitophagy induction, and inflammasome inhibition may be effective therapeutic strategies for enhancing vascular health

NADPH oxidase 4 regulates inflammation in ischemic heart failure: role of soluble epoxide hydrolase

Aims: Oxidative stress is implicated in cardiomyocyte cell death and cardiac remodeling in the failing heart. The role of NADPH oxidase 4 (NOX4) in cardiac adaptation to pressure overload is controversial, but its function in myocardial ischemic stress has not been thoroughly elucidated. This study examined the function of NOX4 in the pathogenesis of ischemic heart failure, utilizing mouse models, cell culture, and human heart samples. Results: Nox4-/- mice showed a protective phenotype in response to permanent left anterior descending coronary artery ligation with smaller infarction area, lower cardiomyocyte cross-sectional area, higher capillary density, and less cell death versus wild-type (WT) mice. Nox4-/- mice had lower activity of soluble epoxide hydrolase (sEH), a potent regulator of inflammation. Nox4-/- mice also showed a 50% reduction in the number of infiltrating CD68+ macrophages in the peri-infarct zone versus WT mice. Adenoviral overexpression of NOX4 in cardiomyoblast cells increased sEH expression and activity and CCL4 and CCL5 levels; inhibition of sEH activity in NOX4 overexpressing cells attenuated the cytokine levels. Human hearts with ischemic cardiomyopathy showed adverse cardiac remodeling, increased NOX4 and sEH protein expression and CCL4 and CCL5 levels compared with control nonfailing hearts. Innovation and Conclusion: These data from the Nox4-/- mouse model and human heart tissues show for the first time that oxidative stress from increased NOX4 expression has a functional role in ischemic heart failure. One mechanism by which NOX4 contributes to ischemic heart failure is by increasing inflammatory cytokine production via enhanced sEH activity

Mitochondrial DAMPs-dependent inflammasome activation during aging induces vascular smooth muscle cell dysfunction and aortic stiffness in low aerobic capacity rats

Introduction: Low aerobic exercise capacity is an independent risk factor for cardiovascular disease (CVD) and a predictor of premature death. In combination with aging, low aerobic capacity lowers the threshold for CVD. Aim: Since low aerobic capacity and aging have been linked to mitochondrial oxidative stress and dysfunction, we investigated whether aged Low-Capacity Runner (LCR) rats (27 months) had vascular dysfunction compared to High-Capacity Runner (HCR) rats. Methods and Results: A significant decrease in aortic eNOS levels and vasodilation as well as an increase in aortic collagen and stiffness were observed in aged LCR rats compared to age and sex-matched HCR rats. There was a correlation between age-related vascular dysfunction and increased levels of ROS and DNA damage in aortas of LCR rats. Moreover, mitochondrial oxygen consumption, membrane potential, ATP levels, and mitophagy were lower in VSMCs of aged LCR rats. VSMCs from older LCR rats showed AIM2 inflammasome activation. VSMCs of young (4 months old) LCR rats treated with purified mitochondrial damage-associated molecular patterns (DAMP) recapitulated an inflammasome activation phenotype similar to that seen in aged rat VSMCs. Rapamycin, a potent immunosuppressant, induced mitophagy, stimulated electron transport chain activity, reduced inflammasome activity, mitochondrial ROS and DAMP levels in VSMCs from aged LCR rats. MitoTEMPO, a mitochondrial ROS scavenger, was similarly effective on VSMCs from aged rats. Conclusion: The findings suggest that impaired mitophagy and inflammasome activation in the vasculature under conditions of low aerobic exercise capacity during aging results in arterial dysfunction and aortic stiffness. In older adults with reduced aerobic capacity, mitochondrial antioxidants, mitophagy induction, and inflammasome inhibition may be effective therapeutic strategies for enhancing vascular health.http://deepblue.lib.umich.edu/bitstream/2027.42/175280/2/Mitochondrial DAMPs-dependent inflammasome activation during aging induces vascular smooth muscle cell dysfunction and aortic stiffness in low aerobic capacity rats_Canugovi-JCA 2022.pdfPublished onlineDescription of Mitochondrial DAMPs-dependent inflammasome activation during aging induces vascular smooth muscle cell dysfunction and aortic stiffness in low aerobic capacity rats_Canugovi-JCA 2022.pdf : Published versio

Probabilistic Security Constrained Fuzzy Power Flow Models

In restructured power systems, generation and commercialization activities became market activities, while transmission and distribution activities continue as regulated monopolies. As a result, the adequacy of transmission network should be evaluated independent of generation system. After introducing the constrained fuzzy power flow (CFPF) as a suitable tool to quantify the adequacy of transmission network to satisfy 'reasonable demands for the transmission of electricity' (as stated, for instance, at European Directive 2009/72/EC), the aim is now showing how this approach can be used in conjunction with probabilistic criteria in security analysis. In classical security analysis models of power systems are considered the composite system (generation plus transmission). The state of system components is usually modeled with probabilities and loads (and generation) are modeled by crisp numbers, probability distributions or fuzzy numbers. In the case of CFPF the component’s failure of the transmission network have been investigated. In this framework, probabilistic methods are used for failures modeling of the transmission system components and possibility models are used to deal with 'reasonable demands'. The enhanced version of the CFPF model is applied to an illustrative case.info:eu-repo/semantics/publishedVersio

RECQL4 localizes to mitochondria and preserves mitochondrial DNA integrity

RECQL4 is associated with Rothmund-Thomson Syndrome (RTS), a rare autosomal recessive disorder characterized by premature aging, genomic instability and cancer predisposition. RECQL4 is a member of the RecQ-helicase family, and has many similarities to WRN protein, which is also implicated in premature aging. There is no information about whether any of the RecQ helicases play roles in mitochondrial biogenesis, which is strongly implicated in the aging process. Here, we used microscopy to visualize RECQL4 in mitochondria. Fractionation of human and mouse cells also showed that RECQL4 was present in mitochondria. Q-PCR amplification of mitochondrial DNA demonstrated that mtDNA damage accumulated in RECQL4-deficient cells. Microarray analysis suggested that mitochondrial bioenergetic pathways might be affected in RTS. Measurements of mitochondrial bioenergetics showed a reduction in the mitochondrial reserve capacity after lentiviral knockdown of RECQL4 in two different primary cell lines. Additionally, biochemical assays with RECQL4, mitochondrial transcription factor A and mitochondrial DNA polymerase γ showed that the polymerase inhibited RECQL4’s helicase activity. RECQL4 is the first 3′ to 5′ RecQ helicase to be found in both human and mouse mitochondria and the loss of RECQL4 alters mitochondrial integrity