CDC42 promotes vascular calcification in chronic kidney disease
Zehua Li1,2,3, †, Ji Wu1,2,3, †, Xiuli Zhang1,2,3, †, Caiwen Ou1,2,3, Xinglong Zhong1,2,3, Yanting Chen4, Lihe Lu4, Hailin Liu1,2,3, Yining Li1,2,3, Xiaoyu Liu1,2,3, Bo Wu1,2,3, Yuxi Wang1,2,3, Pingzhen Yang1,2,3, Jianyun Yan1,2,3,*, Minsheng Chen1,2,3,*
1Department of Cardiology, Laboratory of Heart Center, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong 510280, China
2Guangdong Provincial Biomedical Engineering Technology Research Center for Cardiovascular Disease, Guangzhou, Guangdong 510280, China
3Sino-Japanese Cooperation Platform for Translational Research in Heart Failure, Guangzhou, Guangdong 510280, China
4Department of Pathophysiolgy, Zhongshan Medical School, Sun Yat-Sen University, Guangzhou, Guangdong 510080, China
†These authors contributed equally to this work.
*Correspondence to: Jianyun Yan or Minsheng Chen
E-mail: [email protected] or [email protected]
Department of Cardiology, Laboratory of Heart Center, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong 510280, China
Tel: +86 20 62782264
Running title: CDC42 in vascular calcification
No conflicts of interest were declared.
Abstract
Vascular calcification is prevalent in patients with chronic kidney disease (CKD) and a major risk factor of cardiovascular disease. Vascular calcification is now recognised as a biological process similar to bone formation involving osteogenic differentiation of vascular smooth muscle cells (VSMCs). Cell division cycle 42 (CDC42), a Rac1 family member GTPase, is essential for cartilage development during endochondral bone formation. However, whether CDC42 affects osteogenic differentiation of VSMCs and vascular calcification remains unknown. In the present study, we observed a significant increase in the expression of CDC42 both in rat VSMCs and in calcified arteries during vascular calcification. Alizarin red staining and calcium content assay revealed that adenovirus-mediated CDC42 overexpression led to an apparent VSMC calcification in the presence of calcifying medium, accompanied with up-regulation of bone-related molecules including RUNX2 and BMP2. By contrast, inhibition of CDC42 by ML141 significantly blocked calcification of VSMCs in vitro and aortic rings ex vivo. Moreover, ML141 markedly attenuated vascular calcification in rats with CKD. Furthermore, pharmacological inhibition of AKT signal was shown to block CDC42-induced VSMC calcification. These findings demonstrate for the first time that CDC42 contributes to vascular calcification through a mechanism involving AKT signalling; this uncovered a new function of CDC42 in regulating vascular calcification. This may provide a potential therapeutic target for the treatment of vascular calcification in the context of CKD.
Keywords
CDC42; Vascular calcification; Vascular smooth muscle cell; Osteogenic differentiation; Chronic kidney disease; AKT
Introduction
Vascular calcification is one of the most common comorbidities of chronic kidney disease (CKD) involving mineral metabolism imbalance [1-4]. Vascular calcification is an independent risk factor and a cause of death for cardiovascular disease (CVD), since this lesion contributes to the progressive decrease of vessel wall elasticity and eventually leads to dysfunction of the vessel wall [5-8]. Vascular calcification is now recognized as a cell-regulated biological process involving phenotypic switching from vascular smooth muscle cells (VSMCs) into osteo-/chondro-blast-like cells [9-10]. The hallmark of this pathological process is similar to bone formation, which is accompanied with the upregulation of bone-related proteins including runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP2), and downregulation of contractile proteins such as alpha smooth muscle actin (α-SMA, ACTA2) [11-13]. A variety of factors have been identified as promoters of vascular calcification such as oxidative stress, increased calcium and phosphate levels, and inflammation [14-17]. Accumulating studies have demonstrated that activation of AKT signal plays a critical role in vascular calcification [17-18]. However, the precise mechanisms underlying vascular calcification have not been fully illustrated.
CDC42, a member of the Rho family of GTPases, has been shown to play fundamentally
important roles in the regulation of multiple cellular functions including cell morphology, cytokinesis, cytoskeleton organization, proliferation, and migration [19]. Aberrant activation of CDC42 contributes to a variety of pathological conditions such as senescence-associated inflammation, atherosclerosis, and neointimal formation [20-21]. Conditional knockout studies have revealed that tissue specific deletion of CDC42 inhibits mouse chondrocyte differentiation and bone mineralisation [22-23]. In addition, an in vitro study has shown that inhibition of CDC42 signalling by ML141 reverses VSMC calcification induced by LRP6 deficiency [24], suggesting the potential role of CDC42 in vascular calcification. However, whether and how CDC42 modulates vascular calcification under the condition of CKD remain elusive. In this study, we investigated the role of CDC42 in vascular calcification using in vitro, ex vivo and in vivo models.
Materials and methods Human VSMC culture
This research protocol conformed to the Declaration of Helsinki and was approved by the Ethics Committee of Zhujiang Hospital, Southern Medical University, China. Informed consent was obtained from the patients for use of blood. The general characteristics of the patients are presented in supplementary materials, Table S1. Venous blood samples were drawn from both healthy controls and CKD patients into tubes without anticoagulant. Blood samples were left undisturbed at room temperature for 30 min. Serum was prepared by centrifugation at 2000 x g for 10 min at 4 °C. The supernatant was stored at −80 °C for later use. Human serum from healthy controls and CKD patients were used to treat human VSMCs (ATCC, Manassas, VA, USA, CRL-1999) in growth medium (GM) or calcifying medium (CM, DMEM supplemented with 10 mM beta-glycerophosphate and 3 mM CaCl2) in a 37 °C humidified incubator containing 5% CO2.
Cell culture
VSMCs were obtained from thoracic aortas isolated from healthy Sprague-Dawley (SD) rats (200–220 g) as described previously [25]. VSMCs were maintained in GM supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin/streptomycin in a 37 °C humidified incubator containing 5% CO2. VSMCs from passages 5 to 8 were used for all experiments. To induce VSMC calcification, cells were cultured in CM for 8–10 d. VSMCs were cultured in 35 mm dishes at a density of 4 × 105 cells/dish 24 h prior to the experiments and were replaced with fresh medium every 48 h. To investigate the role of CDC42 in vascular calcification, ML141 (5 μM, Selleck, Houston, TX, USA, #S7686), a CDC42 inhibitor, was used to treat cells in the presence of CM. In some experiments, MK-2206 (0.5 mM, Selleck, #S1078) and perifosine (0.3 mM, Selleck, #S1037) were used to examine the role of AKT in vascular calcification.
Cell transfection
VSMCs were seeded in 35 mm dishes and grown in DMEM supplemented with 10% FBS to reach 80% confluence. VSMCs were transfected with adenovirus encoding CDC42 (Ad/CDC42) or control virus (Ad/GFP) at an optimal multiplicity of infection (MOI=10) as described previously [26]. Transfected cells were harvested at indicated time points for further analysis.
Ex vivo aortic ring culture
Rat thoracic aortas were cut into 4–5 mm rings, which were incubated in GM at 37 °C in a humidified 5% CO2 incubator. To induce aortic calcification, aortic rings were cultured in CM for 7 d. In some experiments, ML141 (5 μM, Selleck, USA) was used to treat aortic segments in CM. At the indicated time points, aortic rings were collected for further analysis.
CKD rat model
All animal experiments were performed in accordance with the US National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee at Southern Medical University (Guangzhou, China). Adult male SD rats aged 8 weeks (weighing 200–250g) were obtained from Guangdong Medical Lab Animal Center (Guangzhou, China) and were allowed free access to standard rat chow and tap water in temperature and humidity control conditions with a 12 h dark/light cycle. CKD was induced by renal mass reduction with 5/6 nephrectomy as previously performed [27–28]. Sham-operated controls also received surgical procedure, in which the kidneys were exteriorized and returned to the abdominal cavity. Vascular calcification was induced by a high calcium and phosphate diet (4% calcium and 1.8% phosphate, Guangdong Medical Lab Animal Center, China), combined with 1,25-dihydroxyvitamin D3 subcutaneous injection (1 µg/kg, 3 times per week, Aladdin, Shanghai, China, #C120126) for 4 weeks (Ca/P/VitD). Plasma creatinine (CRE) concentrations of rats were determined using a standard colorimetric assay (Creatinine Assay Kit, Nanjing Jiancheng Bioengineering institute, Nanjing, China, #C011-2) 2 weeks after the surgery. Based on the concentration of CRE, all rats were separated into three groups (n=6): (1) sham + normal chow; (2) CKD + Ca/P/VitD; (3) CKD + Ca/P/VitD+ML141 (Intraperitoneal injection of 3.5 mg/kg/d, Selleck, USA). At the end of experiments, the rats were sacrificed after 4 weeks of treatment with Ca/P/VitD and animal aortic tissues were collected for further analysis.
Micro-computed tomography imaging of aortic calcification
Micro-computed tomography (Micro-CT) scanning was used to analyse aortic calcification. At the end of experiment, rat aortas were harvested, and scanned by Micro-CT (Siemens Inveon, Siemens, Munich, Germany) at a resolution of 0.079 mm. The Inveon research workplace software (Siemens Inveon) was used to analyse Micro-CT images.
Alizarin red staining and determination of calcium content
To assess the calcium deposition, VSMCs were fixed with 4% paraformaldehyde for 10 min and stained with 2% alizarin red solution (PH 4.2) for 5 min at room temperature. When a red-orange colour appeared, excess dye was removed and then the calcium deposition was imaged using a Leica Microsystems microscope. For ex vivo aortic ring assay, rat aortic segments were fixed in 4% paraformaldehyde and embedded in paraffin. Aortic samples were cut into 6 μm thick sections that were then prepared for 2% alizarin red staining. The sections were dehydrated, then cleared in xylene and mounted in Permount medium before microscopy. For whole mount staining, rat aortas were fixed in 95% ethanol overnight, and incubated with 0.003% alizarin red dissolved in 1% potassium hydroxide overnight. Then, the samples were washed in 2% potassium hydroxide 2 times, before being photographed. The degree of calcification was quantified using the methyl thymol blue microplate method, which was determined by absorbance measurement at 610 nm on a microplate reader (Thermo Fisher Scientific) using a standard calcium curve in the same solution. The final calcium level in each group was normalised with the total protein concentrations prepared from a duplicate plate.
RT-qPCR
The effect of CDC42 on the expression of bone-related and smooth muscle-specific gene markers was determined by RT-qPCR. Total RNA was isolated from VSMCs using Trizol reagent (TaKaRa, Japan, #9109) and reverse-transcribed into cDNA. PCR was performed using specific primers and was performed on a 7500 FAST Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using a Real-Time PCR Assay Kit (TakaRa, Japan, #RR420A). The sequences of primers used for amplification were as follows: CDC42, 5’-GCCTATTACTCCAGAGACTGCT-3’ and 5’-TGATGACAACACCAGCTGTGC-3’;
RUNX2, 5’-GCCGGGAATGATGAGAACTA-3’ and 5’-GGACCGTCCACTGTCACTTT-3’; BMP2, 5’-GTTTGGCCTGAAGCAGAGAC-3’ and 5’-CTCGATGGCTTCTTCGTGAT-3’; GAPDH, 5’-GGCAAGGTCATCCCAGAGCT-3’ and 5’-CCCAGGATGCCCTTTAGTGG-3’.
GAPDH was used as an internal control. Data were analysed using the comparative Ct (ΔΔCt)
method for relative quantification.
Western blot analysis
Proteins extracted from VSMCs or rat aortas were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA, IPVH00010). After being blocked with 5% skimmed milk powder for 2 h, the membranes were incubated at 4 °C overnight with the following primary antibodies: anti-BMP2 antibody (1:1000, Abcam, Cambridge, MA, USA, ab6285), anti-α-SMA antibody (1:1000, Abcam, ab32575); anti-GAPDH antibody (1:5000, Bioworld, St. Louis Park, MN, USA, AP0063); anti-RUNX2 antibody (1:1000, Cell Signaling Technology, Danvers, MA, USA, #12556), anti-AKT antibody (1:1000, Cell Signaling Technology, #9272), anti-Phosphorylated AKT antibody (p-AKT) (1:2000, Cell Signaling Technology, #4060) and anti-CDC42 antibody (1:1000, Cell Signaling Technology, #2462). Then, the membranes were washed and incubated with the secondary antibodies (1:5000, BOSTER, Wuhan, China, #BA1054) at room temperature for 2 h, and the signals were detected using the Imaging System (GE, Amersham Imager 600, USA). Protein bands were quantified by Image Pro Plus 6.0 and normalised to GAPDH expression.
Immunofluorescence assay
VSMCs were fixed in 4% paraformaldehyde and then permeabilized with 0.5% Triton X-100 for 20 min at room temperature. After being blocked with 1% Bovine Serum Albumin (BSA) in PBS at room temperature for 30 min, VSMCs were incubated with anti-Phosphorylated AKT antibody (p-AKT) (1:400, Cell Signaling Technology, #4060) overnight at 4 °C
followed by Cy3-labeled Goat Anti-Rabbit IgG (1:400, Bioworld, USA, BS10007) for 2 h at room temperature. After extensive washing in PBS, the nuclei were counterstained in blue with 4, 6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology, Shanghai, China, #C1002). VSMCs were imaged using a microscope (Leica Microsystem, Wetzlar, Germany) and p-AKT fluorescent intensity was determined using ImageJ software (NIH Bethesda, MD, USA).
Statistical analysis
All results were expressed as the mean ± SEM. Statistical analysis was performed using SPSS Statistical software (version 20.0, SPSS Inc., Chicago, IL, USA). Independent Student’s t-tests were used to test the differences between two groups, and comparisons among multiple groups were performed by one-way ANOVA. Differences were accepted as statistically significant at p<0.05.
Results
Expression of CDC42 was increased during vascular calcification
To determine the expression of CDC42 during vascular calcification, rat VSMCs were cultured in CM for 8 days to stimulate VSMC calcification and CKD rats induced by nephrectomy were treated with a high calcium and phosphate diet (4% calcium and 1.8% phosphate), combined with 1,25-dihydroxyvitamin D3 to induce aortic calcification. Both RT-qPCR and western blotting analysis revealed that CDC42 expression was significantly increased in CM-treated cells compared to GM-treated cells (Figure 1A,B). Consistently, we also found that CDC42 protein expression was significantly increased in aortic arteries of CKD rats in comparison with sham controls (Figure 1C). Furthermore, to investigate the effect of CDK serum on CDC42 expression, we also used CDK serum to treat human VSMCs in the presence of GM or CM. Western blotting analysis showed that CDC42 expression was
significantly increased in CDK serum-treated cells compared to healthy serum-treated cells in the presence of CM, but not in GM (Figure 1D).
CDC42 overexpression promotes VSMC calcification
To determine the role of CDC42 in VSMC calcification, rat VSMCs were transfected with Ad/CDC42 in the presence of GM or CM for 6 or 8 d. As shown in Figure 2A, western blotting analysis revealed that the protein level of CDC42 was highly elevated in Ad/CDC42-infected cells in comparison with control Ad/GFP-infected cells at day 6, suggesting that VSMCs were successfully infected with Ad/CDC42. As shown in supplementary material, Figure S1, CDC42 overexpression had no effect on calcification of rat VSMCs in the presence of GM at day 8. Of note, alizarin red staining showed CDC42 overexpression markedly promoted calcification of VSMCs in the presence of CM at day 8 (Figure 2B). Consistently, calcium content assay revealed that Ad/CDC42-infected cells showed increased calcium levels compared to Ad/GFP-infected cells (Figure 2C). Additionally, we found that CDC42 overexpression promoted osteogenic differentiation of rat VSMCs, as indicated by the increased expression of BMP2 and RUNX2 in both transcription and protein levels (Figure 2D,E). Furthermore, given that CKD serum up-regulated the expression of CDC42 in human VSMCs, we also examined the effect of CKD serum on human VSMC calcification. Interestingly, CKD serum markedly accelerated human VSMC calcification in the presence of CM, but not in GM (supplementary material, Figure S2). Collectively, these data suggested a key role of CDC42 in regulating VSMC calcification.
CDC42 inhibition by ML141 attenuates VSMC calcification
To further confirm the effect of CDC42 on VSMC calcification, ML141, a potent and selective inhibitor of CDC42 [29], was used to treat rat VSMCs. As shown in Figure 3A and supplementary material, Figure S1, alizarin red staining showed that ML141 reduced calcium
deposition in VSMCs in the presence of CM at day 8, but this effect was not found in the presence of GM. ML141-treated cells showed lower calcium levels than CM-treated cells (Figure 3B). In addition, we found that ML141 inhibited osteogenic differentiation of rat VSMCs, as indicated by decreased RUNX2, BMP2 mRNA and protein expression in VSMCs at day 6 (Figure 3C,D). These findings further confirmed the positive role of CDC42 in regulation of VSMC calcification in vitro.
CDC42 inhibition by ML141 attenuates calcification of rat aortic rings
To further investigate the role of CDC42 in vascular calcification, we next used ex vivo aortic ring culture assay. ML141 was used to treat rat aortic rings in the presence of CM for 7 days. In accord with in vitro experiment results, alizarin red staining of aortic ring sections also showed that ML141 reduced calcium deposition in aortic rings in the presence of CM at day 7 (Figure 4A). Under this condition, ML141 treatment significantly reduced calcium content in aortic rings (Figure 4B). Additionally, we found that ML141 significantly decreased RUNX2 mRNA and protein expression in aortic rings (Figure 4C). These findings suggest that CDC42 facilitates vascular calcification ex vivo.
CDC42 inhibition by ML141 attenuates calcification in CKD rats
To verify the effect of CDC42 on vascular calcification in vivo, ML141, CDC42 inhibitor, was used to treat CKD rats. As shown in Figure 5A, the levels of CRE were significantly increased in model rats two weeks after 5/6 kidney nephrectomy compared with sham-operated rats, suggesting that rat CKD model was successfully induced by renal surgery. By the termination of the experiment, micro-CT analysis showed a smaller area of calcification of the aortas in ML141-treated rats, compared with model group (Figure 5B). Consistently, alizarin red staining of aortas and aortic sections showed decreased aortic calcification in ML141-treated group compared with model group (Figure 5C and 5E). In
addition, calcium content assay further confirmed that ML141 decreased calcium content in aortic arteries by 52% (p<0.01, Figure 5D). Moreover, western blotting analysis showed that ML141 treatment increased the expression of smooth muscle marker protein, ACTA2, while decreased the expression of bone specific marker protein, RUNX2 (Figure 5F). These results altogether demonstrate that the administration of ML141 is effective for the amelioration of vascular calcification of CKD rats, suggesting that inhibition of CDC42 attenuates vascular calcification in CKD rats.
CDC42 activates AKT signal in VSMCs
The AKT signalling pathway has been shown to play important roles in vascular calcification [17,18]. Therefore, we decided to test whether AKT signal is involved in CDC42-induced VSMC calcification. We first examined the effect of CDC42 on the activation of AKT signal during VSMC calcification. As revealed by western blot analysis, protein levels of p-AKT were significantly increased in Ad/CDC42-infected cells compared with Ad/GFP-infected cells in the presence of CM (Figure 6A). By contrast, inhibition of CDC42 by ML141 decreased protein levels of p-AKT in rat VSMCs (Figure 6B). In addition, immunofluorescence staining further confirmed increased expression of phosphorylated AKT in Ad/CDC42-infected cells compared to Ad/GFP-infected cells (Figure 6C and supplementary material, Figure S3). These results indicated that CDC42 overexpression stimulates the phosphorylation of AKT and activates AKT signal during VSMC calcification.
Inhibition of AKT signal attenuates CDC42-induced VSMC calcification
Next, we investigate the role of AKT signal in CDC42-induced VSMC calcification. perifosine and MK-2206, pharmacological AKT inhibitors [30-31], were used to treat VSMCs infected with Ad/CDC42 in the presence of CM. As shown in Figure 6D, CDC42 overexpression stimulated the phosphorylation of AKT, which can be inhibited by perifosine
and MK-2206 treatment. Of note, alizarin red staining showed that CDC42 overexpression enhanced CM-induced mineral deposition in VSMCs, which can be blocked by both perifosine and MK-2206 treatment (Figure 6E). Consistently, both perifosine and MK-2206 treatment decreased calcium content in VSMCs (Figure 6F). In addition, perifosine and MK-2206 treatment abrogated CDC42-induced up-regulation of RUNX2 in VSMCs (Figure 6G). Taken together, these results demonstrated that AKT signalling pathway participates in CDC42-induced VSMC calcification.
Discussion
Vascular calcification, characterized by reduced vascular wall elasticity and compliance, is very common in patients with CKD and considered as an independent risk factor and an established predictor for CVD [1,2]. Vascular calcification has now been considered as an active cell-regulated process resembling bone formation, and osteogenic differentiation of VSMCs plays a critical role in the progression of vascular calcification [32]. Therefore, it is of extreme importance to uncover the molecular mechanisms responsible for vascular calcification.
CDC42 has been shown to regulate bone development [22,23]. It is unclear whether CDC42 contributes to the development of vascular calcification in the context of CKD. In this study, we found that CDC42 expression is up-regulated in VSMCs and aortas during vascular calcification. CDC42 overexpression promotes osteogenic differentiation and calcification of VSMCs. By contrast, inhibition of CDC42 by ML141 attenuates calcification of VSMCs and aortic rings. Furthermore, ML141 remarkably attenuates aortic calcification in rats with chronic kidney disease. Finally, we found that AKT signal is involved in CDC42-mediated VSMC calcification. To our knowledge, this is the first report showing that CDC42 promotes vascular calcification in the context of CKD via activation of AKT signalling, suggesting
CDC42 could become the potential therapeutic target for the treatment of vascular calcification in CKD. These findings provide a new insight into the pathogenesis of vascular calcification.
CDC42 has been shown to participate in a variety of cellular processes including cell growth, migration, and proliferation [33]. Previous studies have demonstrated that CDC42 is essential for chondrogenesis and bone mineralisation [22,23]. It has been reported that CDC42, accelerated the rate of chondrocyte differentiation and apoptosis, thereby regulating endochondral bone growth and development [34,35]. An animal study has revealed that CDC42 activation has also been implicated in the development of atherosclerosis [36]. In addition, CDC42 antagonist ML141 has been shown to attenuate LRP6 deficiency-induced VSMC calcification in vitro [24]. In this study, we found that CDC42 expression is up-regulated in VSMCs and aortas under osteogenic condition, suggesting the potential role of CDC42 in vascular calcification. In agreement with a previous report [24], our study showed that inhibition of CDC42 by ML141 inhibits calcification of VSMCs and aortic rings. Conversely, adenovirus-mediated CDC42 overexpression accelerates VSMC calcification under the condition of high calcium and phosphate, but not under normal condition. Furthermore, ML141 treatment inhibits rat aortic calcification in CKD rats. Collectively, our data suggest that CDC42 promotes vascular calcification under the condition of CKD.
CDC42 has been shown to regulate osteoclast precursor proliferation via the AKT pathway [37]. In addition, our previous study has shown that AKT signalling pathway plays an essential role in the regulation of VSMC migration and proliferation [26], suggesting the role of AKT signalling in vascular disease. Furthermore, a number of studies have demonstrated that AKT activation is involved in the regulation of vascular calcification. In vitro experiments have shown that inhibition of AKT signalling prevents oxidative stress-induced
VSMC calcification [17]. In vivo studies also have demonstrated that activation of AKT signalling pathway is essential for vascular calcification [38]. Therefore, we speculate that AKT signalling regulates CDC42-induced vascular calcification. Consistent with these findings, we found that the phosphorylation of AKT was stimulated in VSMCs transfected with Ad/CDC42, indicating that AKT signal was activated by CDC42. Moreover, CDC42-induced VSMC calcification was attenuated by pharmacological inhibition of AKT signal. Taken together, this study has supported the fact that AKT signal mediates CDC42-mediated vascular calcification in CKD. In addition, vascular extracellular matrix (ECM) degradation induced by elevated Ca and P in CKD actively regulates vascular calcification. A predominant feature of arterial calcification in CKD is the accumulation of mineral deposits along the elastic lamina [39]. Mineralized elastin is associated with degradation of elastin and the degraded elastin has a high affinity for calcium. Elastin degradation has been shown to regulate vascular calcification in the uremic milieu [40,41].
In summary, this study has uncovered a novel function of CDC42 in the regulation of vascular calcification under the condition of CKD. We demonstrated that activation of AKT signalling by CDC42 promoted vascular calcification and we highlighted the critical role of CDC42 in vascular calcification, suggesting that CDC42 may become a novel potential therapeutic target for the prevention and treatment of vascular calcification in CKD. Since bone defects usually occur in parallel to the development of vascular calcification in CKD, future studies using CDC42 inhibitor are required to determine the effect of CDC42 on bone defects in CKD.
Acknowledgements
This work was supported by the Natural Science Foundation of China (81770280, 81870190, 31771060, U1501222, 31671025, 81871504), the Guangdong Natural Science Foundation,
China (2018A030313577, 2016A030313226), the Science and Technology Planning Project of Guangdong Province (2017A020215184), and the Guangzhou science and technology planning project (201704020143).
Statement of Author contributions
MC and JY designed the study and supervised the experiments. ZL, JW, XZ, CO, XZ, YC, LL, HL, YL, XL, and BW performed the experiments. ZL, PY and JY analysed the data. ZL and YW wrote the original draft. JY edited the manuscript.
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Figure legends
Figure 1 Expression of CDC42 in VSMCs and in calcified arteries during vascular calcification. Confluent rat VSMCs were cultured in growth medium (GM) or calcifying medium (CM) for 8 d (n=4). (A) mRNA expression levels of CDC42 in VSMCs were analysed by RT-qPCR. (B) Western blotting analysis of CDC42 protein expression in VSMCs.
(C) SD rats subjected to 5/6 kidney nephrectomy were treated with a high calcium and phosphate diet (4% calcium and 1.8% phosphate) combined with 1,25-dihydroxyvitamin D3 subcutaneous injection for 4 weeks (n=6). Western blotting analysis of CDC42 protein expression in aortic arteries. (D) Human VSMCs were treated with serum from healthy controls or CKD patients in growth medium (GM) or calcifying medium (CM) for 6 days (n=4). Western blotting analysis of CDC42 protein expression in human VSMCs. *p<0.05 and **p<0.01. NS=no significance.
Figure 2 Effect of CDC42 overexpression on VSMC calcification. Rat VSMCs were transfected with Ad/GFP or Ad/CDC42 in growth medium (GM) or calcifying medium (CM) for 6 or 8 days. (A) Protein expression of CDC42 was analysed by western blotting at day 6.
(B) Mineral deposition in VSMCs was detected by alizarin red staining at day 8, Scale bar,
500 μm. (C) Quantitative analysis of calcium content. (D) RUNX2 and BMP2 mRNA expression levels in VSMCs were analysed by RT-qPCR. (E) Western blotting analysis of RUNX2 protein expression in VSMCs. n=4. *p<0.05, **p<0.01.
Figure 3 Effect of ML141 on VSMC calcification. Confluent rat VSMCs were treated with CDC42 inhibitor, ML141 (5 μM) for 8 days. (A) Mineral deposition in VSMCs was detected by alizarin red staining (Scale bar, 500 μm). (B) Calcium content was measured. (C) Expression of BMP2 and RUNX2 was determined by RT-qPCR. (D) The protein expression of RUNX2 and BMP2 was analysed by western blotting. n=4. *p<0.05 and **p<0.01.
Figure 4 Effect of ML141 on calcification of rat aortic rings. Rat aortic rings were cultured in growth medium (GM) or calcifying medium (CM) with or without CDC42 inhibitor, ML141 (5 μM) for 7 days. (A) Mineral deposition in aortas was detected by alizarin red staining. High magnification images of areas outlined in black rectangle are shown in the lower panel (Scale bar, 100 μm). (B) Calcium content was measured. (C) Expression of RUNX2 was determined by western blotting. n=4. *p<0.05 and **p<0.01.
Figure 5. Effect of ML141 on vascular calcification in CKD rats. Two weeks after 5/6 kidney nephrectomy, SD rats were treated with a high calcium and phosphate diet (4% calcium and 1.8% phosphate) combined with 1,25-dihydroxyvitamin D3 subcutaneous injection for 4 weeks, with or without intraperitoneal injection of CDC42 inhibitor, ML141 (3.5 mg/kg/d).
(A) Levels of CRE were measured using a standard colorimetric assay at week 2 post surgery.
(B) Calcified aortas of CKD rats were examined by micro-CT. (C) Alizarin red staining of aorta tissues. (D) Calcium content was quantified using the methyl thymol blue microplate method. (E) Representative images of aortic sections stained with alizarin red. Boxed areas at higher magnifications are shown to the below (Scale bar, 100 μm). (F) Expression of ACTA2 and RUNX2 was determined by western blotting. n=6. *p<0.05 and **p<0.01.
Figure 6. AKT signal is required for CDC42-induced vascular calcification. (A) Rat VSMCs were transfected with Ad/GFP or Ad/CDC42 in growth medium (GM) or calcifying medium (CM) for 6 days. Western blotting analysis of protein expression of p-AKT in VSMCs. (B) VSMCs were treated with CDC42 inhibitor, ML141 (5 μM) for 6 days. Protein expression of p-AKT in VSMCs was analysed by western blotting. (C) Expression of p-AKT was analysed by immunostaining (Scale bar = 100 μm). p-AKT inhibitors perifosine (0.3 mM) and MK-2206 (0.5 mM) were used to treat rat VSMCs transfected with Ad/CDC42 for 8 days. (D)
Protein expression of p-AKT and AKT were analysed by western blotting. (E) Mineral deposition in VSMCs was detected by alizarin red staining (Scale bar, 500 μm). (F) Calcium content was measured. (G) Protein expression ML141 of RUNX2 was analysed by western blotting. n=4. *p<0.05 and **p<0.01.