VDAC1 and RhoA in Plasma Exosomes Inuence the Severity of Adolescent Idiopathic Scoliosis

Background: Adolescent idiopathic scoliosis (AIS) is the most common spine deformity, but biomarkers for its condition are lacking. Rhodopsin A (RhoA) and voltage-dependent anion-selective channel 1 (VDAC1) in plasma exosomes were dened as differentially expressed proteins between AIS patients and healthy controls. The purpose of this study was to assess exosomes as biomarkers for the occurrence and progression of AIS. Methods (cid:0) We recruited 10 AIS patients and 8 healthy controls to detect expressed proteins from plasma by liquid chromatography coupled to tandem mass spectrometry. Plasma samples were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Pathway analysis identied that the VDAC1 and RhoA proteins were alterations expressed in the AIS patients, with the most different alteration was found in extracellular exosomes. Ultracentrifugation was carried out to isolate exosomes from plasma. Verication of the most differentially expressed protein was accessed by Western blot analysis and bioinformatics analysis was performed to predict the pathway of it. Results: 42 of signicantly differentially expressed proteins were found in all subjects, and 17 proteins had signicant difference. The differentially expressed proteins were enriched in plasma exosomes, and some proteins, such as FN1, were upregulated and others, such as VDAC1, RhoA and AHNAK, were downregulated in the AIS patients. Furthermore, ultracentrifugation was carried out to isolate exosomes from plasma, and RhoA and VDAC1 proteins in plasma exosomes were veried to downregulate by western blot. KEGG signaling pathways were used to predict potential pathways involved in the RhoA and VDAC1 proteins in the AIS patients. We found that the RhoA protein inuences AIS probably through the chemokine signaling pathway, platelet activation and cAMP signaling pathway, and the VDAC1 protein is a key factor that participates in the necroptosis pathway, acting on the development of AIS. Conclusions: Consequently, this study mapped a prole of plasma

downregulated in the AIS patients. Furthermore, ultracentrifugation was carried out to isolate exosomes from plasma, and RhoA and VDAC1 proteins in plasma exosomes were veri ed to downregulate by western blot. KEGG signaling pathways were used to predict potential pathways involved in the RhoA and VDAC1 proteins in the AIS patients. We found that the RhoA protein in uences AIS probably through the chemokine signaling pathway, platelet activation and cAMP signaling pathway, and the VDAC1 protein is a key factor that participates in the necroptosis pathway, acting on the development of AIS.
Conclusions: Consequently, this study mapped a pro le of plasma protein, found the differentially expressed protein in AIS, which indicating that plasma exosomes, as a novel biomarker with high speci city, could be associated with the severity of AIS. Background Adolescent idiopathic scoliosis (AIS) is de ned as a lateral curvature of the spine with a Cobb angle of at least 10° [ 1] and affects approximately 0.47-11.1% of the general population [2] . The reported prevalence was 1.8% [3] . AIS is a highly heterogeneous condition, with some patients showing rapid progression [4] . This disease increases the risk of physical and mental symptoms, such as trunk deformity and pulmonary impairment [5,6] . Several previous studies failed to identify biomarkers of AIS [7,8] . Therefore, screening biomarkers that can be used in the early identi cation and diagnosis of AIS is important for monitoring curvature progression before skeletal maturation. Page 3/19 Exosomes are 20-100 nm in size and are enveloped by a phospholipid membrane. These particles are produced by normal as well as malignant cells and are present in all human body uids [9] . Exosomes are considered a biomarker for many diseases [10][11][12][13][14] . Similar to cells, exosomes are composed of lipid bilayers and contain many molecular components of cells, such as proteins, miRNAs, and DNA [15] . We used proteins to con rm the role of exosomes because they usually display a high purity [16] and stable expression.
In this study, in order to investigate novel biomarkers of AIS, we used mass spectrometry (MS) to screen the differentially expressed plasma proteins between AIS patients and healthy controls, and the potential biomarkers of exosomal proteins in AIS patients were con rmed.

Patients and clinical samples
All the participants provided written informed consent. A total of 10 AIS patients and 8 matched healthy individuals with similar baselines (Table 1) were enrolled from the Xinhua Hospital a liated with the Shanghai Jiaotong University School of Medicine from August 2017 to August 2018. According to The Scoliosis Research Society (SRS), all enrolled AIS patients were con rmed when the Cobb angle was 10°o r higher and axial rotation was observed by radiography and a physical examination. The study processes are shown in Fig. 1. Ten milliliters of venous blood were collected in EDTA anticoagulant tubes. Plasma was obtained after a 15-min centrifugation at 300 g to remove the cells and a second 5-mi centrifugation at 1500 g to remove the remaining platelets and apoptotic bodies. The puri ed plasma samples were stored at -80 °C until further use.
Isolation of the exosomes from the blood plasma with ultracentrifugation After centrifugation at 2,000 g and 12,000 g to remove the cells and cellular debris, the exosomal supernatant was centrifuged at 110,000 g for 2 hours to collect the exosomal fraction. Then, the suspension was ltered through a 0.22 µm lter, centrifuged at 110,000 g for 70 min two times, and resuspended in PBS. All centrifugation steps were performed at 4 °C.
Flow cytometry and analysis of particle size To con rm the contents of the puri ed exosomes from the plasma samples, we used an Accuri C6 ow cytometer (BD Instruments) to detect CD63 and CD81 by ow cytometric analysis. The size distribution of both kinds of exosomes was evaluated with a Zetasizer Nano series-Nano-ZS (Malvern Instruments).

Electron microscopy
Fifty microliters of exosomal proteins was adsorbed by copper grids and xed with 1% glutaraldehyde. After staining with 3% phosphotungstic acid solution, the grids and the samples were dried, and a JEM1230 system was used to image the exosome samples.

LC-MS/MS
Sample dilution buffer (100 mM, 10 mM HEPES, pH = 7.4) was added to the blood sample to remove the lipids, and then, cold ethanol solution was used to remove the peak components. Next, we enzymatically hydrolyzed the protein sample by TEAB solution containing trypsin. Then, label reagent was added to each tube, and the sample corresponding to each TMT color was recorded and mixed. The TMT-labeled peptide mixture was separated on an Agilent 1260 HPLC system at a rate of 0.2 ml/min using a Water

Bioinformatics and statistical analyses
Functional gene ontology enrichment analysis of biological processes, molecular functions, and cellular components was performed using DAVID Bioinformatics Resources version 6.7. The protein-protein interaction (PPI) network of differentially expressed proteins was analyzed by STRING. The PPI network was processed by Cytoscape software. The pathway network was further analyzed by Ingenuity Pathway Analysis (IPA).
All statistical analyses were performed using SPSS 20.0 statistical software. Data are expressed as the mean ± standard deviation. The Pearson correlation test was used for the correlation between the Cobb angle and the differentially expressed proteins. Any difference with a P value less than 0.05 after correction was considered signi cant.

Results
Exosomes proteins were differentially expressed in the AIS patients and the controls After the exosomes were isolated from the plasma of the AIS patients and the controls, we performed MS to screen the differentially abundant proteins in the exosomes of the plasma samples from the AIS patients versus the healthy controls (log 2 ratio > 1.5 or < -1.5). A total of 652 proteins were identi ed, and 626 proteins had quantitative information. Forty-two signi cantly differentially expressed proteins were found in the AIS group and the control group, and 17 proteins had values greater than 1.5 ( Fig. 2A, B) ( Table 2). The differentially expressed proteins were selected for further analysis. According to the cellular component analysis, the differentially expressed proteins were enriched in extracellular exosomes, followed by cytosol and extracellular spaces (Fig. 2C). These differentially expressed proteins may act on AIS by participating in biological processes such as cell-cell adhesion, proteolysis, and the Fc-epsilon receptor signaling pathway (Fig. 2D). In the AIS patients, some proteins, such as FN1, were upregulated and others, such as VDAC1, RhoA and AHNAK, were downregulated. The FN1, VDAC1, RhoA and AHNAK proteins interacted to in uence the development of AIS (Fig. 2E). These results suggest that the plasma exosomal proteins FN1, VDAC1, RhoA and AHNAK are likely biomarkers of AIS.
The RhoA and VDAC1 proteins were downregulated in the AIS patients To further determine whether the proteins were signi cantly different in the AIS patients, we selected FN1, RhoA, VDAC1 and AHNAK for veri cation in vitro. Proteomic analysis con rmed that the differentially expressed proteins in the AIS and control groups were mostly distributed in the extracellular exosomes; thus, the characteristics and target proteins of the plasma exosomes were further veri ed and analyzed in this study.
Extracellular vehicles (EVs) isolated from the plasma of the AIS patients and the controls were identi ed by using transmission electron microscopy and particle size detection (Fig. 3A, B). Additionally, western blotting was performed to detect the expression of the exosomal marker CD63 and the endoplasmic reticulum-speci c marker calnexin but not the transmembrane protein TSG101 (Fig. 3C). Flow cytometric analysis of the exosomal markers CD63 and CD81 con rmed that the isolated EVs were puri ed exosomes (Fig. 3D).
To determine whether the RhoA, VDAC1, FN1 and AHNAK protein levels differed between the AIS and control groups, we performed western blot analyses of the two groups. The results showed that the RhoA and VDAC1 protein levels in the AIS group were signi cantly lower than those in the control group, consistent with the MS results (Fig. 3F). However, the FN1 protein was downregulated in the AIS patients (Fig. 3E), contrary to the MS results. After analyzing the grayscale values, we found that the RhoA protein expression in the controls was greater than twice that of the AIS patients. The AHNAK protein did not differ between the AIS patients and the controls. Taken together, the results showed that RhoA and VDAC1 may play a vital role in AIS patients.
The RhoA and VDAC1 were associated with the severity in the AIS patients To determine whether RhoA and VDAC1 expression correlates with the severity of AIS patients, we further analyzed the clinical characteristics and protein expression. Comparing the patients' baseline values, we observed that the Cobb angle had a signi cant association with the RhoA and VDAC1 proteins. The Cobb angle is used for quantitative assessment of the lateral curvature of the spine, representing the severity of AIS. After Pearson analysis, the results showed that the Cobb angle was negatively correlated with the RhoA and VDAC1 proteins (Fig. 4), and the formulas were shown as below: The related pathways were involved in the RhoA and VDAC1 proteins in the AIS patients To explore how the RhoA and VDAC1 proteins are involved in AIS, we next performed an analysis of the KEGG signaling pathways for Homo sapiens (human). The RhoA protein in uences AIS primarily through the chemokine signaling pathway, platelet activation and cAMP signaling pathway, and the VDAC1 protein is a key factor that participates in the necroptosis pathway, acting on the development of AIS. These data showed that the RhoA and VDAC1 proteins are likely predictors of AIS. The pathway networks are shown (Fig. 5-6).

Discussion
This is the rst report con rming the role of exosomes in AIS. We identi ed the differentially expressed proteins VDAC1, RhoA, FN1 and AHNAK in AIS, and VDAC1 and RhoA were downregulated, which is related to the severity and progression of the disease. Exosomes are likely potential biomarkers and predictors of AIS.
Our study rst screened the differentially expressed proteins in the total plasma proteins, and the differentially expressed proteins were concentrated in the exosomes. To further identify biomarkers, we chose to verify the proteins in the exosomes. The results demonstrated that the RhoA and VDAC1 proteins were downregulated in AIS, consistent with the MS data. RhoA is an important regulator of platelet function and development [16] . RhoA directs cytoskeletal reorganization to promote shape changes and particle release during hemostasis, activating [16] platelets and the signaling pathways of damaged vessel walls. Previous studies have con rmed the elevated levels of calcium and phosphorus in the platelets from AIS patients [17] , and the level of platelet calmodulin is a predictor of progression of the curve [18] , and some proteins over or under expressed by AIS Lenke type 5 patients were involved in the blood coagulation pathway [21] . In the platelet-skeleton hypothesis, platelet activation is thought to be accompanied by changes in calmodulin in the dilated vessels of AIS, which are related vertebral deformation, while activated platelets release growth factors in the blood vessels of the near-growth plate, stimulating the development of the vertebral endplate after mechanical injury and affecting the progression of AIS [19] . Therefore, we believe that RhoA affected the scoliosis progression of AIS by regulating platelet function. As a key factor in mitochondrial apoptosis, VDAC1 is involved in the release of mitochondrial proapoptotic proteins into the cytosol, and overexpression and dysregulation of this pathway induce apoptosis and other related diseases. The asymmetry of the paravertebral muscles has been observed in AIS. We hypothesize that under different dynamic cell conditions, the difference between activated chondrocyte proliferation and apoptosis in the vertebral body and the convex side growth plate leads to lateral curvature and rotation. TheVDAC1 protein may in uence the growth and imbalance of muscles and bones by promoting the apoptosis and proliferation of chondrocytes, resulting in scoliosis. This result is consistent with the previous study, which given that vitamin D binding protein (DBP) is associated with bone metabolism and play an important role in the pathogenesis of AIS or disease severity [21] .
However, the results for FN1 were inconsistent in MS and western blotting. Most likely, total plasma proteins were mixed with other components detected by MS, but western blotting focused only on the exosomes. Previous studies have shown that FN is increased in AIS patients with Lenke type 5 curvature [21] . Therefore, more studies are needed to demonstrate the role of FN1 in AIS in the future, but we believe that exosomes are useful in our study because these particle achieve intercellular material transfer and information exchange and are involved in regulating cellular physiological functions and certain pathological processes [20] .
This study has some limitations. First, the validation of the biomarkers was performed in a small cohort of patients, and only 18 subjects including 10 patients with primary moderate to severe AIS were enrolled. Second, considering that AIS is more common among females than males, this study needed more female subjects. Third, the selection of the target proteins may be biased. Previous studies have reported that the VDAC1, RhoA, FN1 and AHNAK proteins are related to AIS. Moreover, other related differentially expressed proteins should be studied. Exosomes should be examined in animals in the future.

Conclusions
In this study, the rst to examine exosomes in AIS, we demonstrated that exosomes are potential biomarkers of AIS, and RhoA and VDAC1, as target proteins in AIS, were downregulated, predicting the severity of AIS. Therefore, exosomes can probably be used for the early diagnosis of AIS.

Ethics approval and consent to participate
The study was approved by the Ethics Committee of Xinhua Hospital A liated to Shanghai Jiao Tong University School of Medicine and written informed consent was obtained from each patient.
Shanghai Jiao Tong University School of Medicine (17CSK02). The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Authors' Contributions
QD, NS and XZ participated in the study design, data collection, analysis of data and preparation of the manuscript.LM , NC, XL and YY carried out the experimental work and the data collection. HL, MC, JL participated in the interpretation of data and drafted the manuscript. QD critically revised the manuscript. All authors read and approved the nal manuscript. Figure 1 Study enrollment and study processes.    The predicted interactions in the differentially expressed genes related to RhoA, AHNAK and FN1 in the AIS patients.