Hypoxia-Inducible Factor-1α Regulates High Phosphate-Induced Vascular Calcification via Type III Sodium-Dependent Phosphate Cotransporter 1

Vascular calcification (VC) has a high incidence in patients with chronic kidney disease, which is a worldwide public health problem and presents a heavy burden to society. Hypoxia-inducible factor (HIF)-1α, the active subunit of HIF-1, has been reported to play a vital role in high phosphate-induced VC. However, the underlying mechanism is still undetermined, and effective treatment is unavailable. In the present study, human aortic smooth muscle cells (HASMCs) were cultured under normal or high phosphate media conditions. HIF-1α small interfering RNA and overexpression plasmids were employed to regulate HIF-1α expression. Phosphonoformic acid was employed to restrain the function of type III sodium-dependent phosphate cotransporter 1 (Pit-1). The expression levels of HIF-1α, Pit-1, runt-related transcription factor 2 (Runx2), and smooth muscle 22 alpha (SM22α) were evaluated, and the calcium contents were also examined. Cell growth was assessed using an MTT assay. High phosphate stimulation caused an upregulation in HIF-1α and Pit-1 expression levels and induced calcium depositions in HASMCs. Upregulation of Runx2 expression accompanied by downregulation of SM22α expression was observed in the high phosphate group. Following the suppression of HIF-1α expression, there was a concomitant attenuation in Pit-1 expression, calcium deposition, the alteration of phenotypic transition marker genes, and vice versa. The most serious calcium deposition was noted in HASMCs cultured under high phosphate conditions which were pretreated with a HIF-1α overexpression plasmid. However, when the biological functions of Pit-1 were restrained, the putative serious calcium deposition was not formed even in HASMCs transfected with a HIF-1α overexpression plasmid. The findings confirmed that HIF-1α regulated Pit-1 expression and exerted its pro-calcifying effect through Pit-1, which identified HIF-1α and Pit-1 as therapeutic targets for high phosphate-induced VC.


Introduction
Vascular calcifcation (VC) has a high incidence in patients with chronic kidney disease (CKD), which is a worldwide public health problem and presents a heavy burden to society [1][2][3].Hyperphosphatemia is an essential triggering factor of VC; however, the underlying mechanism is still uncertain, and efective treatment is absent [4,5].Te investigation of the molecular mechanism in VC that can be used to develop therapies has become a major focus of attention [6].
It has been previously shown that VC gradually progresses passively with the deposition of minerals.However, it has been widely accepted that VC is an active cell-mediated process, which includes the vascular smooth muscle cell (VSMC) phenotypic transition, calcifcation prompting factors and inhibitor disorders, apoptosis, and the dysfunction of the extracellular matrix [7][8][9].In human VSMCs, type III sodium-dependent phosphate cotransporter 1 (Pit-1) is the major sodium-dependent inorganic phosphorus (NaPi) cotransporter, which plays a key role in high phosphate-induced VC [10,11].When Pit-1 is activated, intracellular inorganic phosphorus transport in VSMCs will be increased, and the downstream signal of Pit-1, such as RUNX2, will be activated, eventually leading to the occurrence of VC [10][11][12][13].However, little is known regarding the regulation of Pit-1.
Hypoxia-inducible factor 1 (HIF-1) is a pivotal protein produced under hypoxic conditions, which participates in various biological processes, such as hematopoiesis, angiogenesis, infammation, and tumor formation [14].Te activity of HIF-1 is mainly determined by its alpha subunit (HIF-1α) [15].Although HIF-1α has been reported to perform a vital catalytic role in high phosphate-induced VC, the precise mechanism of this process remains unknown [16].Based on existing literature and our previous research achievements, we hypothesized that HIF-1α may play a role in the regulation of VC through Pit1, which has not been reported yet.
Te present study aimed to explore the interaction between HIF-1α and Pit-1 in high phosphate-induced VC and to examine the mechanisms involved in this process.

Cell Culture and Calcifcation
Model.Te human aortic smooth muscle cells (HASMCs) were purchased from Procell Life Science & Technology Co., Ltd.(Wuhan, China, cat.No. CL-0517), and their culture conditions were the same as described previously [12].Na 2 HPO 4 •12H 2 O and NaH 2 PO 4 •2H 2 O were employed to simulate high phosphate conditions (2.5 mM; pH 7.2-7.4) in Dulbecco's modifed Eagle's medium (HyClone; Cytiva) and induce VC as described previously [12].HASMCs were divided into fve groups as follows: (1) control (CNT) group, which contained HASMCs treated with normal inorganic phosphorus concentration (Pi; 0.9 mM); (2) high Pi group (HP), including HASMCs treated with high Pi (2.5 mM); (3) small interfering RNA (siRNA) and high Pi group (HPSI), which contained HASMCs transfected with HIF-1α siRNA that were treated with high Pi; (4) overexpression and high Pi group (HPOE), which included HASMCs transfected with HIF-1α overexpression plasmid that were treated with high Pi; (5) overexpression high Pi and phosphonoformic acid (PFA) group (HPOEPFA), which contained HASMCs transfected with HIF-1α overexpression plasmid that were treated with high Pi and 0.5 mM PFA. Te media were renewed every other day, and the cells were cultured for a maximum period of 7 days.

Quantifcation of Calcifcation.
Te Ca 2+ concentration of the cells was examined by a commercially available kit (Calcium assay kit; Nanjing Jiancheng Bioengineering Institute) as described previously [12].

Alizarin Red Staining.
HASMCs were analyzed using standard Alizarin red staining as described previously [12].Red or brown staining, as viewed under a light microscope (Olympus Corporation; magnifcation ×200), indicated positive staining of calcium nodules.

Western Blotting.
Te western blotting procedure and conditions and the primary and secondary antibodies were the same as described previously [12,17].
2.6.Cell Transfection.siRNA was employed to knock down the expression levels of HIF-1α, the sense sequence used was 5′-CUAUGACCUGCUUGGUGCUGAUTT-3′, and the antisense sequence used was 5′-AUCAGCACCAAG CAGGUCAUAGTT-3′.Te transfections were performed using HiPerFect transfection reagent (Qiagen AB) following the manufacturer's instructions.Both normal cells and cells transfected with scramble siRNA were used as controls.
Te overexpression plasmid of HIF-1α, pcDNA3.0-HA-HIF1A(human)-1,was purchased from the MiaoLing Plasmid Sharing Platform (P23864; MiaoLingbio), and the cells were transfected with X-tremeGene Transfection Reagent (Roche Diagnostics) according to the manufacturer's instructions.Both normal cells and cells transfected with empty vector plasmid were used as controls.

MTT Assay.
Te cells in each group were seeded at a density of 6000 cells per well in 96-well plates and cultured under either normal or high phosphate conditions.Te culture media were refreshed every other day, and the cells were maintained for a maximum period of 7 days.Prior to testing, 10 μl of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide) was added to each well followed by an additional incubation period of 4 h.Subsequently, the culture medium was discarded, and the cells were treated with 150 μl DMSO.Te colorimetric analysis of samples was conducted using an enzymatic reader at a wavelength of 490 nm.All experiments were performed in triplicate.Te diferences between groups were evaluated using oneway ANOVA followed by Tukey's post-hoc test.p < 0.05 was considered to indicate a statistically signifcant diference.

Efects of siRNA and Overexpression Plasmid on HIF-1α
Expression.HIF-1α siRNA and overexpression plasmid sequences were employed to regulate HIF-1α expression;   Cardiology Research and Practice western blotting was used to detect HIF-1α protein expression levels in cultured HASMCs for 24 h following transfection (Figure 1).Te results indicated that the relative expression levels of HIF-1α were signifcantly inhibited by HIF-1α siRNA compared with those in the CNT and scramble siRNA groups (p < 0.01) (Figure 1(a)).Moreover, HIF-1α expression was signifcantly upregulated following treatment of the cells with a HIF-1α overexpression plasmid compared with that noted in the CNT and empty plasmid groups (p < 0.05; Figure 1(b)), indicating the validity of the HIF-1α gene regulation.Te knockdown efcacy was also detected on day 7.As shown in Figures 2(a) and 2(b), both the expression levels of HIF-1α protein and mRNA were signifcantly inhibited (p < 0.01).

Efects of HIF-1α Regulation on Cell Growth and Pit-1
Expression in High Phosphate-Stimulated HASMCs.Cell growth was assessed using an MTT assay (Figure 2(c)).Te results indicated that the addition of 2.5 mM phosphate or upregulation of HIF-1α promoted cell growth (p < 0.05), while the knockdown of HIF-1α expression inhibited cell growth (p < 0.05).Te RT-qPCR assay was performed to analyze the mRNA expression levels of HIF-1α and Pit-1 on day 7 (Figure 3), and western blotting was used to detect HIF-1α and Pit1 protein expression levels (Figure 4).Te results indicated that the expression levels of HIF-1α and Pit-1 were signifcantly upregulated in the HP group compared with those in the CNT group (p < 0.05).It is interesting to note that Pit-1 expression levels were significantly suppressed in the HPSI group and signifcantly increased in the HPOE group compared with those in the HP group (p < 0.05), which indicated a molecular regulation between HIF-1α and Pit-1.Terefore, the regulatory capacity of HIF-1α on Pit-1 was demonstrated under high phosphate conditions.

Efects of HIF-1α Regulation on the Phenotypic Transition
Marker Genes in High Phosphate-Stimulated HASMCs.In addition to the previous fndings, the expression levels of the phenotypic transition marker genes, runt-related transcription factor 2 (Runx2), and smooth muscle 22 alpha (SM22α) were evaluated in high phosphate-treated HASMCs on day 7. RT-qPCR analysis (Figure 5) and western blotting (Figure 4) demonstrated the upregulation of Runx2 (p < 0.05) and the downregulation of SM22α (p < 0.01) in the HP group compared to the corresponding levels noted in the CNT group, which indirectly refected the phenotypic transition of the cultured cells to a certain extent.Knockdown of HIF-1α expression resulted in a signifcant attenuation of the alterations in Runx2 and SM22α expression levels induced by high phosphate (p < 0.05).In the HPOE group, notable changes in the expression levels of Runx2 (p � 0.19) and SM22α (p < 0.05) were noted compared with those of the HP group.

HIF-1α Regulates High Phosphate-Induced VC via Pit-1.
Alizarin red staining (Figure 6(a)) and the o-cresolphthalein complexone method (Figure 6(b)) were employed to determine the calcifcation degree and investigate the underlying mechanism of cultured HASMCs on day 7. Te morphological and quantitative measurements indicated that high phosphate conditions induced signifcant calcifcation compared with normal conditions (p < 0.01).Moreover, the calcifcation degree was signifcantly alleviated in the HPSI group (p < 0.05) and aggravated in the HPOE group (p < 0.05) compared with that noted in the HP group.Most notably, PFA, a specifc antagonist of Pit-1, signifcantly disrupted the calcifcation of HASMCs transfected with a HIF-1α overexpression plasmid (p < 0.01).

Discussion
In the present study, the expression levels of HIF-1α and Pit-1 were upregulated in high phosphate-stimulated HASMCs.Upon inhibition of HIF-1α expression, a corresponding decrease in Pit-1 expression was observed, and vice versa.
Tese fndings provide evidence for the regulatory capacity of HIF-1α on Pit-1, suggesting that the activation of the HIF-1α/Pit-1 signaling pathway may occur upon high phosphate stimulation.Further investigations are required to clarify these fndings.To the best of our knowledge, the present study is the frst to explore the interaction between HIF-1α and Pit-1.Hyperphosphatemia has been shown to accelerate VC [19,20]; however, the underlying mechanisms require clarifcation [4].Mokas et al. [16] initially reported the procalcifying characteristic of HIF-1α in high phosphateinduced VC.To date, the mechanistic link between HIF-1α and high phosphate-induced VC is still unclear.Pit-1 is the predominant NaPi cotransporter in human VSMCs [21], which has been identifed as a pivotal transporter in phosphate-induced VC [11].Phosphate can upregulate Pit-1 expression and its activity in VSMCs [22].Previous studies conducted by our group have also supported the signifcant role of Pit-1 in high phosphate-induced VC [12,13,17].Te present fndings indicate that HIF-1α may fulfll its procalcifying characteristic via its regulation on Pit-1.It is concluded that HIF-1α as well as Pit-1 may become therapeutic targets for high phosphate-induced VC.In addition, it is inferred that HIF-1 strengthening agents may exert negative efects on the cardiovascular health of patients with CKD and that their clinical use should be cautious in the specifc population subgroups.Consequently, high-quality real-world studies should be carried out to evaluate this efect.
To further verify the fndings of the present study, the expression levels of the phenotypic transition marker genes, Runx2 and SM22α, were investigated in cultured HASMCs.As demonstrated in our previous investigations, the upregulation of Runx2 concomitant with the downregulation of SM22α specifcally occurs during the phenotypic transition of HASMCs from smooth muscle cells to osteoblast-like cells [12,13,17].Te expression of phenotypic transition marker genes in HASMCs cultured under high phosphate conditions was blunted upon transfection with HIF-1α siRNA, while it was exacerbated following transfection with a HIF-1α overexpression plasmid.Tese results support the notion that HIF-1α can modulate the phenotypic transition of vascular smooth muscle cells, which was reported by other investigations [23,24].However, whether HIF-1α modulates the phenotypic transition through Pit-1 or other molecules requires additional verifcation.
Te lack of experiments checking the efect of PFA alone on calcifcation is a limitation of the present study, as it is beyond our scope.Additionally, Villa-Bellosta R. and Sorribas V. reported that PFA alone prevents high phosphate-induced Cardiology Research and Practice calcifcation [21].Terefore, we believe that PFA alone can restrict calcifcation in our experiments.Te key role of Pit-1 in high phosphate-induced VC has been extensively investigated [10-13, 17, 25]; therefore, we did not assess the impact of Pit-1 overexpression without HIF-1a activation, which may be considered a limitation of this study.Another limitation is that we did not check the mRNA expression of Runx2 and SM22α in the HPOEPFA group, which represents a potential avenue for future investigation.
To the best of our knowledge, the present study is the frst to confrm that HIF-1α regulates Pit-1 expression and exerts its pro-calcifying efect through Pit1.Te fndings unveil HIF-1α and Pit-1 as therapeutic targets for high phosphate-induced VC.

.
Every experiment was performed at least in triplicate.Te data are shown as mean ± standard deviation.Statistical analyses were 2 Cardiology Research and Practice performed using SPSS 18.0 software (SPSS, Inc.).

Figure 1 :
Figure1: Changes in HIF-1α protein expression.HASMCs were cultured in normal DMEM media and transfected with HIF-1α siRNA (a) or an overexpression plasmid (b).Te expression levels of the HIF-1α protein were examined by western blot analysis at 24 h following transfection.Te levels were normalized to those of tubulin.Te data shown are indicative of mean ± SD. * p < 0.05 vs. control; * * p < 0.01 vs. control.HIF-1α, hypoxia-inducible factor 1 alpha; HASMCs, human aortic smooth muscle cells; DMEM, Dulbecco's modifed Eagle's medium; siRNA, small interfering RNA.