DOI: https://doi.org/10.21203/rs.2.19789/v1
Background: Spinal dural arteriovenous fistula (SDAVF) is the most common spinal vascular shunt lesion. Although pathological changes in the SDAVF draining vein (SDAVF-DV) have been elucidated, protein changes remain enigmatic. We investigated protein changes in the SDAVF-DV.
Methods: Three SDAVF-DV samples were collected, and superficial temporal artery (STA) and superficial temporal vein (STV) samples were used as controls. After quantification and enzymolysis of the proteins, label-free quantitative proteomics was performed, and the peptide mixture was fractionated and analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify the differentially expressed proteins. Bioinformatics analysis of the differentially expressed proteins was also performed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) network analyses.
Results: Compared with the STA, the SDAVF-DV had 195 upregulated proteins and 303 downregulated proteins. GO analysis showed that the most differential GO terms in each category were the adenylate cyclase-modulating G protein-coupled receptor signalling pathway, U6 snRNP and SH3 domain binding. KEGG pathway analysis showed that the most differentially expressed protein pathway was focal adhesion. Compared with the STV, the SDAVF-DV had 158 upregulated proteins and 362 downregulated proteins. GO analysis showed that the most differential GO terms in each category were lamellipodium assembly, U6 snRNP, and SH3 domain binding. KEGG pathway analysis showed that the most differentially expressed protein pathway was dilated cardiomyopathy. The PPI analysis revealed PPIs among the top 300 proteins.
Conclusions: We demonstrated that the SDAVF-DV showed specific protein expression changes under long-period venous hypertension. The results of the present study will provide insights into the pathogenesis of SDAVF formation at the protein level. The proteomic results provide a scientific foundation for further study to explore the pathophysiological mechanism of SDAVF.
Spinal dural arteriovenous fistula (SDAVF) is the most common spinal vascular shunt lesion characterized by an abnormal connection between a radicular meningeal artery and a radicular medullary vein. As venous connections drain to radicular veins, the draining vein shows gradual arterialization. Because of venous hypertension, clinical presentations and progressive myelopathy can be assessed.
In SDAVF, venous drainage is provided by longitudinal spinal veins linked together and to the epidural network1, 2. In many clinical case reports, the arterialized SDAVF draining vein (SDAVF-DV) was identified easily after opening the dura during an operation. The pathology of the arterialized SDAVF-DV was mentioned in a previous study. However, the protein changes in this arterialized vein under high intravascular pressure remain enigmatic.
In the present study, we used quantitative proteomics to compare the SDAVF-DV with the superficial temporal artery (STA) and superficial temporal vein (STV) to show different protein expression levels under venous hypertension. The results of our present study might provide insights into the pathogenesis of SDAVF formation at the protein level.
Ethics statement
The current study was examined and approved by the Ethics Committee of Huashan Hospital, Fudan University. Each participant provided their written informed consent to participate in this study.
Patients and tissue sample preparation
Three SDAVF-DVs were removed after microsurgery ligation. Three STAs and three STVs were obtained from patients with intracranial tumours via the extended pterional approach3, 4. We used the samples from each group for the comparative proteomics analysis. The tissues used for the proteomics analysis were immediately frozen in liquid nitrogen and stored at -80°C. The SDAVF-DVs, STAs and STVs were homogenized in a 4% SDS, 100 mM Tris‐HCl and 100 mM DTT solution. Then, a fluorescence assay was conducted to determine the total protein concentration. Approximately 200 μg of total protein from the tissues was proteolysed on a 10-kDa filter (PALL Life Sciences, Shanghai, China) using a Filter Aided Sample Preparation (FASP) protocol as described in detail elsewhere5. The peptide solution was transferred to a Solid Phase Extraction Cartridge (Empore 7 mm/3 mL) for desalting and clean-up. The peptide samples were resuspended in water with 0.1% formic acid (v/v), and the protein content was estimated by UV light spectral density at 280 nm6 prior to analysis by nano-liquid chromatography tandem mass spectrometry (N-LC-MS/MS).
Label-free quantitative analysis and data processing
Trypsin-digested peptides from the tissues were analysed by LC-MS/MS; each sample was analysed twice. All raw Xcalibur files acquired from the MS runs were analysed using the default settings of MaxQuant software (version 1.3.0.5) with minor modifications as previously described7. Hierarchical clustering was performed with MEV software (v4.6, TIGR). The differentially expressed proteins (p < 0.05) were analysed by hierarchical clustering to identify potential markers capable of classifying all samples.
The clustering pattern and expression analyses and volcano plots were based on R software according to standardized data. Venn diagrams of the characteristics of each of the three groups of differentially expressed proteins were generated.
The Gene Ontology (GO) and enrichment analyses of the dysregulated proteins in this experiment were based on the publicly available databases DAVID 6.7 (http://david.abcc.ncifcrf.gov/) and QuickGO (http://www.ebi.ac.uk/QuickGO/).
The genomic, chemical and systemic functions of the dysregulated proteins were analysed and enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (http://www.kegg.jp/kegg/pathway.html). The significance of differential protein enrichment in each pathway entry was calculated using the hypergeometric distribution test and is expressed as the p value.
Predicted protein-protein interaction (PPI) networks for these differentially expressed proteins were constructed using the STRING database (http://string.embl.de/) and Cytoscape software (http://www.cytoscape.org/).
Statistical analysis
The statistical analysis was performed with IBM SPSS, and the graphs were generated with GraphPad Prism software. The significance of differences between two groups in the proteomics analysis was assessed using one-way analysis of variance (ANOVA). Proteins were defined as significantly differentially expressed when the ratio was ³2 or £0.5 in the SDAVF-DV compared with normal tissues (p <0.01).
Identification of differentially expressed proteins in the SDAVF-DVs, STAs and STVs
Three paired SDAVF-DV, STA and STV tissue samples were analysed in the initial discovery phase.
An equal amount of protein from each tissue was digested. Then, the peptides were analysed by N-LC-MS/MS. Using MaxQuant (version 1.3.0.5), we identified 2829 non-redundant proteins with a local false discovery rate (FDR) < 1% and at least two unique peptides per protein. The label-free quantification (LFQ) intensity ratios for the 2829 proteins were calculated, and significant differences in the protein expression levels between two tissues were determined using a t-test (p< 0.05). Compared with the STA, the SDAVF-DV had 195 significantly upregulated proteins and 303 significantly downregulated proteins. Compared with the STV, among the 520 proteins that exhibited significant differences, 158 were significantly upregulated and 362 were significantly downregulated in the SDAVF-DV (Figure 1A). When the three groups were combined, 480 differentially expressed proteins were identified (shown in the heatmap in Figure 1B). Venn analysis showed the variation and commonalities of different proteins in each group. A total of 1026 proteins were expressed in all groups, and 150 proteins were identified only in the SDAVF-DV (Figure 1C).
GO analysis
We performed GO analysis to analyse the differentially expressed proteins. When comparing the SDAVF-DV with the STA, most of differential GO terms expressed in each category were the adenylate cyclase-modulating G protein-coupled receptor signalling pathway, U6 snRNP and SH3 domain binding. We examined the top ten up- and downregulated GO terms in the biological processes, cellular components and molecular functions categories with 2.0-fold (p<0.05) differential gene expression (Table 1).
When comparing the SDAVF-DV with the STV, and the most differential GO terms expressed in each category were lamellipodium assembly, U6 snRNP, and SH3 domain binding.
The top ten up- and downregulated GO terms based on the comparison of the SDAVF-DV and STV are also listed in Table 2. In GO classification, 93 differentially enriched GO terms were found between the SDAVF-DV and STA: 60 GO terms were upregulated, and 33 were downregulated (Figure 2A). Compared with the STV, the SDAVF-DV had 109 differentially enriched GO terms: 31 terms were upregulated, and 78 were downregulated (Figure 2B).
KEGG pathway analysis
The KEGG pathway analysis of these differentially expressed proteins also demonstrated related pathways. Figure 3 shows the number of proteins in each KEGG pathway and the p value of the top 20 pathways. Compared with the STA, the top three differentially expressed protein pathways were focal adhesion, the PI3K-Akt signalling pathway and the extracellular matrix (ECM)-receptor interaction. Compared with the STV, the top three differentially expressed pathways were dilated cardiomyopathy, hypertrophic cardiomyopathy and adrenergic signalling in cardiomyocytes.
PPI analysis
We used the STRING database to analyse the differentially expressed proteins, obtain the interactions/relationships among the differentially expressed proteins and calculate the combined score. We selected the top 300 proteins and found significant PPIs among them (Figures 5 and 6). Compared with the STA and STV, the SDAVF-DV showed up- and downregulated proteins, and the top three interaction proteins are listed in Table 3.
SDAVF is a common arteriovenous shunt located inside the dura mater close to the spinal nerve root8. Venous hypertension, which induces medullar venous outflow disturbances, results in chronic hypoxia and congestive myelopathy9. The direct intraoperative measurement of the vascular pressure in the fistula can be as high as 74% of the systemic arterial pressure10, 11. This finding may explain why, in some patients, symptoms become worse during physical activity with a concomitant increase in arterial pressure12, 13. Under long-period venous hypertension, draining vein arterialization begins.
A clear understanding of the mechanism of SDAVF development is still lacking. Our study was the first to perform a comparative proteome analysis and show the differential expression of proteins in arterialized SDAVF-DVs compared with normal arteries and veins. In general, most of the proteins were the same between the three groups. Because of its special pathophysiology, the SDAVF-DV showed specific protein expression compared with the STA and STV.
In the intraoperative observation, the SDAVF-DV showed arterial morphology. A. Thron proposed a hypothesis based on spine arteriovenous shunt anatomy9. Keisuke Takai showed that the vessel wall of the proximal subarachnoid portion of the intradural draining vessels was irregularly thickened by collagen and exhibited elastic fibrosis and was without a continuous internal elastic lamina and a regular smooth muscle layer. The diameter of the vessels was significantly enlarged14.
After GO analysis, the SDAVF-DV showed a decrease in smooth muscle contractile fibres, which might indicate smooth muscle cell dysfunction. This might be induced by long-range venous hypertension stretching on the SDAVF-DV. Stretch plays an important role in maintaining smooth muscle cell function and regulating inflammation. A former study showed that mechanical stretch-induced endoplasmic reticulum stress, apoptosis and inflammation contribute to thoracic aortic aneurysm and dissection15. In our research, we also identified that mechanical stretching could induce smooth muscle cell changes from a contract phenotype to an inflammatory phenotype16, 17. The regulation of inflammatory factors is related to the hypothesis on SDAVF formation. In THE KEGG and PPI analyses, the ECM and focal adhesion showed obvious changes. Degeneration of the ECM is primarily induced by the secretion of inflammatory cytokines and cell infiltration in cerebral vascular disease18, 19. During SDAVF formation, inner vessel wall inflammation might contribute to an insufficient ECM and trigger changes in pathological proteins.
To our knowledge, few studies have focused on the SDAVF-DV. However, several researchers have investigated its pathological characteristics14, 20. We first examined protein changes to determine whether the lesion vessel was an artery, vein or vein-to-artery transition.
Most previous studies have revealed the histology and anatomy of the SDAVF-DV. Based on intraoperative findings, we demonstrated protein changes in the arterial SDAVF-DV. Our study adds new information on the formation of SDAVF to the realm of protein changes in the draining vein using proteomics. This finding may shed light on the mechanism of SDAVF formation.
SDAVF= Spinal dural arteriovenous fistula
SDAVF-DV= Spinal dural arteriovenous fistula draining vein
STA= superficial temporal artery
STV= superficial temporal vein
KEGG= Kyoto Encyclopedia of Genes and Genomes (http://www.genome.jp/kegg/)
Availability of data and materials:
The datasets used in the current study are available from the corresponding author on reasonable request.
Declaration:
Table 1 Top 10 GO term (SDAVF-DV vs STA)
Category |
Top 10 upregulated GO term |
Top 10 downregulated GO term |
Biological process |
||
|
adenylate cyclase-modulating G-protein coupled receptor signaling pathway |
translation reinitiation |
|
lamellipodium assembly |
IRES-dependent translational initiation |
|
lamellipodium morphogenesis |
ribosome disassembly |
|
regulation of epithelial cell migration |
regulation of growth |
|
regulation of microtubule-based process |
regulation of cardiac muscle hypertrophy |
|
wound healing |
positive regulation of calcineurin-NFAT signaling cascade |
|
regulation of receptor recycling |
plasma membrane repair |
|
ESCRT III complex disassembly |
regulation of vascular endothelial growth factor receptor signaling pathway |
|
regulation of osteoclast differentiation |
prostaglandin biosynthetic process |
|
trophectodermal cell differentiation |
secretion |
Cellular component |
||
|
nuclear pore outer ring |
U6 snRNP |
|
GATOR2 complex |
Lsm1-7-Pat1 complex |
|
mitochondrial envelope |
ESCRT III complex |
|
elastic fiber |
cytoplasmic side of plasma membrane |
|
GAIT complex |
membrane coat |
|
intermediate filament |
endoplasmic reticulum exit site |
|
neurofilament cytoskeleton |
nuclear outer membrane |
|
microfibril |
rough endoplasmic reticulum |
|
membrane raft |
LINC complex |
|
late endosome membrane |
smooth muscle contractile fiber |
Molecular function |
||
|
SH3 domain binding |
phospholipid binding |
|
antioxidant activity |
protein disulfide oxidoreductase activity |
|
ATP-dependent NAD(P)H-hydrate dehydratase activity |
lyase activity |
|
ADP-dependent NAD(P)H-hydrate dehydratase activity |
glutathione binding |
|
phosphatidylinositol phospholipase C activity |
prostaglandin-E synthase activity |
|
phospholipase C activity |
catalytic activity |
|
metallopeptidase activity |
protein serine/threonine phosphatase inhibitor activity |
|
G-protein coupled serotonin receptor binding |
peroxidase activity |
|
3-hydroxyacyl-CoA dehydrogenase activity |
interleukin-1 receptor antagonist activity |
|
acetyl-CoA C-acyltransferase activity |
proteinase activated receptor binding |
Table 2 Top 10 GO term (SDAVF-DV vs STV)
Category |
Top 10 upregulated GO term |
Top 10 downregulated GO term |
Biological process |
||
|
lamellipodium assembly |
exonucleolytic nuclear-transcribed mRNA catabolic process involved in deadenylation-dependent decay |
|
lamellipodium morphogenesis |
ubiquitin homeostasis |
|
neuromuscular synaptic transmission |
endosomal vesicle fusion |
|
dGTP catabolic process |
positive regulation of pinocytosis |
|
regulation of innate immune response |
regulation of cardiac muscle hypertrophy |
|
dATP catabolic process |
positive regulation of calcineurin-NFAT signaling cascade |
|
regulation of receptor recycling |
early endosome to Golgi transport |
|
ESCRT III complex disassembly |
regulation of cardiac muscle contraction |
|
positive regulation of epithelial to mesenchymal transition |
protein targeting to plasma membrane |
|
positive regulation of glycogen biosynthetic process |
positive regulation of voltage-gated calcium channel activity |
Cellular component |
||
|
zonula adherens |
U6 snRNP |
|
cell-substrate adherens junction |
Lsm1-7-Pat1 complex |
|
sarcoplasm |
Cajal body |
|
mitochondrial small ribosomal subunit |
macropinosome |
|
mitochondrial oxoglutarate dehydrogenase complex |
ciliary pocket membrane |
|
mitochondrial envelope |
ESCRT III complex |
|
phosphopyruvate hydratase complex |
cytoplasmic side of plasma membrane |
|
alphav-beta3 integrin-vitronectin complex |
membrane coat |
|
cytosolic ribosome |
smooth muscle contractile fiber |
|
microtubule minus-end |
integrin alpha8-beta1 complex |
Molecular function |
||
|
SH3 domain binding |
phospholipase A2 activator activity |
|
dGTPase activity |
PDZ domain binding |
|
dGTP binding |
phosphate ion binding |
|
insulin receptor binding |
phosphatidylinositol phosphate binding |
|
3-hydroxyacyl-CoA dehydrogenase activity |
phospholipid binding |
|
acetyl-CoA C-acyltransferase activity |
cysteine-type peptidase activity |
|
enoyl-CoA hydratase activity |
catalytic activity |
|
long-chain-3-hydroxyacyl-CoA dehydrogenase activity |
peroxidase activity |
|
3-hydroxyacyl-CoA dehydrogenase activity |
interleukin-1 receptor antagonist activity |
|
acetyl-CoA C-acyltransferase activity |
muscle alpha-actinin binding |
Table 3 Top 3 interaction protein number proteins
|
Gene ID |
Upregulated protein |
SDAVF-DV vs STA |
||
|
P63261 |
Actin, cytoplasmic 2 |
Upregulated |
P07814 |
Bifunctional glutamate/proline--tRNA ligase |
|
P62750 |
60S ribosomal protein L23a |
|
|
|
|
P02751 |
Fibronectin |
Downregulated |
Q15149 |
Plectin |
|
P02452 |
Collagen alpha-1(I) chain |
|
||
SDAVF-DV vs STV |
||
|
P00738 |
Haptoglobin |
Upregulated |
P02652 |
Apolipoprotein A-II |
|
P02765 |
Alpha-2-HS-glycoprotein |
|
|
|
|
P19652 |
Alpha-1-acid glycoprotein 2 |
Downregulated |
P10909 |
Clusterin |
|
P14780 |
Matrix metalloproteinase-9 |