TMT-Based Quantitative Proteomic Analysis Identification of Integrin Alpha 3 and Integrin Alpha 5 as Novel Biomarkers in Pathogenesis of Acute Aortic Dissection

Background Acute aortic dissection (AAD) is a devastating cardiovascular disease with a high rate of disability and mortality. This disease often rapidly progresses to fatal multiple organ hypoperfusion, and the incidence has been increasing in recent years. However, the molecular mechanisms have yet to be clarified. This study is aimed at identifying the differential abundance proteins (DAPs) of aortic arch tissues in patients with AAD by proteomics and select possible proteins involved in AAD pathogenesis. Methods The fresh aortic arch tissues obtained from 5 AAD patients and 1 healthy donor were analyzed by amine-reactive tandem mass tag (TMT) labelling and mass spectrometry; then, the pathological sections of another 10 healthy donors and 20 AAD patients were chosen to verify the proteomic results by immunohistochemistry. Results Of 809 proteins identified by proteomic analysis, 132 differential abundance proteins (DAPs) were screened, of which 100 proteins were significantly downregulated while 32 upregulated. Among 100 downregulated proteins, two proteins with known function, integrin alpha 3 (ITGA-3) and ITGA-5, were selected as target proteins involved in AAD pathogenesis. Two target DAPs were verified by immunohistochemisty, and the results showed that the integrated option density (IOD) of ITGA-3 and ITGA-5 in AAD patients was significantly lower than that in healthy donors, which were consistent with the proteomic results (P < 0.001). Conclusion ITGA-3 and ITGA-5 represent novel biomarkers for the pathogenesis of AAD and might be a therapeutic target in the future.


Introduction
Acute aortic dissection (AAD) is one of the most serious acute aorta syndrome characterized by severe chest and/or back pain. AAD of the ascending aorta is highly lethal (a mortality rate of 1%-2% per hour early after symptom onset) that requires prompt diagnosis and surgical intervention to optimize outcomes [1][2][3]. The etiologies include not only mechanical stimulation such as hypertension and trauma but also vascular injury caused by atherosclerosis, inflammation, and connective tissue disease [4]. Despite recent progress in recognition of the diagnostic and therapeutic advances, clinicians are far from comfortable in defining an optimal therapy to manage aortic dissection, and AAD is still a focus and key point in the field of cardiovascular surgery and critical care so far.
AAD is a surgical emergency occurring when an intima of the aorta is damaged and ruptured and blood enters the media creating a false lumen between the two layers. The typical pathophysiological changes include large modifications to the extracellular matrix, oxidative stress, inflammatory response, vascular smooth muscle (VSM) hypertrophy, and high expression of metalloproteinase, which directly lead to the compensatory hyperplasia of collagen fiber, apoptosis and phenotypic transformation of vascular smooth muscle cells (VSMCs), and steady degradation of the extracellular matrix proteins in the aortic wall [5][6][7][8][9][10][11][12][13]. Current research showed that the activation of the TGF-β1 signaling pathway and high expression of its downstream matrix metalloproteinase (MMPs) are involved in the pathogenesis of AAD [14][15][16]. Studies also have found that the PI3K/AKT signaling pathway contributes to phenotypic switching of vascular smooth muscle cell (VSMC) and induces apoptosis [17][18][19]. However, detailed molecular mechanisms still need to be explored.
Quantitative mass spectrometry (MS) is an adjunctive tool to help understand the mechanisms of many diseases [20]. Large-scale identification of differential abundance proteins (DAPs) can be used to identify key proteins, and immunohistochemistry is an important traditional technique used to verify the selected proteins. The objective of this study was to identify and quantify DAPs of aortic arch tissues in patients with AAD using TMT-labelled MS and further to verify the selected DAPs of interest using immunohistochemistry, by which we hope to find some possible proteins involved in the pathogenesis of AAD. A flowchart to illustrate this study is shown in Figure 1.

Tandem Mass Tag Labelling
2.1.1. Sample. The dissected aortic tissues (aortic arch including all layers of the wall: intima, media, and adventitia) from 5 AAD patients were extracted during surgery. In addition, one piece of healthy aortic arch tissue was obtained from a 44-year-old male donor. CTA was performed in all patients to confirm the diagnosis (all were Stanford type A dissection). Four out of them with hypertension history complained of chest and/or back pain, while one 22-year-old patient, with no history of hypertension and trauma, no family history of Marfan syndrome, and no typical clinical symptoms, was diagnosed with AAD. The clinical features and more detailed description of dissected aortic samples are summarized in Supplementary Table 1.
All tissues were collected as typical lesion specimens of approximately 1:0 × 1:0 cm 2 in size. Specimens were immediately placed in an ice-bath, RNA-free specimen box and transferred to a −80°C refrigerator for preservation. The study was approved by the ethics committee of Zhongshan Hospital, Fudan University (Shanghai, China). Informed consent was obtained from patients or their legal surrogates before enrolment.

Sample Pretreatment.
Tissues were carefully pulled out from the frozen tubes, cut into pieces, weighed about 50 mg, added 10 times extraction buffer, and extracted protein with a grinding mill. The extracted samples were spun to remove the residue of tissues. The protein concentration of the extracted sample solution was measured with a BCA Protein Assay kit (Pierce). Proteins extracted from tissue were precipitated with a TCA Protein Precipitation Kit (QYBIO). The protein pellet was dried out by SpeedVac. The pellet was subsequently dissolved with 8 M urea in 100 mM Tris-Cl (pH 8.5, Sigma). Tris(2-carboxyethyl)phosphine (TCEP, final concentration is 5 mM, Thermo Scientific) and iodoacetamide (final concentration is 10 mM, Sigma) were added to the solution and incubated at room temperature for 20 and 15 minutes for reduction and alkylation, respectively. The solution was digested with LysC at 1 : 100 (w/w) (Promega) for 4 h and then diluted four times and digested with trypsin at 1 : 50 (w/w) (Promega) for 16 h. The digested peptide mixtures were labelled with the TMT kit (6-plex, Thermo).

Liquid Chromatography Tandem Mass Spectrometry
Analysis of Peptides. For multidimensional protein identification technology (MudPIT), total peptide mixtures were pressure loaded onto a biphasic-fused silica capillary column. The entire column setting (biphasic column-union-analytical column) was placed in line with an Agilent 1200 quaternary high-performance liquid chromatography pump (Palo Alto, CA) for MS analysis. The digested proteins were analyzed using a 12-step MudPIT separation method [21] as described previously.

Mass Spectrometry
Condition. Data-dependent tandem mass spectrometry (MS/MS) analysis was performed with an Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA). Peptides eluted from the liquid chromatography system were directly electrosprayed into the mass spectrometer with a distal 1.5 kV spray voltage. One acquisition cycle included one full-scan MS spectrum (m/z, 300-1800), followed by the top 20 MS/MS events.
2.1.5. Data Analysis. The acquired MS/MS data were analyzed against a Uniprot Homo sapiens database (database released on Sep. 24, 2015) using Integrated Proteomics Pipeline (IP2, http://integratedproteomics.com/). To estimate peptide probabilities and false discovery rates accurately, we used a decoy database containing the reversed sequences of all the proteins appended to the target database. Carbamidomethylation of cysteine was considered as a static modification.  Table 3. The study was also approved by the ethics committee of Zhongshan Hospital of Fudan University.

Immunohistochemistry
Method. The expression of integrin alpha 3 (ITGA-3) and integrin alpha 5 (ITGA-5) were compared stained with DAB staining and hematoxylin. Briefly, paraffin sections were dewaxed, and antigen was retrieved by citric acid buffer. The primary antibody of ITGA-3 and ITGA-5 is the rabbit antibody (Abcam, ab131055 and ab239400, respectively, 1 : 100). The corresponding secondary antibody is HRP-goat anti-rabbit IgG (H+L) (GXYbio, S8002, 1 : 100). The sections were reacted with the DAB kit (ZSGB-BIO, China, ZLI-9019). Eight separate views (magnification = original × 400) were randomly selected. The integrated option density (IOD) of ITGA-3 and ITGA-5 was chosen to determine the protein semiquantitative expression. Free ImageJ software (version 1.2; WS Rasband, National Institute of Health, Bethesda, MD) was used to conduct deconvolution and downstream analysis [22], and then, the Mann-Whitney test of the IOD was performed using SPSS software (version 25.0, IBM). A total of 809 proteins were identified from individual unique peptides (Supplementary Table 2). Limited by the number of the healthy control, we defined the identified proteins as DAPs if there was a log 2 FC in excess of 2 or in less of -2.132 proteins changed significantly (32 upregulated and 100 downregulated) in AAD patients compared with the healthy donor (Tables 1 and 2).

BioMed Research International
To explain the cellular localization and associated functions of DAPs, gene Ontology analysis (GO analysis) was performed by Blast2GO software (version 4) to provide further understanding of these results by biological processes, cell components, and molecular function ( Figure 3). According to the GO results, most DAPs were located in the extracellular region part and participated in several functions, such as cell junction, metabolic process, and developmental process, indicating that proteins involved in extracellular activities were important in AAD tissues. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was used to classify the functional annotations of DAPs. Interestingly, among the 30 significantly enriched pathways, most of downregulated DAPs (n = 16/51) were found in the PI3K-AKT signaling pathway ( Figure 4). Protein-protein interaction (PPI) analysis further suggested that ITGA-3 and ITGA-5 were important nodes in the PI3K-AKT signaling pathway ( Figure 5).

Verification of the Levels of ITGA-3 and ITGA-5 by
Immunohistochemistry. The levels of ITGA-3 and ITGA-5 in pathological sections of 20 AAD patients and 10 healthy donors were further assessed by immunohistochemistry (Figure 6(a)). The basic information of them is shown in Supplementary Table 3. The IOD of ITGA-3 and ITGA-5 in AAD patients was significantly lower compared with healthy donors (P < 0:001) (Figure 6(b)).

Discussion
AAD of the ascending aorta is a life-threatening cardiovascular emergency with a mortality rate of 1% to 2% per hour early after the symptom onset [1,3]. With increasing incidence of hypertension, the morbidity of AAD has increased annually in recent years [23]. However, the detailed molecular mechanisms of this disease have yet to be clarified. Finding out important proteins in dissected aorta tissues of patients with AAD can help to elucidate the pathogenesis of this disease. In this study, the quantitative proteomics showed that 100 proteins were significantly downregulated while 32 upregulated in dissected aorta tissues of AAD patients compared with those in ascending aorta tissue of the healthy donor. Bioinformatic analysis revealed that most DAPs were primarily located in the extracellular region part and their biological functions mainly focused on cell junction, metabolic process, and developmental process. Interestingly, the PI3K-AKT signaling pathway was selected by KEGG analysis, which has been reported involved in a series of biological processes by transducing stimulatory extracellular signals to the nucleus, including proliferation, apoptosis, angiogenesis, and tumor growth [24,25]. Liu and his colleagues demonstrated that the PI3K/AKT signaling transduction pathway was involved in rat vascular smooth muscle cell proliferation induced by apelin-13 [18]. Other studies also have shown that the 0PI3K/AKT signaling pathway contributes to VSMC dysfunction, vasoconstriction, and vascular remodeling [19,26]. However, few studies reported the molecule or protein in the PI3K/AKT signaling pathway contributing to the VSMC dysfunction in human aorta tissues.
In this study, we found that integrins, especially ITGA-3 and ITGA-5, were significantly downregulated in dissected aorta tissues of AAD patients. As expected, ITGA-3 and ITGA-5 showed a strong downexpression in the cytomembrane and cytoplasm. As a kind of cell surface adhesion molecule with signal transduction function, integrins are key transmembrane protein upstream in the P13K-AKT signaling pathway. By connecting the extracellular matrix with actin cytoskeleton, it can maintain cell morphology and mediate cell adhesion, proliferation, differentiation, and other physiological processes according to the types of extracellular ligands. Downregulation of integrin may affect the stability of cytoskeleton, thus ultimately affecting the physiological function of cells [27,28]. Taken together, downregulation of ITGA-3 and ITGA-5 might be involved in the pathogenesis of AAD by reducing the adhesion between cell and extracellular matrix and modulating the focal adhesion pathway.
The proteomic approach provides an exciting platform for determining the pathogenesis of aortic dissection. As an initial step, our study identified the downregulation of ITGA-3 and ITGA-5, both of which may participate in AAD pathogenesis via the focal adhesion pathway. To make up for the control sample shortage in the proteomic study, we 7 BioMed Research International obtained the pathological sections of another 10 healthy donors and 20 AAD patients from the Pathological Sample Library of Zhongshan Hospital to further make our results credible in the following validation test. The performance of the PI3K-AKT signaling pathway needs to activate downstream proteins, which provides insights into the specific molecule or signaling pathway contributing to the pathogen-esis of aortic dissection. It needs to be further explored in animals and humans.

Conclusions
The aortic arch tissues of patients with AAD show a large number of DAPs, the molecular functions of which were  (18) Transcription regulator activity (14) Antioxidant activity (12) Translation regulator activity (10) Cargo receptor activity (4)