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

Authors: Lingyu Xing; Yuan Xue; Yilin Yang; Ping Wu; Catherine C. L. Wong; Haojun Wang; Zhenju Song; Dongwei Shi; Chaoyang Tong; Chenling Yao; Guorong Gu
Journal: BioMed Research International
 (2020)

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## 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 (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.


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## Body

## 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 [1–3]. 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 [5–13]. 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 [14–16]. Studies also have found that the PI3K/AKT signaling pathway contributes to phenotypic switching of vascular smooth muscle cell (VSMC) and induces apoptosis [17–19]. 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.Figure 1
Experimental flowchart. The aortic arch tissues of 5 patients with AAD were extracted during surgery. The peptides labelled with TMT were prepared, and a MS/MS analysis was performed. The proteomic data were analyzed, and the DAPs were selected and verified by immunohistochemistry.


## 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 Table1.All tissues were collected as typical lesion specimens of approximately1.0×1.0 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 (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).


#### 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 (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.


### 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: approximately1.0×1.0×0.2 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 (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).


## 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 Table1.All tissues were collected as typical lesion specimens of approximately1.0×1.0 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 (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).


### 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 (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.


## 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 Table1.All tissues were collected as typical lesion specimens of approximately1.0×1.0 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 (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).


## 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 (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.


## 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: approximately1.0×1.0×0.2 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 (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).


## 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: approximately1.0×1.0×0.2 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 (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).


## 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 (Figure2). Patient 1 might fall in different subtypes of AAD compared with the other patients.Figure 2
Principal component analysis (PCA) and heat map of proteome data. (a) Principal component analysis of the proteome data in a 2D graph of PC1 and PC2. (b) Expression heat map of the proteome data in the samples.
(a)
(b)A total of 809 proteins were identified from individual unique peptides (Supplementary Table2). 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).Table 1
Upregulated differential abundance proteins in AAD patients (n=32).
Accession
Expression of healthy donor
Mean expression of AAD patients (n=5)
log2FC
Description
Q14315
324.67
7870.896
4.599
Filamin-C
Q5U0D2
204
3628.784
4.153
Transgelin
P37802
2044.82
28287.816
3.79
Transgelin-2
B0AZV6
471.5
5283.69
3.486
cDNA, FLJ79546, highly similar to SH3 domain-binding glutamic acid-rich-like protein
J3KND3
180
1960.68
3.445
Myosin light polypeptide 6
A8KAH9
565
6078.1
3.427
RAP1A, member of RAS oncogene family
A0A024QZQ2
267
2674.5
3.324
Prosaposin (Variant Gaucher disease and variant metachromatic leukodystrophy), isoform CRA_b
A0A024RAB6
368.77
3658.2
3.31
Heparan sulfate proteoglycan 2 (Perlecan), isoform CRA_b
P08294
718
6862.38
3.257
Extracellular superoxide dismutase [Cu-Zn]
F2RM37
315.5
2933.168
3.217
Coagulation factor IX
A0A024R321
236.44
2162.786
3.193
Filamin-B, beta (actin binding protein 278), isoform CRA_a
B4DHX4
241.5
2181.672
3.175
cDNA FLJ52902, highly similar to Rab GDP dissociation inhibitor alpha
A6NIZ1
565
5073.772
3.167
Ras-related protein Rap-1b-like protein
A0A024QZX3
180
1546.584
3.103
Serpin peptidase inhibitor, clade B (Ovalbumin), member 6, isoform CRA_a
Q53HQ0
270
2227.7
3.045
Flotillin 1 variant (fragment)
A6NLG9
252.8
2077.26
3.039
cDNA FLJ36740 fis, clone UTERU2013322, highly similar to Biglycan
A5YM53
229
1812.1875
2.984
ITGAV protein
V9HWF6
287.5
2256.258
2.972
Alpha 1-acid glycoprotein
B2RDY9
190
1426.9175
2.909
Adenylyl cyclase-associated protein
B4DLV7
311.17
2185.602
2.812
cDNA FLJ60299, highly similar to Rab GDP dissociation inhibitor beta
P01031
461
3067.166667
2.734
Complement C5
Q9NZM1
201
1334.328
2.731
Myoferlin
P13645
870.5
5446.79
2.645
Keratin, type I cytoskeletal 10
P08779
1462.67
8870.308
2.6
Keratin, type I cytoskeletal 16
B1N7B8
201
1028.0625
2.355
Cryocrystalglobulin CC1 kappa light chain variable region (fragment)
A0A024RBX9
256
1281.3
2.323
Pyruvate dehydrogenase E1 component subunit alpha
Q7Z7J6
241.25
1205.772
2.321
Actin alpha 1 skeletal muscle protein
B7Z6P1
266.05
1306.056
2.295
cDNA FLJ53662, highly similar to actin, alpha skeletal muscle
A8K4W0
347
1656.5
2.255
40S ribosomal protein S3a
A0A024R821
451
1989.75
2.141
Eukaryotic translation initiation factor 3 subunit B
P07357
947
4078
2.106
Complement component C8 alpha chain
G3V4G1
274
1140
2.057
Neuroguidin (fragment)
Note: based on protein abundance. A protein was defined as DAPs if there was a log2FC in excess of 2. FC: fold change.Table 2
Downregulated DAPs in AAD patients (n=100).
Accession
Expression of healthy donor
Mean expression of AAD patients (n=5)
log2FC
Description
H3BQZ7
2286.5
69.33333333
-5.04344799
HCG2044799
Q6GMX6
24204.11
1024.248
-4.56261507
IGH@ protein
Q6MZQ6
21830.2
939.36
-4.53850338
Putative uncharacterized protein DKFZp686G11190
Q6N089
19877.09
909.564
-4.4497876
Putative uncharacterized protein DKFZp686P15220
V9HW68
19889.09
1004.376
-4.3076059
Epididymis luminal protein 214
Q92626
2750
187.25
-3.87639399
Peroxidasin homolog
A0A024R971
5098
368.058
-3.79192634
Fibromodulin, isoform CRA_a
A0A087X2C0
5633
410.31
-3.77911725
Ig mu chain C region
A0A024R9G4
1679
122.6666667
-3.77478706
Family with sequence similarity 49, member B, isoform CRA_a
A0A0G2JPD4
6460.29
505.912
-3.67464056
Uncharacterized protein
B0YJ88
4307
350.696
-3.61839044
Radixin
Q96C32
9160.43
752.5633333
-3.60553042
Polyubiquitin-C
A8K3Q7
19376
1632.302
-3.56929086
Annexin
P43652
2496.5
211.875
-3.55862163
Afamin
A0A024R0S6
5116.29
460.624
-3.47343655
EH-domain containing 2, isoform CRA_a
Q9NZU5
5750
529.346
-3.44127902
LIM and cysteine-rich domains protein 1
F2ZC06
3779.75
349.452
-3.43512461
Thyroid hormone receptor interacting protein 6 isoform 1
P26006
4296
414.25
-3.37442039
Integrin alpha 3/beta 1
A8MX94
5754.4
581.104
-3.30779722
Glutathione S-transferase P
B2R6V9
1398
142
-3.29940153
cDNA, FLJ93141, highly similar to Homo sapiens coagulation factor XIII, A1 polypeptide (F13A1), mRNA
P01023
3824.29
397.764
-3.26520731
Alpha 2-macroglobulin
O94832
1636
170.944
-3.25857706
Unconventional myosin-Id
B4DKT9
4013
422.1666667
-3.24879658
cDNA FLJ54052, highly similar to alpha 1 catenin (cadherin-associated protein)
D6RA82
5307.67
564.375
-3.23335269
Annexin
B4DQG5
2126.25
227.9175
-3.22172763
cDNA FLJ54122, highly similar to Cytosol aminopeptidase (EC 3.4.11.1)
P12235
1117
125.3333333
-3.15578712
ADP/ATP translocase 1
A0A024QZV0
1431
160.625
-3.15525531
HCG1811539, isoform CRA_b
Q6UVK1
1875
210.564
-3.15455989
Chondroitin sulfate proteoglycan 4
A0A0B4J1R6
1682.75
191.546
-3.13505805
Transketolase
B4DRV4
2956
340.5
-3.11791957
cDNA FLJ55667, highly similar to secreted protein acidic and rich in cysteine
A0A024RC65
4177
531.732
-2.97369595
HCG1991735, isoform CRA_a
A0A024RDE1
1252
161.95
-2.95061419
SPARC-like 1 (Mast9, hevin), isoform CRA_a
Q8TCD0
14216
1858.354
-2.93541833
Uncharacterized protein
A0A024RAC9
253
34
-2.89553073
Zinc finger, UBR1 type 1, isoform CRA_c
O76024
2314
313
-2.8861543
Wolframin
B4DQX8
3990
547.875
-2.86447007
cDNA FLJ51723, highly similar to DCC-interacting protein 13 alpha (fragment)
V9HWK1
2754
387.9425
-2.82761382
Triosephosphate isomerase
P49327
1231.5
174.842
-2.81629294
Fatty acid synthase
B4DNM8
360
51.75
-2.79836614
cDNA FLJ53395, highly similar to Prolyl 3-hydroxylase 1 (EC 1.14.11.7)
A0A087WZW8
14216
2052.316
-2.7921908
Protein IGKV3-11
A0A087WUS7
2422
354.7
-2.77152763
Ig delta chain C region
B4E3A8
1457
216.3333333
-2.75167299
cDNA FLJ53963, highly similar to leukocyte elastase inhibitor
A8K3B6
406
60.5
-2.74647268
Nonspecific protein-tyrosine kinase
S6C4R6
12602.88
1884.586
-2.74143392
IgG L chain
A0A087WYL9
14216
2132.92
-2.73661383
Ig kappa chain C region
Q6GMX0
11199.11
1698.368
-2.72116309
Uncharacterized protein
Q6PJF2
9283.64
1452.93
-2.67572538
IGK@ protein
P27824
1292.5
204.5
-2.65999153
Calnexin
Q6P5S8
14216
2252.75
-2.65775646
IGK@ protein
A0A087X130
12702.62
2022.842
-2.65067055
Ig kappa chain C region
A0A087WWT3
646
104
-2.63495064
Serum albumin
V9HW34
9283.64
1502.036
-2.62777119
Epididymis luminal protein 213
Q8NCL6
2879.25
479.72
-2.58542857
cDNA FLJ90170 fis, clone MAMMA1000370, highly similar to Ig alpha 1 chain C region
Q9NPP6
3076.09
515.486
-2.57709256
Immunoglobulin heavy chain variant (fragment)
Q96K68
2872.08
483.564
-2.57031719
cDNA FLJ14473 fis, clone MAMMA1001080, highly similar to Homo sapiens SNC73 protein (SNC73) mRNA
B4DEA3
218
37
-2.55873096
cDNA FLJ56531, highly similar to UV excision repair protein RAD23 homolog B
Q96CD0
228.5
41
-2.47849835
F-box/LRR-repeat protein 8
V9HWF2
1684.5
306.8175
-2.45686775
Malate dehydrogenase
A8K4C8
5080
925.6666667
-2.45626382
60S ribosomal protein L13
A0A024RDY2
860
158.994
-2.43536434
Tumor protein, translationally controlled 1, isoform CRA_a
Q6MZV6
2675.38
500.17
-2.41925339
Putative uncharacterized protein DKFZp686L19235
A0A024RB01
2564
484.4
-2.40412549
Integrin, alpha 5 (fibronectin receptor, alpha polypeptide), isoform CRA_b
A6NLN1
652
123.9175
-2.39549202
Polypyrimidine tract binding protein 1, isoform CRA_b
P07204
989
190.33
-2.37746754
Thrombomodulin
F6U211
253
49.46666667
-2.35460879
40S ribosomal protein S10
P63267
1432
283.6666667
-2.33576296
Actin, gamma-enteric smooth muscle
A0A0C4DFX3
829
164.6666667
-2.33182356
EMILIN-1
A6NNI4
571
114.3333333
-2.32024467
Tetraspanin
A0A024RA21
843
170.8
-2.30322466
Secernin 1, isoform CRA_a
B2R5H0
1174
245.915
-2.25520077
Protein S100
B4DPU3
7412
1556.5
-2.2515573
cDNA FLJ56548, highly similar to elongation factor 2
Q6MZU6
6876.26
1445.532
-2.25002355
Putative uncharacterized protein DKFZp686C15213
A0A024R1N1
1066
224.5
-2.24742009
Myosin, heavy polypeptide 9, nonmuscle, isoform CRA_a
A0A075B6N8
6872.24
1450.324
-2.24440519
Ig gamma-3 chain C region (fragment)
B7Z539
898
192.05
-2.22523348
cDNA FLJ56954, highly similar to interalpha trypsin inhibitor heavy chain H1
A0A024R930
232
49.75
-2.22135637
Proteoglycan 4, isoform CRA_a
A8K9M5
1651.17
354.448
-2.21984277
cDNA FLJ77947, highly similar to human complement protein C8 beta subunit mRNA
A0A024R7Z5
412.5
89.2075
-2.20915721
Syndecan binding protein (Syntenin), isoform CRA_c
B4DDS8
846
184.5
-2.19703685
cDNA FLJ56686, moderately similar to FADD protein
A0A024R3W7
239
52.5
-2.18662129
Eukaryotic translation elongation factor 1 beta 2, isoform CRA_a
A0A087WUN8
371
82
-2.17772337
Syntaxin-binding protein 2
B4DTX5
1412
314.816
-2.16515932
cDNA FLJ60072, highly similar to Homo sapiens sorbin and SH3 domain containing 1 (SORBS1), transcript variant 6, mRNA
Q9BVK6
267
59.75
-2.15982912
Transmembrane emp24 domain-containing protein 9
Q9Y217
430
96.33333333
-2.15822967
Myotubularin-related protein 6
Q8IWB1
194
43.5
-2.15696935
Inositol 1,4,5-trisphosphate receptor-interacting protein
A0A075B6G3
818.5
183.5766667
-2.15659972
Dystrophin
K7EQQ3
6367
1439.5
-2.14504598
Keratin, type I cytoskeletal 9
B4E2F9
1999.32
455.736
-2.13323916
cDNA FLJ57038, highly similar to Filamin-A
C9JAK5
1520
346.7
-2.13231158
ADP-ribosylation factor 4
B7Z9B7
1412
322.75
-2.12925109
cDNA FLJ54732, moderately similar to sorbin and SH3 domain-containing protein 1
B2ZZ89
884
202.512
-2.12603897
Epididymis luminal protein 102
A0M8W4
902
207.5
-2.1200161
Ubiquitin-conjugating enzyme E2 variant 2
B0QZ18
3015
723.296
-2.05949992
Copine-1
A0A024RAX0
676
162.25
-2.05880477
Matrix Gla protein
B0QY01
225
54.125
-2.05555798
Target of Myb protein 1 (fragment)
A0A087WU08
2574
619.875
-2.05396283
Haptoglobin
A0A024R6P0
2748
666.416
-2.04388706
Serpin peptidase inhibitor, clade A (alpha 1 antiproteinase, antitrypsin), member 3, isoform CRA_c
A0A024RD41
3012
731.6666667
-2.04146333
RAB23, member RAS oncogene family, isoform CRA_a
A0A024R1Z6
242.5
59
-2.03919789
Vesicle amine transport protein 1 homolog (T. californica), isoform CRA_a
Q59EQ1
478
119.5
-2
Cadherin 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 (Figure3). 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).Figure 3
The Gene Ontology (GO) enrichment of 132 differential abundance proteins (DAPs). (a) Sorted by descending order of the number of protein associated with the listed GO ID. (b) Sorted by descending order ofP value for the GO enrichment terms. Because of the limited number of patients, we considered DAPs as meaningful only as strong differently expressed (∣log2FC∣>2).
(a)
(b)Figure 4
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for 132 DAPs between health donor vs. patients. (a) Top 30 significantly enriched pathways were shown in the senior bubble chart. The rich factor is the ratio of DAP numbers annotated in this pathway term to all gene numbers annotated in this pathway term (P<0.01). (b) Most of downregulated DAPs (n=16/51) were found in the PI3K-AKT signaling pathway.
(a)
(b)Figure 5
The protein-protein interaction networks is analyzed by string software. Different line colours represent the types of evidence used in predicting the associations: gene fusion (red), neighborhood (green), cooccurrence across genomes (blue), coexpression (black), experimental (purple), association in curated databases (light blue), or comentioned in PubMed abstracts (yellow).


### 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 (Figure6(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)).Figure 6
The biomarkers (ITGA-3, ITGA-5) verified by immunohistochemistry (IHC). (a) The black arrows present positive signals of ITGA-3 and ITGA-5 staining by DAB in healthy donors. Negative control: the section was stained with secondary antibodies only. Scalebars=200 μm. (b) The IODs of ITGA-3 and ITGA-5 in AAD patients were significantly lower compared with healthy donors (∗∗∗P<0.001).
(a)
(b)


## 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 (Figure2). Patient 1 might fall in different subtypes of AAD compared with the other patients.Figure 2
Principal component analysis (PCA) and heat map of proteome data. (a) Principal component analysis of the proteome data in a 2D graph of PC1 and PC2. (b) Expression heat map of the proteome data in the samples.
(a)
(b)A total of 809 proteins were identified from individual unique peptides (Supplementary Table2). 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).Table 1
Upregulated differential abundance proteins in AAD patients (n=32).
Accession
Expression of healthy donor
Mean expression of AAD patients (n=5)
log2FC
Description
Q14315
324.67
7870.896
4.599
Filamin-C
Q5U0D2
204
3628.784
4.153
Transgelin
P37802
2044.82
28287.816
3.79
Transgelin-2
B0AZV6
471.5
5283.69
3.486
cDNA, FLJ79546, highly similar to SH3 domain-binding glutamic acid-rich-like protein
J3KND3
180
1960.68
3.445
Myosin light polypeptide 6
A8KAH9
565
6078.1
3.427
RAP1A, member of RAS oncogene family
A0A024QZQ2
267
2674.5
3.324
Prosaposin (Variant Gaucher disease and variant metachromatic leukodystrophy), isoform CRA_b
A0A024RAB6
368.77
3658.2
3.31
Heparan sulfate proteoglycan 2 (Perlecan), isoform CRA_b
P08294
718
6862.38
3.257
Extracellular superoxide dismutase [Cu-Zn]
F2RM37
315.5
2933.168
3.217
Coagulation factor IX
A0A024R321
236.44
2162.786
3.193
Filamin-B, beta (actin binding protein 278), isoform CRA_a
B4DHX4
241.5
2181.672
3.175
cDNA FLJ52902, highly similar to Rab GDP dissociation inhibitor alpha
A6NIZ1
565
5073.772
3.167
Ras-related protein Rap-1b-like protein
A0A024QZX3
180
1546.584
3.103
Serpin peptidase inhibitor, clade B (Ovalbumin), member 6, isoform CRA_a
Q53HQ0
270
2227.7
3.045
Flotillin 1 variant (fragment)
A6NLG9
252.8
2077.26
3.039
cDNA FLJ36740 fis, clone UTERU2013322, highly similar to Biglycan
A5YM53
229
1812.1875
2.984
ITGAV protein
V9HWF6
287.5
2256.258
2.972
Alpha 1-acid glycoprotein
B2RDY9
190
1426.9175
2.909
Adenylyl cyclase-associated protein
B4DLV7
311.17
2185.602
2.812
cDNA FLJ60299, highly similar to Rab GDP dissociation inhibitor beta
P01031
461
3067.166667
2.734
Complement C5
Q9NZM1
201
1334.328
2.731
Myoferlin
P13645
870.5
5446.79
2.645
Keratin, type I cytoskeletal 10
P08779
1462.67
8870.308
2.6
Keratin, type I cytoskeletal 16
B1N7B8
201
1028.0625
2.355
Cryocrystalglobulin CC1 kappa light chain variable region (fragment)
A0A024RBX9
256
1281.3
2.323
Pyruvate dehydrogenase E1 component subunit alpha
Q7Z7J6
241.25
1205.772
2.321
Actin alpha 1 skeletal muscle protein
B7Z6P1
266.05
1306.056
2.295
cDNA FLJ53662, highly similar to actin, alpha skeletal muscle
A8K4W0
347
1656.5
2.255
40S ribosomal protein S3a
A0A024R821
451
1989.75
2.141
Eukaryotic translation initiation factor 3 subunit B
P07357
947
4078
2.106
Complement component C8 alpha chain
G3V4G1
274
1140
2.057
Neuroguidin (fragment)
Note: based on protein abundance. A protein was defined as DAPs if there was a log2FC in excess of 2. FC: fold change.Table 2
Downregulated DAPs in AAD patients (n=100).
Accession
Expression of healthy donor
Mean expression of AAD patients (n=5)
log2FC
Description
H3BQZ7
2286.5
69.33333333
-5.04344799
HCG2044799
Q6GMX6
24204.11
1024.248
-4.56261507
IGH@ protein
Q6MZQ6
21830.2
939.36
-4.53850338
Putative uncharacterized protein DKFZp686G11190
Q6N089
19877.09
909.564
-4.4497876
Putative uncharacterized protein DKFZp686P15220
V9HW68
19889.09
1004.376
-4.3076059
Epididymis luminal protein 214
Q92626
2750
187.25
-3.87639399
Peroxidasin homolog
A0A024R971
5098
368.058
-3.79192634
Fibromodulin, isoform CRA_a
A0A087X2C0
5633
410.31
-3.77911725
Ig mu chain C region
A0A024R9G4
1679
122.6666667
-3.77478706
Family with sequence similarity 49, member B, isoform CRA_a
A0A0G2JPD4
6460.29
505.912
-3.67464056
Uncharacterized protein
B0YJ88
4307
350.696
-3.61839044
Radixin
Q96C32
9160.43
752.5633333
-3.60553042
Polyubiquitin-C
A8K3Q7
19376
1632.302
-3.56929086
Annexin
P43652
2496.5
211.875
-3.55862163
Afamin
A0A024R0S6
5116.29
460.624
-3.47343655
EH-domain containing 2, isoform CRA_a
Q9NZU5
5750
529.346
-3.44127902
LIM and cysteine-rich domains protein 1
F2ZC06
3779.75
349.452
-3.43512461
Thyroid hormone receptor interacting protein 6 isoform 1
P26006
4296
414.25
-3.37442039
Integrin alpha 3/beta 1
A8MX94
5754.4
581.104
-3.30779722
Glutathione S-transferase P
B2R6V9
1398
142
-3.29940153
cDNA, FLJ93141, highly similar to Homo sapiens coagulation factor XIII, A1 polypeptide (F13A1), mRNA
P01023
3824.29
397.764
-3.26520731
Alpha 2-macroglobulin
O94832
1636
170.944
-3.25857706
Unconventional myosin-Id
B4DKT9
4013
422.1666667
-3.24879658
cDNA FLJ54052, highly similar to alpha 1 catenin (cadherin-associated protein)
D6RA82
5307.67
564.375
-3.23335269
Annexin
B4DQG5
2126.25
227.9175
-3.22172763
cDNA FLJ54122, highly similar to Cytosol aminopeptidase (EC 3.4.11.1)
P12235
1117
125.3333333
-3.15578712
ADP/ATP translocase 1
A0A024QZV0
1431
160.625
-3.15525531
HCG1811539, isoform CRA_b
Q6UVK1
1875
210.564
-3.15455989
Chondroitin sulfate proteoglycan 4
A0A0B4J1R6
1682.75
191.546
-3.13505805
Transketolase
B4DRV4
2956
340.5
-3.11791957
cDNA FLJ55667, highly similar to secreted protein acidic and rich in cysteine
A0A024RC65
4177
531.732
-2.97369595
HCG1991735, isoform CRA_a
A0A024RDE1
1252
161.95
-2.95061419
SPARC-like 1 (Mast9, hevin), isoform CRA_a
Q8TCD0
14216
1858.354
-2.93541833
Uncharacterized protein
A0A024RAC9
253
34
-2.89553073
Zinc finger, UBR1 type 1, isoform CRA_c
O76024
2314
313
-2.8861543
Wolframin
B4DQX8
3990
547.875
-2.86447007
cDNA FLJ51723, highly similar to DCC-interacting protein 13 alpha (fragment)
V9HWK1
2754
387.9425
-2.82761382
Triosephosphate isomerase
P49327
1231.5
174.842
-2.81629294
Fatty acid synthase
B4DNM8
360
51.75
-2.79836614
cDNA FLJ53395, highly similar to Prolyl 3-hydroxylase 1 (EC 1.14.11.7)
A0A087WZW8
14216
2052.316
-2.7921908
Protein IGKV3-11
A0A087WUS7
2422
354.7
-2.77152763
Ig delta chain C region
B4E3A8
1457
216.3333333
-2.75167299
cDNA FLJ53963, highly similar to leukocyte elastase inhibitor
A8K3B6
406
60.5
-2.74647268
Nonspecific protein-tyrosine kinase
S6C4R6
12602.88
1884.586
-2.74143392
IgG L chain
A0A087WYL9
14216
2132.92
-2.73661383
Ig kappa chain C region
Q6GMX0
11199.11
1698.368
-2.72116309
Uncharacterized protein
Q6PJF2
9283.64
1452.93
-2.67572538
IGK@ protein
P27824
1292.5
204.5
-2.65999153
Calnexin
Q6P5S8
14216
2252.75
-2.65775646
IGK@ protein
A0A087X130
12702.62
2022.842
-2.65067055
Ig kappa chain C region
A0A087WWT3
646
104
-2.63495064
Serum albumin
V9HW34
9283.64
1502.036
-2.62777119
Epididymis luminal protein 213
Q8NCL6
2879.25
479.72
-2.58542857
cDNA FLJ90170 fis, clone MAMMA1000370, highly similar to Ig alpha 1 chain C region
Q9NPP6
3076.09
515.486
-2.57709256
Immunoglobulin heavy chain variant (fragment)
Q96K68
2872.08
483.564
-2.57031719
cDNA FLJ14473 fis, clone MAMMA1001080, highly similar to Homo sapiens SNC73 protein (SNC73) mRNA
B4DEA3
218
37
-2.55873096
cDNA FLJ56531, highly similar to UV excision repair protein RAD23 homolog B
Q96CD0
228.5
41
-2.47849835
F-box/LRR-repeat protein 8
V9HWF2
1684.5
306.8175
-2.45686775
Malate dehydrogenase
A8K4C8
5080
925.6666667
-2.45626382
60S ribosomal protein L13
A0A024RDY2
860
158.994
-2.43536434
Tumor protein, translationally controlled 1, isoform CRA_a
Q6MZV6
2675.38
500.17
-2.41925339
Putative uncharacterized protein DKFZp686L19235
A0A024RB01
2564
484.4
-2.40412549
Integrin, alpha 5 (fibronectin receptor, alpha polypeptide), isoform CRA_b
A6NLN1
652
123.9175
-2.39549202
Polypyrimidine tract binding protein 1, isoform CRA_b
P07204
989
190.33
-2.37746754
Thrombomodulin
F6U211
253
49.46666667
-2.35460879
40S ribosomal protein S10
P63267
1432
283.6666667
-2.33576296
Actin, gamma-enteric smooth muscle
A0A0C4DFX3
829
164.6666667
-2.33182356
EMILIN-1
A6NNI4
571
114.3333333
-2.32024467
Tetraspanin
A0A024RA21
843
170.8
-2.30322466
Secernin 1, isoform CRA_a
B2R5H0
1174
245.915
-2.25520077
Protein S100
B4DPU3
7412
1556.5
-2.2515573
cDNA FLJ56548, highly similar to elongation factor 2
Q6MZU6
6876.26
1445.532
-2.25002355
Putative uncharacterized protein DKFZp686C15213
A0A024R1N1
1066
224.5
-2.24742009
Myosin, heavy polypeptide 9, nonmuscle, isoform CRA_a
A0A075B6N8
6872.24
1450.324
-2.24440519
Ig gamma-3 chain C region (fragment)
B7Z539
898
192.05
-2.22523348
cDNA FLJ56954, highly similar to interalpha trypsin inhibitor heavy chain H1
A0A024R930
232
49.75
-2.22135637
Proteoglycan 4, isoform CRA_a
A8K9M5
1651.17
354.448
-2.21984277
cDNA FLJ77947, highly similar to human complement protein C8 beta subunit mRNA
A0A024R7Z5
412.5
89.2075
-2.20915721
Syndecan binding protein (Syntenin), isoform CRA_c
B4DDS8
846
184.5
-2.19703685
cDNA FLJ56686, moderately similar to FADD protein
A0A024R3W7
239
52.5
-2.18662129
Eukaryotic translation elongation factor 1 beta 2, isoform CRA_a
A0A087WUN8
371
82
-2.17772337
Syntaxin-binding protein 2
B4DTX5
1412
314.816
-2.16515932
cDNA FLJ60072, highly similar to Homo sapiens sorbin and SH3 domain containing 1 (SORBS1), transcript variant 6, mRNA
Q9BVK6
267
59.75
-2.15982912
Transmembrane emp24 domain-containing protein 9
Q9Y217
430
96.33333333
-2.15822967
Myotubularin-related protein 6
Q8IWB1
194
43.5
-2.15696935
Inositol 1,4,5-trisphosphate receptor-interacting protein
A0A075B6G3
818.5
183.5766667
-2.15659972
Dystrophin
K7EQQ3
6367
1439.5
-2.14504598
Keratin, type I cytoskeletal 9
B4E2F9
1999.32
455.736
-2.13323916
cDNA FLJ57038, highly similar to Filamin-A
C9JAK5
1520
346.7
-2.13231158
ADP-ribosylation factor 4
B7Z9B7
1412
322.75
-2.12925109
cDNA FLJ54732, moderately similar to sorbin and SH3 domain-containing protein 1
B2ZZ89
884
202.512
-2.12603897
Epididymis luminal protein 102
A0M8W4
902
207.5
-2.1200161
Ubiquitin-conjugating enzyme E2 variant 2
B0QZ18
3015
723.296
-2.05949992
Copine-1
A0A024RAX0
676
162.25
-2.05880477
Matrix Gla protein
B0QY01
225
54.125
-2.05555798
Target of Myb protein 1 (fragment)
A0A087WU08
2574
619.875
-2.05396283
Haptoglobin
A0A024R6P0
2748
666.416
-2.04388706
Serpin peptidase inhibitor, clade A (alpha 1 antiproteinase, antitrypsin), member 3, isoform CRA_c
A0A024RD41
3012
731.6666667
-2.04146333
RAB23, member RAS oncogene family, isoform CRA_a
A0A024R1Z6
242.5
59
-2.03919789
Vesicle amine transport protein 1 homolog (T. californica), isoform CRA_a
Q59EQ1
478
119.5
-2
Cadherin 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 (Figure3). 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).Figure 3
The Gene Ontology (GO) enrichment of 132 differential abundance proteins (DAPs). (a) Sorted by descending order of the number of protein associated with the listed GO ID. (b) Sorted by descending order ofP value for the GO enrichment terms. Because of the limited number of patients, we considered DAPs as meaningful only as strong differently expressed (∣log2FC∣>2).
(a)
(b)Figure 4
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for 132 DAPs between health donor vs. patients. (a) Top 30 significantly enriched pathways were shown in the senior bubble chart. The rich factor is the ratio of DAP numbers annotated in this pathway term to all gene numbers annotated in this pathway term (P<0.01). (b) Most of downregulated DAPs (n=16/51) were found in the PI3K-AKT signaling pathway.
(a)
(b)Figure 5
The protein-protein interaction networks is analyzed by string software. Different line colours represent the types of evidence used in predicting the associations: gene fusion (red), neighborhood (green), cooccurrence across genomes (blue), coexpression (black), experimental (purple), association in curated databases (light blue), or comentioned in PubMed abstracts (yellow).


## 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 (Figure6(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)).Figure 6
The biomarkers (ITGA-3, ITGA-5) verified by immunohistochemistry (IHC). (a) The black arrows present positive signals of ITGA-3 and ITGA-5 staining by DAB in healthy donors. Negative control: the section was stained with secondary antibodies only. Scalebars=200 μm. (b) The IODs of ITGA-3 and ITGA-5 in AAD patients were significantly lower compared with healthy donors (∗∗∗P<0.001).
(a)
(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.



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*Source: 1068402-2020-07-02.xml*