Proteomic Analysis of tPVAT during Ang II Infusion: Comparison
Please note this is a comparison between Version 1 by Wenjuan Yao and Version 2 by Bruce Ren.

In this study, we provided the most comprehensive proteome profile of mice tPVAT and some novel insights into Ang II-mediated tPVAT dysfunction. We identified some novel proteins, potentially associated with Ang II infusion, such as acyl-CoA carboxylase α , very long-chain acyl-CoA synthetase (ACSVL), uncoupling protein 1 (UCP1), perilipin, RAS protein-specific guanine nucleotide-releasing factor 2 (RasGRF2), and hypoxia inducible factor 1 α (HIF-1 α ). Importantly, the key pathways were involved in fatty acid biosynthesis, FABP3-PPAR α / γ , RasGRF2-ERK-HIF-1 α , RasGRF2-PKC-HIF-1 α , and STAT3-HIF-1 α axis.

  • perivascular adipose tissue
  • renin-angiotensin system
  • angiotensin II
  • proteomic analysis
  • tandem mass tags

1. Introduction

The renin–angiotensin–aldosterone system (RAAS) is involved in systemic blood pressure regulation and renal electrolyte homeostasis. It is well accepted that Ang II mediates vascular smooth muscle cell (VSMC) proliferation, migration, and vascular remodeling [1][2][1,2]. We also previously reported that Ang II regulates VSMC proliferation and vascular remodeling by RhoGDI signaling [3]. However, there is no detailed report on the effect of Ang II on PVAT (perivascular adipose tissue) structure and function.
PVAT is an adipose tissue with endocrine and paracrine functions surrounding the blood vessels, such as the small mesenteric and femoral arteries or the large abdominal aorta (AA), and plays wide-ranging physiological roles on vascular environment stability and influences vascular pathogenesis [4][5][4,5]. Under physiological conditions, PVAT has strong anti-atherosclerosis properties, induces non-shivering thermogenesis (NST), and metabolizes fatty acids by secreting various bioactive factors [6]. Under pathological conditions (such as obesity), PVAT loses its thermogenic ability and secretes pro-inflammatory adipocytokines that induce endothelial dysfunction and inflammatory cell infiltration and promotes the development of atherosclerosis [6]. The PVAT in the thoracic aorta is more similar to brown adipose tissue (BAT), while the PVAT in the abdominal aorta is similar to white adipose tissue (WAT). WAT primarily stores energy in the form of triglycerides, while excessive lipid accumulation in WAT leads to adipocyte hypertrophy and dysfunction. This leads to increased secretion of harmful fat factors and inflammatory cytokines into circulation, thus impairing the function of the vascular endothelium [7][8][7,8]. Unlike WAT, BAT can absorb lipids by uncoupling oxidation in the mitochondrial electron transport chain to generate heat, and this is characterized by the expression of the heat-producing gene uncoupling protein 1 (UCP1) [9]. In addition, WAT can be browned under the stimulation of low temperature and catecholamine [10][11][10,11]. The browning of WAT is characterized by a high expression of the brown adipocyte marker UCP1 [12], which is now called the third adipose tissue-beige adipose tissue (BEt). In general, changes in the phenotype of PVAT correlate with disease progression.
Many studies have shown that there is local RAAS activity in PVAT [13]. It has been reported that Ang II inhibits adipogenic differentiation of human preadipocytes in vitro via the AT1 receptor [14]. The AngII-AT2 or Ang (1–7)-Mas activation stimulates adipogenesis and antagonizes the antiadipogenic effect of AngII-AT1 activation [15][16][15,16].

2. Pathological Conversion of tPVAT after Ang II Infusion

To determine the effects of Ang II on the tPVAT phenotype, male C57BL/6J mice aged 42–48 days were infused with Ang II or normal saline for two weeks. Mice that were infused with Ang II were significantly heavier than the controls by 7 and 12 days (Figure 1A). The tPVAT was successfully separated (Figure 1B,C). Figure 1C shows that Ang II infusion made the tPVAT morphology significantly irregular and caused hypertrophy, when compared with the saline group. The tPVAT weight of the Ang II infusion group was significantly higher than that of the control group (Figure 1D). Next, we histologically analyzed tPVAT after Ang II infusion. Mice infused with Ang II had a marked phenotypic conversion of tPVAT from a regular cell size to an irregular size (Figure 1E). Compared with the saline group, the Ang II-infused mice showed a substantial decrease in adipocyte size in tPVAT (Figure 1E).
Figure 1. Phenotypic differences between saline and Ang II infused mice. Mice infused with saline were used as control. (A) Body weights of the mice perfused with saline and Ang II, during 2 weeks of perfusion (n = 20, biological replicates); * p < 0.05, vs. the control group (saline infusion). (B) Simplified schematic showing thoracic PVAT (tPVAT) in mice; PVAT, perivascular adipose tissue. (C) Representative picture illustrating isolation of tPVAT. (D) tPVAT weights from saline and Ang II infused mice (n = 20, biological replicates); * p < 0.05, vs. the control group. (E) Hematoxylin and eosin staining of tPVAT and the quantification of cell diameter between saline and Ang II infusion group (n = 8, biological replicates); * p < 0.05, vs. the control group.

3. Molecular Changes during tPVAT Pathogenesis Analyzed by LC-MS/MS

To elucidate the molecular events occurring in tPVAT during Ang II infusion, a quantitative proteomic analysis, based on TMT labeling, was executed in the in vivo Ang II infusion model. A total of 30,079 peptide fragments, of which 27,160 were unique peptides corresponding to a total of 5037 proteins, were used. We received a good quality deviation during the data acquisition process using a high-quality Q Exactive mass spectrometer. The mass deviations of all the identified peptides were primarily distributed within 10 ppm, indicating that the identification results were accurate and reliable. We obtained an ideal score with a median of 27.49, and more than 65.64% of the peptides scored higher than 20 when evaluating each MS2 spectrogram. A 1.2-fold change cut-off, with a p-value < 0.05, was used to indicate significant changes in the abundance of the differentially expressed proteins (DEPs) between the saline and Ang II-infused groups.

3.1. Identification of DEPs

In our study, the changes in expression of the DEPs between the saline and Ang II-infused groups were determined using a volcano plot and K-means clustering heatmaps as shown in Figure 2A,B. A total of 291 proteins were found to be significantly differentially expressed between the two groups, of which 145 proteins were upregulated and 146 proteins were downregulated (Figure 2C), indicating a drastic phenotypic alteration of tPVAT during Ang II infusion, when compared with the saline-infused group. The abbreviated lists of the up- and down-regulated proteins are provided in Table 1 and Table 2. As demonstrated in Table 1, most of the top 10 up-regulated proteins have been shown to be involved in epigenetic modification, cell phenotype changes, and differentiation. These proteins include KDM1A, PKN1, and ENGase [17][18][19][18,19,20]. In addition, CtBP1 participates in BAT differentiation [20][21], and TMEM41B is a novel ER-localized regulator of lipid mobilization and lipid droplets [21][22]. Regarding the down-regulated proteins, as demonstrated in Table 2, many of the top 10 proteins have been shown to participate in lipid metabolism, brown adipogenesis, and various pathological processes, such as coronary heart disease (CHD) and robust adiposity [22][23][24][25][26][27][23,24,25,26,27,28].
Figure 2. Differentially expressed proteins (DEPs) of tPVAT were identified in Ang II infused mice. (A) The volcano plot showed the distribution and number of DEPs between the saline and Ang II infused tPVAT. The red dots represent the up-regulated proteins, while the blue dots represent the down-regulated proteins. (B) Hierarchical clustering of DEPs from the Ang II group, compared with the saline group (three biological replicates per group). Red indicates upregulation and blue represents downregulation. (C) The number of DEPs. There are 145 up-regulated proteins and 146 down-regulated proteins.
Table 1. List of up-regulated proteins during Ang II infusion.
Accession NO. Gene Name Protein Name Accession NO. Gene Name Protein Name
List of down-regulated proteins during Ang II infusion.
Accession NO. Gene Name Protein Name Accession NO. Gene Name Protein Name
Q6ZQ88 KDM1A Lysine-specific histone demethylase 1A Q9CPV3 MRPL42 39S ribosomal protein L42, mitochondrial
P70268 PKN1 Serine/threonine-protein kinase N1 Q8VCW8 ACSF2 Medium-chain acyl-CoA ligase ACSF2, mitochondrial
D) KEGG pathway enrichment bubble chart. The horizontal axes represent the richFactor (richFactor ≤ 1), which represents the ratio of the number of DEPs annotated to the KEGG pathway to the number of all identified proteins annotated to the KEGG category. The vertical axis represents the statistical results of DEPs under Top 20 KEGG pathways. The color of the bubble represents the significance of the enriched KEGG pathways. The color gradient from green to red represents the p value; the closer to red color, the lower the p value and the higher the significance level corresponding to the enrichment.
Table 3. Distribution of proteins and signaling pathways response to Ang II infusion, based on GO and KEGG analysis.
Terms Count p Value FDR Gene NamerichFactor Accession NO.
Protein Name Degree Up/Down Regulation
P18406 CCN1 CCN family member 1 A2RTL5 RSRC2 Arginine/serine-rich coiled-coil protein 2
GO (gene ontology)
P11276 FN1 Fibronectin 28 up
Q09098 Pate4 Prostate and testis expressed protein 4 Q8BL86 MBLAC2 Metallo-beta-lactamase domain-containing protein 2
Gene silencing (BP) 12 0.0011 0.5010 0.1600 Q91YE5, Q9Z2D6, O88712, Q6ZQ88, Q6Y7W8, Q9CY73, Q80VL1, P15864, P43276, O55135, P42227, P68433
P60710 ACTB Actin, cytoplasmic 1 25 down Q8BX80 ENGase Cytosolic endo-beta-N-acetylglucosaminidase Q9D0F9 PGM1 Q9ERR7Phosphoglucomutase-1
Selenof Selenoprotein F Q6ZWY8 TMSB10 Thymosin beta-10
Monosaccharide binding (MF) 9 0.0029 0.5010 0.1698 P41317, Q8K2I4, O08528, Q60715, Q64237, Q9D136, P12382, P06745, Q9JK38
Q63844 MAPK3 Mitogen-activated protein kinase 3 17 down O88712 CtBP1 C-terminal-binding protein 1 P53986 O54916SLC16A1 Reps1 RalBP1-associated Eps domain-containing protein 1 Q9D5V6 Syap1Monocarboxylate transporter 1
Synapse-associated protein 1
Extracellular matrix (CC) 22 0.0012 0.5010 0.1164 P07356, P11276, Q9WVH9, P97429, Q8K4G1, Q62009, P97384, A2ASQ1, C0HKD8, P06797, P41317, Q8BPB5, Q80YX1, O08746, P82198, P18406, Q61704, Q8R054, Q60754, O70503, P09242, Q9CXD9
Q9CRB2 NHP2 H/ACA ribonucleoprotein complex subunit 2 17 down Q8K1A5 Tmem41b Q9CRB2 NHP2Transmembrane protein 41B Q8BGQ1 VIPAS39 H/ACA ribonucleoprotein complex subunit 2 Q8BK08Spermatogenesis-defective protein 39 homolog
P42227 STAT3 Signal transducer and activator of transcription 3 16 up P70392 RasGRF2 Ras-specific guanine nucleotide-releasing factor 2 Q9D2R0 AACS Acetoacetyl-CoA synthetase
Transmembrane protein 11, mitochondrial
KEGG (kyoto encyclopedia of genes and genomes) pathways Q6U7R4 TIMD4 T-cell immunoglobulin and mucin domain-containing protein 4 Q80X80 C2CD2L Q61096
Fatty acid biosynthesisPhospholipid transfer protein C2CD2L
4 0.0069 0.5894 0.2857 Q5SWU9, Q8JZR0, Q8R3F5, Q91WC3
P50580 PA2G4 PRTN3 Myeloblastin Q9CXR1 Q9JKP5 MBNL1 Muscleblind-like protein 1DHRS7 Dehydrogenase/reductase SDR family member 7
Q9WV85 NME3 Nucleoside diphosphate kinase 3
EGFR tyrosine kinase inhibitor resistance 6 0.0076 0.5894 0.1935 Q63844, Q8BMB3, P63073, Q64373, P20444, P42227 Q8VD66 ABHD4 (Lyso)-N-acylphosphatidylethanolamine lipase Q8K3K7 Q61285 ABCD2
Q62348 TSN Translin Q9DCL2AGPAT2 1-acyl-sn-glycerol-3-phosphate acyltransferase beta
CIAO2A Cytosolic iron-sulfur assembly component 2A
PPAR signaling pathway 8 0.0092 0.5894 0.1538 P12242, Q8CGN5, O88492, Q8JZR0, P11404, Q64516, O35488, Q91WC3 Q6Y7W8 GIGYF2 GRB10-interacting GYF protein 2 Q924N4 Q8VCH7 RDH10 Retinol dehydrogenase 10SLC12A6 ATP-binding cassette sub-family D member 2
Proliferation-associated protein 2G4 15 up
Q99ME9 GTPBP4 Nucleolar GTP-binding protein 1 14 HIF-1 signaling pathwaySolute carrier family 12 member 6
8 0.0103 0.5894 0.1509 P12382, O08528, P11798, Q63844, Q8BMB3, P63073, P20444, P42227 Q61490 ALCAM CD166 antigen Q922Z0 DDO D-aspartate oxidase
O55135 eIF6 Eukaryotic translation initiation factor 6 14 down Q3THF9
P61202 COQ10B Coenzyme Q-binding protein COQ10 homolog B, mitochondrial P70333 HNRNPH2 Heterogeneous nuclear ribonucleoprotein H2 Q8CHD8 RAB11FIP3 Rab11-family interacting protein 3 Q14DH7
Galactose metabolism 4 0.0114 0.5894 0.2500 P12382, Q9D0F9, O08528, P70699 ACSS3 Acyl-CoA synthetase short-chain family member 3, mitochondrial
Q9JK38 GNPNAT1 Glucosamine 6-phosphate N-acetyltransferase Q69ZP3 PNKD Probable hydrolase PNKD Q8R054 SRPX2 Sushi repeat-containing protein SRPX2 Q00898 SERPINA1E Alpha-1-antitrypsin 1–5
Q05144 Rac2 Ras-related C3 botulinum toxin substrate 2 O35864 COPS5 COP9 signalosome complex subunit 5 Q3UVG3 FAM91A1 Protein FAM91A1 Q9JJF9 SPPL2a
P60710Signal peptide peptidase-like 2A
ACTB Actin, cytoplasmic 1 Q61464 ZNF638 O89017 LGMN Legumain Q9CS72 FILIP1 Filamin-A-interacting protein 1
COPS2 Zinc finger protein 638
Q9Z140 CPNE6 Copine-6 Q9JM14 NT5C 5’(3’)-deoxyribonucleotidase, cytosolic type Q8R1R3 StarD7 StAR-related lipid transfer protein 7, mitochondrial Q9QXG4 ACSS2 Acetyl-coenzyme A synthetase, cytoplasmic
Q924W5 Smc6 Structural maintenance of chromosomes protein 6 P28063 PSMB8 Proteasome subunit beta type-8
COP9 signalosome complex subunit 2 Q64516 GK Glycerol kinase Q5RKZ7 MOCS1 Molybdenum cofactor biosynthesis protein 1
P47955 RPLP1 60S acidic ribosomal protein P1 Q9D1L0 CHCHD2 Coiled-coil-helix-coiled-coil-helix domain-containing protein 2
P63073 eIF4E Eukaryotic translation initiation factor 4E Q3TMW1 CCDC102A Coiled-coil domain-containing protein 102A
12 Starch and sucrose metabolism 4 TRIM32 E3 ubiquitin-protein ligase TRIM32 Q9Z0V7 TIMM17B Mitochondrial import inner membrane translocase subunit Tim17-BQ9D023 MPC2 Mitochondrial pyruvate carrier 2 P67984 Rpl22 60S ribosomal protein L22
P83870 PHF5A PHD finger-like domain-containing protein 5A O88895 HDAC3 Histone deacetylase 3 E9Q1P8 IRF2BP2 Interferon regulatory factor 2-binding protein 2 Q99KF1 TMED9 Transmembrane emp24 domain-containing protein 9
P41317 MBL2 Mannose-binding protein C P97370 ATP1B3 Sodium/potassium-transporting ATPase subunit beta-3 P12242 UPC1 Mitochondrial brown fat uncoupling protein 1 Q9CY73 MRPL44 39S ribosomal protein L44, mitochondrial
up 0.2353
Q64373 BCL2L1 Bcl-2-like protein 1 11 down Q9D0F9, O08528, P06745, P70699 P15864 H1-2 Histone H1.2 P26645FABP3 Fatty acid-binding protein, heart Q8BHJ6 SERINC5 Serine incorporator 5
P09470 ACE Angiotensin-converting enzyme P70699 GAA Lysosomal alpha-glucosidase
Q8BMB3 eIF4E2 Eukaryotic translation initiation factor 4E type 2Ras-related protein Rab-2B
0.0143 0.6137 MARCKS Myristoylated alanine-rich C-kinase substrate Q3U4G3 XXYLT1 Xyloside xylosyltransferase 1
Q9D136 OGFOD3 2-oxoglutarate and iron-dependent oxygenase domain-containing protein 3 Q8C7H1 MMAA Methylmalonic aciduria type A homolog, mitochondrial
Q62009 POSTN Periostin Q80VW7
Q91WU5AKNA Microtubule organization protein AKNA
AS3MT Q78JN3 Eci3 Enoyl-CoA delta isomerase 3, peroxisomal O54940 BNIP2 BCL2/adenovirus E1B 19 kDa protein-interacting protein 2
P68433 H3C1 Histone H3.1 Q6PEE2 CTIF CBP80/20-dependent translation initiation factor
Arsenite methyltransferase Q3TEA8 HP1BP3 Q5SF07 IGF2BP2 Insulin-like growth factor 2 mRNA-binding protein 2 Q9D4H1 EXOC2 Exocyst complex component 2
Q9JM90 STAP1 Signal-transducing adaptor protein 1 Q8CFV9 RFK Riboflavin kinase
Q80YX1 TNC Tenascin P29595 NEDD8 NEDD8
O35488 SLC27A2 Very long-chain acyl-CoA synthetase 11 down Other glycan degradation 3 Q9DCE5 PAK1IP1 p21-activated protein kinase-interacting protein 1 Q9Z0P5 TWF2 Twinfilin-2
Q8JZR0 ACSL5 Long-chain-fatty-acid--CoA ligase 5 Q9CXJ1 EARS2 Probable glutamate--tRNA ligase, mitochondrial
0.0169 Q9CXD9 LRRc17 Leucine-rich repeat-containing protein 17 Q8VHQ9 ACOT11 Acyl-coenzyme A thioesterase 11
Q8BWJ3 PHKA2 Phosphorylase b kinase regulatory subunit alpha, liver isoform Q920B9 SUPT16H FACT complex subunit SPT16
Q811L6 MAST4 Microtubule-associated serine/threonine-protein kinase 4 Q9DCV4 RMDN1 Regulator of microtubule dynamics protein 1
P61202 COPS2 COP9 signalosome complex subunit 2 P06745 GPI Glucose-6-phosphate isomerase
Q1W617 SHROOM4 Protein SHROOM4 O08600 EndoG Endonuclease G, mitochondrial
P63158 HMGB1 High mobility group protein B1 O08746 MATN2 Matrilin-2
Heterochromatin protein 1-binding protein 3 Q78HU7 GYPC Glycophorin-C Q8R332 NUP58 Nucleoporin p58/p45
Q9QZB0 RGS17 Regulator of G-protein signaling 17 P97384 ANXA11 Annexin A11
Q8BGX2 TIMM29 Mitochondrial import inner membrane translocase subunit Tim29 Q5XG73 ACBD5 Acyl-CoA-binding domain-containing protein 5
P97429 ANXA4 Annexin A4 Q60754
Q5SWU9 ACACA0.6230 0.3000 Q8BX80, Q69ZF3, Q8K2I4
Acetyl-CoA carboxylase 1 11 up Peroxisome
P679848 0.0210 0.6757 0.1333 Rpl2260S ribosomal protein L22 11 upMARCO Macrophage receptor MARCO
P08228, Q8JZR0, P34914, Q78JN3, Q61285, O35488, Q91WC3, Q922Z0
Lysosome 8 O55186 CD59a CD59a glycoprotein A2AJB2 TMEM141 Transmembrane protein 141
Q8BLV3 SLC9A7 Sodium/hydrogen exchanger 7 Q8K2I4 MANBA Beta-mannosidase
Q0VBL1 TIGD2 Tigger transposable element-derived protein 2 P20444 PRKCA Protein kinase C alpha type
O09117 SYPL1 Synaptophysin-like protein 1 P43276 H1.5 Histone H1.5
Q9WTR5 CDH13 Cadherin-13 Q8CGN5 PLIN1 Perilipin-1
Q8VCF0 MAVS Mitochondrial antiviral-signaling protein Q63844 MAPK3 Mitogen-activated protein kinase 3
O88492 PLIN4 Perilipin-4 Q3UMY5 EML4 Echinoderm microtubule-associated protein-like 4
Q80US4 ACTR5 Actin-related protein 5 Ndufb11 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 11, mitochondrial      
Table 2.
Barrier-to-autointegration factor
Q61207, Q9Z1T1, P06797, O89017, P12265, P70699, O70370, Q8K2I4
Caspase recruitment domain-containing protein 19
O70451 SLC16A7
Thiamine metabolism 2 0.0427 0.7758 0.3333 P09242, Q9R0Y5 Monocarboxylate transporter 2 Q3UEZ8 SLC10A4 Sodium/bile acid cotransporter 4
P26011 ITGB7 Integrin beta-7 Q99ME9 GTPBP4 Nucleolar GTP-binding protein 1
Q9JHH9 COPZ2 Coatomer subunit zeta-2 Q0VGM9 RTEL1 Regulator of telomere elongation helicase 1
O54788 DFFB DNA fragmentation factor subunit beta Q9JLQ0 CD2AP CD2-associated protein
P82348 SGCG
Apoptosis 7 0.0443 0.7758 0.1228 P60710, O08600, P06797, Q63844, Q64373, O70370, O54788 Gamma-sarcoglycan O70370 CTSS Cathepsin S
Ras signaling pathway 8 0.0472 0.7758 0.1143 P62874, Q05144, Q9DAS9, Q63844, Q64373, P20444, P70392, Q9D4H1 B1AVZ0 UPRT Uracil phosphoribosyltransferase homolog Q69ZF3 GBA2 Non-lysosomal glucosylceramidase
Q9JHI7 EXOSC9 Exosome complex component RRP45 Q8K124 PLEKHO2 Pleckstrin homology domain-containing family O member 2
Q61207 PSAP Prosaposin P19973 LSP1 Lymphocyte-specific protein 1
O35638 STAG2 Cohesin subunit SA-2 P62874 GNB1 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
Q91YE5 BAZ2A Bromodomain adjacent to zinc finger domain protein 2A Q80VL1 TDRKH Tudor and KH domain-containing protein
Q9CY57 Chtop Chromatin target of PRMT1 protein Q9Z2D6 MECP2 Methyl-CpG-binding protein 2
O55135 eIF6 Eukaryotic translation initiation factor 6 Q61503 NT5E 5’-nucleotidase
Q8R3B7 BRD8 Bromodomain-containing protein 8 Q8K2Q5 CHCHD7 Coiled-coil-helix-coiled-coil-helix domain-containing protein 7
Q8R143 PTTG1IP Pituitary tumor-transforming gene 1 protein-interacting protein Q8BP48 MetAP1 Methionine aminopeptidase 1
Q9D2V5 AAR2 Protein AAR2 homolog Q64237 DBH Dopamine beta-hydroxylase
P28798 GRN Progranulin O35226 PSMD4 26S proteasome non-ATPase regulatory subunit 4 P36536 SAR1A
Q9R0Y5 AK-1GTP-binding protein SAR1A Q78J03 MsrB2 Methionine-R-sulfoxide reductase B2, mitochondrial
Adenylate kinase isoenzyme 1 E9PZM4 CHD2 Chromodomain-helicase-DNA-binding protein 2 Q8BJ03 COX15 Cytochrome c oxidase assembly protein COX15 homolog
P03899 MTND3Q99L45 EIF2S2 Eukaryotic translation initiation factor 2 subunit 2
NADH-ubiquinone oxidoreductase chain 3 P26369 U2AF2 Splicing factor U2AF 65 kDa subunit Q8BHC0 LYVE-1 Lymphatic vessel endothelial hyaluronic acid receptor 1 P97452
Q8JZS0 LIN7ABOP1 Ribosome biogenesis protein BOP1
Protein lin-7 homolog A P56379 ATP5MPL ATP synthase subunit ATP5MPL, mitochondrial Q8VE11 MTMR6 Myotubularin-related protein 6 O08528
Q91V12 ACOT7 Cytosolic acyl coenzyme A thioester hydrolaseHK2 Hexokinase-2
E9Q5C9 NOLC1 Nucleolar and coiled-body phosphoprotein 1 Q91V76 C11orf54 Ester hydrolase C11orf54 homolog Q9Z1T1 Ap3B1 AP-3 complex subunit beta-1
Q64008 Rab34 Ras-related protein Rab-34 Q920A5 SCPEP1 Retinoid-inducible serine carboxypeptidase
Q00724 RBP4 Retinol-binding protein 4 P59279 P30993 C5aR1 C5a anaphylatoxin chemotactic receptor 1 Q91VU0 FAM3C Protein FAM3C
Q91WC3 ACSL6 Long-chain-fatty-acid--CoA ligase 6 Q9CQE7 ERGIC3 Endoplasmic reticulum-Golgi intermediate compartment protein 3
Q7TPM3 TRIM17 E3 ubiquitin-protein ligase TRIM17 P50580 PA2G4 Proliferation-associated protein 2G4
P70122 SBDS Ribosome maturation protein SBDS Q9CZD5 MTIF3 Translation initiation factor IF-3, mitochondrial
Q8CHG3 GCC2 GRIP and coiled-coil domain-containing protein 2 P01631   Ig kappa chain V-II region 26–10
RAB2B Q80X95 RRAGA Ras-related GTP-binding protein A Q9D125 MRPS25 28S ribosomal protein S25, mitochondrial
Q8R3F5 MCAT Malonyl-CoA-acyl carrier protein transacylase, mitochondrial Q5SW19 CLUH Clustered mitochondria protein homolog
Q99LJ6 GPX7 Glutathione peroxidase 7 Q7TPE5 SLC7A6OS Probable RNA polymerase II nuclear localization protein SLC7A6OS
O70503 HSD17B12 Very-long-chain 3-oxoacyl-CoA reductase P42227 STAT3 Signal transducer and activator of transcription 3
P12382 PFKL ATP-dependent 6-phosphofructokinase, liver type Q9Z0S1 BPNT-1 3′(2′),5′-bisphosphate nucleotidase 1
Q61704 ITIH3 Inter-alpha-trypsin inhibitor heavy chain H3 P09242 ALPL Alkaline phosphatase, tissue-nonspecific isozyme

4.2. KEGG Pathway Analysis

By searching the major biological pathways and relevant regulatory processes involved in the KEGG, we analyzed all of the DEPs in the saline and Ang II infusion groups. The results of the KEGG pathway analysis showed that the significant enrichment pathways included fatty acid (FA) biosynthesis (4 proteins, richFactor = 0.29), EGFR tyrosine kinase inhibitor resistance (6 proteins, richFactor = 0.19), PPAR signaling pathway (8 proteins, richFactor = 0.15), HIF-1 signaling pathway (8 proteins, richFactor = 0.15), galactose metabolism (4 proteins, richFactor = 0.25), starch and sucrose metabolism (4 proteins, richFactor = 0.24), other glycan degradation (3 proteins, richFactor = 0.30), peroxisome (8 proteins, richFactor = 0.13), lysosome (8 proteins, richFactor = 0.12), thiamine metabolism (2 proteins, richFactor = 0.33), apoptosis (7 proteins, richFactor = 0.12), and Ras signaling pathway (8 proteins, richFactor = 0.11) (Figure 4D and Table 3).
Next, we connected these significant enrichment pathways and found that Ang II infusion deeply affected the fat metabolism and adipocyte differentiation (Figure 5A,B). Figure 5A shows that the up-regulation of acetyl-CoA carboxylase α and acyl-CoA synthetase long-chain family member 5 significantly affected the synthesis of long-chain FAs. In addition, Figure 5B shows that Ang II infusion affected the PPAR signaling pathway by regulating very long-chain acyl-CoA synthase (ACSVL) and FA binding protein 3 (FABP3), and thereby affected lipid metabolism, thermogenesis, and adipocyte differentiation. Additionally, we also found that Ang II infusion could affect many downstream signaling pathways, such as Rac, PI3K/Akt, MAPK, and the HIF-1α pathways, by regulating the Ras signaling that, in turn, affected many cell functions and participated in pathogenesis (Figure 5C).
Figure 5. Diagram of the important signaling pathways that have changed after Ang II infusion. Up-regulated DEPs after Ang II infusion are shown in red. Down-regulated DEPs after Ang II infusion are shown in green. (A) Fatty acid biosynthetic pathway. (B) PPAR signaling pathway that affects lipid metabolism, themogenesis, and adipocyte differentiation. (C) Ras and its downstream signaling pathways. Ras-Rac signaling affects cell motility. Ras-PI3K-Akt and Ras-ERK1/2 signaling affects cell apoptosis and gene expression. Ras-HIF1α pathway affects cell protein degradation and oxygen delivery.

4.3. PPI Analysis

In the PPI network, there were 20 high-connectivity degree DEPs with a large circle and a degree value of more than 10 between the saline and Ang II groups (Figure 6). These high-connectivity DEPs were identified as fibronectin (P11276), actin (P60710), mitogen-activated protein kinase (MAPK) 3 (Q63844), H/ACA ribonucleoprotein complex subunit 2 (Q9CRB2), signal transducer and activator of transcription (STAT) 3 (P42227), proliferation-associated protein 2G4 (P50580), nucleolar GTP-binding protein 1 (Q99ME9), eukaryotic translation initiation factor 6 (O55135), COP9 signalosome complex subunit 2 (P61202), Bcl-2-like protein 1 (Q64373), ACSVL (O35488), acetyl-CoA carboxylase 1 (Q5SWU9), 60S ribosomal protein L22 (P67984), hexokinase-2 (O08528), superoxide dismutase [Cu-Zn] (P08228), eukaryotic translation initiation factor 4E (P63073), high mobility group protein B1 (P63158), eukaryotic translation initiation factor 2 subunit 2 (Q99L45), lysine-specific histone demethylase 1A (Q6ZQ88), and ubiquitin carboxyl-terminal hydrolase isozyme L5 (Q9WUP7) (Table 4). Some high-connectivity node proteins in Table 4 were related to the MAPK-Erk, STAT, FA biosynthesis, PPAR, and apoptosis signaling pathways, and these were highly consistent with those obtained using KEGG.
Figure 6. Protein protein interaction (PPI) analysis of DEPs between saline and Ang II infusion group. The circled nodes represent DEPs, and the lines represent protein-protein interactions. Red circles represent up-regulation proteins. Blue circles represent down-regulation proteins. The size of the circle indicates the degree of protein connectivity.
Table 4. DEPs with high connectivity degree in PPI analysis between saline and Ang II infusion group.
Accession NO.
Superoxide dismutase [Cu-Zn] 10 up
P63073 eIF4E Eukaryotic translation initiation factor 4E 10 down
P63158 HMGB1 High mobility group protein B1 10 up
Q99L45 EIF2S2 Eukaryotic translation initiation factor 2 subunit 2 10 up
Q6ZQ88 KDM1A Lysine-specific histone demethylase 1A 10 up
Q9WUP7 UCHL5 Ubiquitin carboxyl-terminal hydrolase isozyme L5 10 up
Probable global transcription activator SNF2L2
Cathepsin L1
Lipase maturation factor 2
Annexin A2
P99028 UQCRHVolume-regulated anion channel subunit LRRC8A Q61391 MME Neprilysin
Cytochrome b-c1 complex subunit 6, mitochondrial Q9WVH9 FBLN5 Fibulin-5 Q9CQV4 RETREG3 Reticulophagy regulator 3 P82198 TGFBI Transforming growth factor-beta-induced protein ig-h3
P58742 AAAS Aladin P43135 NR2F2 COUP transcription factor 2 Q8BGR6 ARL15 ADP-ribosylation factor-like protein 15 Q9WUP7
Q6PCP5 MffUCHL5 Ubiquitin carboxyl-terminal hydrolase isozyme L5
Mitochondrial fission factor Q8R4R6 NUP35 Nucleoporin NUP35 P11276 FN1 Fibronectin P52196 TST Thiosulfate sulfurtransferase
O35488 P70460 VASP Vasodilator-stimulated phosphoprotein Q9QZA0 CA5B Carbonic anhydrase 5B, mitochondrial
P06339 H2-T23 H-2 class I histocompatibility antigen, D-37 alpha chain Q8QZY9 SF3B4 Splicing factor 3B subunit 4
Q65Z40 Wapl Wings apart-like protein homolog P34914 EPHX2 Bifunctional epoxide hydrolase 2
P10126 Eef1a1 Elongation factor 1-alpha 1 O54946 DNAJB6 DnaJ homolog subfamily B member 6
P54823 Ddx6 Probable ATP-dependent RNA helicase DDX6 Q9CQ86 MIEN1 Migration and invasion enhancer 1
Q8BUE4 Aifm2 Apoptosis-inducing factor 2 Q8CG46 SMC5 Structural maintenance of chromosomes protein 5
O35143 ATP5IF1 ATPase inhibitor, mitochondrial Q60715 P4HA1 Prolyl 4-hydroxylase subunit alpha-1
SLC27A2 Very long-chain acyl-CoA synthetase P11798 CAMK2A Calcium/calmodulin-dependent protein kinase type II subunit alpha
Q9DAS9 GNG12 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-12 Q8BHL3 TBC1D10B TBC1 domain family member 10B
Q9D1G5 LRRC57 Leucine-rich repeat-containing protein 57 Q8BTI8 SRRNM2 Serine/arginine repetitive matrix protein 2
Q8BGD8 COA6 Cytochrome c oxidase assembly factor 6 homolog P97952 SCN1B Sodium channel subunit beta-1
Q9CRB8 MTFP1 Mitochondrial fission process protein 1 P51912 SLC1A5 Neutral amino acid transporter B (0)
Q99MS7 EHBP1L1 EH domain-binding protein 1-like protein 1 Q8BPB5 EFEMP1 EGF-containing fibulin-like extracellular matrix protein 1
Q61029 TMPO Lamina-associated polypeptide 2, isoforms beta/delta/epsilon/gamma Q64373 BCL2L1 Bcl-2-like protein 1
Q91YU6 LZTS2 Leucine zipper putative tumor suppressor 2 Q6P9Q4 FHOD1 FH1/FH2 domain-containing protein 1 Q9Z0V8 Timm17a Mitochondrial import inner membrane translocase subunit Tim17-A
Q8K212 PACS1P15105 GLUL Glutamine synthetase
Phosphofurin acidic cluster sorting protein 1 Q99L88 SNTB1 Beta-1-syntrophin Q9CWX9 Ddx47 Probable ATP-dependent RNA helicase DDX47 P08228
Q3U3R4 LMF1SOD1 Superoxide dismutase [Cu-Zn]
Lipase maturation factor 1 Q80UZ0 FGD5 FYVE, RhoGEF and PH domain-containing protein 5 O35344 Kpna3 Importin subunit alpha-4 Q5SWU9
Q9JI46 NUDT3 Diphosphoinositol polyphosphate phosphohydrolase 1ACACA Acetyl-CoA carboxylase 1
O35704 SPTLC1 Serine palmitoyltransferase 1 O54950 Prkag1 5’-AMP-activated protein kinase subunit gamma-1 Q91Z96
Q8K4G1 LTBP-4 Latent-transforming growth factor beta-binding protein 4 A2ASQ1BMP2K BMP-2-inducible protein kinase
AGRN Agrin Q99P65 Slc29a3
P70429 EVLEquilibrative nucleoside transporter 3 Q9CYK1 WARS2 Tryptophan--tRNA ligase, mitochondrial
Ena/VASP-like protein Q9JIG8 PRAF2 PRA1 family protein 2
C0HKD8 MFAP1a Microfibrillar-associated protein 1A Q8R323 RFC3 Replication factor C subunit 3
In addition, in order to verify the results of the TMT proteomics, we selected the top 10 up- or down-regulated proteins for immunohistochemical analyses. Figure 3 shows that the expressions of KDM1A, PKN1, CtBP1, TMEM41B, PRTN3, GIGYF2, and ALCAM were significantly up-regulated, while that of CCN1, MBNL1, NHP2, RDH10, TIMD4, and TSN were significantly decreased after Ang II infusion. The immunohistochemical results confirmed the expression pattern observed in the quantitative proteomics analysis. Since there were no suitable antibodies for ENGase, RasGRF2, ABHD4, Pate4, Selenof, Reps1, and COQ10B, we did not perform immunohistochemical verification of these proteins.
Figure 3. Immunohistochemical verification of DEPs. (A) Immunohistochemistry staining of up-regulated proteins, such as lysine-specific histone demethylase 1A (KDM1A), serine/threonine-protein kinase N1 (PKN1), C-terminal-binding protein 1 (CtBP1), transmembrane protein 41B (TMEM41B), myeloblastin (PRTN3), CD166 antigen (ALCAM), and GRB10-interacting GYF protein 2 (GIGYF2). Positive staining was indicated by brown coloration, and nuclei were stained with hematoxylin in blue. (B) Immunohistochemistry staining of down-regulated proteins, such as CCN family member 1 (CCN1), muscleblind-like protein 1 (MBNL1), H/ACA ribonucleoprotein complex subunit 2 (NHP2), retinol dehydrogenase 10 (RDH10), T-cell immunoglobulin and mucin domain-containing protein 4 (TIMD4), and Translin (TSN). Positive cells are indicated by brown coloration.

4. Bioinformatics Analysis of the Altered Proteins during Ang II Infusion

4.1. GO Functional Annotation and Analysis

We used the subcellular structure prediction software CELLO to analyze the subcellular location of all the DEPs. As shown in Figure 4A, most of the proteins were distributed in the nucleus, cytoplasm, extracellular matrix, and mitochondria. The DEPs were then analyzed against the GO database using three sets of ontologies: biological process (BP), molecular function (MF), and cellular component (CC). The most enriched GO terms of BP, MF, and CC were annotated as a gene silencing (12 proteins, richFactor = 0.16), monosaccharide binding (9 proteins, richFactor = 0.17), extracellular matrix (22 proteins, richFactor = 0.12), respectively (Figure 4B,C and Table 3). Other important BPs were included in DNA packaging, monocarboxylic acid transport, positive regulation of immunoglobulin secretion, organic acid transmembrane transport, carboxylic acid transmembrane transport, etc. (Figure 4C). Other important MFs included CoA-ligase activity, acid-thiol ligase activity, medium-chain fatty acid-CoA ligase activity, ligase activity, forming carbon-sulfur bonds, and organic hydroxy compound transmembrane transporter activity (Figure 4C). Other important CCs included host intracellular organelles, host intracellular membrane-bounded organelles, SUMO ligase complex, and Smc5–Smc6 complex (Figure 4C).
Figure 4. Functional analysis of DEPs. (A) Subcellular location of DEPs between saline and Ang II group. The numbers show the proteins located in different subcellular components. (B) Overall enriched GO terms. The vertical axis with different colors of the bar charts represents the significance of enrichment of the three categories (biological processes, molecular functions, cellular components). The horizontal axes represent the number of DEPs. (C) Top 20 enriched GO analyses of Ang II-altered proteins. BP: biological processes; MF: molecular functions; CC: cellular components. The color gradient from orange to red represents the p value. The numbers above the bar charts represent the richFactor (richFactor ≤ 1). (
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