In recent years, the accelerating spread of processed foods and a Westernized diet have caused concerns regarding excessive phosphoric acid intake in Japan. Inorganic phosphate (Pi) functions as an energy transporter and is an essential building block of cell membranes and bones. The excessive intake of Pi, or chronic kidney disease (CKD), leads to hyperphosphatemia that eventually causes ectopic calcification [1
]. Cardiovascular disease, due to vascular calcification, is one of the leading causes of death among CKD patients [3
]. Cardiovascular events, including vascular calcification, account for 50% of all deaths in patients with CKD [3
]. Hyperphosphatemia, which is a common CKD symptom, is a risk factor for arterial calcification, and serum Pi levels correlate with cardiovascular morbidity and mortality in CKD patients [6
]. Although the kidney is a common site of ectopic calcification in CKD, neurodegenerative diseases, in which ectopic calcification occurs in the brain, have also been reported. Idiopathic basal ganglia calcification (IBGC)—also known as Fahr’s disease or, more recently, primary familial brain calcification (PFBC)—is a rare neuropsychiatric disease characterized by ectopic bilateral calcifications, mainly in the basal ganglia, but also the cerebellum, brain stem, and subcortical white matter [8
encoding Pi transporter 2 (PiT2) has been reported as a causative gene. This mutated transporter was predicted to be unable to transport Pi from the extracellular environment [9
]. We previously reported that Pi levels in the cerebral spinal fluid of IBGC patients with SLC20A2
variants were significantly higher than in healthy controls [11
]. These results suggest that defects in Pi homeostasis results in ectopic calcification even in the brain.
The formation of ectopic calcification is associated with various other factors such as oxidative stress, endoplasmic reticulum stress, increased apoptosis, increased DNA damage response, and decreased calcification regulator [12
]. However, the mechanism by which a high concentration of Pi loading causes calcification is not fully understood. The main calcification component is thought to be calcium-Pi deposition [13
]. The mineralization process is similar to bone formation and is due to transdifferentiation into osteoblast-like cells by an oversupply of Pi influx [16
]. Indeed, calcification has been reported to occur in vascular smooth muscle cells (VSMCs) when Pi is excessive. In addition, inflammatory factor tumor necrosis factor (TNF)-α, a known risk factor for CKD, and bone morphogenetic protein (BMP)-2, an osteogenic factor, reportedly induce calcification by promoting phenotypic changes of VSMCs [19
]. It is also known that inflammatory cells such as macrophages and T lymphocytes are involved in the formation of vascular calcification. Macrophages promote calcification by activating the osteogenic differentiation in VSMCs through the release of cytokines such as TNF-α and interleukin-6 (IL-6) [22
]. Among cytokines produced and secreted by inflammatory cells, interferon-γ (IFN-γ) and TNF-α have been shown to induce Alkaline Phosphatase (ALP) expression in VSMCs [24
]. Furthermore, TNF-α has been shown to promote osteoblast differentiation through induction of transcription factors such as runt-related transcription factor 2 (Runx2) and muscle segment homeobox 2 (Msx2) [25
]. On the other hand, in the inflammatory reaction of the blood vessel wall, reactive oxygen species (ROS) is generated by the action of NADPH oxidase (NOX). It is known that ROS promotes apoptosis of VSMCs and differentiation into osteoblasts directly or through the production of oxidized low-density lipoprotein (LDL). Elucidating and controlling the mechanism of VSMCs phenotypic changes are considered useful for preventing vascular calcification and developing therapeutic strategies.
To further elucidate the vascular calcification mechanism, understanding the involvement of ROS, induced by a high concentration of Pi, is essential. ROS are produced during the general absorption and metabolism processes, but excess ROS accumulation causes various disorders in cells and tissues [27
]. Indeed, previous studies have reported that ROS are induced by high Pi concentrations that are associated with vascular calcification [29
]. ROS also inhibit the action of nitric oxide (NO) in vascular endothelial cells, thereby suppressing vasodilation and cell proliferation in VSMCs. ROS are also involved in the onset and progression of arteriosclerosis by inducing calcification [32
]. Therefore, suppressing ROS may be a therapeutic strategy to treat vascular calcification induced by a high concentration of Pi loading.
-dihydroperoxides (DHPs) are known to exert various beneficial effects [33
]. Recently, a method for generating new DHPs that can be easily adjusted from commercially available compounds was reported [36
]. Among these DHPs, 12AC3O leaded to apoptosis in K562 leukemia cells by scavenging intracellular ROS, without affecting the growth of peripheral blood monocytes (PBMCs) and fibroblasts [38
]. In a previous study, we also found that the DHP 12AC2O exerted a neuroprotective effect by inhibiting abnormal protein accumulation via the direct trapping of intracellular ROS in amyotrophic lateral sclerosis model cells [39
]. These results suggest that DHPs act as ROS scavengers to reduce intracellular ROS damage. In the present study, we investigated the effect of 12AC3O against high concentrations of Pi-induced calcification in VSMCs.
Ecotopic calcification is a high prevalent vascular phenotype that has associated with aging, atherothrombotic cardiovascular disease, diabetes mellitus, and CKD. It is known that ecotopic calcification is promoted by transdifferentiation of VSMCs. Complex VSMCs biology important in the pathogenesis of atherosclerosis, however, remains poorly understood. Although the pathogenesis of calcification is not known, hypoxia signaling in the cardiovascular system has been revealed as an upper activation pathway in VSMC in myocardial infraction patients [48
The calcification pathways, caused by a high concentration of Pi, have been gradually elucidated. VSMCs exposed to procalcifying levels of phosphate, akin to what may occur in patients with CKD, lose expression of the smooth muscle contractile proteins SM22α and SM α-actin and express the bone markers Runx2, osteopontin, osteocalcin, and alkaline phosphatase [50
]. The incorporation of excess extracellular Pi into cells, mainly through the Pi transporter PiT1, leads to ROS production [51
]. Oxidative stress has been implicated in vascular calcification and shown to promote SMC differentiation. Increased activity of NADPH oxidase and elevated levels of hydrogen peroxide initiate SMC differentiation by upregulating Runx2 expression [12
]. The disruption of Pi homeostasis causes ROS dysregulation via NADPH and mitochondria dysfunction [21
]. Several studies have reported the relationship between calcification and ROS [29
]. These studies revealed that ROS promote calcification by inducing the differentiation to osteoblasts via the activation of Protein Kinase B (AKT) signaling [29
]. In addition, nuclear factor-kappa B (NF-kB) [53
] and mitogen-activated protein kinase (MAPK) [55
] signaling pathways have been shown to be involved in calcification. Downstream transcription factors of the MAPK signaling pathway, including Runx2 and Msx2, play an important role in the calcification of VSMCs [22
Furthermore, ROS are known to accumulate with aging. Since accumulated ROS increases calcification, risk factors, such as CKD, leading to calcification include aging [57
]. In the present study, high concentrations of Pi-induced calcification by increasing ROS production in VMSCs, although the detailed mechanisms remain to be elucidated. Additionally, 12AC3O decreased ROS by heavy loading of Pi and subsequently inhibited calcification. Thus, antioxidants could prevent calcification by inhibiting ROS, and 12AC3O (or its derivative) may be a candidate for developing agents to prevent calcification.
IBGC is a rare genetic disease, characterized by symmetric calcification in the basal ganglia and other brain regions. Previously, SLC20A2
, which encodes a Pi transporter, has been identified as the causative gene for IBGC [9
]. In addition, XPR1
, another gene encoding a Pi transporter, has been identified. Therefore, it is presumed that the high concentration of Pi surrounding blood vessels contributes to ectopic calcification in the pathological condition of IBGC. Subsequently, disrupted Pi homeostasis inside and outside cells is expected to occur in IBGC. In our previous study, we demonstrated that mitochondrial-related ROS were produced by the disruption of Pi homeostasis [59
]. In the present study, we showed that 12AC3O reduced the signal of Mito SOX, which evaluates mitochondrial oxidative stress. Therefore, 12AC3O may show potential beneficial effects on vascular calcification in IBGC.
12AC3O is new compound which can be easily synthesized. In this research, we showed that 12AC3O has the effect of suppressing oxidative stress similar to other antioxidants and directly scavenging oxidative stress. However, the inhibitory effect of 12AC3O against calcification may be insufficient as a fundamental strategy for treating vascular calcification. The pathological cascade gradually progresses as long as the high phosphate state is maintained. Endoplasmic reticulum stress and autophagy are also involved in the vascular calcification process [60
]. It is also known that oxidative stress induces autophagy to protect cells. It is necessary to continue to investigate how the ROS levels produced by a high concentration of Pi loading affect cellular functions. Indeed, the reduction of Pi levels in vivo is essential for suppressing calcification.
In conclusion, 12AC3O, one of the novel DHPs, inhibited high Pi-induced calcification in VSMCs. Additionally, 12AC3O obviously attenuated Pi-induced ROS and exerted a direct scavenging activity against superoxide anion and hydroxyl radical. It has been reported that various pathways are involved in calcification, caused by a high concentration of Pi loading, and ROS production is an important factor. Therefore, 12AC3O may effectively inhibit the formation of pathological calcifications. These results support 12AC3O as a potential candidate to attenuate ROS-related ectopic calcification.
4. Materials and Methods
4.1. Cell Culture
The P53LMACO1 cell line (JCRB0150) used in this study was purchased from (Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank, Osaka, Japan). p53LMAco1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and maintained at 37 °C in humidified 5% CO2/95% air. For the high Pi experiment, p53LMAco1 cells were seeded on a 12-well plate in DMEM containing 10% FBS for 24 h. The medium was then replaced with 10% FBS-DMEM containing the appropriate amounts of sodium phosphate buffer (0.1 M Na2HPO4/NaH2PO4, pH 7.4) to produce final Pi concentrations of 3.0 mM. Ten percent FBS-DMEM medium was used as the control in this study.
4.2. Neurotoxicity Assays
p53LMAco1 cells were seeded at a density of 5.0 × 103 cells/well in 96-well plates in DMEM containing 10% FBS. p53LMAco1 cells were incubated with or without 12AC3O (1.0, 3.0, or 10 µM) for 24 h. Triton-X was used as a negative control. The amount of LDH was measured in the culture supernatant. Cell toxicity was measured using an LDH assay kit following the protocol (Wako Pure Chemical Industries Ltd., Osaka, Japan).
4.3. 32Pi Transport Assays
A Pi uptake assay was performed using 3.0 mM Pi- and 12AC3O-treated cells grown to confluency in 24-well plastic plates, as previously described. The transport rate was expressed as nmol Pi per minute per mg protein [61
Cells were seeded at the density of 1.0 × 105 cells/well in a 12-well multiplate and cultured for 24 h. After that, p53LMAco1 was treated with 1.0 mM Pi as control or 3.0 mM Pi as vehicle and 12AC3O (0.1, 1.0, 3.0 µM) for 24 h. Total RNA was extracted using TriPure Isolation reagent (Sigma, St. Louis, MO, USA), following the manufacturer’s protocols. cDNA was prepared with ReverTra Ace® qPCR RT Master Mix (Toyobo, Osaka, Japan) from 0.5 µg of total RNA, following the manufacturer’s protocols. An aliquot of diluted cDNA was applied to qRT-PCR. qRT-PCR analysis was performed using THUNDERBIRD® SYBR qPCR Mix (Toyobo) and amplified using a StepOne Real-Time PCR System (Thermo Fisher Scientific Inc., Waltham, MA, USA). Primers used in the real time RT-PCR analysis were: β-actin (forward: 5′- GGCCAACCGGGAGAAAA-3′, reverse: 5′-GAGGCATAGAGGGACAGCACA-3′), Runx2 (forward: 5′-TGCAAGCAGTATTTACAACAGAGG-3′; reverse: 5′-GGCTCACGTCGCTCATCTT-3′). The level of β-actin cDNA in the sample was used as an internal control for all PCR amplification reactions.
4.5. Alizarin Red Staining
Alizarin S (100 mg) was dissolved in 10 mL of purified water and adjusted to pH 6.4 using a 0.1% KOH solution. Previously treated cells were washed with 1×Phosphate Buffered Saline (PBS), fixed with 4% paraformaldehyde, washed with purified water, and then stained with alizarin solution for 10 min. After washing three times with purified water, the sample was observed with an all-in-one fluorescence microscope, and image analysis was performed using ImageJ (NIH, Bethesda, MD, USA).
4.6. Von Kossa Staining
Treated cells were washed with 1×PBS, fixed with 4% paraformaldehyde, washed with purified water, treated with 5% aqueous silver nitrate solution, and incubated at room temperature for 2 h. UV (365 nM) was irradiated for 30 min on a Benchtop 2UV Transilluminators (UVP) (Analytik Jena, Jena, Germany). After washing with purified water, a 5% aqueous sodium thiosulfate solution was added over 3 min. After washing with purified water, the sample was observed with a fluorescence microscope, and image analysis was performed using ImageJ (NIH).
4.7. ROS Detection
To detect intracellular ROS production, we used the redox-sensitive dyes CellROX Green (Thermo Fisher Scientific Inc.) and MitoSOX Red (Thermo Fisher Scientific Inc.). CellROS was used to detect intracellular ROS, and MitoSOX was used to detect mitochondrial superoxide. After p53LMAco1 cells were prepared in uncoated glass-bottomed microwells, CellROX was added to the medium to a final concentration of 2.5 µM, and MitoSOX was added to the medium to a final concentration of 5 µM. Cells were incubated with CellROX or MitoSOX at 37 °C for 30 min or 10 min, respectively. Treated cells were fixed with 4% paraformaldehyde. After fixation, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1:2000), and then cells were encapsulated with VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA). The sample was observed and photographed with a fluorescence microscope (Zeiss LSM 700, Carl Zeiss, Oberkochen, Germany), and fluorescence intensity was measured using Image J (NIH).
4.8. ESR Analysis
The ESR analysis (JES-FA 200, JEOL) was performed as The ESR analysis (JES-FA 200, JEOL) was performed as described previously [39
Data were presented as the mean ± S.E.M. The significance of differences was determined by an analysis of variance. Further statistical analysis for post-hoc comparisons was performed using the Bonferroni/Dunn test (SigmaPlot 11, Systat Software Inc., San Jose, CA, USA).