BioMed Research International

BioMed Research International / 2020 / Article

Research Article | Open Access

Volume 2020 |Article ID 1068402 | https://doi.org/10.1155/2020/1068402

Lingyu Xing, Yuan Xue, Yilin Yang, Ping Wu, Catherine C. L. Wong, Haojun Wang, Zhenju Song, Dongwei Shi, Chaoyang Tong, Chenling Yao, Guorong Gu, "TMT-Based Quantitative Proteomic Analysis Identification of Integrin Alpha 3 and Integrin Alpha 5 as Novel Biomarkers in Pathogenesis of Acute Aortic Dissection", BioMed Research International, vol. 2020, Article ID 1068402, 12 pages, 2020. https://doi.org/10.1155/2020/1068402

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

Academic Editor: Aijun Wang
Received20 Jan 2020
Revised27 Apr 2020
Accepted04 Jun 2020
Published02 Jul 2020

Abstract

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 (). Conclusion. ITGA-3 and ITGA-5 represent novel biomarkers for the pathogenesis of AAD and might be a therapeutic target in the future.

1. 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 [13]. 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 [513]. 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 [1416]. Studies also have found that the PI3K/AKT signaling pathway contributes to phenotypic switching of vascular smooth muscle cell (VSMC) and induces apoptosis [1719]. 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.

2. Materials and Methods

2.1. 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  cm2 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.

2.1.2. 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 () (Promega) for 4 h and then diluted four times and digested with trypsin at 1 : 50 () (Promega) for 16 h. The digested peptide mixtures were labelled with the TMT kit (6-plex, Thermo).

2.1.3. 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.

2.1.4. 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 (, 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.

2.2. Immunohistochemistry
2.2.1. Sample

The aortic arch tissue’s paraffin sections of another 20 patients with AAD and 10 healthy donors were performed and provided by the Pathological Sample Library of Zhongshan Hospital of Fudan University. The standardized method to fix the tissues would be operated by professional staff as follows: approximately  cm3 of tissue would be extracted during surgery and fixed with formalin rapidly for 24 h; paraffin sections are performed and stored in the sample library. The basic clinical information is shown in Supplementary Table 3. The study was also approved by the ethics committee of Zhongshan Hospital of Fudan University.

2.2.2. 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 () 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).

3. Results

3.1. Identification Differential Abundance Proteins (DAPs) of Ascending Aorta Tissues in Patients with AAD Using TMT Quantitative Proteomics

TMT-labelled MS analysis was performed on fresh aortic arch tissues of 5 AAD patients and one healthy donor. Principal component analysis (PCA) clustergram was created using OmicsBean software from proteins identified by quantitative MS. Except patient 1, other patients have consistent clustering as shown in PCA and heat map analysis (Figure 2). Patient 1 might fall in different subtypes of AAD compared with the other patients.

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 log2FC 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).


AccessionExpression of healthy donorMean expression of AAD patients ()log2FCDescription

Q14315324.677870.8964.599Filamin-C
Q5U0D22043628.7844.153Transgelin
P378022044.8228287.8163.79Transgelin-2
B0AZV6471.55283.693.486cDNA, FLJ79546, highly similar to SH3 domain-binding glutamic acid-rich-like protein
J3KND31801960.683.445Myosin light polypeptide 6
A8KAH95656078.13.427RAP1A, member of RAS oncogene family
A0A024QZQ22672674.53.324Prosaposin (Variant Gaucher disease and variant metachromatic leukodystrophy), isoform CRA_b
A0A024RAB6368.773658.23.31Heparan sulfate proteoglycan 2 (Perlecan), isoform CRA_b
P082947186862.383.257Extracellular superoxide dismutase [Cu-Zn]
F2RM37315.52933.1683.217Coagulation factor IX
A0A024R321236.442162.7863.193Filamin-B, beta (actin binding protein 278), isoform CRA_a
B4DHX4241.52181.6723.175cDNA FLJ52902, highly similar to Rab GDP dissociation inhibitor alpha
A6NIZ15655073.7723.167Ras-related protein Rap-1b-like protein
A0A024QZX31801546.5843.103Serpin peptidase inhibitor, clade B (Ovalbumin), member 6, isoform CRA_a
Q53HQ02702227.73.045Flotillin 1 variant (fragment)
A6NLG9252.82077.263.039cDNA FLJ36740 fis, clone UTERU2013322, highly similar to Biglycan
A5YM532291812.18752.984ITGAV protein
V9HWF6287.52256.2582.972Alpha 1-acid glycoprotein
B2RDY91901426.91752.909Adenylyl cyclase-associated protein
B4DLV7311.172185.6022.812cDNA FLJ60299, highly similar to Rab GDP dissociation inhibitor beta
P010314613067.1666672.734Complement C5
Q9NZM12011334.3282.731Myoferlin
P13645870.55446.792.645Keratin, type I cytoskeletal 10
P087791462.678870.3082.6Keratin, type I cytoskeletal 16
B1N7B82011028.06252.355Cryocrystalglobulin CC1 kappa light chain variable region (fragment)
A0A024RBX92561281.32.323Pyruvate dehydrogenase E1 component subunit alpha
Q7Z7J6241.251205.7722.321Actin alpha 1 skeletal muscle protein
B7Z6P1266.051306.0562.295cDNA FLJ53662, highly similar to actin, alpha skeletal muscle
A8K4W03471656.52.25540S ribosomal protein S3a
A0A024R8214511989.752.141Eukaryotic translation initiation factor 3 subunit B
P0735794740782.106Complement component C8 alpha chain
G3V4G127411402.057Neuroguidin (fragment)

Note: based on protein abundance. A protein was defined as DAPs if there was a log2FC in excess of 2. FC: fold change.

AccessionExpression of healthy donorMean expression of AAD patients ()log2FCDescription

H3BQZ72286.569.33333333-5.04344799HCG2044799
Q6GMX624204.111024.248-4.56261507IGH@ protein
Q6MZQ621830.2939.36-4.53850338Putative uncharacterized protein DKFZp686G11190
Q6N08919877.09909.564-4.4497876Putative uncharacterized protein DKFZp686P15220
V9HW6819889.091004.376-4.3076059Epididymis luminal protein 214
Q926262750187.25-3.87639399Peroxidasin homolog
A0A024R9715098368.058-3.79192634Fibromodulin, isoform CRA_a
A0A087X2C05633410.31-3.77911725Ig mu chain C region
A0A024R9G41679122.6666667-3.77478706Family with sequence similarity 49, member B, isoform CRA_a
A0A0G2JPD46460.29505.912-3.67464056Uncharacterized protein
B0YJ884307350.696-3.61839044Radixin
Q96C329160.43752.5633333-3.60553042Polyubiquitin-C
A8K3Q7193761632.302-3.56929086Annexin
P436522496.5211.875-3.55862163Afamin
A0A024R0S65116.29460.624-3.47343655EH-domain containing 2, isoform CRA_a
Q9NZU55750529.346-3.44127902LIM and cysteine-rich domains protein 1
F2ZC063779.75349.452-3.43512461Thyroid hormone receptor interacting protein 6 isoform 1
P260064296414.25-3.37442039Integrin alpha 3/beta 1
A8MX945754.4581.104-3.30779722Glutathione S-transferase P
B2R6V91398142-3.29940153cDNA, FLJ93141, highly similar to Homo sapiens coagulation factor XIII, A1 polypeptide (F13A1), mRNA
P010233824.29397.764-3.26520731Alpha 2-macroglobulin
O948321636170.944-3.25857706Unconventional myosin-Id
B4DKT94013422.1666667-3.24879658cDNA FLJ54052, highly similar to alpha 1 catenin (cadherin-associated protein)
D6RA825307.67564.375-3.23335269Annexin
B4DQG52126.25227.9175-3.22172763cDNA FLJ54122, highly similar to Cytosol aminopeptidase (EC 3.4.11.1)
P122351117125.3333333-3.15578712ADP/ATP translocase 1
A0A024QZV01431160.625-3.15525531HCG1811539, isoform CRA_b
Q6UVK11875210.564-3.15455989Chondroitin sulfate proteoglycan 4
A0A0B4J1R61682.75191.546-3.13505805Transketolase
B4DRV42956340.5-3.11791957cDNA FLJ55667, highly similar to secreted protein acidic and rich in cysteine
A0A024RC654177531.732-2.97369595HCG1991735, isoform CRA_a
A0A024RDE11252161.95-2.95061419SPARC-like 1 (Mast9, hevin), isoform CRA_a
Q8TCD0142161858.354-2.93541833Uncharacterized protein
A0A024RAC925334-2.89553073Zinc finger, UBR1 type 1, isoform CRA_c
O760242314313-2.8861543Wolframin
B4DQX83990547.875-2.86447007cDNA FLJ51723, highly similar to DCC-interacting protein 13 alpha (fragment)
V9HWK12754387.9425-2.82761382Triosephosphate isomerase
P493271231.5174.842-2.81629294Fatty acid synthase
B4DNM836051.75-2.79836614cDNA FLJ53395, highly similar to Prolyl 3-hydroxylase 1 (EC 1.14.11.7)
A0A087WZW8142162052.316-2.7921908Protein IGKV3-11
A0A087WUS72422354.7-2.77152763Ig delta chain C region
B4E3A81457216.3333333-2.75167299cDNA FLJ53963, highly similar to leukocyte elastase inhibitor
A8K3B640660.5-2.74647268Nonspecific protein-tyrosine kinase
S6C4R612602.881884.586-2.74143392IgG L chain
A0A087WYL9142162132.92-2.73661383Ig kappa chain C region
Q6GMX011199.111698.368-2.72116309Uncharacterized protein
Q6PJF29283.641452.93-2.67572538IGK@ protein
P278241292.5204.5-2.65999153Calnexin
Q6P5S8142162252.75-2.65775646IGK@ protein
A0A087X13012702.622022.842-2.65067055Ig kappa chain C region
A0A087WWT3646104-2.63495064Serum albumin
V9HW349283.641502.036-2.62777119Epididymis luminal protein 213
Q8NCL62879.25479.72-2.58542857cDNA FLJ90170 fis, clone MAMMA1000370, highly similar to Ig alpha 1 chain C region
Q9NPP63076.09515.486-2.57709256Immunoglobulin heavy chain variant (fragment)
Q96K682872.08483.564-2.57031719cDNA FLJ14473 fis, clone MAMMA1001080, highly similar to Homo sapiens SNC73 protein (SNC73) mRNA
B4DEA321837-2.55873096cDNA FLJ56531, highly similar to UV excision repair protein RAD23 homolog B
Q96CD0228.541-2.47849835F-box/LRR-repeat protein 8
V9HWF21684.5306.8175-2.45686775Malate dehydrogenase
A8K4C85080925.6666667-2.4562638260S ribosomal protein L13
A0A024RDY2860158.994-2.43536434Tumor protein, translationally controlled 1, isoform CRA_a
Q6MZV62675.38500.17-2.41925339Putative uncharacterized protein DKFZp686L19235
A0A024RB012564484.4-2.40412549Integrin, alpha 5 (fibronectin receptor, alpha polypeptide), isoform CRA_b
A6NLN1652123.9175-2.39549202Polypyrimidine tract binding protein 1, isoform CRA_b
P07204989190.33-2.37746754Thrombomodulin
F6U21125349.46666667-2.3546087940S ribosomal protein S10
P632671432283.6666667-2.33576296Actin, gamma-enteric smooth muscle
A0A0C4DFX3829164.6666667-2.33182356EMILIN-1
A6NNI4571114.3333333-2.32024467Tetraspanin
A0A024RA21843170.8-2.30322466Secernin 1, isoform CRA_a
B2R5H01174245.915-2.25520077Protein S100
B4DPU374121556.5-2.2515573cDNA FLJ56548, highly similar to elongation factor 2
Q6MZU66876.261445.532-2.25002355Putative uncharacterized protein DKFZp686C15213
A0A024R1N11066224.5-2.24742009Myosin, heavy polypeptide 9, nonmuscle, isoform CRA_a
A0A075B6N86872.241450.324-2.24440519Ig gamma-3 chain C region (fragment)
B7Z539898192.05-2.22523348cDNA FLJ56954, highly similar to interalpha trypsin inhibitor heavy chain H1
A0A024R93023249.75-2.22135637Proteoglycan 4, isoform CRA_a
A8K9M51651.17354.448-2.21984277cDNA FLJ77947, highly similar to human complement protein C8 beta subunit mRNA
A0A024R7Z5412.589.2075-2.20915721Syndecan binding protein (Syntenin), isoform CRA_c
B4DDS8846184.5-2.19703685cDNA FLJ56686, moderately similar to FADD protein
A0A024R3W723952.5-2.18662129Eukaryotic translation elongation factor 1 beta 2, isoform CRA_a
A0A087WUN837182-2.17772337Syntaxin-binding protein 2
B4DTX51412314.816-2.16515932cDNA FLJ60072, highly similar to Homo sapiens sorbin and SH3 domain containing 1 (SORBS1), transcript variant 6, mRNA
Q9BVK626759.75-2.15982912Transmembrane emp24 domain-containing protein 9
Q9Y21743096.33333333-2.15822967Myotubularin-related protein 6
Q8IWB119443.5-2.15696935Inositol 1,4,5-trisphosphate receptor-interacting protein
A0A075B6G3818.5183.5766667-2.15659972Dystrophin
K7EQQ363671439.5-2.14504598Keratin, type I cytoskeletal 9
B4E2F91999.32455.736-2.13323916cDNA FLJ57038, highly similar to Filamin-A
C9JAK51520346.7-2.13231158ADP-ribosylation factor 4
B7Z9B71412322.75-2.12925109cDNA FLJ54732, moderately similar to sorbin and SH3 domain-containing protein 1
B2ZZ89884202.512-2.12603897Epididymis luminal protein 102
A0M8W4902207.5-2.1200161Ubiquitin-conjugating enzyme E2 variant 2
B0QZ183015723.296-2.05949992Copine-1
A0A024RAX0676162.25-2.05880477Matrix Gla protein
B0QY0122554.125-2.05555798Target of Myb protein 1 (fragment)
A0A087WU082574619.875-2.05396283Haptoglobin
A0A024R6P02748666.416-2.04388706Serpin peptidase inhibitor, clade A (alpha 1 antiproteinase, antitrypsin), member 3, isoform CRA_c
A0A024RD413012731.6666667-2.04146333RAB23, member RAS oncogene family, isoform CRA_a
A0A024R1Z6242.559-2.03919789Vesicle amine transport protein 1 homolog (T. californica), isoform CRA_a
Q59EQ1478119.5-2Cadherin 11, type 2 isoform 1 preproprotein variant (fragment)

Note: based on protein abundance. A protein was defined as DAPs if there was a log2FC in less of -2.

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 () 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).

3.2. 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 () (Figure 6(b)).

4. 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 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 pathogenesis of aortic dissection. It needs to be further explored in animals and humans.

5. Conclusions

The aortic arch tissues of patients with AAD show a large number of DAPs, the molecular functions of which were primarily cell junction. In particular, ITGA-3 and ITGA-5 were highly differentially expressed in these tissues. Downregulation of the two proteins may contribute to the progression of AAD, which may serve as a diagnostic biomarker and a novel therapeutic target in AAD.

Abbreviations

AAD:Acute aortic dissection
DAPs:Differential abundance proteins
TMT:Tandem mass tag
ITGA-3:Integrin alpha 3
ITGA-5:Integrin alpha 5
MMPs:Matrix metalloproteinase
VSMC:Vascular smooth muscle cell
MS:Mass spectrometry
CTA:Computed tomography angiography
TCEP:Tris(2-carboxyethyl)phosphine
MudPIT:Multidimensional protein identification technology
IOD:Integrated option density
PCA:Principal component analysis
GO analysis:Gene Ontology analysis
KEGG:Kyoto Encyclopedia of Genes and Genomes
PPI analysis:Protein-protein interaction analysis.

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval

The study was approved by the ethics committee of Zhongshan Hospital, Fudan University (Shanghai, China) (record number 2010-12).

Informed consent was obtained from patients or their legal surrogates before enrolment.

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this article.

Authors’ Contributions

GRG and CLY conceived, designed, and coordinated the study. LYX, YX, and YLY drafted this manuscript. ZJS and CYT revised it. GRG, CLY, and CYT were involved in the collection of tissue samples of AAD patients and healthy donors. HJW, PW, and Catherine CL Wong carried out the TMT proteomic analysis and immunohistochemistry. YLY and DWS were responsible for data analysis. All authors read, approved, and contributed to the final manuscript. Lingyu Xing, Yuan Xue, and Yilin Yang contributed equally to this work as co-first authors.

Acknowledgments

We thank the patients and staff of Zhongshan Hospital, Fudan University. We gratefully acknowledge the support and cooperation of Shanghai key laboratory of organ transplantation. This study was supported by the Key Project of Shanghai Municipal Health Bureau (2016ZB0202) and the Scientific Research Project of Shanghai Municipal Health Bureau (201940163).

Supplementary Materials

Supplementary 1. Supplementary Table 1 Basic data between AAD patients and healthy control in the TMT method. Note: a“0” is no; “1” is yes. bAccording to the more popular Stanford system, dissections involving the ascending aorta are classified as type A, whereas those involving only the descending aorta are classified as type B.

Supplementary 2. Supplementary Table 2 List of expreesion of all identified proteins between the two groups in the TMT method.

Supplementary 3. Supplementary Table 3 Basic data between AAD patients and healthy controls in immunohistochemistry. Note: a“0” is no; “1” is yes. bAccording to the more popular Stanford system, dissections involving the ascending aorta are classified as type A, whereas those involving only the descending aorta are classified as type B.

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