Critical Limb Ischaemia Exacerbates Mitochondrial Dysfunction in ApoE–/– Mice Compared with ApoE+/+ Mice, but N-acetyl Cysteine still Confers Protection

Abstract

The study shows that critical limb ischaemia (CLI) exacerbates mitochondrial dysfunction and that antioxidant therapies such as N-acetyl cysteine (NAC) still confer protection, even if comorbidities are known to reduce the protective effects of many drugs. Clinical studies using antioxidant strategies have been mostly disappointing and antioxidant therapies are still far from being integrated into treatment algorithms for peripheral arterial disease, but these results suggest that oxidative stress needs to be considered as a target for protective strategies in the setting of CLI. Objectives: The current study was performed in order to determine the influence of hypercholesterolaemia on critical limb ischaemia (CLI) and whether targeting oxidative stress by antioxidant therapies such as N-acetyl cysteine (NAC), considered to be a direct scavenger of reactive oxygen species, could confer muscle protection. Methods: Apolipoprotein E (ApoE)e/e mice (n ¼ 9, 29 weeks old) and their genetic controls ApoEþ/þ mice (n ¼ 9, 29 weeks old) were submitted to sequential right femoral and iliac ligations; the left limb served as control. ApoEþ/þ mice were divided into two groups: Group 1 (n ¼ 4) and Group 2 (n ¼ 5); as well as ApoEe/e mice: Group 3 (n ¼ 3), and Group 4 (n ¼ 6). NAC treatment was administered to Groups 2 and 4 in drinking water. Mice were sacrificed on Day 40 and gastrocnemius muscles were harvested to study mitochondrial respiration by oxygraphy, calcium retention capacity by spectrofluorometry, and production of reactive oxygen species by electron paramagnetic resonance. Results: CLI associated with ApoE deficiency resulted in more severe mitochondrial dysfunction: maximum oxidative capacity and calcium retention capacity were decreased (À42.9% vs. À25.1%, p ¼ .010; and À73.1% vs. À40.3%, p ¼ .003 respectively) and production of reactive oxygen species was enhanced (þ63.6% vs. þ41.4%, p ¼ .03) in ApoEe/e mice compared with ApoEþ/þ mice respectively. Antioxidant treatment restored oxidative capacity, calcium retention capacity and decreased production of reactive oxygen species in both mice strands. Conclusions: In this small murine study, hypercholesterolaemia exacerbated mitochondrial dysfunction, as clinically expected; but antioxidant therapy appeared protective, which is counter to clinical experience. Further work is clearly needed.

The Rise of Mitochondria in Peripheral Arterial Disease Physiopathology: Experimental and Clinical 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.…”

“…Rodent models of PAD displayed elevated protein expression of the apoptotic factors cleaved-caspase 3, cleaved-poly (ADP-robose) polymerase (PARD) and mitochondrial and cytosolic Bcl2-associated X (Bax), and reduced protein expression of the anti-apoptotic factor Bcl-2, compared with controls [21,54,55]. Furthermore, a decrease in mitochondrial calcium retention capacity was observed in ischemic limbs compared with contralateral ones (Table 5) [25,44,47,56,57]. IR increased Bax (63.4%, p = 0.020) and Bax/Bcl-2 ratio (+84.6%, p = 0.029).…”

Skeletal Muscle Mitochondrial Dysfunction and Oxidative Stress in Peripheral Arterial Disease: A Unifying Mechanism and Therapeutic Target

“…Lejay et al [128] Swiss mice-femoral artery ligation 40 days post-surgery N-acetylcysteine -decrease tissue damage -improved mitochondrial respiration and calcium retention -decreased ROS levels Lejay et al [165] Apolipoprotein E deficient mice-femoral artery ligation 40 days post-surgery N-acetylcysteine -improved mitochondrial respiration and calcium retention -decreased ROS production Miura et al [127] Mice-femoral artery ligation 21 days post-surgery MitoTEMPO -improved limb perfusion recovery -decreased ROS production -decreased mtDNA damage…”

“…Recent preclinical evidence has demonstrated that treatment with antioxidant therapies show promise in both chronic ischemia and acute ischemia-reperfusion models ( Table 1). For example, Lejay et al reported that N-acetylcysteine, a global antioxidant and precursor to glutathione, improved mitochondrial respiratory function and reduced oxidative stress in mice subjected to chronic severe ischemia in both normal Swiss mice [128] and apolipoprotein E-deficient mice [165]. Another study employed a transgenic mouse overexpressing a mitochondrial-targeted catalase (scavenger of hydrogen peroxide), which was shown to decrease ischemia muscle injury and improve mitochondrial function in diet-induced diabetic mice [102].…”

Prolonged Cold Ischemia Did Not Impair Mitochondrial Oxygen Consumption or Reactive Oxygen Species Production in Human Uterine Fundus and Horn Myometrium

“…Tissues were finally harvested and dried at 150 • C for 15 min. ROS production was expressed in µmol/min/mg dry weight, as previously reported [29].…”