PRMT3 methylates HIF-1α to enhance the vascular calcification induced by chronic kidney disease

Background Medial vascular calcification is commonly identified in chronic kidney disease (CKD) patients and seriously affects the health and life quality of patients. This study aimed to investigate the effects of protein arginine methyltransferase 3 (PRMT3) on vascular calcification induced by CKD. Methods A mice model of CKD was established with a two-step diet containing high levels of calcium and phosphorus. Vascular smooth muscle cells (VSMCs) were subjected to β-glycerophosphate (β-GP) treatment to induce the osteogenic differentiation as an in vitro CKD model. Results PRMT3 was upregulated in VSMCs of medial artery of CKD mice and β-GP-induced VSMCs. The inhibitor of PRMT3 (SGC707) alleviated the vascular calcification and inhibited the glycolysis of CKD mice. Knockdown of PRMT3 alleviated the β-GP-induced osteogenic transfomation of VSMCs by the repression of glycolysis. Next, PRMT3 interacted with hypoxia-induced factor 1α (HIF-1α), and the knockdown of PRMT3 downregulated the protein expression of HIF-1α by weakening its methylation. Gain of HIF-1α reversed the PRMT3 depletion-induced suppression of osteogenic differentiation and glycolysis of VSMCs. Conclusion The inhibitory role of PRMT3 depletion was at least mediated by the regulation of glycolysis upon repressing the methylation of HIF-1α.


Introduction
Chronic kidney disease (CKD), an irreversible and progressive kidney disease, is linked with the changes of renal structure and degradation of kidney function (Websteret al. 2017).The premature death risk of CKD patient is 5-10 times higher than that of general population, which is largely resulted from the death of cardiovascular diseases (Di Angelantonio et al. 2007;Sarnak et al. 2003;Webster et al. 2017).Notably, medial vascular calcification is commonly identified in CKD patients (Düsinget al. 2021).It causes the decreased compliance of artery walls, leading to the elevation of pulse-wave velocity and systolic pressure.The changes of mechanical and hemodynamic properties contribute to left ventricular hypertrophy, reduced coronary perfusion, and ultimately heart failure as time goes on (Lanzer et al. 2014;Nelson et al. 2020).Moreover, it has been reported that CKD patients exhibit dysregulation of phosphorus and calcium metabolism.High content of serum phosphorus dives vascular calcification, including the transformation of vascular smooth muscle cells (VSMC) into osteoblastic phenotype and extracellular matrix degradation (McCartyet al. 2014, Shanahanet al. 2011).The regulation of osteogenic differentiation of VSMCs may be the key process for the prevention of vascular calcification.
Glycolysis acts as a vital part in glycometabolism and is associated with the osteogenic differentiation of VSMCs (Liet al. 2018).The glycolysis pathway is activated by various calcification factors and overproduces the products of glycolysis including lactate (Yanget al. 2017).Niu et al. have testified that the inhibition of fructose-2,6-biphosphatase 3 (PFKFB3, a key enzyme in glycolysis) could mitigate the glycerophosphate (β-GP)-induced osteogenic differentiation of VSMCs (Niuet al. 2021).The similar effects of glycolysis suppression mediate the repression of vascular calcification could be found in previous studies (Ma et al. 2020;Wang et al. 2022).These researches imply that targeting glycolysis might be a great strategy for the treatment of medial vascular calcification.
Protein arginine methylation is a frequent post-translation modification that is catalyzed by protein arginine methyltransferases (PRMTs) family proteins, which transfer a methyl group to the arginine residues of substrates (Blancet al. 2017).Nine members are identified in PRMTs that could be divided into three types (Blancet al. 2017, Hwanget al. 2021).One of the type I proteins named PRMT3 evoke us much attention.Unlike other members of the PRMT family, PRMT3 exhibits a unique substrate-binding C2H2 zinc finger motif at its N-terminal and a catalytic domain at its C-terminal (Shiet al. 2023).PRMT3 could catalyze the formation of ω-mono-or asymmetric dimethyl arginine, and it mainly locates in cytoplasm rather than distributes in nucleus and cytoplasm (Bachandet al. 2004).Accumulating evidences have demonstrated that the substrates of PRMT3 including RPS2 and HMG1Aα/β (Swiercz et al. 2005;Zou et al. 2007).Lei et al. have reported that PRMT3 facilitates glycolysis by promoting the arginine methylation of lactate dehydrogenase A (LDHA, a key factor in glycolysis) in liver cancer cells (Leiet al. 2022).PRMT3 inhibitor represses the osteogenesis and calcium deposition of mesenchymal stem cells (Minet al. 2019).Based on the vital role of glycolysis and calcification of VSMCs in CKD, it is possible that PRMT3 has the potential on vascular calcification.Furthermore, PRMT3 has been found to drives tumor progressions by the methylation and stabilization of hypoxia-induced factor 1α (HIF-1α) (Liao et al. 2022;Zhang et al. 2021), which could regulate the metabolic switch by upregulating the expression of glycolytic enzymes and flux (Kieranset al. 2021).These studies implied the potential of PRMT3 on vascular calcification might be associated with glycolysis by HIF-1α.
This study aimed to investigate the effects of PRMT3 on vascular calcification.Herein, a CKD model of mice was established, and β-GP-induced VSMCs were used for in vitro assessment.Then, the underlying mechanism was studied.

CKD induction and treatment
Animal experiment was approved by the Medical Ethic Committee of Shengjing Hospital of China Medical University (2022PS997K) following The Guideline for the Care and Use of Laboratory Animals.Eight-week old C57BL/6 mice (male) were housed at 22 ± 1 °C and feed ad libitum for a week of acclimation.They were randomly divided into three groups: Control, CKD, and CKD plus SGC707 (CKD + SGC707).CKD model was established as previously described (Tani et al. 2017;Tóth et al. 2022).Briefly, the mice in CKD and CKD + SGC707 group received a diet with 0.8% phosphate and 0.2% adenine for six weeks and then a diet with 1.8% phosphate and 0.2% adenine for four weeks.Meanwhile, mice in CKD + SGC707 group were intraperitoneal injected with 10 mg/kg SGC707.The dose of SGC707 was chosen according to the previous study (Hoekstraet al. 2019).Mice in control group were fed with 0.8% phosphate for ten weeks.Subsequently, mice were sacrificed, and the serum and aortic tissues were collected for further experiments.

Biochemical testing
Tissues were mixed with normal saline (1:9, g/v) and then mechanically homogenizing in ice bath.After centrifugation at 430 g for 10 min, the supernatant was collected.Meanwhile, cells resuspended with phosphate buffered saline were subjected to ultrasonication, followed by the collection of cell supernatant.The supernatant of tissues and cells, as well as serum of mice were used for further detection, respectively.The levels of blood urea nitrogen (BUN), creatinine, alkaline phosphatase (ALP), phosphorous, and calcium were detected using the Urea Assay Kit, Creatinine Assay kit, Alkaline Phosphatase Assay kit, Phosphate Assay Kit, and Calcium Assay Kit.All the kits were purchased from Nanjing Jiancheng Bioengineering.

Histological examination
Paraffin-embedded sections of aorta (5 μm) were deparaffinized in xylene, and rehydrated in an ethanol gradient with distilled water.For the assessment of aortic calcium deposition, the sections were stained with Alizarin Red S reagent (Solarbio, Beijing, China) according to the user's protocols.For the evaluation of PRMT3 expression, after block with 1% bovine serum albumin for 15 min at room temperature, the sections were incubated with the primary antibody anti-PRMT3 (1:50; Proteintech, Wuhan, China) at 4 °C overnight, followed by the incubation with the secondary antibodies horseradish peroxidaseconjugated goat anti-rabbit IgG (1:500; ThermoFisher Scientific, Pittsburgh, PA, USA) at 37 °C for 60 min.Subsequently, the sections were counterstained with hematoxylin for 3 min.For the measurement of pathological changes, the sections were conducted to hematoxylin and eosin staining.

Cell culture and cell calcification model
Human VSMCs were purchased from Procell (Wuhan, China).VSMCs were cultured with F12K medium supplemented with 10% fetal bovine serum at 37 °C in a 5% CO 2 incubator.VSMCs were conducted to a detection using the short tandem repeat profiling and PCR Mycoplasma Test Kit (Wanleibio, Shenyang, China) to confirm that the cells were not contaminated with other cell lines or mycoplasma contamination.After infected with/ without lentivirus for 48 h, cells were treated with 10 mM β-glycerophosphate (β-GP) for the induction of calcification for 1, 3, 7, and 14 d.Then, treatment with β-GP for 7d was selected for further experiments.

Adenovirus preparation
For the knockdown of PRMT3, short hairpin RNA targeting PRMT3 (shPRMT3) was designed and synthetized by GenScript (Nanjing, China).shPRMT3 or negative control was inserted into pShuttle-CMV-H1.The recombinant vector Ad-shPRMT3 (PRMT3 (−) ) and Ad-negative control shRNA (NC), was confirmed by enzymes digestion and sequence.Next, PRMT3 (−) was transfected into HEK293A cells with helper plasmid pAdEasy-1.The virus was collected and filtered using a 0.45-µm filter.Subsequently, VSMCs were infected with the particles with the multiplicity of infection at 100.

Quantitative RT-PCR
Total RNA was extracted from tissues or cells using the TRIpure Reagent (BioTeke, Beijing, China) and then conducted to cDNA synthesis using the BeyoRT II M-MLV reverse transcriptase (Beyotime, Shanghai, China) in accordance with manufacturers' instructions.Amplification was performed by employing the 2×Taq PCR MasterMix (Solarbio) in the presence of SYBR Green (Solarbio).Beta-actin was used as the internal control.The primer sequence was shown in Table 1.

Co-immunoprecipitation (Co-IP)
Protein samples were subjected to immunoprecipitation using a Pierce Co-IP Kit (Pierce, immunoprecipitation, IL, USA) in accordance with the users' protocols.In brief, anti-PRMT3 or anti-HIF-1α antibodies were covalently linked to an amine-active resin in a column.Next, the samples was added to the column and then incubated at 4 °C overnight.The immunoprecipitate of the protein samples with IgG was employed as a negative control.The co-precipitated protein was further subjected to western blot analysis.

Statistical analysis
Data was presented with mean and standard deviation of three independent experiments in vitro and six independent experiments in vivo.Statistical analysis was performed using GraphPad Prism 8.0 software (La Jolla, California, America).Comparsion between two groups was performed using Student's t-test.Differences among multiple groups were compared with one-way ANOVA analysis.Differences with p value < 0.05 were considered statistically significant.

PRMT3 is upregulated in thoracic aorta of CKD mice
To investigate the pathological changes of CKD, a mice model was established with high phosphorus diet as described above (Fig. 1A).As shown in Fig. 1B-E, the levels of BUN, creatinine, ALP, and phosphorus were elevated in the serum of CKD mice, indicating the renal injury and vascular calcification of CKD mice.Alizarin Red S staining confirmed that the aortic sections in CKD mice exhibited severe aortic calcification compared to control (Fig. 1F).Subsequently, the mRNA and protein expression of PRMT3 was detected.The results revealed that PRMT3 was upregulated in aorta of CKD mice (Fig. 1G).The results of immunohistochemical staining further confirmed the upregulation of PRMT3 in VSMCs of medial artery (Fig. 1H).

PRMT3 inhibitor SGC707 represses the vascular calcification and glycolysis of CKD mice
The effects of PRMT3 on the CKD-induced renal function and vascular calcification were further explored.SGC707 (PRMT3 inhibitor) attenuated the CKD-induced renal injury of CKD mice, as evidenced by the low levels of creatinine and BUN (Fig. 2A-B).There was a trend that the increased phosphorus content was reduced by SGC707 (Fig. 2C).Then, histological changes were assessed by ematoxylin-eosin and Alizarin Red S staining.Hematoxylin-eosin staining demonstrated that neatly arranged VSMCs and elastic lamellae were shown in control group, whereas disorder, vacuolization, and loss of elastic lamellae and VSMCs were observed in artery of CKD mice (Fig. 2D).SGC707 alleviated the pathological injuries (Fig. 2D).Next, SGC707 reduced the CKDinduced calcium deposition of artery (Fig. 2E-F).The decrease of calcium content in CKD mice with SGC707 treatment echoed the results indicated above (Fig. 2G).
Next, SGC707 inhibited the osteogenic differentiation, as evidenced by the reduction of ALP content (Fig. 2H), upregulation of α-SMA, SM22α, SMMHC and the downregulation of OPN and OCN (Fig. 2I).SGC707 decreased the CKD-induced upregulation protein levels of GLUT1 and PKM2 (Fig. 2I), indicating the repressed function in glycolysis.

PRMT3 is upregulated in the β-GP-induced VSMCs and facilitates the calcification
To deeply investigate the underlying mechanism of the promoted role of PRMT3 in media calcification, a widely used cell model was established with β-GP treatment (Fig. 3A).PRMT3 was upregulated in VSMCs with β-GP in a time-dependent manner (Fig. 3B).mRNA and protein expression of PRMT3 was decreased in VSMCs infected with adenovirus PRMT3 (−) compared to VSMCs infected with NC (Fig. 3C).Knockdown of PRMT3 reduced the β-GP-induced upregulation of PRMT3 and osteogenic marker RUNX family transcription factor 2 (RUNX2) (Fig. 3D-E).Then, Loss of PRMT3 weakened the ALP and calcium content of VSMCs in the presence of β-GP (Fig. 3F-G).The similar results were confirmed by Alizarin Red S staining (Fig. 3H-I).PRMT3 silencing elevated the protein levels of α-SMA, SM22α, SMMHC, and decreased the expression of OPN and OCN (Fig. 3J).These results implied that PRMT3 downregulation dampened the calcification of VSMCs induced by β-GP.

PRMT3 knockdown downregulates HIF-1α and represses the glycolysis of VSMCs induced by β-GP
HIF-1α could promote the key enzymes in glycolysis, which might act as an essential part in vascular calcification.The role of HIF-1α evoke us much attention.
As shown in Fig. 4A, the protein expression of HIF-1α was upregulated in VSMCs with β-GP treatment in a time-dependent manner.Interfering in the expression of PRMT3 reduced the upregulation of HIF-1α protein expression in VSMCs induced by β-GP (Fig. 4B).Then, PRMT3 depletion decreased the β-GP-induced upregulation of GLUT1, PFKFB3, PKM2, and LDHA (Fig. 4C-E).
The content of lactic acid was also declined by PRMT3 silencing (Fig. 4F).The data suggested that PRMT3 knockdown repressed the glycolysis in VSMCs induced by β-GP.

HIF-1α overexpression reversed the PRMT3 knockdowninduced repression of VSMCs calcification and glycolysis in the presence of β-GP
To better investigate the role of HIF-1α, HIF-1α (+) or Vec was infected into VSMCs with PRMT3 downregulation.Infection with HIF-1α (+) reversed the downregulated expression of HIF-1α, and OPN, and the upregulated levels of α-SMA in VSMCs in the presence of PRMT3 deficiency (Fig. 5A-D).HIF-1α overexpression reverted the PRMT3 knockdown-induced downregulation of RUNX2 mRNA expression and the decrease of ALP content (Fig. 5E-F).Unsurprisingly, HIF-1α overexpression aggravated the calcium deposition (Fig. 5G).Subsequently, the effects of HIF-1α on glycolysis were explored.High expression of HIF-1α reversed the reduction of lactic acid content and the downregulation of GLUT1 and PKM2 (Fig. 5H-J).The results indicated that the inhibitory effects of PRMT3 knockdown on β-GP-induced calcification in VSMCs might be at least partly mediated by

PRMT3 enhances the methylation of HIF-1α
The regulation of PRMT3 on HIF-1α expression was further explored.As shown in Fig. 6A, a physical interaction between PRMT3 and HIF-1α was observed.The interaction was enhanced in VSMCs with the β-GP induction (Fig. 6B-C).PRMT3 was downregulated in VSMCs infected with PRMT3 (−) in the presence of β-GP (Fig. 6D-E).PRMT3 dampened the asymmetric dimethylarginine levels of HIF-1α (Fig. 6F).
Next, the molecular mechanism was testified in vivo.
The CKD-induced upregulation of HIF-1α and the asymmetric dimethylarginine levels of HIF-1α were weakened by SGC707 (Fig. 6G-H).The results indicated that PRMT3 methylated HIF-1α both in vivo and in vitro.

Discussion
Vascular calcification is an actively regulated process, which can be found in the media and intima layer of arteries.Intimal calcification usually occurs in atherosclerotic lesions, whereas medial calcification is Because the calcification induced by medial VSMCs is dominant in CKD progression, this study aimed to investigate the effects of PRMT3 on CKD-induced medial calcification.Herein, we induced CKD employing a twophase diet, with the results of calcification of both media and intima layer of arteries being comparable to previous studies (Taniet al. 2017).Interestingly, the PRMT3 inhibitor SGC707 was found to mitigate the medial calcification, but not the intimal calcification according to the histopathological results.Our previous main focus was on CKD induced vascular calcification, whereas the changes of renal function evokes us much attention at the present.The renal function was declined in the CKD mice, as evidenced by the elevation of the metabolic waste including serum creatinine and BUN.The changes were further reversed by SGC707 treatment.However, the effects of SGC707 on (F) Whole-cell lysis was collected for co-immunoprecipitation employing anti-HIF-1α antibodies, and the samples were further subjected to western blot analysis using anti-HIF-1α and anti-Pan-Rme2a antibodies.(G) Protein levels of HIF-1α in aorta of mice.(H) Whole-tissue lysis was collected for coimmunoprecipitation employing anti-HIF-1α antibodies, and the samples were further subjected to western blot analysis using anti-HIF-1α and anti-Pan-Rme2a antibodies.Data is represented as mean ± SD. p < 0.01 vs. β-GP + NC or CKD.β-GP: β-glycerophosphate; HIF-1α: hypoxia inducible factor 1 subunit alpha; NC: negative control; PRMT3: protein arginine methyltransferase 3; VSMCs: vascular smooth muscle cells other kidney injury markers including urine biochemistries and physiological parameters as well as the injury markers in kidney remain unknown, which is another limitation of the manuscript.Subsequently, the levels of serum phosphate were detected.High levels of serum phosphate induce transformation of VSMC into osteoblast-like cells, this transformation having been closely associated to medial arterial calcification (Giachelli 2003;Lomashvili et al. 2008;Reynolds et al. 2004).Herein, there was a trend that the increased phosphorus and calcium contents in serum of CKD mice were reduced by SGC707 induction.We preferred that SGC707 exerted the protective function in vascular calcification might at least partly mediated by the reduction of phosphorus and calcium content.
PRMT3 was confirmed to be upregulated in the VSMCs in the medial artery of CKD mice by immunohistochemical staining.Interestingly, we found that VSMCs in medial artery of control mice exhibited irregular in shape, whereas VSMCs of CKD mice exhibited long shuttle in shape.We speculated that the differences between them might be associated with the changes of elastin and α-actin filaments as well as transdifferentiation of cells.Under non pathological conditions, VSMCs are arranged between elastic lamellae (Karimiet al. 2016).The elastin has oblique extension that connects with the surfaces of cells, and the smooth muscle α-actin extends obliquely across the VSMCs (Karimiet al. 2016).The crowd of abundance of elastin and α-actin might lead to the irregular appearance of VSMCs.It has been reported that elastin loss in media artery enhances the osteogenic transformation of VSMCs and is involved in medial vascular calcification (Zhouet al. 2019).Transdifferentiation of VSMCs into osteoblastic phenotype is the key cellular mechanism of medial calcification (Patelet al. 2019).It is possible that VSMCs exhibited differenced appearance between control and CKD mice due to the differences of arterial microenvironment, which is consistent with the previous study (Maet al. 2020).
Glucose metabolism is clarified to be closely linked with vascular calcification (Nakahara et al. 2017;Zhu et al. 2020).Glycolysis is involved in the osteogenic differentiation and phenotype switch of VSMCs.The inhibition of glycolysis mitigates the VSMC calcification by the regulation of PFKFB3-lactate signaling (Niuet al. 2021).The promoted effects of PRMT3 on glycolysis have been found in tumorigenesis (Leiet al. 2022), implying the potential of PRMT3 on calcification might be related to glycolysis.Herein, PRMT3 knockdown inhibited the β-GP-induced calcification of VSMCs via the repression of glycolysis.The similar role of PRMT3 loss was observed in CKD-induced vascular calcification in vivo.
Moreover, glycolysis is the major energetic source of cells that is stimulated by hypoxia.Hypoxia is a primary condition leading to vascular calcification (Baloghet al. 2019).The activation of hypoxic genes including HIF-1α is related to CKD and triggers osteogenesis (Araldiet al. 2010).Herein, β-GP induced the upregulated the expression of HIF-1α in VSMCs.Nomoto et al. have testified that HIF-1α activation contributes to elevated PFKFB3 (the key enzyme in glycolysis) levels, causing the increased glycolysis products including LDH and lactic acid in diabetes (Nomotoet al. 2020).The similar results were obtained in the current study.HIF-1α overexpression reversed the PRMT3 depletion -induced calcification and glycolysis of VSMCs in the presence of β-GP.Subsequently, the underlying mechanism of the regulation of PRMT3 on HIF-1α was explored.The expression of HIF-1α could be regulated by several posttranslational modifications including ubiquitination, acetylation, and methylation (Geng et al. 2012;Kim et al. 2016;Zhang et al. 2021).This study revealed a HIF-1α modification by methylation.Loss of PRMT3 downregulated the expression of HIF-1α both in vivo and in vitro.However, the sites of methylation HIF-1α remain unknown, which is a limitation of this study.In colorectal cancer cells, R282 of HIF-1α is asymmetrically di-methylated by PRMT3 protein, and the dimethylation of HIF-1α reduces its ploy-ubiquitination levels (Zhanget al. 2021).The similar mechanism might be existed in the progression of CKD-induced vascular calcification, which will be studied in our further investigation.

Conclusion
The expression of PRMT3 was upregulated in VSMCs of medial artery of CKD mice and β-GP-induced VSMCs.In vivo, the inhibitor of PRMT3 (SGC707) attenuated the vascular calcification and repressed the glycolysis of CKD mice.In vitro, loss of PRMT3 alleviated the β-GP-induced osteogenic transfomation of VSMCs by the repression of glycolysis.Next, PRMT3 interacted with HIF-1α, and the knockdown of PRMT3 downregulated the protein expression of HIF-1α by inhibiting its methylation levels.Gain of HIF-1α reverted the PRMT3 depletion-induced suppression of osteogenic differentiation and glycolysis of VSMCs.The results suggest that PRMT3 boosts the CKD-induced vascular calcification by enhancing the glycolysis upon the arginine methylation of HIF-1α (Fig. 7).

Fig. 1 Fig. 2
Fig.1PRMT3 is upregulated in thoracic aorta of CKD mice C57BL/6 mice were received a diet containing 0.8% phosphorus and 0.2% adenine for six weeks, followed by a diet containing 1.8% phosphorus and 0.2% adenine for four weeks.(A) Experimental protocol of CKD mouse model.The levels of BUN (B), creatinine (C), ALP (D), and phosphorus (E) were elevated in the serum of mice.(F) Aorta calcification was evaluated by Alizarin Red S. Scar bar is 50 μm.(G) mRNA and protein levels of PRMT3 were detected in aorta of mice.(H) PRMT3 expression was assessed by immunohistochemical staining.Scar bar is 50 μm.Data is represented as mean ± SD. p < 0.05, p < 0.01 vs. Control.ALP: alkaline phosphatase; BUN: blood urea nitrogen; CKD: chronic kidney disease; PRMT3: protein arginine methyltransferase 3

Fig. 6
Fig. 6 PRMT3 knockdown suppresses the methylation of HIF-1α (A-B) VSMCs were incubated with/without 10 mM β-GP.Whole-cell lysis was collected for co-immunoprecipitation employing anti-PRMT3 antibodies, and the samples were further subjected to western blot analysis using identified antibodies.(C) Quantification of HIF-1α upon IP normalization in VSMCs.(D-E) Protein levels of PRMT3 in VSMCs infected with Ad-shPRMT3 (shown as PRMT3 (−) ) or Ad-NC shRNA (shown as NC) in the presence of β-GP.(F)Whole-cell lysis was collected for co-immunoprecipitation employing anti-HIF-1α antibodies, and the samples were further subjected to western blot analysis using anti-HIF-1α and anti-Pan-Rme2a antibodies.(G) Protein levels of HIF-1α in aorta of mice.(H) Whole-tissue lysis was collected for coimmunoprecipitation employing anti-HIF-1α antibodies, and the samples were further subjected to western blot analysis using anti-HIF-1α and anti-Pan-Rme2a antibodies.Data is represented as mean ± SD. p < 0.01 vs. β-GP + NC or CKD.β-GP: β-glycerophosphate; HIF-1α: hypoxia inducible factor 1 subunit alpha; NC: negative control; PRMT3: protein arginine methyltransferase 3; VSMCs: vascular smooth muscle cells

Table 1
Sequences of primers used in quantitative RT-PCR