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  • br Competing interests declaration br Introduction N acetylc


    Competing interests declaration
    Introduction N-acetylcysteine (NAC) is widely used in a variety of diseases and presents a good safety profile. Some of its therapeutic effects are supposed to be secondary to its antioxidant effects. NAC is readily hydrolyzed to cysteine, a precursor of glutathione (GSH), or can act directly as a free radical scavenger [1], [2]. It has been reported to be effective for different diseases, not only in experimental studies, but also in humans [3]. Oxidative damage is described both in animal models and humans with chronic limb ischemia [4], [5]. Increased xanthine oxidase activity, activated neutrophils and mitochondrial dysfunction are the main source of oxidative species during the development of this condition [5], [6]. Despite of this there are few reports determining the effectiveness of antioxidants for the adjuvant treatment of chronic limb ischemia. Besides oxidative damage, the decrease of tissue oxygen levels is followed by several adaptive responses, which include optimization of oxygen utilization by the mitochondria, mitochondrial regeneration, and generation of new blood vessels to increase blood perfusion [7]. The first response is mediated mainly by hypoxia inducible factors (HIFs); the second one is mediated mainly by autophagy/mitophagy and mitochondrial biogenesis; and the third one is driven by the up-regulation of a variety of angiogenic growth factors [8], [9], [10]. HIFs are transcription factors that promote AMG 925 to hypoxia through regulation of >150 genes involved in angiogenesis, metabolism, cell proliferation and cell migration [11]. The mitochondrial adaptation to ischemia includes modifications in the composition of electron transfer chain (ETC) proteins [12], increased ETC activity, and increased oxygen consumption [13]. Additionaly, angiogenesis is the process by which new blood vessels develop from pre-existing vessels [11]. This physiological response helps to preserve tissue integrity and/or function in settings of local hypoxia or reduced O2 availability [17]. Besides HIFs, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis [18]. Ligation of the femoral artery also induces another vascular response known as arteriogenesis. Arteriogenesis is defined as the process of artery maturation or the de novo growth of collateral conduits [19]. Moreover, these vessels undergo remodeling and growth into parallel conductance vessels, thereby establishing a collateral circuit [20]. VEGF is one of the most important proangiogenic factors involved in therapeutic angiogenesis during and after ischemia [21], [22]. All these evidences come predominately from acute models of muscle ischemia/reperfusion (I/R), adaptations during but in chronic limb ischemia are not well described. For example, in humans acute-on-chronic ischemia induced the up-regulation of VEGF, HIF and proinflammatory genes, in contrast to chronic ischemia that had attenuated HIF and VEGF expression [23]. Inflammation is also differently when comparing intermittent claudication and critical limb ischemia. The first induces only a minor up-regulation of circulating cytokines, in contrast to critical limb ischemia that induces a cytokine profile similar to severe medical conditions such as sepsis [24]. Mitochondrial autophagy/mitophagy and mitochondrial biogenesis consist of two opposing cellular pathways that adjust mitochondrial content to sustain energy metabolism in response to different signals, such as stress and metabolic state. Mitochondrial biogenesis is controlled through transcription factors, which include the peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) and the nuclear respiratory factors (NRF1 and NRF2) [14]. Mitochondrial autophagy, on the other hand, consists of the degradation of mitochondrial macromolecules by a structure called autophagosome [15]. The autophagosome is dependent of several autophagy-related proteins (ATG) and specific mitochondrial proteins are required to identify and mark the mitochondria for mitophagy, including PINK and Parkin [16]. Mitochondrial dynamics was studied in I/R models mainly in heart and brain [25], and in skeletal muscle mainly during exercise [26], but not during chronic ischemic conditions.