The Rise of Mitochondria in Peripheral Arterial Disease Physiopathology: Experimental and Clinical Data

Peripheral arterial disease (PAD) is a frequent and serious condition, potentially life-threatening and leading to lower-limb amputation. Its pathophysiology is generally related to ischemia-reperfusion cycles, secondary to reduction or interruption of the arterial blood flow followed by reperfusion episodes that are necessary but also—per se—deleterious. Skeletal muscles alterations significantly participate in PAD injuries, and interestingly, muscle mitochondrial dysfunctions have been demonstrated to be key events and to have a prognosis value. Decreased oxidative capacity due to mitochondrial respiratory chain impairment is associated with increased release of reactive oxygen species and reduction of calcium retention capacity leading thus to enhanced apoptosis. Therefore, targeting mitochondria might be a promising therapeutic approach in PAD.


Introduction
Peripheral arterial diseases (PAD) is a major concern for public healthcare, affecting more than 200 million individual worldwide [1,2]. Its prevalence varies from 3% to 10%, but can reach up to 20% in the elderly population [3].
PAD is defined by a narrowing of the peripheral arterial vasculature. It mostly affects lower limbs, leading to overall functional disability and reduced quality of life. Initially asymptomatic, PAD progressively compromises lower limb vascularization, leading to obstruction of the vessels by atheroma. PAD includes all stages of the disease, from asymptomatic with abolition of distal pulses, to intermittent claudication or critical limb threatening ischemia (CLTI) characterized by rest pain and/or ulcers. PAD is also often associated with cognitive dysfunction characterized by reduced performance in nonverbal reasoning, reduced verbal fluency, and decreased information processing speed [4]. Thus, PAD is a serious condition threatening both limb (risk of amputation) and vital prognosis of the patients. Indeed, despite recent therapeutic progress, morbidity and mortality rates remain incompressible around 20% and 15% five years after a diagnosis of symptomatic or asymptomatic PAD [5]. On average, the life expectancy of claudicating patients is reduced by 10 years, with a majority of death attributable to cardiovascular causes.
The treatment of PAD is mainly based on revascularization of the ischemic limb [6]. Nevertheless, this surgical procedure is not always possible, notably when the vascular state is too precarious or when the local evolution is too advanced. It appears therefore important to better understand PAD pathophysiology to offer optimal patient care. Indeed, insufficient oxygen supply was long presumed to be the main and sole cause for PAD symptoms. However, recent advances in understanding PAD physiopathology identified mitochondria as a key element in the deleterious process of PAD [7,8].
Thus, skeletal muscles alterations significantly participate in PAD injuries, modulating its prognosis, and this review aims to describe muscle mitochondrial dysfunctions. Particularly, we will analyze data focused on mitochondrial respiratory chain respiration, on reactive oxygen species release and on mitochondrial calcium retention capacity which decrease is associated with enhanced apoptosis ( Figure 1).

Normal Condition
Life requires energy, and this energy is stored in adenosine triphosphate (ATP) molecules, that are produced in the mitochondria by oxidative phosphorylation and assessed through mitochondrial respiration determination. Specifically, the oxidation of nutrients through the Krebs cycle provides reduced coenzymes (the reduced form of nicotinamide adenine dinucleotide (NADH)) and to a lesser extent flavin adenine dinucleotide (FADH 2 ), which are electron donors. This flow of electrons is supported by different redox reactions provided by the four complexes of the mitochondrial respiratory chain, up to the reduction of molecular oxygen in water. The respiratory complexes use the energy generated by this electron transfer to allow an active translocation of protons from the matrix to the inter-membrane mitochondrial space. This expulsion of protons results in the generation of a concentration gradient and a mitochondrial membrane potential across the inner membrane. ATP synthases use the transmembrane protonmotive force as a source of energy to drive a mechanical rotary mechanism leading to the chemical synthesis of ATP from ADP and Pi.The F 1 F 0 ATP synthase enzymes allow proton flux and ATP synthesis through a and c subunits of the F 0 domain. The maintenance of this electrochemical gradient, also called protomotive force, is an essential element for the energetic role of the mitochondria [9][10][11].
Mitochondrial respiration generates free radicals derived from oxygen, the reactive oxygen species (ROS). A free radical is a chemical species containing an unpaired electron. Extremely unstable, this compound can react with more stable molecules to match its electron. It can then pull out an electron and behave as an oxidant, usually leading to the formation of new radicals in the chain and causing significant cell damage. Main ROS comprise superoxide anion, the hydroxyl radical and the highly reactive compound hydrogen peroxide (H 2 O 2 ). Detoxification systems exist, enzymatic (superoxide dismutase, catalase, glutathione peroxidase) or not (vitamins and trace elements).
Nevertheless, when the radical production remains contained below a certain threshold, the ROS activate defenses pathways involving the development of cellular antioxidants and mitochondrial biogenesis. Also named mitohormesis, these mechanisms constitute one of the therapeutic targets that can limit the lesions linked to repeated cycles of ischemia-reperfusion in PAD [12].

Ischemic Condition
During ischemia, ATP is generated by anaerobic glycolysis, leading to glycogen storage depletion, anaerobic metabolism activation and local lactic acidosis. The resulting depletion of ATP reduces the function of membrane pumps and causes cellular edema. Indeed, the cell tends to correct the acidosis by expelling the H+ ions via the Na+/H+ exchanger, thus saturating the cytoplasm with Na + ions and causing an osmotic shift to the cytoplasm. Cell edema is aggravated by Na+/K+ ATP-dependent exchanger dysfunction due to lack of ATP, which also leads to Na+ accumulation in the cytoplasm. Acidosis also activates mediators, such as phospholipaseA2, that metabolize membrane phospholipids to arachidonic acid, a precursor of inflammatory mediators such as leukotrienes and prostaglandins. Ischemia will also initiate conversion of xanthine dehydrogenase to xanthine oxidase [13].
Reperfusion is able to prevent the irreversible damages of ischemia. Nevertheless, this process also generates lesions that aggravate the pre-existing tissue damages. At the cellular level, reoxygenation interrupts the lesions induced by ischemia, but causes reperfusion injury. During the first few minutes of reperfusion, the rapid correction of acidosis increases the cytosolic Ca 2+ , thus promoting the opening of the mitochondrial permeability transition pore [14]. This opening causes a sudden change in the mitochondrial membrane permeability, resulting in energy collapse incompatible with cell survival and inducing the release of pro-apoptotic factors from the inter-membrane mitochondrial space to the cytosol, leading to cell death. It is the intrinsic mitochondrial apoptosis pathway.
In parallel, reperfusion generates massive oxidative stress since xanthine oxidase and succinate, produced during ischemia, catalyzes the formation of uric acid from hypoxanthine and of ubiquinol, respectively, accompanied by the formation of large amounts of free radicals. Very interestingly, Chouchani et al. demonstrated a conserved metabolic response of tissues to ischemia-reperfusion (IR) revealing that reducing ischemia-induced succinate increase and its oxidation after reperfusion might be an important therapeutic during IR settings. [15][16][17].
The ROS thus produced exceed the cellular antioxidant defenses creating a vicious circle. Production of free radicals will cause a dysfunction of the mitochondrial respiratory chain, which in turn generate more ROS. Such ROS overproduction leads to several deleterious effects: lipid peroxidation, protein oxidation and DNA mutations, but also to the opening of the mitochondrial permeability transition pore.

Experimental Data
Impairments in mitochondrial respiration were observed in both claudicating and CLTI experimental models. Indeed, significant reduction in mitochondrial complexes I, II and IV activities was found in muscles of rats submitted to hindlimb ischemia-reperfusion compared to contralateral muscles [18][19][20][21]. Similarly, mouse models of CLTI presented decreased activities of the complexes I, III and IV in ischemic muscles compared with controls [22].
The respiratory impairments were shown to be strain-, muscle-, age-and disease-specific. Indeed, mitochondrial respiration was affected by hindlimb ischemia-reperfusion in limb muscles of BALB/c mice, but not of C57BL/6 mice [23]. Secondly, ischemia-reperfusion injury was shown to affect more severely the respiration in glycolytic muscles than in oxidative ones [24]. Furthermore, the impairments in mitochondrial respiration observed in young mice were greater in older animals submitted to ischemia-reperfusion injury [25]. Lastly, the decline in mitochondrial oxidative capacity was more severe in diabetic rats compared to non-diabetic animals ( Table 1) [26].

Clinical Data
Oxygraphy measurements in PAD patients revealed significantly altered respiratory activity, notably of complexes I, III and IV, and of the acceptor control ratio [27][28][29][30]. Interestingly, more recent studies reported no difference in the mitochondrial respiration rate between PAD patients and healthy controls, despite alterations in O 2 delivery, tissue-reoxygenation and ATP synthesis rate during exercise [31,32]. These conflicting findings could be explained by disparities in disease severity. Indeed, the alterations in mitochondrial oxidative capacity may have been the result of factors associated with higher morbidity in PAD, such as sarcopenia [33][34][35].
Mitochondrial energy metabolism was shown to decrease in PAD patients compared to controls [36,37]. In contrast, Hou et al. found similar mitochondrial ATP production rate in both PAD patients and healthy controls [38]. Again, such differences might be explained by differences in disease severity or morbidity factors rate.
It is important to note that patients suffering from both PAD and type II diabetes (DT2) are more susceptible to reduced O2 consumption and mitochondrial oxidative phosphorylation compared to patients with PAD alone or to controls (Table 2) [39,40].

Reactive Oxygen Species Production, Proteins, Lipids and DNA Alterations and Impaired Antioxidant Defense, in PAD
The interaction between mitochondria and oxidative stress in skeletal muscle is modulated by repeated cycles of ischemia-reperfusion in the context of PAD. The vascular damages create an imbalance between oxygen supply and demand during efforts, generating a situation of ischemia; followed by a situation of reperfusion when the patient is at rest. Repetition of ischemia and reperfusion cycles are deleterious for skeletal muscle and lead to myopathy and to remote organ damage [7,28,41].

Experimental Data
ROS production was found increased in both animal models of PAD (acute ischemia-reperfusion and CLTI) as compared to controls, using either measurements of 1) free radical species by electron paramagnetic resonance spectroscopy, 2) dihydroethidium (DHE) by epifluorescence microscopy or 3) H 2 O 2 by Amplex Red perioxide assay [24,42,43]. Interestingly, ROS production was greater in PAD animals presenting with hypercholesterolemia or diabetes [26,44]. These evidences suggest an association between PAD comorbidity factors and enhanced mitochondrial dysfunction.

Clinical Data
Similar to the findings on experimental models, PAD patients displayed increased mitochondria-derived ROS production characterized by elevated levels of free radical species [32].
Lastly, evidence of reduced antioxidant defenses has been shown in PAD patients, notably with altered activities of the antioxidant enzymes SOD, catalase and glutathione peroxidase (Table 4) [28].

Clinical Data
Human investigations also showed a clear implication of apoptosis in PAD pathophysiology. Indeed, PAD patients displayed elevated levels of genes mediating apoptosis [58], increased DNA fragmentation and caspase-3 activity [59]. Additionally, multiple studies reported higher levels of apoptotic cells in different cell types, notably endothelial cells and lymphocytes [60,61]. Interestingly, another study reported similar levels of endothelial apoptosis in both PAD and control groups. This result is likely due to disparities in patient's selection, with patients presenting with lower associated risk factors (Table 6) [62]. PAD: peripheral arterial disease; PCR: polymerase chain reaction.

Conclusions
In summary, PAD is a public health issue even when poorly symptomatic [63], and mitochondria are importantly involved in its pathophysiology. Not only because mitochondrial alterations reduce the energy available for cell function, but also because mitochondria participate in the increased ROS production (and therefore to a greater oxidative stress) and in the enhanced opening of the mitochondrial permeability pore which favor the intrinsic pathway of cell apoptosis. These data account for the fact that skeletal muscle mitochondrial function might be considered as a prognostic factor in the setting of PAD in humans. On the other hand, if ROS production remains contained below a certain threshold, mitohormesis and stimulation of the antioxidant defenses can be protective supporting that mitochondrial function modulation might be a therapeutic target. Thus, further studies focused on mitochondria are warranted to optimize the care of patients presenting with PAD.

Conflicts of Interest:
The authors declare no conflict of interest.