Dihydromyricetin ameliorates vascular calcification in chronic kidney disease by targeting AKT signaling

Vascular calcification is highly prevalent in chronic kidney disease (CKD), and characterized by trans-differentiation from contractile vascular smooth muscle cells (VSMCs) into an osteogenic phenotype. However, no effective and therapeutic option to prevent vascular calcification is yet available. Dihydromyricetin (DMY), a bioactive flavonoid isolated from Ampelopsis grossedentata, has been found to inhibit VSMCs proliferation and the injury-induced neointimal formation. However, whether DMY has an effect on osteogenic differentiation of VSMCs and vascular calcification is still unclear. In this study, we sought to investigate the effect of DMY on vascular calcification in CKD and the underlying mechanism. DMY treatment significantly attenuated calcium/phosphate-induced calcification of rat and human VSMCs in a dose-dependent manner, as shown by alizarin red S staining and calcium content assay, associated with down-regulation of osteogenic markers including type I collagen (COL I), RUNX2, BMP2 and osteocalcin (OCN). These results were further confirmed in aortic rings ex vivo. Moreover, DMY ameliorated vascular calcification in rats with CKD. Additionally, we found that AKT signaling was activated during vascular calcification, whereas significantly inhibited by DMY administration. DMY treatment significantly reversed AKT activator-induced vascular calcification. Furthermore, inhibition of AKT signaling efficiently attenuated calcification, which was similar to that after treatment with DMY alone, and DMY had a better inhibitory effect on calcification as compared to AKT inhibitor. The present study demonstrated that DMY has a potent inhibitory role in vascular calcification partially by inhibiting AKT activation, suggesting that DMY may act as a promising therapeutic candidate for patients suffering from vascular calcification.


Introduction
Vascular calcification is a process of hydroxyapatite crystals deposits in the medial or intimal layers of arteries [1]. It is an important risk factor for cardiovascular disease and mostly occurs among the elderly, atherosclerosis, diabetes mellitus, and chronic kidney disease (CKD) patients [2,3]. Initially, vascular calcification was regarded as a passive process of calcium and phosphate deposition. However, accumulating evidence has suggested that vascular calcification is an active cell-mediated regulated process sharing similarities with bone formation [4,5].
Vascular smooth muscle cells (VSMCs) play a pivotal role in mediating vascular calcification by switching from contractile to osteogenic phenotype [6]. This phenotype switch is recognized as osteogenic differentiation. Molecules including runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), osteocalcin (OCN), type I collagen (COL I), Osterix and alkaline phosphatase (ALP) are widely used as markers of osteogenic differentiation [7][8][9]. A variety of triggers and signaling pathways have been identified being involved in regulation of vascular calcification, but the exact molecular mechanisms of vascular calcification still remain elusive [10]. Consequently, no effective and convincing therapeutic strategies to prevent the progression of vascular calcification are yet available [11].

Dihydromyricetin (DMY), a bioactive flavonoid isolated from Ampelopsis
Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20210259/921788/cs-2021-0259.pdf by guest on 20 October 2021 Clinical Science. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/CS20210259 grossedentata, has been found to possess broad pharmacological activities, including anti-inflammatory, anti-oxidant, anti-apoptotic, antibacterial, anti-tumor, anti-alcohol and hepatoprotective properties [12][13][14][15][16]. A study reported that DMY could markedly inhibit VSMCs proliferation and the injury-induced neointimal formation via induction of TR3 [17]. A randomized controlled trial showed that DMY improved glucose and lipid metabolism and relieved inflammation in patients afflicted by nonalcoholic fatty liver disease [18]. In addition, DMY has been demonstrated to ameliorate atherosclerosis in LDL receptor deficient mice [19]. These findings suggest the potential role of DMY in vascular diseases. However, the effect and mechanism of DMY on vascular calcification are still unclear. In this study, we aimed to investigate the effect of DMY on vascular calcification using in vitro, ex vivo, in vivo models, and explore the underlying mechanisms.

Cell culture
Primary aortic VSMCs were isolated from 2-month-old male Sprague-Dawley (SD) rats (200-220g) as described previously [20]. Briefly, the rats were intraperitoneally euthanized with sodium pentobarbital (150 mg/kg), and the descending thoracic aorta was removed. After removal of the external connective tissue, the thoracic aorta was longitudinally cut open and the intima and adventitia were peeled off. Then, the medial layer of vessel was cut into small pieces and cultured in the high glucose (4.5 g/L) Dulbecco's modified eagle medium (DMEM;

Aortic rings culture
Thoracic aortas were dissected from 2-month-old male SD rats. Aortas were cut into 0.5 cm rings, and then incubated in GM, GM with DMY (80 μmol/L), CM or CM with DMY (80 μmol/L) for 7 days, with fresh medium renewed every 2 days. After 7 days incubation, aortic segments were harvested for further analysis. Ex vivo experiments were repeated for 4 times. University. CKD rat model was used in this experiment and was induced by subtotal 5/6 nephrectomy (extirpation of right kidney and ligation of arteries supplying two-thirds of left kidney) as previously described [21,22]. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg, Sigma-Aldrich, USA, #57-33-0). After shaving and disinfection with iodophor, a right ventrolateral incision was performed to expose the right kidney. The right kidney was ligated with non-absorbable thread and cut between the hilum and the ligated portion to remove the kidney. Similarly, the left renal artery was exposed and two-thirds of the arterial supply to the left kidney was ligated and verified by the subsequent homogeneous discoloration. Sham operated-controls were subjected to a sham operation, undergoing a simple laparotomy. Two weeks after the surgery, blood samples were taken to evaluate serum creatinine (CRE) by using Creatinine Assay Kit (Jiancheng Bio, Nanjing, China, C011-2). At the same time, the rats subjected to the 5/6 nephrectomy were fed with a high calcium and phosphate diet (4% calcium and 1.8% phosphate), and supplemented with 1,25-dihydroxy vitamin D3 by gavage (1 μg/kg, three times per week, Aladdin, Shanghai, China, C120126) for 4 weeks (Ca/P/VitD 3 ).

CKD rat model
Rats were randomly divided into three groups: (1) Sham group (Sham, n = 6), oral administration of a normal chow diet and sham-operation without 5/6 nephrectomy; Clinical Science. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/CS20210259 8 experimental study [23]. In some experiments, CKD rats were treated with SC79 (5 mg/kg; MCE, HY-18749) by intraperitoneal injection every 3 days for 4 weeks. At the end of the experiment, the rats were sacrificed under anesthesia by pentobarbital sodium (40 mg/kg, i.p.) and aortas (dissected from the ascending aortic root to the iliac bifurcation) were collected for further analysis.

MTS Assay
The viability of VSMCs was measured using MTS assay kit (Promega, Madison, WI, USA, G3580). Briefly, VSMCs were inoculated into 96-well plates at a density of 5×10 3 cells/well. Cells were then incubated with different concentrations of DMY (20,40 or 80 μmol/L) for 1, 3 or 7 days. 10 µl of MTS solution was then added into each well and incubated for 2 hours at 37℃. The absorbance was measured by a microplate spectrophotometer at the wavelength of 490 nm.

Micro-CT imaging of aortic calcification
Micro-CT analysis was performed to analyze aortic calcification. Rat aortas were collected, and scanned in a micro-CT scanner (SCANCO Medical AG, Switzerland) at a resolution of 34.5 μm. Calcified arterial lesions were defined as CT attenuation equal to or greater than 130 HU in each section [24]. Images were analyzed by micro-CT Evaluation Program V6.6 software (SCANCO Medical AG, Switzerland).

Alizarin red S Staining and quantification of calcium content
Cells were harvested after 7 days incubation for alizarin red S staining and calcium content assay. Alizarin red S staining method was used to determine VSMCs and arterial calcification. To visualize VSMCs calcification, cells were washed 3 times with phosphate buffered saline (PBS) and then fixed with 4% paraformaldehyde for 10 minutes. After that, the cells were washed 3 times with PBS and exposed to 2% alizarin red S (pH 4.2; Solarbio, Beijing, China, G8550) for 10 minutes, and then washed 3 times with deionized water and observed under a Leica Microsystems microscope. Positively stained cells displayed a red color. To quantify the extent of calcification, alizarin red S dye was eluted with 10% formic acid and the absorbance at 405 nm was measured by a spectrophotometer. To visualize aortic tissue calcification, thoracic aortic segments, which were fixed in 4% paraformaldehyde and embedded in paraffin, were cut into 4 μm in thickness and deparaffinized. Aortic sections were stained with 2% alizarin red S for 5 minutes. The sections were visualized by an inverted microscope. Calcified areas of aortic rings were shown as red staining and were analyzed with Image J software. To visualize the whole-mount aorta calcification, aorta samples that dissected from the ascending aortic root to the iliac bifurcation were fixed in 95% ethanol overnight. Afterwards aortas were incubated with 0.004% alizarin red S dissolved in 1% KOH overnight, and then washed twice with 2% KOH before being photographed. The calcium content of cells and aortas was determined by Calcium Content Detection Kit (methyl thymol blue microplate method; Leagene, Beijing, China, TC1021) according to the manufacturer's protocol [25,26]. The method is based on the mechanism that calcium reacts with methyl thymol blue (MTB) to form colored complexes monitored spectrophotometrically. Briefly, cells and aortic tissues were homogenized, and the supernatant was separated by centrifugation. Then, 2.5 μl of supernatant samples were The calcium level was determined by absorbance measurement at 610 nm on a microplate reader (Thermo Scientific, USA). Protein concentration was quantified by BCA protein assay (Pierce, USA). Calcium content was normalized to total protein concentration and expressed as μg/mg protein, and the final results were normalized to control.

Quantitative Real-Time Polymerase Chain Reaction
Total RNA was isolated from VSMCs by the Trizol method (AG, Hunan, China, GAPDH was used as an internal control. Data were processed by using the comparative 2 −ΔΔCT method for relative quantification.

Western blot analysis
Rat aortic tissues and rat/human VSMCs were lysed with RIPA lysis buffer

Statistical Analysis
The SPSS software was used for statistical analysis. One-way analysis of variance followed by the Tukey post hoc test were used to analyze the data. Data were expressed as mean ± standard error of the mean (SEM). P <0.05 was considered significant differences.

DMY inhibits calcification of rat VSMCs
To determine the role of DMY on calcification of rat VSMCs, VSMCs were exposed to different concentrations of DMY (20,40 or 80 μmol/L) in the presence of CM for 7 days. As shown in Fig. 1A 1C). This may be due to the inhibitory role of DMY in VSMCs proliferation.
Moreover, western blot analysis demonstrated that PCNA was downregulated by DMY treatment in comparison to CM-treated cells (Fig. S1). These data revealed that DMY inhibits the proliferation ability of VSMCs at day 7. Calcium content assay further confirmed that DMY reduced CM-induced VSMCs calcification with strongest effects at a concentration of 80 μmol/L (Fig. 1D). Therefore, the concentration of 80 μmol/L of DMY was selected for the subsequent experiments.
Additionally, RT-qPCR analysis revealed that DMY decreased the mRNA expression of osteogenic differentiation related genes including RUNX2 and BMP2 (Fig. 1E).

DMY inhibits calcification of human VSMCs
To further verify the effect of DMY on VSMCs calcification, human VSMCs were cultured in different concentrations of DMY (20,40 or 80 μmol/L) in the presence of CM for 7 days. Alizarin red staining and calcium content assay further confirmed the inhibitory role of DMY in human VSMCs calcification at day 7 ( Fig.   2A-C). Furthermore, RT-qPCR analysis revealed that DMY treatment markedly reduced RUNX2 expression (Fig. 2D). Western blot analysis revealed that DMY markedly decreased osteogenic differentiation markers such as COL I, RUNX2 and OCN (Fig. 2E). Collectively, these data demonstrated that DMY inhibited osteogenic differentiation and calcification of human VSMCs.

DMY attenuates calcification of aortic rings
Next, ex vivo aortic ring culture assay was performed to confirm the role of DMY in vascular calcification. Rat aortic rings were incubated in GM, GM supplemented with DMY (80 μmol/L), CM or CM supplemented with DMY (80 μmol/L) for 7 days. Alizarin red S staining demonstrated that DMY reduced mineral deposits in rat aortic rings ( Fig. 3A and B), and DMY has no effect on mineralization in rat aortic rings under the condition of GM. Calcium content assay also revealed that 80 μmol/L of DMY attenuated calcium content in aortic rings (Fig. 3C). Moreover, as illustrated in Fig. 3D, DMY treatment significantly down-regulated COL I, RUNX2 and BMP2 expression compared with aortic rings cultured in CM alone. Accordingly,

DMY ameliorates aortic artery calcification of CKD rats
To explore the effects of DMY on vascular calcification in vivo, DMY (200 mg/day) was used to treat CKD rats for 4 weeks. The rats were randomly divided into three groups: sham, model, DMY. Rats from model group and DMY group were subjected to 5/6 nephrectomy, while rats from sham group were given laparotomy without 5/6 nephrectomy. The concentration of CRE levels were dramatically elevated 2 weeks after surgery in model group and DMY group rats compared with sham group rats (Fig. 4A), which implied successfully established CKD model rats. Alizarin red S staining of whole aortas and sections confirmed that DMY ameliorated aortic calcification (Fig. 4B, E and F). Similarly, micro-CT showed that the DMY treatment significantly decreased the mineral density in CKD rats (Fig. 4C), which indicating that DMY ameliorated aortic calcification. In accordance with ex vivo experiments, calcium content analysis demonstrated decline of calcium deposits in DMY-treated aortic arteries (Fig. 4D). In addition, western blot analysis showed that the expression of COL I, RUNX2, BMP2 and OCN was up-regulated in calcified arteries, and DMY markedly decreased the expression of these osteogenic differentiation markers (Fig.   4G). In addition, to investigate the effect of DMY on rat vascular calcification under normal conditions, SD rats were randomly divided into sham group and sham+DMY group. Alizarin red S staining of whole aortas and sections and western bot analysis showed that DMY has no effect of vascular calcification under normal conditions ( Fig.   S2 A-D). These findings confirmed the effect of DMY on ameliorating vascular

DMY inhibits AKT signaling in vascular calcification both in vitro and in vivo
Previous studies have illustrated that the AKT signaling pathway plays a pivotal role in vascular calcification [27,28]. In line with these findings, we have previously reported that AKT signaling was up-regulated during vascular calcification [29]. In order to investigate whether AKT signaling is involved in inhibition of vascular calcification by DMY, we performed western blot analysis and immunofluorescence assay for p-AKT and AKT expression after 3-day incubation. Western blot analysis revealed that the protein level of p-AKT was significantly elevated in CM-treated VSMCs, while DMY dramatically down-regulated p-AKT protein expression compared with CM-treated cells (Fig. 5A). Then we examined the effect of DMY on p-AKT expression in vivo. As shown by Fig. 5B, the protein expression of p-AKT was significantly decreased in DMY group compared with the model group. In addition, immunofluorescence analysis showed increased p-AKT expression in VSMCs cultured in CM, which was drastically inhibited by DMY treatment (Fig. 5C). As showed in supplementary figure S3, immunostaining showed that DMY treatment has no effect on total AKT expression in VSMCs.

AKT signaling mediates the inhibitory effects of DMY on VSMCs calcification
To explore the role of AKT signaling in mediating DMY-induced inhibition of VSMCs calcification, SC79, a potent AKT activator was used. Alizarin red S staining showed that SC79 enhanced VSMCs calcification compared with CM-treated cells at day 7. However, this effect was abrogated by DMY treatment (Fig. 6A). Quantification analysis of alizarin red S staining further demonstrated the inhibitory role of DMY in SC79-enhanced VSMCs calcification (Fig. 6B). The similar findings were also demonstrated by calcium content assay (Fig. 6C). Additionally, we evaluated the protein expression of p-AKT and osteogenic markers RUNX2 and BMP2. The results showed that the ratio of p-AKT/total AKT, RUNX2 and BMP2 were increased by SC79 compared with CM. However, this effect was blocked by DMY, indicating the inhibitory effect of DMY on AKT signaling and VSMCs calcification ( Fig. 6D and E). We concluded that DMY ameliorated VSMCs calcification probably by inhibiting AKT signaling. To further confirm the relationship between DMY and AKT on calcification, AKT inhibitor MK2206 was used to treat VSMCs. As shown by Fig.7A

AKT signaling pathway plays a role in DMY-mediated amelioration of vascular calcification in CKD rats
The role of AKT signaling in DMY-inhibited vascular calcification in CKD rats was further evaluated with the AKT activator SC79. As evidenced by micro-CT analysis, more aortic calcification was observed in SC79 group than the model group, whereas the pro-calcific effect of SC79 was significantly retarded by DMY (Fig. 8A).
Simultaneously, Alizarin red S staining of the whole mount aortas and aortic sections revealed that SC79 promoted the aortic calcification of SD rats, but this effect was abolished by DMY treatment (Fig. 8B-D, Figure S4). Moreover, calcium content analysis showed a similar pattern ( Figure 8E). Furthermore, the protein level of p-AKT and osteogenic markers RUNX2 and BMP2 were up-regulated in the SC79 group compared with model group, while this up-regulation was abrogated by DMY treatment (Fig. 8F). According to these data, it suggests that DMY attenuates vascular calcification in CKD rats, at least partly by blocking the AKT signaling pathway.
During osteogenic differentiation, both protein expression ratio of p-AKT1/AKT1 and p-AKT2/AKT2 were elevated in CM-treated cells, whereas this upregulation was abolished by DMY (Fig. S5 A and B). These results suggested that both AKT1 and Western blot analysis confirmed the successful knockdown of AKT1 and AKT2 by siRNA in VSMCs (Fig. S6 A and B). At day 7, alizarin red S staining revealed that both AKT1 and AKT2 knockdown reduced mineralization of VSMCs, DMY+AKT1 siRNA group showed reduced VSMCs mineralization in comparison with CM+AKT1 siRNA group, and the same results existed between DMY+AKT2 siRNA group and CM+AKT2 siRNA group (Fig. 9A), and these results were further confirmed by quantification of alizarin red S staining (Fig. 9B). Consistently, calcium content assay showed similar results (Fig. 9C). In addition, knockdown of AKT1 or AKT2 by siRNA significantly decreased RUNX2 and BMP2 expression in comparison to Control siRNA-treated cells in the presence of CM (Fig. 9D). Altogether, these data indicate that both AKT1 knockdown and AKT2 knockdown suppress calcification of VSMCs, confirming a role for AKT signal in osteogenic differentiation and calcification.

Discussion
Vascular calcification decreases vessel elasticity and compliance, thus impairing cardiovascular hemodynamics, resulting in substantial cardiovascular morbidity and mortality [2,32]. Given the huge medical costs, it is urgent to find effective therapeutic strategies to prevent and treat vascular calcification. DMY, as the most abundant natural flavonoid in vine tea, can also be found in traditional medical plants including Hovenia dulcis and Cedrus deodara and plant-based fruits including grapes and red bayberry [33][34][35]. Several studies showed that DMY had multiple cardiovascular protective effects. DMY has been found to attenuate angiotensin II-induced cardiomyocyte hypertrophy and myocardial hypertrophy induced by transverse aortic constriction via oxidative stress inhibition [36,37]. Recent studies have shown that DMY inhibits rat VSMCs proliferation and migration and angiotensin II-induced cardiac fibroblast proliferation [17,38]. Besides, DMY is capable of protecting vascular endothelial cells, alleviating lipid accumulation, enhancing cholesterol efflux and inhibiting foam cell formation during the process of atherosclerosis [19,39]. Moreover, DMY also regulates glucose metabolism, improves insulin resistance and protects against diabetic cardiomyopathy [40]. According to our knowledge, this is the first report delineating that DMY suppresses vascular calcification partially by inhibiting AKT signaling.
AKT signaling plays a vital role in regulating VSMCs function, including proliferation, differentiation, cell survival, cell death by regulating various downstream signaling effectors [41][42][43]. There are convincing evidence supporting that AKT signaling is deeply involved in osteogenic differentiation and calcification, and acts as an upstream signaling to directly activate RUNX2 [28,44]. A recent publication has showed that AKT signaling drives H 2 O 2 -induced VSMCs calcification by upregulating RUNX2 activity [45]. Inhibition of the AKT signaling resulted in decreased osteogenic differentiation of VSMCs [46]. Additionally, activated AKT was proved sufficiently to promote vascular calcification in animals in vivo [27].
Consistent with these findings, we found that AKT signaling was activated in calcified VSMCs and aortas. DMY has been reported to inhibit AKT phosphorylation in vitro [47,48]. Consistently, our study also showed that DMY down-regulated phosphorylation of AKT both in vitro and in vivo. Therefore, we postulate that AKT signaling mediates the inhibitory effects of DMY on vascular calcification. The role of AKT signaling in DMY-attenuated vascular calcification was further evaluated with the specific AKT activator SC79 and inhibitor MK2206. We identified that AKT activation led to increased RUNX2 activation in VSMCs, as measured by western blot, and AKT deactivation decreased RUNX2 protein expression level in VSMCs. This indicated a potential correlation between AKT signaling and RUNX2 in vascular Emerging evidence suggests that there are distinct functions for AKT isoforms [49,50]. It is unclear whether AKT isoforms exhibit distinct functions in CM-induced

Data availability Statement
The data underlying this study will be shared on request to the corresponding author.

Clinical perspectives
 Vascular calcification is highly prevalent in CKD and is associated with increased cardiovascular morbidity and mortality. However, no effective and therapeutic  DMY may become an effective agent for preventing progression of vascular calcification in CKD.

Competing Interests
None.