α-Lipoic acid attenuates vascular calcification via reversal of mitochondrial function and restoration of Gas6/Axl/Akt survival pathway

Abstract Vascular calcification is prevalent in patients with chronic kidney disease and leads to increased cardiovascular morbidity and mortality. Although several reports have implicated mitochondrial dysfunction in cardiovascular disease and chronic kidney disease, little is known about the potential role of mitochondrial dysfunction in the process of vascular calcification. This study investigated the effect of α-lipoic acid (ALA), a naturally occurring antioxidant that improves mitochondrial function, on vascular calcification in vitro and in vivo. Calcifying vascular smooth muscle cells (VSMCs) treated with inorganic phosphate (Pi) exhibited mitochondrial dysfunction, as demonstrated by decreased mitochondrial membrane potential and ATP production, the disruption of mitochondrial structural integrity and concurrently increased production of reactive oxygen species. These Pi-induced functional and structural mitochondrial defects were accompanied by mitochondria-dependent apoptotic events, including release of cytochrome c from the mitochondria into the cytosol, subsequent activation of caspase-9 and -3, and chromosomal DNA fragmentation. Intriguingly, ALA blocked the Pi-induced VSMC apoptosis and calcification by recovery of mitochondrial function and intracellular redox status. Moreover, ALA inhibited Pi-induced down-regulation of cell survival signals through the binding of growth arrest-specific gene 6 (Gas6) to its cognate receptor Axl and subsequent Akt activation, resulting in increased survival and decreased apoptosis. Finally, ALA significantly ameliorated vitamin D3-induced aortic calcification and mitochondrial damage in mice. Collectively, the findings suggest ALA attenuates vascular calcification by inhibiting VSMC apoptosis through two distinct mechanisms; preservation of mitochondrial function via its antioxidant potential and restoration of the Gas6/Axl/Akt survival pathway.


Introduction
Vascular calcification (VC), a common consequence of aging, atherosclerosis, diabetes and chronic kidney disease (CKD), is a strong independent predictor of increased cardiovascular morbidity and mortality [1][2][3]. VC is an active cellular process regulated by an imbalance between pro-mineralizing factors including inflammation, oxidative stress and high phosphate and calcium, and calcification inhibitory factors including fetuin-A, matrix gla protein and pyrophosphate [1][2][3][4]. Among the pro-mineralizing factors, hyperphosphatemia is a persistent and prevalent problem in CKD patients and has emerged as a major contributor to VC [5,6].
Previous in vitro studies have demonstrated that vascular smooth muscle cell (VSMC) calcification by elevated inorganic phosphate (Pi) uptake via a sodium-dependent phosphate cotransporter (Pit-1) is caused by both phenotypic transition from VSMCs to osteoblast-like cells and apoptotic cell death [7][8][9][10][11][12]. Osteoblastic differentiation of VSMCs is mediated by the up-regulation of several osteogenic genes, including core-binding factor-1 (Cbfa-1, also known as Runx2), osteopontin and osteocalcin [8,12]. In parallel with phenotypic transition of VSMCs into osteoblast-like cells, VSMC apoptosis plays a crucial role in the development of Pi-induced VSMC calcification [7,[9][10][11]. VC is initiated by apoptotic bodies and matrix vesicles, which are derived from apoptotic and viable VSMCs, respectively, and may serve as a calcification nidus [3,9,13]. Apoptotic bodies and matrix vesicles were known to be implicated in VSMC calcification by nucleating insoluble basic calcium phosphate [9,13,14]. Furthermore, recent studies have demonstrated that the Pi-induced VSMC apoptosis and subsequent calcification are dependent on the down-regulation of the Gas6/Axl/Akt survival pathway that inhibits apoptosis and increases survival of VSMCs [10,11]. For instance, 3-hydroxy-3-methylglutaryl CoA reductase inhibitors (statins) protect VSMCs from Pi-induced calcification by suppressing apoptosis via restoration of Gas6/Axl/Akt survival pathway [11].
Mitochondria, in addition to supplying cellular energy, play a central role in the intrinsic apoptotic pathway. Mitochondria-mediated apoptosis involves the release of cytochrome c from the inner membrane space to the cytosol, which in turn triggers the activation of caspase-9 and -3 cascades [15,16]. These apoptotic events are closely linked to mitochondrial dysfunction, which exhibits changed mitochondrial membrane potential (⌬⌿m), increased oxidant generation as a result of the perturbation of electron transport chain reaction, and decreased intracellular ATP content because of oxidant-insulted low respiratory activity [17][18][19]. Although the precise mechanisms for mitochondriamediated apoptosis remain to be elucidated, oxidative stress caused by endogenously and exogenously excessive oxidant insults and/or impaired oxidant defenses is generally believed to be key in both mitochondrial dysfunction and cellular apoptosis [20]. Mitochondria-targeted antioxidants could inhibit the peroxidation of mitochondrial components including cytochrome c and consequently block apoptosis [21]. Among the various antioxidants, ␣-lipoic acid (1,2-dithiolane-3-pentanoic acid, ALA), a naturally occurring antioxidant with anti-apoptotic property [22][23][24][25], is a cofactor for mitochondrial metabolic enzymes, pyruvate dehydrogenase and ␣-ketoglutarate dehydrogenase [22,24,26]. ALA is considered the most potent and ideal antioxidant in that it is soluble in both fat and water and is capable of not only directly scavenging oxidants but also boosting levels of other antioxidants such as glutathione, vitamin C and vitamin E [23,24]. Moreover, ALA has been demonstrated to improve age-associated decline in mitochondrial function and structure and inhibit intrinsic mitochondrial apoptotic pathway in endothelial cells through its antioxidant function [22,25,27]. Owing to the multiple beneficial effects of ALA, this compound has been suggested as a potential therapeutic agent for the prevention and treatment of various pathologies including cardiovascular disease, diabetes, liver damage, atherosclerosis and neurodegenerative diseases [23,24,28,29]. In addition, several studies have reported that oxidants are one of major causative factors of VSMC calcification and antioxidants have beneficial effects on therapy in hypertension and CKD [30][31][32][33]

Measurement of mitochondrial membrane potential and intracellular ATP content
VSMCs were seeded in a 6-well culture plate at a density of 1 ϫ 10 5

Caspase activity assay
After cells were lysed and centrifuged as described in a caspase-9 and -3 colorimetric assay kit (R&D Systems, Minneapolis, MN, USA), the resulting supernatant (100-200 g) was assayed for caspase-9 or -3 activity in the presence of colorimetric substrate and the absorbance was measured at 405 nm.

In situ aortic superoxide anion detection
To estimate oxidant generation in aortic tissues, dihydroethidium (DHE) staining was performed as previously described [31]. DHE 1A and B). Next, to examine whether mitochondrial dysfunction is involved in VSMC calcification, changes in mitochondrial membrane potential and cellular ATP levels were examined during Pi-induced VSMC calcification. Intriguingly, mitochondrial membrane potential was gradually decreased during Pi-induced VSMC calcification, accompanied by decline in intracellular ATP levels ( Fig. 1C and D). Oxygen consumption was also modestly reduced at the late stage of Pi-induced calcification (Fig. S1). These results suggest that mitochondrial metabolic function is perturbed during Pi-induced VSMC calcification.

ALA decelerates Pi-induced VSMC calcification via reversal of mitochondrial dysfunction
It is well known that ALA plays a fundamental role in enhancing mitochondrial metabolism and ATP production [23,24,26,27]. Appropriately, the effect of ALA on disturbed mitochondrial function caused by Pi in VSMCs was evaluated. The decline in mitochondrial membrane potential and ATP production in Pi-induced VSMC calcification was restored by ALA treatment ( Fig. 2A and B). Moreover, electron microscopic analysis revealed that ultrastructural alterations in mitochondria with disrupted cristae by Pi were considerably reversed by ALA (Fig. 2C). Consistent with the beneficial effects of ALA on mitochondrial metabolism and structure, ALA significantly attenuated Pi-induced VSMC calcification (Fig. 2D). Taken together, these results support the suggestion that ALA inhibits Pi-induced VSMC calcification via recovery of mitochondria metabolism and structure.

ALA suppresses Pi-induced mitochondriamediated apoptosis and restores Gas6/Axl/Akt survival signal down-regulated by Pi
It has been demonstrated that mitochondria dysfunction contributes to cell apoptosis [17][18][19] and that apoptosis plays a 277 critical role in Pi-induced VSMC calcification [7,[9][10][11]. Therefore, the effect of ALA on Pi-induced VSMC apoptosis was assessed. As shown in Figure 3A, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining indicated that apoptotic cell death was obviously increased in Pi-treated VSMCs and that apoptosis was significantly inhibited by ALA. It was also observed that ALA blocked Pi-induced sequential activations of the intrinsic mitochondrial apoptotic pathway ( Fig. 3B and C): release of cytochrome c from mitochondria into cytosol, activation of caspase-9, an initiator caspase, and activation of caspase-3, an executioner caspase. Consistent with the inhibitory effect of ALA on Pi-induced caspase activation in mouse aortic VSMCs, ALA inhibited the Pi-induced activation of caspase-9 and -3 in human aortic SMCs (Fig. S2). Pi-induced VSMC apoptosis and calcification have been reportedly shown to be associated with down-regulation of Gas6/Axl/Akt-mediated survival signals [10,11]. Therefore, the next experiment investigated the effect of ALA on survival signals in VSMCs. Pi markedly suppressed the expression of Gas6 and Axl and led to Akt inactivation, and this down-regulation of Gas6/Axl/Akt signalling pathway was almost completely reversed by ALA (Figs S3 and 3D). Taken together, these findings offer evidence that ALA protects VSMCs from Pi-induced calcification through inhibition of mitochondria-mediated apoptotic pathway by reversal of mitochondrial dysfunction as well as restoration of Gas6/Axl survival pathway.

ALA blocks oxidative stress-induced VSMC calcification by inhibiting apoptosis and sustaining survival
Increasing evidence suggests that oxidants induce the perturbation of electron transport chain reaction of mitochondria and vice-versa, resulting in the accumulation of excessive oxidants and, ultimately, mitochondria-mediated apoptosis through an altered mitochondrial membrane potential and release of apoptotic factors including cytochrome c [15,16]. Recent studies have potentially implicated exogenous and endogenous oxidants in VSMC calcification [30,31] Figure 4A (Fig. 4C)

Fig. 3 ALA inhibits mitochondria-mediated apoptosis of VSMCs induced by Pi and restores Gas6-Axl survival pathway. (A-C) Inhibition of Pi-induced VSMC apoptosis by ALA. VSMCs were treated with Pi in the presence or absence of ALA (300 M) for 4 days (A, C) or indicated times (B). Apoptotic cells were detected by TUNEL staining (A). Nuclei were counterstained with propidium iodide and then the two images were merged. TUNEL-positive apoptotic cells were counted as a percentage of the total number of cells. Scale bar indicates 100 m. To assess the release of cytochrome c from mitochondria into cytosol (B), mitochondrial and cytosolic fractions were subjected to Western blotting with antibodies against cytochrome c (Cyt C), cytochrome c oxidase 2 (Cox 2, a mitochondrial marker protein) and ␤-actin (a cytosolic marker protein). Cytosolic extracts were analyzed for caspase-9 or caspase-3 activity. The activity was normalized to protein content and expressed as a fold difference relative to control (C). Data are expressed as the mean Ϯ S.D. (n ϭ 3). (D) ALA restored Gas6 and Axl expression and Akt activation, which are down-regulated by Pi. Whole cell lysates were obtained from VSMCs treated with Pi for the indicated times and subjected to
Western blotting with specific antibodies to Gas6, Axl, ␤-actin, phospho-Akt and Akt. *P Ͻ 0.01; † P Ͻ 0.05. (Fig. 4A-C). ALA totally blocked cytochrome c release by Pi and Pi plus antimycin A, and restored Gas6-Axl, whose expression is down-regulated by Pi and Pi plus antimycin A (Fig. 4D and E). These observations supported the suggestion that ALA possesses antioxidant capacity and the protective effect of ALA on VSMC calcification may be closely connected to the inhibition of mitochondria-mediated apoptosis and restoration of Gas6/Alx survival pathway.

ALA inhibits vitamin D 3 -induced aortic calcification in mice
Supra-physiological dosages of vitamin D analogues are associated with ectopic calcification of soft tissues including the aorta [35,36]. To further confirm the protective effect of ALA against VSMC calcification in vivo, an aortic calcified mouse model was established by subcutaneous administration of vitamin D3 toxic dosages. Vitamin D3-mediated aortic calcification in the mice was visualized by von Kossa staining and quantified by calcium content in a whole aortic tissue. Microscopic examination in the von Kossa-stained cross-sections showed a prominent calcification in the medial layer of the aorta (Fig. 5A and B). This calcification was   considerably diminished by the intraperitoneal injection of ALA at a dosage of 40 mg/kg/day. As shown in Figure 5C and D, the superoxide anion production and TUNEL-positive apoptotic cells were strongly increased and the level of Gas6 and Axl expression was decreased in the aorta of vitamin D3-administered mice compared to control. ALA significantly reduced these vitamin-D3induced superoxide production and apoptotic cell death along with restoration of Gas6 and Axl expression in the aorta (Fig. S4). In line with the beneficial effect of ALA on Pi-induced mitochondrial function in VSMCs, vitamin D3-induced mitochondrial cristae disruption was greatly attenuated by ALA (Fig. 5E).

Discussion
In this study, ALA blocked Pi-induced oxidant generation and thiol oxidation and mitochondrial dysfunction concurrently in VSMCs. Consequently, this affirmative property of ALA prevented Piinduced VSMC calcification by both the restoration of Gas6/Axl survival signals and suppression of mitochondria-mediated apoptosis (Fig. 6). Various diseases including aging, atherosclerosis, hypertension, diabetes and CKD that have been linked to a high prevalence of VC are associated with increased oxidative stress [7,21,37]. Moreover, recent reports have demonstrated that elevated oxidant signals are mainly observed around the calcifying foci of the aortic valve and exogenous hydrogen peroxide accelerates VSMC calcification in the osteogenic media containing ascorbic acid, ␤-glycerophosphate and dexamethasone [30,31]. In general, oxidative stress is generated in cells by diverse sources. One is superoxide anion formation by the reductive reaction of electrons leaked from mitochondria at the level of the respiratory chain with oxygen [34]. Other sources include multiple enzymatic systems including NADPH oxidase, lipooxygenase, cyclooxygenase, xanthine oxidase and uncoupled nitric oxide synthase [38].

transporters, resulting in oxidant generation and thiol oxidation of cellular constituents. Pi overload induced increased intracellular and mitochondria-derived oxidant production and ALA inhibited the intracellular and mitochondrial oxidant generation induced by
Pi. Tempol, a superoxide dismutase-mimetic antioxidant, also showed protective effect against Pi-induced mitochondrial oxidant generation and VSMC calcification (Fig. S5). These results suggest that Pi-induced oxidative stress may trigger mitochondrial oxidant accumulation and VSMC calcification and that there is a causal connection between oxidative stress and the induction of mitochondrial dysfunction [17][18][19]. To prove this, further study is necessary to reveal the precise causal connection between ALA and mitochondrial function. Presently, increased oxidant generation by antimycin A, which is an inhibitor of complex III that is principle in the leakage of electrons during mitochondrial respiratory chain through complex I-IV, accelerated VSMC calcification. Moreover, treatment of VSMCs with Pi showed an increase of oxidant generation and thiol oxidation. In addition, it has been reported that oxidants could trigger mitochondrial dysfunction [15,16].

The combined results indicate that an increase of oxidant generation induced by Pi treatment could directly or indirectly contribute to mitochondrial dysfunction and consequently calcification, even if detailed mechanisms underlying the connection between Pi-induced mitochondrial dysfunction and oxidative stress in the process of calcification await further investigation.
Excessive oxidant damages mitochondrial metabolic enzymes and alters mitochondrial membrane permeability, leading to mitochondria dysfunction and cell apoptosis. There is a close link between oxidative stress, mitochondrial dysfunction and apoptosis [17][18][19]. In the intrinsic mitochondrial-mediated apoptotic pathway, the oxidation of mitochondrial protein thiol groups promotes a decrease in mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-9 and -3, eventually leading to apoptotic cell death [17,39]. In concert with combined data that antioxidants can inhibit apoptotic serial events [21], antioxidants would be anticipated to have protective effects against Pi-induced VSMC apoptosis, one of the causative factors for calcification. For instance, ALA with reduction potential inhibits apoptosis of various cell types including human bone marrow stromal cells, endothelial cells and hepatocytes [25,[40][41][42]. The present results indicate that apoptosis during Pi-induced VSMC calcification is associated with intrinsic mitochondrial-mediated apoptotic sequential events, and that ALA blocks both Pi-induced VSMC apoptosis and calcification as a result of its high reduction capacity. In addition, it has been demonstrated that the expression of Gas6 and Axl that is involved in cell survival in a range of cell types [43] is down-regulated during VC, leading to apoptosis and consequent calcification. To maintain cell survival, the binding of Gas6 to its cognate receptor Axl induces phosphatidylinositol 3-OH kinase (PI3K) activation and subsequent Akt activation, showing sequential events of the Gas6/Axl/PI3K/Akt signal. ALA activates PI3K/Akt through direct binding to the tyrosine kinase domain of the insulin receptor [42], but the nature of the ALAregulated upstream regulators of the PI3K/Akt pathway are unclear. The present results demonstrate that ALA inhibits Pi-induced apoptosis and calcification through the restoration of Gas6 and Axl expression, which is down-regulated by excessive Pi in vitro and vitamin D3 toxicity in vivo and one of the upstream regulators of PI3K/Akt survival pathway. We also found that AMPactivated protein kinase (AMPK) mediates the protective effect of ALA against Pi-induced VSMC calcification through restoration of Gas6 gene expression (Fig. S6). However, further analyses of the possible association of Gas6/Axl/Akt/AMPK survival pathway with mitochondrial dysfunction are necessary.
Both bone-forming osteoblasts and VSMCs are driven from common mesenchymal stem cells [44]. Thus, VSMCs can transdifferentiate into osteoblast-like cells that are able to deposit calcium. Although calcification by osteoblasts in bone is a normal process to maintain bone homeostasis, calcification of VSMCs results in a lethal cardiovascular disease. VC is closely connected to bone decalcification (osteoporosis): (i) the patients with VC who are ageing, diabetic and CKD have a high clinical incidence of osteoporosis [45]; (ii) statins, which are used in the treatment of CKD, have been proven to reduce the coronary calcification as well as to increase bone mineralization [45] and (iii) bisphosphonates, which are used to treat osteoporosis, are effective to prevent VC in CKD patients [3,46]. However, long-term use of bisphosphonate in stage 4 or 5 CKD raises efficacy and safety concerns [3]. Presently, ALA effectively inhibited Pi-induced VSMC calcification in vitro and vitamin D3-induced aortic calcification in mice. Decreased VC by ALA could alleviate vascular stiffness and as a result, may help reduce a high clinical incidence of cardiovascularrelated diseases such as hypertension and cardiac hypertrophy. Moreover, ALA has also been suggested to protect bone loss by inhibition of oxidant and tumour necrosis factor-␣-induced apoptosis of bone marrow stromal cells (osteoblast precursors) and suppression of formation of bone-degrading osteoclasts through its antioxidant and anti-inflammatory function [40,47,48]. Consequently, the present and previous findings suggest a possibility that ALA might be used for the treatment of VC in CKD patients without bone loss or possibly with enhanced bone formation, although further in vivo studies are required to verify this possibility.
In summary, VC could be determined by cellular redox status and changes in mitochondrial function. ALA, which is known to potentiate reduction capacity and mitochondrial metabolic function, blocks Pi-induced VSMC calcification through inhibition of mitochondria-mediated apoptosis and restoration of the Gas6/Axl/Akt survival pathway. This study provides evidence that targeted candidates, which are able to enhance mitochondrial function and to have antioxidant potential, may offer a prospective therapeutic strategy for the prevention and treatment of VC.