Experimental Gerontology 69 (2015) 70–78

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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero

Perspective

Circulating cell-free mitochondrial DNA as the probable inducer of early endothelial dysfunction in the prediabetic patient Noé Alvarado-Vásquez Department of Biochemistry, National Institute of Respiratory Diseases “Ismael Cosío Villegas”, Calz. de Tlalpan 4502, Col. Sección XVI, 14080 Mexico, D.F., Mexico, Mexico

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Article history: Received 24 November 2014 Received in revised form 9 May 2015 Accepted 25 May 2015 Available online 27 May 2015 Section Editor: Holly M Brown-Borg Keywords: Diabetes mellitus type 2 Vascular system Endothelial cell Mitochondria TLR9

a b s t r a c t Recent evidence has shown that 346 million people in the world have diabetes mellitus (DM); this number will increase to 439 million by 2030. In addition, current data indicate an increase in DM cases in the population between 40 and 59 years of age. Diabetes is associated with the development of micro- and macro-vascular complications, derived from chronic hyperglycemia on the endothelium. Some reports demonstrate that people in a prediabetic state have a major risk of developing early endothelial dysfunction (ED). Today, it is accepted that individuals considered as prediabetic patients are in a pro-inflammatory state associated with endothelial and mitochondrial dysfunction. It is important to mention that impaired mitochondrial functionality has been linked to endothelial apoptosis and release of mitochondrial DNA (mtDNA) in patients with sepsis, cardiac disease, or atherosclerosis. This free mtDNA could promote ED, as well as other side effects on the vascular system through the activation of the toll-like receptor 9 (TLR9). TLR9 is expressed in different cell types (e.g., T or B lymphocytes, mastocytes, and epithelial and endothelial cells). It is localized intracellularly and recognizes nonmethylated dinucleotides of viral, bacterial, and mitochondrial DNA. Recently, it has been reported that TLR9 is associated with the pathogenesis of lupus erythematosus, rheumatoid arthritis, and autoimmune diabetes. In this work, it is hypothesized that the increase in the levels of circulating mtDNA is the trigger of early ED in the prediabetic patient, and later on in the older patient with diabetes, through activation of the TLR9 present in the endothelium. © 2015 Elsevier Inc. All rights reserved.

1. Diabetes mellitus (DM) DM involves a group of metabolic diseases characterized by hyperglycemia, derived from an impaired secretion or a diminishing effect of synthesized insulin (ADA, 2014). According to a World Health Organization report, 346 million people exist worldwide with DM (WHO, 2013). Recent estimates suggest that this number will increase up to 439 million in 2030 (Shaw et al., 2010). DM is divided in two types: type 1 (DM1), which is characterized by an absolute lack of insulin; and type 2 (DM2), which includes a diminished insulin secretion and insulin resistance (ADA, 2014). Diabetes type 2, which accounts for 90 to 95% of all cases of DM, encompasses individuals with insulin resistance and with a relative insulin deficiency (ADA, 2014; Kerner and Brückel, 2014). Although, the level of blood insulin in these last patients is considered in some occasions as normal or slightly elevated, the levels of glucose are increased (Simental-Mendía et al., 2014). An important characteristic of DM2 is that it goes undiagnosed for many years because of the gradual increase in glucose levels; this is the reason why in the earlier stages there is an absence of classical symptoms of illness (Kerner and Brückel, 2014). The causes involved in DM2 development E-mail addresses: [email protected], [email protected].

http://dx.doi.org/10.1016/j.exger.2015.05.010 0531-5565/© 2015 Elsevier Inc. All rights reserved.

are various and include: age, obesity, lack of physical activity, and racial or ethnic characteristics (ADA, 2014; Kerner and Brückel, 2014). Genetic predisposition is considered as one of the most important factors to develop DM2; regrettably the genetic bases of this disease are complex and poorly defined yet (DIAGRAM Consortium et al., 2014). Different epidemiological reports have shown that chronic hyperglycemia is a relevant risk factor for cardiovascular disease in patients with DM1 and DM2 (Lehto et al., 1996; Standl et al., 1996; Zhang et al., 2012). In addition, hyperglycemia is considered the primary cause of microvascular (e.g., nephropathy and neuropathy) and macrovascular (amputation and cardiovascular disease) damage in the patient with DM (Klein, 1995; Smith-Palmer et al., 2014). Chronic variations in glucose levels in the prediabetic or diabetic patient are involved also with numerous side effects (Giaccari et al., 2009; Nwose et al., 2014). In a recent analysis by McEwen et al. (2012), these authors found that predictors of death associated with the high levels of glucose found in DM2 patients were: older age, male sex, white race, lower income, smoking, dyslipidemia, insulin treatment, angina/myocardial infarction/other coronary disease/coronary angioplasty/bypass, etc. These last data show the diversity of elements involved in the mortality of the patient with DM, and also highlight the relevance of vascular diseases for diabetic patients.

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1.1. DM, cardiovascular disease (CVD) and aging New evidence shows an increase in the number of DM cases in the population between 40 and 59 years of age and there are 175 million people unaware of their disease (IDF, 2013). On the other hand, the world population is aging, which is associated with an increase in the number of middle aged or elderly individuals with DM (Colagiuri et al., 2005). The world prevalence of diabetes among adults (aged 20–79 years) was 6.4% in 2010, and this will increase to 7.7% of adults in 2030 (Shaw et al., 2010). Diabetes occurrence increases with age in all world regions, and although the group of 60–79 is principally affected (18.6%), the largest number of diabetic patients is in the group of 40– 59 years (Guariguata et al., 2014). Lately, the importance of prevention has been recognized, as well as the need of continuous monitoring of blood glucose levels. Derived from this information, it has been reported that senile diabetes (N65 years) shows only a few symptoms. Additionally, oral glucose tolerance tests in healthy subjects (N65 years of age) revealed a diabetic condition more commonly than in adults below 60 years of age (Motta et al., 2008). Recently, it has been published that the prevalence of DM2 reaches 25% in people older than 65 years (Twito et al., 2015). It is interesting to mention that pre-diabetes is present commonly in the elderly, and is present in half of those aged 75 years and older. The aforementioned facts, underline the presence of DM as an important disease associated with aging. In relation to vascular disease (CVD), a cohort study made during 2004–2010 in older adults (70 to 79 years), with diabetes of short or long duration, showed that cardiovascular complications were one of most commonly observed complications (Huang et al., 2014). Recently, it has been reported that older adults with coronary heart disease show depressive symptoms and poorer exercise capacity that are associated with functional decline over 5 years (Sin et al., 2015). Willey et al. (2014) established that hypertension and diabetes have important effects on the burden of stroke, especially in individuals b 80 years of age and Hispanics. In addition, Dei Cas et al. (2015) reported that the frequency of patients with heart failure and DM will increase with the general aging of the population. A specific cellular type involved with the development of CVD is the endothelial cell. Although the importance of endothelial cells during aging has been continuously ignored, recent evidence suggests the impaired insulin/IGF-1-like signaling as pro-aging agents, inducing endothelial dysfunction (Avogaro et al., 2013). Lately, Donato et al. (2015) reviewed the association between aging and endothelial dysfunction. In this work, we mention that aging of the endothelial cell is affected by different risk factors linked with CVD, and that aging per se is associated with endothelial dysfunction (Donato et al., 2015). It is relevant to mention, that endothelial dysfunction is also observed in the preclinical stage of diabetes (prediabetes) and is manifested as a disturbance of vascular tone regulation (Smirnova et al., 2013). This subclinical cardiovascular disease occurs before the manifestation of cardiovascular disease in diabetes, and involves stasis, endothelial dysfunction, and atherothrombosis (Nwose et al., 2014). Interestingly, results reported by Isordia-Salas et al. (2014) showed that proinflammatory and prothrombotic markers are present in people with DM2, as well as in prediabetic patients, and were associated with the concentration of glucose in plasma. All mentioned evidences support the importance of evaluating the endothelial functionality before and after the clinical diagnosis of diabetes. 1.2. Prediabetes Diabetes mellitus is defined by the association of physiological glucose levels with microvascular complications (ADA, 2014). However, in 1979, the National Diabetes Data Group (NDDG) established a new concept about an intermediate situation, where glucose levels are involved again; this state is called glucose intolerance (NDDG, 1979). Additionally, in 1997 the Expert Committee on the Diagnosis and Classification of diabetes mellitus included another concept known as

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impaired fasting glucose (IFG), which, in association with impaired glucose tolerance (IGT), defines prediabetes; both IFG and IGT are considered important risk factors for the development of diabetes later on (Buysschaert and Bergman, 2011). At this point, it is important to mention that microvascular (e.g., retinopathy, kidney disease, and neuropathy) and macrovascular complications (cardiovascular disease) have been reported in prediabetic individuals (Nwose et al., 2014; Xing et al., 2014). For example, Xing et al. (2014), after evaluating 1585 individuals with normal glucose levels and 755 patients with prediabetes, found a clear association of prediabetes (defined using HbA1c and/or fasting plasma glucose criteria) with markers of micro- and macrovascular disease. However, the authors point out that other risk factors are necessary to worsen the vascular damage. Recently, Nwose et al. (2014) evaluated the presence of stasis, endothelial dysfunction, and atherothrombosis biomarkers in prediabetic patients. Their results showed that all three elements are associated with the vasculopathy in prediabetic patients, when compared to the healthy group. Today, it is thought that the prevalence of prediabetes is increasing in the world, which unfortunately is associated with a high risk of developing cardiovascular diseases (Bwititi and Nwose, 2014). These last authors suggest the importance of using a combination of blood glucose levels with an index of oxidative damage to improve the treatment of prediabetes (Bwititi and Nwose, 2014). Recently, Isordia-Salas et al. (2014) determined the levels of high-sensitivity C-reactive protein (hs-CRP) and fibrinogen in subjects with normal glucose tolerance (NGT), prediabetes, and DM2. Their results showed that these proinflammatory and prothrombotic markers are present in people with DM2, but without cardiovascular disease. Interestingly, the levels of these same markers were associated with the glucose levels in the prediabetic patient. Additionally, determination of interleukin-5 (IL-5), IL-6, IL-7, tumor necrosis factor-α (TNF-α), and granulocyte–monocyte colony-stimulating factor (GM-CSF) associated with inflammatory processes revealed significantly higher values in prediabetic patients (Lucas et al., 2013). Likewise, cytokines such as interferon-γ (IFN-γ), IL-1β, IL-2, IL-4, IL-8, IL-10, IL12p70, and IL-13 showed an increase in their levels, although not statistically significant. All these data support the relevance of the presence of an inflammatory state in diabetic and prediabetic patients. 2. Endothelial dysfunction The endothelium is constituted by a cellular monolayer that lines the inside of blood vessels, which represent 1% of the body mass and cover 4000 to 7000 m2 (Aird, 2004). Endothelial cells play an important role in the regulation of the vasomotor tone and the trafficking of cells and nutrients, and intervene in the regulation of blood fluidity. On the other hand, the endothelium is involved in the formation of new vessels, as well as in the liberation and synthesis of pro- and anti-inflammatory molecules (Aird, 2006). The endothelium could be exerting this diversity of functions by its great capacity to change both phenotype and functions (Aird, 2006). When physiological conditions change in the organism, by shifts in the levels of lipids, presence of oxidative stress, hypertension, environmental toxins, or diabetes mellitus, a change in the phenotype of endothelial cells is observed (Piarulli et al., 2013). This latter leads to a lesser synthesis of nitric oxide (NO), a greater synthesis of reactive oxygen species (ROS), an increase in the expression of adhesion molecules, as well as in cell permeability (Patel et al., 2013; Piarulli et al., 2013). An increase in the synthesis of cytokines linked to the migration and proliferation of muscle cells and fibroblasts is also observed (Lim and Park, 2014). All these changes in endothelial cells are defined as endothelial dysfunction (ED) (Sena et al., 2013). 2.1. Endothelial dysfunction in diabetes mellitus (DM) DM, where hyperglycemia, insulin resistance, and dyslipidemia are present, meets all conditions that predispose to ED, a condition associated with vascular and cardiac stiffness and atherosclerosis (Jia and

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Sowers, 2014). ED has been linked also to hypertension, myocardial infarct, stroke, and heart failure, and is considered a relevant factor for the evolution of cardiovascular disease (CVD) (Jia and Sowers, 2014). It is interesting to mention that results obtained in cross-sectional studies suggest that ED could predict the frequency of DM2, independently from other elements like obesity, abnormal glucose metabolism, or inflammation (Frankel et al., 2008). A prospective, case control study, evaluating a cohort of postmenopausal women, reported that elevated levels of circulating E-selectin and intercellular adhesion molecule-1 were linked to a higher risk of DM2 (Song et al., 2007). Recently, van Sloten et al. (2014) evaluated the probable association among ED, DM2, impaired glucose metabolism (IGM), and insulin resistance with the probability of developing cardiovascular events. Results obtained show that ED, DM2, IGM, or insulin resistance could act synergistically and increase the probability of cardiovascular events. Today, it is recognized that diminished endothelial nitric oxide synthase (eNOS) activity, reduced nitric oxide (NO) synthesis, as well as an increased synthesis of reactive oxygen species (ROS) are characteristics of DM that favor development of pro-atherogenic changes (Tousoulis et al., 2013). Additional evidence supports that endothelial cells from healthy individuals, but with a strong familial history of DM2, show alterations in the synthesis of NO and ROS in presence of high glucose (Alvarado-Vásquez et al., 2007a, 2007b). The latter is probably associated with the numerous changes observed in molecules involved in endothelial functionality (e.g., VCAM-1, vWF), or in the proinflammatory state (e.g., C-reactive protein, TNF-α, E-selectin) (Gogitidze et al., 2010; Gómez et al., 2008; Tousoulis et al., 2013). At this point, it is important to mention that variations in the expression of these molecules (VCAM-1 and E-selectin), as well as changes in NO and ROS synthesis in the endothelial cell, have been linked to the presence of high glucose concentrations (Haubner et al., 2007; Patel et al., 2013).

2.2. Mitochondrial dysfunction in the DM In DM, one of the major targets seems to be the vascular system, both at the micro- and macro-vascular levels (e.g., kidney, retina, coronary arteries) (Zoungas et al., 2014). At present, the critical role of endothelial dysfunction for the onset of vascular disease has been accepted. In addition, it is interesting to comment that recent evidences support the importance of mitochondrial dynamics in the development of both ED and vascular disease (Tang et al., 2014). Mitochondria are organelles involved in ATP synthesis, regulation of calcium concentration, cellular proliferation, immune response, apoptosis, and with the synthesis of some components of the oxidative phosphorylation chain (Li et al., 2012; Nagy et al., 2003; Sobek and Boege, 2014; Tang et al., 2014). In addition, mitochondria depict a great morphological and functional diversity. Previously, Collins and Bootman (2003) showed that the characteristics of mitochondria in pancreatic acinar cells, porcine aortic endothelial (PAE) cells, COS-7 cells, SH-SY5Y cells, and neocortical astrocytes are very heterogeneous in both distribution and size. Their results showed also a clear aggregation of mitochondria in the perinuclear zone. In diseases of the vascular system, mitochondria of endothelial cells show variation in shape and function (Tang et al., 2014). In hyperglycemic conditions, an impaired ROS synthesis is induced by mitochondria of endothelial cells (Alvarado-Vásquez et al., 2007a). Mitochondrial Ca2+ concentration and the morphology of mitochondria in endothelial cells, incubated in high glucose concentrations, are increased and modified (Paltauf-Doburzynska et al., 2004). Moreover, high glucose concentrations may induce endothelial apoptosis, probably by their negative effect on the mitochondrial permeability transition pore (mPTP) (Sasaki et al., 2012). On the other hand, chronic hyperglycemia induces the change from oxidative phosphorylation to glycolysis, a decrease of mitochondrial membrane potential, as well as an uncoupled respiration associated with a reduced ATP/ADP ratio (Moruzzi et al., 2014). Currently, we know that the ATP/ADP ratio is a

critical element for functionality and endothelial viability (CappelliBigazzi et al., 1997). In the diabetic patient, the concentration of lipids is considered also an important factor for the development of endothelial and cardiovascular damage. However, in the prediabetic patient, the concentration of lipids has not shown relevant variations. For example, determinations of levels of glucose, lipids, and hormones after an oral glucose tolerance test, in subjects with prediabetes, showed that only the glucose levels are altered in these individuals (Sonnier et al., 2014). In the work of Healy et al. (2015), it was reported that mean levels of fasting glucose, insulin, and C-peptide were increased in prediabetic patients; in contrast, the levels of triglycerides remained without changes. In a recent work, the utility of the ADA risk test or of body composition measurements to recognize individuals with prediabetes was evaluated. Anthropometric measures, fasting glucose and lipids, and percentage of hemoglobin A1c were determined. The results obtained showed that non-invasive methods were highly sensitive, although their specificity was low, to detect the presence of prediabetes (Vanderwood et al., 2015). However, the levels of glucose and percentage of hemoglobin A1c were statistically significant. Interestingly, some recent papers show that high levels of lipids have some beneficial effects on mitochondria. For example, evaluation of age-related molecular changes, in rat brain mitochondria fed with monounsaturated fatty acids (Ochoa et al., 2011), showed that this type of diet prevented the presence of mtDNA deletions in brain mitochondria of aged animals. In another work, rats fed with a high-fat diet and given daily injections of heparin to raise free fatty acid (FFA) (Garcia-Roves et al., 2007) showed an increase in mitochondrial biogenesis in muscles, as well as an increase in the mitochondrial enzymes involved in fatty acid oxidation, citrate cycle, and respiratory chain. Likewise, an increase in mitochondrial DNA copy number was observed. In human volunteers, consumption of a high-fat diet for 3 days decreases the PGC-1α protein level (which induces oxidative phosphorylation and mitochondrial biogenesis) by approximately 20% (Sparks et al., 2005). The mentioned evidences support the role of glucose in prediabetes as an important element to start the damage in endothelial cells, and, although, it is not possible to discard a possible role of lipids, the current data do not support this possibility. 2.2.1. Mitochondrial deoxyribonucleic acid (mtDNA) Recently, S. Wang et al. (2013) and Y. Wang et al. (2013) showed that mutations of mitochondrial DNA (mtDNA) can be related to DM. These authors propose that the mitochondrial gene tRNA(Leu(UUR)) 3243 A → G mutation is a risk factor for the development of DM, and suggest its importance to indicate the clinical status of the patient. In other pathological conditions, such as sepsis, the mtDNA is considered a key activator of inflammation and immune response (Nakahira et al., 2013). In this last work, analyses of blood mtDNA levels in intensive care unit (ICU) patients showed an association between high mtDNA levels and the mortality rate in ICU patients. In a retrospective cohort study, Jiménez-Ibáñez et al. (2012) showed that hyperglycemia is a common finding in patients admitted to the ICU. The levels of mtDNA have been evaluated and compared between patients with traumatic damage and those with severe sepsis, and the analysis revealed the importance of mtDNA in the clinical evolution of these patients. However, the prognostic significance and the mechanism involved in the liberation of mtDNA in the patients with traumatic damage or sepsis are different (Yamanouchi et al., 2013). Liberation of mtDNA has been reported in myocardial infarction, which correlated with the degree of myocardial damage (Bliksøen et al., 2012). Based on the aforementioned, Arnalich et al. (2012) suggest the probable utility of determining plasma mtDNA levels for outcome prediction after cardiac arrest, and its relevance as an indicator of apoptosis in cardiac tissue. In an experimental model, using endothelial cells of the umbilical cord and LOX-1/LDLR double KO mice as an atherosclerosis model, the mtDNA damaged by oxidative stress acts as a promoter of the inflammatory response

Perspective

(Ding et al., 2013). In human retinal vascular endothelial cells, incubation with high glucose induces an mtDNA oxidative damage, as well as a fall in the mitochondrial membrane potential (MMP), associated to an increase in ROS synthesis, and the presence of early apoptosis. Based on these data, the authors suggest that mtDNA oxidative damage, in the presence of high glucose, is the “trigger” of cell dysfunction (Xie et al., 2008). Evidence provided by Botto et al. (1995) shows that mtDNA lesions are present in circulating cells and hearts of patients with coronary disease, supporting the relevance of mtDNA in the origin of atherosclerosis. Recently, a review on the role of mtDNA in the development of atherosclerosis was published (Yu and Bennett, 2014). In this review, additional evidence on the role of damaged mtDNA by oxidative stress as an inducer of mitochondrial dysfunction and promoter of atherosclerosis is provided. However, many questions on the role of mtDNA in the onset of vascular damage still remain and need to be clarified. 3. Toll like-receptors The above-mentioned evidences give support to suggest that mtDNA could be released in hyperglycemic or pro-inflammatory conditions, similarly as has been observed in sepsis, cardiac disease, and atherosclerosis. To this regard, it is important to take into account that DM and prediabetes are considered pro-inflammatory states (Lucas et al., 2013; Nwose et al., 2014) that could be favoring the onset of ED by oxidative stress and by impaired regulation of calcium metabolism (Haubner et al., 2007; Patel et al., 2013; Piarulli et al., 2013; Tousoulis et al., 2013). An interesting but scarcely studied mechanism, probably associated with the onset of ED in the prediabetic patient, could be linked to toll like-receptors. Toll like-receptors (TLRs) are a group of proteins, recognized as a subclass of pathogen-associated patternrecognition receptors (PRRs) (Tilich and Arora, 2011). TLRs are part of the innate immune system and play an important role in the detection and categorizing of infectious agents, as well as in the activation and type of immune response against aggressor agents (Kawasaki and Kawai, 2014). The innate immune system uses PRRs for the identification of infectious agents. They recognize molecules expressed by the microbial agents and are known as pathogen-associated molecular patterns (PAMPs), and recognize molecules released from injured or transformed cells that are referred to as damage-associated molecular patterns (DAMPs) (Kawasaki and Kawai, 2014). Vertebrates are endowed with different types of PRRs, including the TLRs, as well as RIG-I-like receptors (RLRs), nod-like receptors (NLRs), AIM2-like receptors (ALRs), and C-type lectin receptors (CLRs) (Gürtler and Bowie, 2013; Kawasaki and Kawai, 2014). TLRs are expressed in different cell types such as macrophages, B and T cells, plasmacytoid dendritic cells (pDC), and epithelial and endothelial cells. In humans, 10 TLRs have been recognized and classified according to their cellular localization; for example, TLR1, -2,- 4, -5, -6, and -10 are localized in the cell membrane, while TLR3, -7, -8, and -9 are expressed in intracellular vesicles or organelles (Tilich and Arora, 2011).

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Also, the activation of TLR9 using CpG oligodeoxynucleotides (CpG ODN) prevents and reverses eosinophilic airway inflammation in animal models, which suggest its usefulness in the treatment of human allergic airway disease (Kline and Krieg, 2008). Additionally, subcutaneous injection of the TLR9 agonist (CpG ODN) in newborn lambs gives protection against the parainfluenza-3 virus (Nichani et al., 2010). The use of CpG ODN to activate TLR9 and the synthesis of interferons (alpha, beta) in humans infected with the HIV-1 has been proposed (Becker, 2005). The activation of TLR9 in patients with basal cell carcinoma using synthetic ODN induces an increase in the levels of interleukin-6 and -12, and tumor necrosis factor. In addition, one complete regression and four partial regressions were observed after the use of synthetics ODN in these patients (Hofmann et al., 2008). Recently, the role of TLR9 as a therapeutic agent in the treatment of cancer was reviewed (Fűri et al., 2013). The evidence analyzed supports the importance of TLR9 activation for the treatment of systemic and colonic inflammatory conditions, as well as in the treatment of colorectal cancer, with minimal side effects. The beneficial effects observed can be linked to the diverse cytokines (e.g., IL-1β, IL-6, IL-8, IL-15, IL-32, IFNβ, IFN-γ, TNF-α) or molecules involved in the immune response (iNOS, MIP-1β and NFκB) and expressed after activating TLR9 (Peng and Zhang, 2015). Regrettably, the impaired expression or chronic activation of TLR9 has been associated with negative effects in either animal models or humans. In patients with chronic obstructive pulmonary diseases (COPD), determination by immunoelectron microscopy of TLR9 in lungs showed a clear increase in the number of TLR9-positive cells, which suggests a possible role of this TLR in this disease (Schneberger et al., 2013). The dysregulation of TLR9 has been linked also to abnormal activation of mucosal immunity in patients with ulcerative colitis (Tan et al., 2014). In patients with chronic hepatitis B virus infection (CHB), expressions of TLR9, MYD88, IRAK1, TRAF6, and NFκB in peripheral mononuclear cells were diminished. These results suggest that CHB patients are incapable of activating the TLR9 pathway, leading to an inadequate response against the hepatitis B virus (Sajadi et al., 2013). In a murine model of severe peripheral tissue injury, the results showed the importance of TLR2, TLR4, and TLR9 in the T cell-associated immune dysfunction after traumatic tissue injury (Darwiche et al., 2013). In a murine model of nonalcoholic steatohepatitis, the authors reported that TLR9 activation was associated with IL-1β synthesis, presence of steatosis, inflammation, and fibrosis (Miura et al., 2010). In colon carcinoma cells, activation of TLR9 using CpG ODN was associated with cellular proliferation, and with a reduced response to the anticancer drug Adriamycin. Additionally, the expression of TLR9 was linked to TLR3 expression; suggesting a role of both in the survival of cancer cells (Nojiri et al., 2013). In a murine sepsis model, animals showed septic heart failure, a diminished end-systolic pressure, stroke, and cardiac output. Interestingly, TLR9-deficient mice showed lesser cardiac inflammation associated to a better cardiac function (Lohner et al., 2013). 3.2. TLR9 in autoimmune or chronic diseases

3.1. Toll-like receptor 9 (TLR9) In particular, TLR9 is localized in intracellular compartments and detects unmethylated CpG dinucleotides of bacterial and viral genomes; however, it can also detect dinucleotides of mitochondrial origin (Zhang et al., 2014). Although, TLR9 is expressed in human B cells and inactive plasmacytoid dendritic cells (pDC), some works report its expression in neutrophils and activated pulmonary epithelial cells (Platz et al., 2004). On the other hand, an increasing number of works evaluating the therapeutic potential of TLR9 activation in different pathologies can be found in the literature (Krieg, 2006). For example, Bhan et al. (2008) reported that TLR9 is essential in the immune response against the bacterium Legionella pneumophila, suggesting its probable utility to increase the immune response mediated by TLR9 in pneumonia cases.

In autoimmune or chronic diseases, evaluation of TLR9's role has been pursued for many years. In systemic lupus erythematous (SLE), the use of an antagonist of TLR9, such as chloroquine, hydroxychloroquine, and quinacrine dates back to the 1950s. However, although some evidence suggests its probable utility for SLE treatment, results are still not conclusive (Sun et al., 2007). TLR9 activation in patients with SLE has been associated with the production of antibodies and overproduction of IFN-α, which exacerbate the clinical symptomatology of SLE (Pradhan et al., 2012). It is interesting to mention, that the use of ODN inhibitors decreases the synthesis of IFN-α (Pradhan et al., 2012). However, genotyping of TLRs using single-nucleotide polymorphisms (SNPs), as well as mRNA expression of TLRs in peripheral blood mononuclear cells (PBMCs) from patients with SLE and individual controls, showed that

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variations in the gene expression of TLR9 was linked to variations in TLR3 and TLR8 gene expression (Laska et al., 2014). In rheumatoid arthritis (RA), which is a chronic inflammatory disease, synovial fibroblasts and T cells play a central role in the evolution of the disease. Recently, the expression of TLR2, -3, -4, and TLR9 in osteoarthritis synovial fibroblasts has been reported (Hu et al., 2014). McKelvey et al. (2011) evaluated the expression of five known isoforms of human TLR9, in 15 samples of joint synovial tissue and 15 samples of rheumatoid subcutaneous nodules obtained from patients with RA. The results showed that B lymphocytes and pDC contribute to the high TLR9-A transcript levels in the inflamed synovium. In comparison, macrophages and T-lymphocytes are the main origin of high levels of TLR9-C transcripts in subcutaneous nodules. These data underline the importance of different isoforms of TLR9 for the inflammatory damage observed in patients with RA. 3.3. TLR9 in diabetes mellitus Although diabetes and prediabetes are considered pro-inflammatory states (Lucas et al., 2013; Nwose et al., 2014), the role of TLR9 has been especially studied in type 1 diabetes. For example, in 2007, using biobreeding of diabetes-resistant rats (BBDR) that were later on infected with the parvovirus, Kilham rat virus (KRV), 25% of total animals infected with KRV developed DM1 (Zipris et al., 2007). Interestingly, administration of chloroquine (antagonist of TLR9) decreases both the rate of diabetes and IL-12p40 levels, suggesting the participation of TLR9 in the onset of diabetes in the BBDR. In the work of Wong et al. (2008) using a NOD mice model deficient in TLR3 and TLR9, the authors found that TLR3deficient mice show no differences in developing spontaneous DM1, in comparison to control mice; in contrast, TLR9-deficient mice showed a clear protection against the development of DM1. In fulminant DM1, analyses of pancreatic autopsy samples have revealed the presence of TLR3, TLR7, and TLR9 (Shibasaki et al., 2010). Other data obtained in that work support the importance of macrophage-dominated insulitis but not T-cell autoimmunity as the origin of beta cell destruction in this type of diabetes. Additional evidence, using microarray, has shown important variations in the expression of TLR9, as well as in the expression of transcription factors ELF4 and IL-1RAP (interleukin 1 receptor accessory protein) in fulminant type 1 diabetes. Moreover, other experimental findings involve IL-1 and TNF-α dependent pathways in β-cell dysfunction (Wang et al., 2011). However, in the case of DM2, which accounts for 90–95% of all cases of DM (ADA, 2014; Kerner and Brückel, 2014), information on the role of TLR9 is limited. Previous evidence indicates that the innate immune response, where TLRs play a central role, is impaired in diabetic patients. Some reports have shown the relevance of TLR4 in the development of DM2 in the induction of the synthesis of TNF-α and IL-1β and by inhibiting the insulin signaling pathways (Rempel et al., 2013). Another TLR, previously associated with the pathology of DM2, is TLR3. This TLR can respond to signals of nutrients and has been linked to the loss of β-cell mass, promoting GI cycle arrest (S. Wang et al., 2013; Y. Wang et al., 2013). However, reports about the role of TLR9 in the development and onset of DM2 are scarce. Evidences about the role of TLR9 in the endothelial damage or cardiovascular disease are inexistent. One of these works is the report of Rojo-Botello et al. (2012). The authors reported high expressions, assessed by immunohistochemistry, of TLR2, TLR3, and TLR9 in gingival samples obtained from patients with DM2. In another recent report, Liu et al. (2012) genotyped TLR2 Arg677Trp and Arg753Gln, TLR4 Asp299Gly and Thr399Ile, TLR9-1486T/C and -1237T/C, all gene polymorphisms of TLRs. Although the authors did not find a statistically significant association between the TLR9-1486T/ C and DM2, they mention that this polymorphism depicts a higher expression in DM2 patients (Liu et al., 2012). In spite of this lack of information about the association between TLR9 and the evolution of DM2 or with endothelial dysfunction, there are data that permit hypothesizing about the probable association of mtDNA with TLR9 at the onset of ED in prediabetic patients.

4. Perspective Fukagawa et al. (1999) showed that aging and high glucose concentrations increase the damage to mitochondrial DNA of muscle cells in an experimental model. In the case of endothelial cells, the deficient regulation in the transport of glucose has been linked to the presence of micro- and macro-vascular damage in high glucose concentrations. It is a consequence of overproduction of mitochondrial ROS and inhibition of the enzyme glyceraldehyde-3-phosphate dehydrogenase (Hermans, 2007). In another work, human umbilical endothelial cells incubated with high glucose showed a higher synthesis of ROS, a decrease of MMP, and endothelial apoptosis (Bhatt et al., 2013). Currently, it is accepted that oxidative stress derived from mitochondrial ROS generation is linked to damage of the mitochondrial DNA, mitochondrial dysfunction, and micro- and macro-vascular disease. However, this oxidative stress favors a high expression of pro-inflammatory and pro-coagulant factors, as well as an impaired nitric oxide synthesis and the start of endothelial apoptosis (Fiorentino et al., 2013). The analysis of mtDNA levels in peripheral blood of patients with DM2 or prediabetes showed that levels of mtDNA were 35% lower in patients with DM2; similar results have been reported in prediabetic individuals, when compared to control individuals (Lee et al., 1998). In spite of these evidences, the issue is still very controversial. Weng et al. (2009) evaluated the content of mtDNA in leukocytes of patients with DM2, leukocytes of patients with impaired fasting glucose, and healthy subjects. Results showed a progressive increase in both mtDNA copy numbers and markers of oxidative stress. Interestingly, this increase was associated to a gradual dysregulation of glucose metabolism, as well as to the family history of diabetes mellitus (Weng et al., 2009). At this point, it is relevant to mention that both total number of mitochondria and copy number of mtDNA change during oxidative stress (Liu et al., 2003). Malik et al. (2009) reported that mtDNA copy numbers show a significant increase in peripheral blood of patients with diabetic nephropathy, which was associated also with the presence of oxidative stress markers. Song et al. (2007) reported that mtDNA content is linked to the sensitivity to insulin in offspring from type 2 diabetic patients. It is interesting to mention that mtDNA levels in these subjects tended to increase according to the levels of glucose. At present, it is unknown what percentage of damaged mtDNA is released by the cell during the stress derived from high glucose levels. The large heterogeneity of size and distribution of mitochondria in the different tissues has made this task very difficult. However, previously it has been reported that mtDNA plasma levels correlate with proinflammatory cytokine levels (tumor necrosis factor alpha, IL-6, RANTES and IL-1ra) (Pinti et al., 2014), which maintain an inflammatory state whose final effect is the release of mtDNA. Although, our knowledge is still incomplete in this regard, some evidences support the importance of mitochondrial changes in endothelial cells to influence the levels of mtDNA, and to maintain a proinflammatory state (Pinti et al., 2014; Olivieri et al., 2013). In the heart, amassing of undigested mitochondria induces the escape of mtDNA to the cytosol, activating the TLR9dependent inflammatory response and favoring the development of cardiomyopathy (Oka et al., 2012). Thus, how does glucose favor the release of mtDNA? Today, it is known that mechanical trauma releases mtDNA into circulation (Zhang et al., 2014). In cells treated with pathogen-associated molecular patterns (DAMPs) mitochondrial dysfunction has been observed, as well as the increase in ROS synthesis. These events activate mitochondrial permeability transition, which causes the leakage of mtDNA to the cytosol (Oka et al., 2012; Ney, 2015). Sun et al. (2013) reported that mitochondrial DAMPs induce changes in endothelial permeability, suggesting that release of mitochondrial factors from dead or dying cells can be a critical factor in the early endothelial response to damage. In diabetes, the increase of glucose levels leads to enhancing endothelial cell permeability via the activation of the PKC isoform alpha (Hempel et al., 1997). In addition, in human retinal vascular endothelial

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cells, incubation with high glucose induces mtDNA oxidative damage, as well as a fall in the mitochondrial membrane potential (MMP), associated to an increase in ROS synthesis and the presence of early apoptosis (Xie et al., 2008). In their work, Bhatt et al. (2013) showed that human umbilical endothelial cells incubated with high glucose had a higher synthesis of ROS, a decrease of MMP, and endothelial apoptosis. Based on the aforementioned, it is possible that the conditions associated with the leakage of mtDNA could be observed in endothelial cells of both the diabetic and prediabetic patient. But, what role does mtDNA play in the onset of ED in the prediabetic patient? This is an interesting question. Results reported from Egawhary et al. (1995) show an increase in the number of detached vascular circulating endothelial cells (CECs) in diabetic patients, who have as a characteristic a 4977 bp deletion in their mtDNA. This deletion has been linked with the presence of hyperglycemia, and associated with the detachment of endothelial cells from the basement membrane (Swoboda et al., 1995). Additionally, now we know that fluctuating glucose concentrations (as happens in the prediabetic patient) exert numerous side effects on the endothelial cells (Liu et al., 2013). These shifts in the levels of glucose have been associated with high levels of IL-6, TNF-α, and molecule ICAM-1, which have been related to coronary heart disease, atherosclerosis, and vascular inflammation (Liu et al., 2013). Here, it is convenient to mention that mtDNA content varies in the different tissues of the body, which can help explain the different levels of damage observed in the diabetic or prediabetic patient (Hsieh et al., 2011). In a recent and interesting publication Pinti et al. (2014) reported an increase in the levels of plasma mtDNA associated with the age of the studied people. In addition, this was associated with low-grade or chronic inflammation present in the population studied, and derived probably from the relation between mtDNA levels and the TNF-α, IL-6, RANTES, and IL-1Ra concentrations. However, can the mtDNA present in plasma affect endothelial cells through activation of TLR9? Although this point is still scarcely studied, previous evidences have shown that endothelial cells stimulated with ODN express TLR9 mRNA, and proinflammatory molecules IL-8 and ICAM-1 (Li et al., 2004). Additionally, Fitzner et al. (2011) found changes in the expression of TLR9 in resting or activated endothelial cells. On the other hand, Pegu et al. (2008) reported the presence of several functional TLRs in primary human dermal and lung endothelial cells, where TLR9 is highly expressed. The expression of TLR9 in the aortic endothelium, as well as its importance for angiogenesis, was confirmed by Aplin et al. (2014). Although, the data reported show variations in the levels of mtDNA, these differences support the relevance of mitochondrial functionality to maintain the health of endothelial cells, as well as the significance of assessing under different conditions and cell types the levels of mtDNA. For example, the increase in mtDNA levels has been associated with the proinflammatory state (Pinti et al., 2014; Olivieri et al., 2013), and with changes in the endothelial permeability and liberation of mtDNA (Sun et al., 2013). Besides, the age-related mtDNA depletion could be a marker of aging, but is also a risk factor for the development of type 2 diabetes (Nile et al., 2014). On the other hand, the increase in mtDNA levels has been considered a biomarker of the inflammatory state (Wang et al., 2015) but, in contrast, also as a protection factor in patients or in models of acute myocardial infarction (Muravyeva et al., 2014). Despite that the panorama is of great complexity, the relevance of mtDNA to maintain endothelial stability could be considered as essential. Here, it is important to remember that prediabetic patients remain undiagnosed for many years, because of the gradual increase in the physiological levels of glucose. The latter is an event considered a critical element for the development of endothelial dysfunction and for the maintenance of the pro-inflammatory state (Giaccari et al., 2009; Nwose et al., 2014; Xing et al., 2014). Based on the above mentioned evidences, this increase in the levels of glucose induces the release of mtDNA in the prediabetic patient (favored by its pro-inflammatory condition and by chronic elevation in glucose levels) (Bwititi and Nwose,

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2014; Weng et al., 2009; Xie et al., 2008), maintaining chronically activated the TLR9 expressed by endothelial cells (Fitzner et al., 2011; Li et al., 2004). This release and circulation of free mtDNA can be privileged by an autophagy- and mitophagy-altered mechanism. Mitophagy is a critical piece to maintain mitochondrial quality, so shifts in the physiological state (e.g., excess of nutrient or presence of oxidative stress) would gradually lead to mitochondrial dysfunction and inadequate removal of mitochondria (Liesa and Shirihai, 2013). This impaired elimination of mitochondria is associated with a deficient elimination of mtDNA, which has been related to metabolic and nervous disorders (Jung and Lee, 2010), including diabetes mellitus (Andreux et al., 2013). As an additional example, in β-cells of βTsc2(−/−) mice, impaired autophagy is linked to hyper-activation of mTORC1, which induces mitochondrial dysfunction and contributes to β-cell malfunction (Bartolomé et al., 2014). Recently, accumulation of fragmented mitochondria in cells from the renal cortex of humans and animals has been related to diabetic nephropathy, suggesting an impaired mitochondrial clearance in these conditions (Higgins and Coughlan, 2014). The establishment of an inflammatory condition linked to high levels of mtDNA would induce synthesis, release or expression of proinflammatory cytokines or molecules associated with the inflammatory process regulated by the TLR9 pathway in endothelial cells, and, in consequence, favor the start of early endothelial dysfunction in the prediabetic patient (Alvarado-Vásquez et al., 2007a, 2007b; Frankel et al., 2008; Isordia-Salas et al., 2014; Lucas et al., 2013; van Sloten et al., 2014). If this hypothesis is confirmed, it would help to understand why endothelial damage and, later on, endothelial dysfunction develop in the prediabetic patient even at discrete variations in glucose levels. Besides, it would emphasize the relevance of evaluating early and continuously the levels of mtDNA and glucose concentration, in patients with a familial history of type 2 diabetes, IFG, IGT, and those individuals with diagnosis of diabetes. All these are aimed at providing an early treatment, and preventing the development of endothelial dysfunction in the individuals at risk, or the development and maintenance of ED in the older diabetic patient. References Aird, W.C., 2004. Endothelium as an organ system. Crit. Care Med. 32 (5 Suppl.), S271–S279. Aird, W.C., 2006. Mechanisms of endothelial cell heterogeneity in health and disease. Circ. Res. 98, 159–162. Alvarado-Vásquez, N., Páez, A., Zapata, E., Alcázar-Leyva, S., Zenteno, E., Massó, F., Montaño, L.F., 2007a. HUVECs from newborns with a strong family history of diabetes show diminished ROS synthesis in the presence of high glucose concentrations. Diabetes Metab. Res. Rev. 23, 71–80. Alvarado-Vásquez, N., Zapata, E., Alcázar-Leyva, S., Massó, F., Montaño, L.F., 2007b. Reduced NO synthesis and eNOS mRNA expression in endothelial cells from newborns with a strong family history of type 2 diabetes. Diabetes Metab. Res. Rev. 23, 559–566. American Diabetes Association, 2014. Diagnosis and classification of diabetes mellitus. Diabetes Care 37 (Suppl. 1), S81–S90. Andreux, P.A., Houtkooper, R.H., Auwerx, J., 2013. Pharmacological approaches to restore mitochondrial function. Nat. Rev. Drug Discov. 12, 465–483. Aplin, A.C., Ligresti, G., Fogel, E., Zorzi, P., Smith, K., Nicosia, R.F., 2014. Regulation of angiogenesis, mural cell recruitment and adventitial macrophage behavior by Toll-like receptors. Angiogenesis 17, 147–161. Arnalich, F., Codoceo, R., López-Collazo, E., Montiel, C., 2012. Circulating cell-free mitochondrial DNA: a better early prognostic marker in patients with out-of-hospital cardiac arrest. Resuscitation 83, e162–e163. Avogaro, A., de Kreutzenberg, S.V., Federici, M., Fadini, G.P., 2013. The endothelium abridges insulin resistance to premature aging. J. Am. Heart Assoc. 2, e000262. Bartolomé, A., Kimura-Koyanagi, M., Asahara, S., Guillén, C., Inoue, H., Teruyama, K., Shimizu, S., Kanno, A., García-Aguilar, A., Koike, M., Uchiyama, Y., Benito, M., Noda, T., Kido, Y., 2014. Pancreatic β-cell failure mediated by mTORC1 hyperactivity and autophagic impairment. Diabetes 63, 2996–3008. Becker, Y., 2005. CpG ODNs treatments of HIV-1 infected patients may cause the decline of transmission in high risk populations—a review, hypothesis and implications. Virus Genes 30, 251–266. Bhan, U., Trujillo, G., Lyn-Kew, K., Newstead, M.W., Zeng, X., Hogaboam, C.M., Krieg, A.M., Standiford, T.J., 2008. Toll-like receptor 9 regulates the lung macrophage phenotype and host immunity in murine pneumonia caused by Legionella pneumophila. Infect. Immun. 76, 2895–2904. Bhatt, M.P., Lim, Y.C., Kim, Y.M., Ha, K.S., 2013. C-peptide activates AMPKα and prevents ROS-mediated mitochondrial fission and endothelial apoptosis in diabetes. Diabetes 62, 3851–3862.

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Circulating cell-free mitochondrial DNA as the probable inducer of early endothelial dysfunction in the prediabetic patient.

Recent evidence has shown that 346million people in the world have diabetes mellitus (DM); this number will increase to 439million by 2030. In additio...
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