Supplement of exogenous inorganic pyrophosphate inhibits atheromatous calcification in Apolipoprotein E knockout mice

Abstract

Inorganic pyrophosphate (PPi) is the endogenous inhibitor for vascular calcification (VC). The present study was to investigate the effects of adenosine disodium triphosphate (ADTP) and alendronate sodium (AL), two exogenous PPi sources, on the atheromatous calcification (AC) in Apolipoprotein E knockout (ApoE KO) mice. ApoE KO mice were randomly divided into five groups: ApoE KO group, ApoE KO + ADTP (Low) group, ApoE KO + ADTP (High) group, ApoE KO + AL (Low) group and ApoE KO + AL (High) group. The mice in ApoE KO + ADTP (Low) group and ApoE KO + ADTP (High) group were intraperitoneally injected with ADTP with dose of 0.5 and 1.0 mg/kg/day for 2 months respectively. The mice in ApoE KO + AL (Low) group and ApoE KO + AL (High) group were intraperitoneally injected with AL with dose of 0.6 and 1.2 mg/kg/day for 2 months respectively. The age matched C57 mice were used as control group. All ApoE KO and C57 mice were fed with normal chow throughout the experiment. The calcification was evaluated using von Kossa method. The contents of PPi, triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL) and low density lipoprotein (LDL), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), interferon-γ (IFN-γ) and interleukin-10 (IL-10) as well as the activity of alkaline phosphatase (ALP) in serum were measured. The results showed that compared with C57 mice, ApoE KO mice developed severe AC accompanied with high levels of TC, TG, LDL, IL-6, TNF-α and IFN-γ in serum and with low levels of PPi and IL-10 in serum. Both ADTP and AL dose-dependently reduced the AC in ApoE KO mice compared with that of ApoE mice, without affecting the contents of lipid profiles. In addition, ADTP and AL increased the contents of PPi and IL-10 while decreased the contents of TNF-α, IL-6 and IFN-γ in serum of ApoE KO mice, having no affection on ALP activity. The results suggested that ADTP and AL reduced AC in ApoE KO mice by increasing the PPi level and regulating the inflammation.

1. Introduction

Vascular calcification (VC) is an independent risk factor for cardiovascular events [1]. VC has been previously reported to be a process of “passive” mineralization. However, a growing evidences has shown that VC is an “active” biological process similar with osteochondrogenic phenotype [2]. VC can be categorized as medial calcification (MC) and atheromatous calcification (AC) [3]. MC mainly occurs in medial layer of arteries involving vascular smooth muscle cells (VSMCs) [4]. AC is mainly located in atherosclerosis (AS) involving VSMCs, vascular endothelial cells (ECs), macrophage-derived foam cells (FCs) as well as the other inflammatory cells, also known as intimal calcification (IC) [5]. AC occurs when macrophages infiltrate the lipid pool and then gradually undergo apoptosis and possibly release matrix vesicles in early fibrous AS [6]. AC has become a high risk factor for acute vascular events including ischemic stroke and myocardial infarction because of the instability and rupture of atheromatous plaque [7]. In the previous study, we found that hypercholesterolemia accelerates MC in rats induced by excessive vitamin D through oxidative stress [8,9]. We also found that cholesterol lowering agent simvastatin, antioxidant agent vitamin E and Traditional Chinese Medicine component Tanshinone IIA inhibited MC through inhibition of oxidative stress [8,9]. These results suggested that oxidative stress plays a critical role in MC induced by hypercholesterolemia.

Inorganic pyrophosphate (PPi) has been found to be a major endogenous inhibitor of VC [10]. PPi can attach to the surface of nascent hydroxyapatite, occupying phosphate binding sites and blocking the further development of VC. Extracellular PPi is derived primarily from adenosine triphosphate (ATP) hydrolysis by ectonucleotide pyrophosphatase/phosphodiesterase-1 (eNPP-1) [11,12], the primary enzyme for extracellular PPi production in VSMCs and aorta. Mutations in the gene encoding eNPP-1 can lead to all-pervasive arterial calcification in infancy [13]. In addition, eNPP-1-deficient mice showed extensive ectopic arterial calcification [12]. It was found that a gradual increase in VSMCs calcification was accompanied by a gradual increase in alkaline phosphatase (ALP) activity [5], which was verified in our previous study [8]. ALP can degrade PPi and make it lose its anti-calcification ability [13]. The previous study was shown that PPi ameliorated excessive VC in prematurely aged mice [10]. We previously reported that MC rat model demonstrated the decreased content of PPi in serum and calpain-1 inhibitor reduced the MC by increasing the level of PPi [14]. However, whether defective synthesis of PPi is also involved in the AC remains largely unclear.

Several reports indicate that inflammation is an important trigger for VC [15,16]. Inflammatory mediators such as tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), interferon-γ (IFN-γ) and interleukin-10 (IL-10) are implied in the pathogenesis of VC [[17], [18], [19], [20]]. TNF-α and IFN-γ were reported to enhance the calcification of VSMCs induced by secondary calciprotein particles and IL-6 was shown to increase the transdifferentiation of VSMC into chondrocytes and calcium deposition [[17], [18], [19], [20]]. High level of IL-10 was shown to be involved in the protective effect of IL-37 on VC in apolipoprotein E knockout (ApoE KO) [21]. A study demonstrated a strong association between macrophage property and osteogenic activity in the arteries of ApoE KO mice [22]. PPi was reported to inhibit the inflammation, which might be another mechanism for the reduction of VC besides its direct inhibition of phosphate binding sites [23]. Taken together, these results suggest that inflammation is the critical driver of VC and that PPi might inhibit VC through different mechanisms.

The purpose of the present study was to investigate the effects of adenosine disodium triphosphate (ADTP) and alendronate sodium (AL), two extragenous PPi sources, on AC in ApoE KO mice and to explore the potential mechanism by evaluating PPi content, lipid profiles and inflammation status.

2. Materials and methods

2.1. Chemicals and reagents

ADTP and AL were purchased from Solarbio Biotech Co. (Beijing, China). ADTP was prepared as 0.05 mg/mL and 0.1 mg/mL being the low and high concentration respectively. AL was prepared as 0.06 mg/mL and 0.12 mg/mL being the low and high concentration respectively. Pyrophosphate assay kit was purchased from Abcam Co. (Shanghai, China). The kits for lipid profiles, the ELISA kits for inflammatory factors and the ALP activity kit were purchased from Nanjing Jiancheng Bioengineering Co. (Nanjing, China).

2.2. Animals and groups

The Ethics Committee on Animal Study of Lanzhou University Second Hospital approved the protocol. ApoE KO mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Eight-week-old male C57BL/6J mice were purchased from the Animal Experimental Center of Lanzhou University, Lanzhou, China. Forty male 8-week-old ApoE KO mice were randomly divided into five groups: ApoE KO group, ApoE KO + ADTP (Low) group, ApoE KO + ADTP (High) group, ApoE KO + AL (Low) group and ApoE KO + AL (High) group. The mice in ApoE KO + ADTP (Low) group and ApoE KO + ADTP (High) group were intraperitoneally injected with ADTP with dose of 0.5 and 1.0 mg/kg/day for 2 months respectively. The mice in ApoE KO + AL (Low) group and ApoE KO + AL (High) group were intraperitoneally injected with AL with dose of 0.6 and 1.2 mg/kg/day for 2 months respectively. Eight age-matched male C57BL/6J mice were used as a C57 group. Mice in ApoE KO group and C57 group were intraperitoneally injected with normal saline. All mice received normal chow for 8 weeks.

2.3. Evaluation of calcified lesion

After the experiment, blood was collected from which serum was isolated. A 10-min systemic perfusion was performed from the left ventricle using ice-cold phosphate-buffered saline (PBS) containing 1 mM EDTA, after which the aortic root was collected. The frozen cross-sectional samples of the aortic root were stained with von kossa staining kit (Sigma). Briefly, von kossa staining including the procedure of staining the sections in silver nitrate solution for 25 min and then exposing to ultraviolet light for 5 min, subsequently turning the calcified areas into black. The images were analyzed with ImageJ. The specific approach incorporates the procedure of importing the image into ImageJ, performing a THRESHOLD operation on the vascular region, adjusting the threshold to just exclude non-specific sites, and applying the same threshold to all images. The size of the calcified lesions in aortic root was expressed in μm².

2.4. Serum lipid profiles

The serum concentration of triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL) and high density lipoprotein (HDL) in 8 mice of each group were examined using the commercially available kits.

2.5. Contents of IL-6, TNF-α, IFN-γ and IL-10 in serum

The contents of IL-6, TNF-α, IFN-γ and IL-10 in serum in 8 mice of each group were determined using ELISA kits according to the manufacturer's protocol.

2.6. Concentration of PPi and ALP activity in serum

Concentration of PPi in serum in 8 mice of each group was detected by spectrophotometric method with pyrophosphate assay kit. Briefly, the standard solution was diluted in a gradient according to the manufacturer's protocol. The 25 μl of the assay solution and diluted standard solution or the serum samples were added to 384-well plates. After being thoroughly mixed followed by 20 min of reaction at room temperature, the solution was measured in a microplate reader at Ex/Em 316/456 nm and a standard curve was graphed from which the PPi was calculated. ALP activity in serum in 8 mice of each group was tested with commercially available kit following the manufacturer's protocol.

2.7. Statistical analysis

Data are expressed as the mean ± SD. The statistical analysis was performed by one-way analysis of variance (ANOVA) using SPSS 27.0 software. Tukey's test was used to perform statistical analysis between two groups. p < 0.05 showed the statistically significant difference.

3. Results

3.1. Effect of ADTP and AL on body weight of ApoE KO mice

At the start of the experiment when the mice were 8-week old, the average body weight of mice in C57 control, ApoE KO, ApoE KO + ADTP (Low), ApoE KO + ADTP (High), ApoE KO + AL (Low) and ApoE KO + AL (High) groups were 18.9g, 19.0 g, 18.4 g, 18.5 g, 18.6 g and 18.2 g, respectively. At the end of the experiment when the mice were 16-week old, the average body weight of mice in five corresponding groups was 26.7 g, 27.4 g, 27.4 g, 27.6 g, 27.4 g and 27.4 g, respectively. No significant differences in average body weight at the start and end of the experiment were found among the groups.

3.2. Effect of ADTP and AL on calcified lesions in the aortic root of ApoE KO mice

After eight weeks of experiment, no calcified plaque was observed in the aortic root of C57 group, whereas mice in ApoE KO group formed severe calcification in the atheromatous plaque in the aortic root (Fig. 1A and B). Compared with that in ApoE KO group, ADTP and AL dose-dependently reduced the aortic calcification lesions by 34.9% and 61.2% for ADTP (Low and High) and by 24.1% and 50.0% for AL (Low and High) respectively. The results suggested that ApoE KO mice developed AC at the age of 16 week fed with normal chow. In addition, ADTP and AL, as exogenous sources of PPi, have been shown to exert a protective effect against aortic calcification, suggesting that the balance of PPi metabolism may be a critical factor in the regulation of AC in ApoE KO mice.

Fig. 1.

ADTP and AL reduced calcified lesions in the aortic root of ApoE KO mice. ApoE KO mice were randomly divided into five groups: ApoE KO group, ApoE KO + ADTP (Low) group, ApoE KO + ADTP (High) group, ApoE KO + AL (Low) group and ApoE KO + AL (High) group. The mice in ApoE KO + ADTP (Low) group and ApoE KO + ADTP (High) group were intraperitoneally injected with ADTP with dose of 0.5 and 1.0 mg/kg/day for 2 months respectively. The mice in ApoE KO + AL (Low) group and ApoE KO + AL (High) group were intraperitoneally injected with AL with dose of 0.6 and 1.2 mg/kg/day for 2 months respectively. The age matched C57 mice were used as control group. All ApoE KO and C57 mice were fed with normal chow throughout the experiment. Data are expressed as the means ± SD. N = 8; *P < 0.05 was considered statistically significant. ADTP: adenosine disodium triphosphate; AL: alendronate sodium; ApoE KO: Apolipoprotein E knockout.

3.3. ADTP and AL had no affection on lipid profiles in the serum of ApoE KO mice

We previously reported that hyperlipidemia accelerated the aortic calcification in rats fed with high lipid diet and excessive dose of vitamin D and that simvastatin inhibited the calcification by decreasing the contents of TC and TG [8]. Therefore, we investigated whether ADTP and AL could regulate lipid profiles and, consequently, inhibit aortic calcification in ApoE KO mice. The obtained results showed that, in comparison with the C57 mice, the ApoE KO mice presented the increases of 117.9%, 411.5% and 92.1% in the levels of TC, LDL-C and TG, respectively, without alterations in the HDL levels (Fig. 2A–D). However, ADTP and AL had no effect on the contents of the lipid profiles. These results suggest that the inhibitory effects of ADTP and AL on AC may not be related to the contents of lipid profiles.

Fig. 2.

ADTP and AL had no affection on lipid profiles in the serum of ApoE KO mice. ApoE KO mice were randomly divided into five groups: ApoE KO group, ApoE KO + ADTP (Low) group, ApoE KO + ADTP (High) group, ApoE KO + AL (Low) group and ApoE KO + AL (High) group. The mice in ApoE KO + ADTP (Low) group and ApoE KO + ADTP (High) group were intraperitoneally injected with ADTP with dose of 0.5 and 1.0 mg/kg/day for 2 months respectively. The mice in ApoE KO + AL (Low) group and ApoE KO + AL (High) group were intraperitoneally injected with AL with dose of 0.6 and 1.2 mg/kg/day for 2 months respectively. The age matched C57 mice were used as control group. All ApoE KO and C57 mice were fed with normal chow throughout the experiment. Data are expressed as the means ± SD. N = 8; *P < 0.05 was considered statistically significant. ADTP: adenosine disodium triphosphate; AL: alendronate sodium; ApoE KO: Apolipoprotein E knockout; TC: total cholesterol; TG: triglyceride; LDL: low density lipoprotein and HDL: high density lipoprotein.

3.4. ADTP and AL increased PPi content without affecting ALP activity in the serum of ApoE KO mice

After observing that ADTP and AL reduced AC in ApoE KO mice, we next measured the PPi content and ALP activity in serum to explore the pathogenesis of AC and the mechanism by which both PPi sources inhibited AC. We observed that mice from the ApoE KO group had significantly lower serum content of PPi (decreased by 24.1%) and increased ALP activity (increased by 133.9%) in compared to the C57 group (Fig. 3A and B). Compared with that in ApoE KO group, ADTP and AL dose-dependently increased the PPi content by 15.6% and 22.3% for ADTP (Low and High) and by 18.4% and 23.5% for AL (Low and High) respectively. However, both ADTP and AL had no action on ALP activity. The results suggested that deficiency of PPi in serum of ApoE KO mice might contribute to the AC and that complementation of PPi with ADTP and AL might be the important mechanism underlying their inhibition of AC.

Fig. 3.

ADTP and AL increased PPi content without affecting ALP activity in the serum of ApoE KO mice. ApoE KO mice were randomly divided into five groups: ApoE KO group, ApoE KO + ADTP (Low) group, ApoE KO + ADTP (High) group, ApoE KO + AL (Low) group and ApoE KO + AL (High) group. The mice in ApoE KO + ADTP (Low) group and ApoE KO + ADTP (High) group were intraperitoneally injected with ADTP with dose of 0.5 and 1.0 mg/kg/day for 2 months respectively. The mice in ApoE KO + AL (Low) group and ApoE KO + AL (High) group were intraperitoneally injected with AL with dose of 0.6 and 1.2 mg/kg/day for 2 months respectively. The age matched C57 mice were used as control group. All ApoE KO and C57 mice were fed with normal chow throughout the experiment. Data are expressed as the means ± SD. N = 8; *P < 0.05 was considered statistically significant. ADTP: adenosine disodium triphosphate; AL: alendronate sodium; ApoE KO: Apolipoprotein E knockout; PPi: inorganic pyrophosphate; ALP: alkaline phosphatase.

3.5. ADTP and AL regulated the content of inflammatory factors in the serum of ApoE KO mice

Inflammation is implied in the development of AC and PPi was reported to inhibit inflammation. Therefore, we hypothesized that the inhibition of AC by ADTP and AL might be also contributed to the inhibition of inflammation. We next examined the content of IL-6, TNF-α, IFN-γ and IL-10, four mediators of inflammation. The results showed that compared to that in C57 mice, the contents of TNF-α, IL-6 and IFN-γ in ApoE KO increased significantly by 53.9%, 44.2% and 44.5% respectively, and that of IL-10 decreased significantly by 48.3%. However, TNF-α content were decreased by 15.0% and 23.5% for ADTP (Low and High) and by 14.6% and 22.2% for AL (Low and High) respectively (Fig. 4A). In addition, IL-6 content were decreased by 10.4% and 18.6% for ADTP (Low and High) and by 9.6% and 14.1% for AL (Low and High) respectively (Fig. 4B), and IFN-γ content were decreased by 10.7% and 20.1% for ADTP (Low and High) and by 10.5% and 19.6% for AL (Low and High) respectively (Fig. 4C), while IL-10 content were increased by 27.8% and 53.5% for ADTP (Low and High) and by 24.2% and 46.4% for AL (Low and High) respectively (Fig. 4D). The results suggested that regulation of inflammation by ADTP and AL might be another critical mechanism underlying their attenuation of AC.

Fig. 4.

ADTP and AL decreased the content of TNF-α and IL-6 in the serum of ApoE KO mice. ApoE KO mice were randomly divided into five groups: ApoE KO group, ApoE KO + ADTP (Low) group, ApoE KO + ADTP (High) group, ApoE KO + AL (Low) group and ApoE KO + AL (High) group. The mice in ApoE KO + ADTP (Low) group and ApoE KO + ADTP (High) group were intraperitoneally injected with ADTP with dose of 0.5 and 1.0 mg/kg/day for 2 months respectively. The mice in ApoE KO + AL (Low) group and ApoE KO + AL (High) group were intraperitoneally injected with AL with dose of 0.6 and 1.2 mg/kg/day for 2 months respectively. The age matched C57 mice were used as control group. All ApoE KO and C57 mice were fed with normal chow throughout the experiment. Data are expressed as the means ± SD. N = 8; *P < 0.05 was considered statistically significant. ADTP: adenosine disodium triphosphate; AL: alendronate sodium; ApoE KO: Apolipoprotein E knockout; IL-6: interleukin-6; TNF-α: tumor necrosis factor α.

4. Discussions

We found in the present study that ApoE KO mice developed severe AC accompanied with hyperlipidemia, inflammation and with low level of PPi in serum. Both ADTP and AL reduced the AC in ApoE KO mice in the dose-dependent manner. In addition, ADTP and AL increased the contents of PPi and IL-10 content and decreased the contents of TNF-α, IL-6 and IFN-γ. These results suggested that ADTP and AL, two exogenous PPi sources, reduced AC in ApoE KO mice by increasing the PPi level and regulating the inflammation.

ApoE KO mice spontaneously develop hypercholesterolemia, subsequently inducing AS even under normal dietary conditions. VC is a very common complication of AS, involving VSMCs, monocyte infiltration and macrophage accumulation within the arterial wall [24,25]. Pathological examinations reveal the co-localization of VC with atherosclerotic lesion, which is termed as AC [9]. We presented in the present study that ApoE KO mice developed AC within the AS plaque accompanied with high levels of TC, TG and LDL, suggesting that hyperlipidemia plays an important role in both AS and AC. These findings are consistent with the reports showing the calcification in atherosclerotic lesions in ApoE KO mice, which were used to observe the effect of Rutin [7], 5-methoxytryptophan [26] and vitamin K2 [27] on AC respectively.

Extracellular PPi is the major endogenous physiological inhibitor of VC, which binds to nascent hydroxyapatite crystals and inhibits VC at micromolar concentration [10,12,24,28]. However, deficiency of PPi synthesis leads to VC due to a lack of inhibitory capacity [10]. Therefore, PPi deficiency is a critical risk factor for VC, suggesting a critical role of PPi homeostasis in VC [29]. We previously reported that deficiency of PPi contributed to the MC in a rat model and to the calcification in VSMCs, which was inhibited by correction of the PPi metabolism balance through inhibition of calpain-1 activity [14]. ADTP releases PPi under the catalysis of eNPP-1 and AL provides PPi directly. The effect of ADTP and AL on AC was not found in the literatures. However, several studies reported the protective effects of sodium pyrophosphate on MC by showing that sodium pyrophosphate inhibits the VC in a mouse model of uraemia [30] and ameliorates VC in a mouse model of Hutchinson-Gilford progeria syndrome [10,31]. It was found that a gradual increase in VSMCs calcification was accompanied by a gradual increase in ALP activity [5], which was verified in our previous study [8]. ALP can degrade PPi and make it lose its anti-calcification ability [13]. Whether PPi is deficient in AC model of ApoE KO mice, subsequently contributing to the AC remains largely unknown. By using this AC model, we investigated the effect of ADTP and AL, two exogenous PPi sources, on AC in ApoE KO mice and found that the content of PPi in serum of ApoE KO mice with AC significantly decreased compared with that of control mice without AC, indicating the potential role of PPi metabolism in AC. We also found that both ADTP and AL inhibited the AC in ApoE KO mice by supplying the PPi directly or indirectly, confirming the essential role of PPi in AC. In addition, the results showed that both ADTP and AL had no affection on the lipid profiles and ALP activity, suggesting that inhibition of AC is independent of the lipid level and ALP activity and directly complementation of PPi might be responsible for their inhibition to AC.

Several studies have demonstrated that vascular inflammation is an important trigger for AC, even the notion that inflammation plays a primordial role in AC has gained ascendency. Vascular wall cells can produce cytokines/protein mediators of inflammation e.g. IL-1, IL-6, TNF-a and IFN-γ, which may stimulate the recruitment of inflammatory cells to the lesion at an early stage of AC [32]. Macrophages can promote AC by releasing pro-inflammatory cytokines (e.g. IL-6 and TNF-α) that induce differentiation of smooth muscle cells towards an osteogenic phenotype and increase apoptosis [[33], [34], [35]] and mast cells thought to be participants in AC produce IL-6 and IFN-γ, promoting lesion progression. Furthermore, cleavage of inflammatory vesicles by caspase-1 promotes IL-1β production, inflammatory vesicle activators such as palmitic acid have been shown to enhance calcification by generating reactive oxygen species and stimulating bone morphogenetic protein-2 expression. Also lymphocytes are enriched at the AC site, and although T lymphocytes are in the minority, they are thought to play a decisive role in inflammatory regulation, and Th1 CD4⁺ cells have been reported to produce IFN-γ [32,36]. In the present study, ADTP and AL reduced the levels of TNF-α, IL-6, IFN-γ and increased the level of IL-10 in ApoE KO mice, suggesting that the regulation of inflammation may be another mechanism by which ADTP and AL attenuates VC. Consistently, thiamine pyrophosphate improved vascular complications of diabetes in rats with type 2 diabetes by reducing the contents of TNF-α and IL-6, two inflammation markers [23].

In summary, the present study demonstrates that ADTP and AL inhibit VC in ApoE KO mice, which can be largely explained by elevation of PPi and regulation of inflammation. The limitation as well as the future perspective of the present study will be to do further experiment to investigate the molecular mechanism and signaling transduction underlying the effect of ADTP and AL on calcification.

Author contribution statement

Wenjiao Gu: Yujie Wei: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Yu Tang: Shining Zhang: Shuangyi Li: Youming Shi: Fenxia Tang: Ali Mohamed Awad: Performed the experiments; Analyzed and interpreted the data.

Xiaowei Zhang: Futian Tang: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper

Data availability statement

Data will be made available on request.

Ethics statement

The Ethics Committee on Animal Study of Lanzhou University Second Hospital approved the protocol (No. D2022-191).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.