Mitochondrial Dysfunction Signature in Diabetic Vascular Endothelium

Mitochondrial dysfunction/malfunction is an important risk factor for the onset and progression of endothelial cells dysfunction, a key contributor to diabetes mellitus associated cardiovascular disease. The topics discussed here are: (i) The regulatory role of mitochondrial ROS in normal, healthy vascular endothelium, (ii) The adverse alteration of mitochondrial function in diabetes-related endothelial dysfunction, (iii) The endeavors towards correction of mitochondrial malfunction in dysfunctional endothelium, (iv) The key points of mitochondria-targeted interventions in diabetic patients, and (v) The pending, incompletely understood issues that open new basic research directions and may conduct to innovative therapeutic strategies in diabetes.


Introduction
Endothelial cells (ECs) cover the lumen of all blood vessels and ensure homeostasis preservation. In physiologic conditions, ECs mitochondria have unique functional traits: (i) a reduced engagement in generation of the energy-rich molecule adenosine-5'-triphosphate (ATP) [1,2]; instead, the source of ATP required for the development of the normal metabolic activity of ECs is the anaerobic glycolysis [3][4][5][6][7]; (ii) a commitment to sense the local environmental signals, and to respond accordingly by regulating the intracellular signaling pathways that assure ECs survival; (iii) a rather modest content per endothelial cell (less than 5% of cytosol volume), a level compatible to the low energy requirements imposed by the ECs normal, quiescent phenotype [3]. ECs mitochondria perform also functions in common with those exerted in cells with higher energy demands (e.g. the cardiomyocytes), such as: the generation of mitochondria-derived reactive oxygen species (mtROS) and the regulation of their level by antioxidant mechanisms [8,9], the dynamic shape changes via fusion and fragmentation (fission) events, and the specific autophagic degradation of the malfunctioning mitochondria by "mitophagy", associated with the execution of the "quality control" activity [10,11].
In diabetes mellitus, hyperglycemia triggers mitochondrial dysfunction/malfunction characterized by the decrease in membrane potential, the dysfunction of the respiratory chain, and the overproduction of mtROS that signal the switch of ECs phenotype from quiescent to "activated" [10]. This change engages the luminal aspect of ECs plasmalemma (where novel molecules are expressed), the cytosol (where AMP kinase becomes upregulated), and mitochondria, where the glycolytic ATP production is replaced by lipid-derived free fatty acids (FFA) β-oxidation [4,12]. The latter pathway provides the carbons for de novo nucleotide biosynthesis, linked to ECs proliferation [7,13]. The "activated" ECs are constantly challenged by the high glucose concentration and the circulating oxidative stress that stimulate the influx of extracellular Ca 2+ into the cytosol, where accumulate within microdomains close to the inner aspect of ECs plasmalemma. As mitochondria are anchored in this particular location, Ca 2+ enter their matrix, and activate the key Ca 2+sensitive enzymes (e.g. Ca 2+ -sensitive dehydrogenases, ATP synthases) involved in ATP synthesis via oxidative phosphorylation pathway (OXPHOS) [3,12]. The aerobic mitochondrial metabolism provides the ATP essential for the biosynthesis of vasoconstrictor molecules, inflammatory cytokines, adhesion molecules, and growth factors. Diabetes induces also the uncoupling of endothelial NO synthase (eNOS) associated with increased levels of superoxide anions (O 2 •-) and with a diminished formation of nitric oxide ( • NO) an endothelium-dependent vasorelaxant [14]. The low • NO levels are caused also by the rapid reaction with O 2 •and formation of the potent oxidant peroxinitrite anions (OONO -) that can damage proteins, lipids and nucleic acids. Moreover, OONOgenerates reactive nitrogen species (RNS) that induce modification of mitochondrial Complexes I and III, and exacerbate mtROS production [8]. Ultimately, the overproduction of mtROS along with the intracellular oxidative stress (ROS and RNS) overwhelms the antioxidant defense mechanisms, the cytochrome c and mitochondrial DNA (mtDNA) are released, and finally ECs apoptosis is installed [6,15]. The interrelated processes exemplified above endorse mitochondrial dysfunction/ malfunction as an important risk factor for the onset and progression of endothelial dysfunction, a key contributor to diabetes-associated cardiovascular disease [16]. The details of these relationships are examined in the following sections: (i) The regulatory role of mtROS in normal, healthy vascular endothelium, (ii) The adverse alteration in mitochondrial function in correlation with diabetes-related ECs dysfunction, (iii) The endeavors towards correction of mitochondrial malfunction in dysfunctional ECs, and (iv) The key points of mitochondria-targeted interventions in diabetic patients. The review is concluded by the pending, incompletely understood issues that may open promising new directions in the diabetes treatment.

Mitochondrial ROS Regulate the Normal Functions of Endothelium
The major mtROS in vascular endothelium are: (i) the free radicals of short half-life that do not diffuse across membranes, such as O 2 •-(half-life in the range of nano-to milliseconds) and hydroxyl radicals Taken together, the implications outlined above ascertain the regulatory and controller role of moderate mtROS levels in the normal and healthy ECs. In contrast, the overproduction of mtROS uncovers traits of mitochondrial dysfunction installment, with multiple consequences on the pathology of the vascular endothelium, as discussed next.

The Adverse Alterations in Mitochondrial Function Contribute to Type 2 Diabetes Mellitus-Related Endothelial Dysfunction
Mitochondrial dysfunction is biochemically described by ETC malfunction characterized by impaired OXPHOS, reduced ATP generation, and overproduction of mtROS. Based on its triggers, mitochondrial dysfunction is classified into two types: (a) the "primary mitochondrial dysfunction", caused by mutations in either nuclear or mitochondrial DNA and central for the largest group of inborn errors of metabolism known as "mitochondrial disorders" and (b) the "secondary/acquired mitochondrial dysfunction", caused by malfunctions that originate outside mitochondria, and are shared by the common forms of diabetes mellitus, inflammation, neoplasia, neurodegenerative and ischemic diseases, as well as by the ageing process [30][31][32][33][34][35].
In endothelium, mitochondrial dysfunction is installed under the influences of several circulating risk factors. An important endanger is the shear stress, defined as the tangential frictional force of blood flow acting on the vessel wall [36]. The perturbation of local hemodynamics engages the ECs mechano-sensors (such as G protein-coupled receptor), mechano-activated ion channels, growth factor receptors, glycocalyx, caveolae, membrane lipids, junction proteins, cytoskeleton network, integrins, focal adhesion kinase, etc. that all trigger the mecano-transduction process [37]. Among the biochemical consequences of the latter process, the literature quotes the modification of redox balance [11], activation of eNOS leading to increased • NO bioavailability, and S-nitrosation of redox-sensitive cysteine-containing ECs proteins [36]. Interestingly, the steady laminar or the pulsatile shear stress and the flow subsequent a period of ischemia causes enhanced production of mtROS and RNS, mitochondrial fission and ECs propensity to apoptosis [21].
Other risk factors for mitochondrial dysfunction installment are the unusual biochemical compounds present in the blood in pathophysiologic circumstances. In diabetes mellitus, circulating hyperglycemia upregulates mtROS production, inhibits the intracellular buffering system of ROS, damages the mtDNA [1,38] and stimulates mitochondrial apoptosis via ROS generated from electrons transfer between cytochrome c and the 66-kDa Src homology 2 domain-containing protein (p66Shc) [39]. The recent data show that high glucose concentration additionally induces p66Shc lysine acetylation, followed by its phosphorylation on serine 36 and the subsequent translocation to the mitochondria; at this specific location, acetylated p66Shc promotes H 2 O 2 generation, augmenting mtROS levels [40]. The excessive mitochondrial O 2 •generation may alter histone methylation in ECs leading to chronic upregulation of nuclear factor kappa B (NF-κB) and to vascular inflammation [41]. Interestingly, the pre-diabetic condition is pro-inflammatory and associated with the release of mtDNA (a probable inducer of early endothelial dysfunction) and activation of the endothelial toll-like receptor 9 (TLR9) [42]. Furthermore, overproduction of mtROS shunts glucose to the hexosamine biosynthetic pathway (HBP); enhanced HBP activity inhibits protein subunits of mitochondrial respiratory complexes I, III, and IV and results in ATP depletion, along with the long-term activation of pro-inflammatory signaling pathways [43].
Published reports indicate that hyperglycemia causes defective mitochondrial biogenesis, mitochondrial fragmentation, excessive autophagy, and accumulation of toxic waste (such as ubiquitin and irreversibly depolarized mitochondria) within ECs [15,44,45]. The high concentration of FFA (like palmitic acid) or oxidized lipoproteins (like oxLDL) are also reported as risk factors for mitochondrial dysfunction induction. Thus, the growth of ECs under high palmitic acid conditions leads to a shift of cells metabolism toward the intensified oxidation of FFAs associated with mitochondrial oxidative dysfunction [4]. Other reports on diabetic retinopathy show that lipotoxicity induced by high palmitic acid damages mtDNA, decreases cytochrome b transcripts, and augments glucotoxicity effects [40]. Furthermore, oxLDL promotes accumulation of mtROS and induces damage to the mtDNA, thus contributing to ECs dysfunction [46,47].
Based on the recent findings, it is important to update the latest data on the biomarkers of mitochondrial and endothelial dysfunction. It is generally admitted that mitochondrial dysfunction biomarkers consist in excessive mtROS production, altered IMM potential (Δψm), an indicator of mitochondrial respiratory chain function/malfunction [48], mitochondrial DNA (mtDNA) damage [10], and ATP depletion; the latter process activates AMP-activated protein kinase (AMPK) signaling pathway leading to excessive autophagy and to ECs apoptosis The mitochondrial fission role consists in the removal of malfunctioning segment within fused mitochondria, preserving a population of healthy mitochondria. Recent reports emphasize that exposure of the human arterioles to low-glucose concentration activates the mitochondrial fission process [52]. In ECs, mitochondrial fission was found to be associated with oxidative stress, activation of the NLRP3 inflammasome, diabetes [38,53,54], and diabetesaccelerated atherosclerosis [55]. According to the novel developments, the occurrence of mitochondrial dysfunction in ECs is one of the causative factors in the pathophysiology of cardiovascular diseases [6,56-58].
Endothelial dysfunction was traditionally described by reduced • NO bioavailability, and impairment of the vascular wall relaxation in response to blood flow, agonists (acetylcholine, bradykinin), and several diseases such as diabetes mellitus, atherosclerosis, hypertension, heart failure, ischemia-reperfusion injury [2]. Moreover, ECs dysfunction is the first event in cardiovascular disease [21] and is a major stimulus of vascular aging [59].
The contemporary data point out to several factors/processes associated with endothelial dysfunction, as follows: (i) The oxidative stress developed under pathophysiologic circumstances. In these conditions, besides mitochondria, a considerable number of enzymatic sources are engaged in endothelial ROS production, such as the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase isoforms (Nox) 2 and 4, the uncoupled eNOS, the cytosolic xanthine oxidase, and the decreased levels and/or activities of antioxidant superoxide dismutase(SOD) and catalase (CAT) [21,36,48]. The imbalance between aberrant ROS generation and antioxidant defense systems represents the primary cause of endothelial dysfunction, leading to vascular damage [60].
(ii) The nitrosative stress, characterized by increased RNS levels; the mechanisms involved consist in uncoupling of the NOS and oxidative damage to lipids, proteins and DNA [61]. At the crossroad between Snitrosation and ROS, the S-nitrosating agent S-nitrosocysteine (CysNO) was found to induce mitochondrial dysfunction in ECs, to cause nearly complete depletion of ATP and NAD + and to trigger cells death [62].
(iii) The ER stress induces endothelial dysfunction by enhancement of NADPH oxidase activity along with oxidative stress and by the diminishment of eNOS activity associated with the impairment of endothelial-dependent vasorelaxation [63-65].
(iv) Endothelial dysfunction mobilizes also the coupling factor 6 (CF6, a component of mitochondrial ATP synthase), the Humanin (a 24-amino-acid peptide encoded by the 16S rRNA region of the mitochondrial genome that specifically influences mtROS generation), the cardiolipin (a phospholipid located at the IMM); when shifted to the OMM, cardiolipin activates the autophagic degradation of mitochondria [2].

The Endeavours towards Correction of Mitochondrial Malfunction in Dysfunctional ECs
An outlook on dysfunction inductors (ordered according to their sources, i.e. the circulating blood, the ECs mitochondria, and ECs metabolism) in parallel with the specific modulators/suppressors involved in correction of each distinctive harmful effect is reviewed in Table 1. The insight into the mechanisms beyond alleviation of hyperglycemic mitochondrial and endothelial dysfunction outlined in Table 1 emphasizes two main correcting approaches: to point a distinct endothelial pathway and/or to target simultaneously several intracellular mechanisms employing a single modulator/suppressor. The distinct endothelial pathways targeted are: signaling through eNOS-dependent mammalian target of rapamycin complex 1 (mTORC1) pathway [91]. Moreover, the physiological substrate of AMPK is glutamine: fructose-6-phosphate amidotransferase 1 (GFAT1), the rate-limiting enzyme in the HBP that controls the post-translational modification of proteins by O-GlcNAcylation [92,93].
An ongoing endeavor points toward the amelioration of mitochondrial ROS overproduction by delivery of antioxidants that selectively target mitochondria, and concentrate at the matrix surface of IMM [94][95][96]. Encouraging results were reported for mitoquinone (MitoQ 10) [97,98] and plastoquinonyl decyltriphenylphosphonium1 (SkQ1) [99] that ameliorate antioxidant status, and for several other compounds and strategies aiming regulation of the mitochondrial antioxidant defense system (Table 1).
A recent example is the silencing of FIS1 or DRP1 protein expression (with siRNA) that conducts to inhibition of mitochondrial fission, of high glucose-induced alterations in mitochondrial networks, and of ROS generation; these effects were concurrent with eNOS activation and cGMP production [38].
Another strategy consists in suppressing the expression of endogenous voltage-gated K + (K V ) channel pore subunit K V 1.5 (Kv1.5); the outcome is inhibition of two intracellular pathways: mitochondria-mediated ROS generation and the apoptotic signaling pathway [108]. Furthermore, activation of AMPKα by C-peptide corrects mitochondrial dynamics alterations (by inhibiting mitochondrial fission and mitochondrial membrane potential collapse), prevents ROS generation induced by high glucose concentration, and impedes ECs apoptosis [109].
The targets and the intracellular mechanisms described above and in Table 1 emerge from studies on ECs in culture and in vivo (animal models or human vessels). However, for a target-to-treat strategy clinicians should be aware that mitochondrial dysfunction signature may vary according to the vascular bed under study, heterogeneity of endothelium, and diversity of (patho) physiological risk factors involved. Consequently, the current efforts to prevent and/or to correct the mitochondrial malfunction in dysfunctional ECs are still an ongoing endeavor.

The Key Points of Mitochondria-Targeted Interventions in Diabetic Patients
Initially, mitochondrial medicine anticipated the application of mitochondrial-directed interventions in the treatment of patients affected by "primary mitochondrial dysfunction" [31,110]. The use of mitochondriotropics, mitochondriotoxics, mitocancerotropics, and mitocans (mitochondria-targeted anticancer drugs) to treat "mitochondrial diseases" has been discussed in excellent reviews [35,111].
However, besides the management of symptoms, there are no effective therapies for "mitochondrial disorders" mitigation [34].
The "secondary mitochondrial dysfunction" associated with diabetes mellitus and insulin resistance is a distinct topic that requires appropriate alleviators able to slow down the associated cardiovascular pathology and their symptoms (Table 1). A caution related to the mitochondria-targeted therapeutic interventions emphasizes that some diabetes-related pathologies might not have solely a mitochondrial origin, but mitochondria might interfere either at their inception or during their development [115].
Thus, mitochondria-targeted antioxidants have been found efficient in all the major vascular beds disturbed by diabetes, i.e. myocardium (preventing cardiomyocytes loss and the reduced energy supply), kidney, neurodegenerative and eye diseases [56,118-121]. Other mitochondrial-based potential treatments of renal diseases include genetic therapies, thiazolidinediones, Sirtuins, and resveratrol [34,56]. However, the therapeutic procedures directed specifically towards dysfunctional mitochondria of diabetic vascular endothelium in humans are still waiting to be uncovered.

From circulating blood
High glucose concentration L-arginine

From Current Pending Issues to New Research Directions and Further Innovative Therapeutic Strategies in Diabetes
The two main malfunctioning targets in diabetes, mitochondria, and endothelium are currently addressed by strategies aiming both dysfunction alleviation and a potential therapeutic benefit. An urgent call is directed towards mitochondrial interventions, which have "the momentum" now [110].
Among the mitochondria-associated pending issues, the following have been quoted: 3-The metabolomic approach on mitochondrial metabolites outlines a promising novel direction in diabetes early detection and treatment [1,34,130].
Related to dysfunctional endothelium, the incompletely understood issues that may stress promising innovative directions in the diabetes treatment are: 1. The characterization of endothelial metabolism [131,132] including by metabolomic approaches [133]. Treatments targeting perturbations of ECs metabolism are not available now [134]. In this context, inhibition of ER stress [135] and the use of H 2 S to balance the 3. The still unclear mechanisms behind the novel coined "glycoredox" interaction, i.e. the association between the redox responses and the glycan function [137].

Conclusion
This review discusses the latest insights on diabetes mellitusassociated vascular endothelial cell dysfunction and highlights the novel regulatory and controller function of mitochondria. The basic knowledge provided here may encourage the teamwork of bench side and bedside scientists to advance into the domain of mitochondrial medicine, with significant potential for treatment of diabetic vascular dysfunctions.