Mesenchymal state of intimal cells may explain higher propensity to ascending aortic aneurysm in bicuspid aortic valves

Individuals with a bicuspid aortic valve (BAV) are at significantly higher risk of developing aortic complications than individuals with tricuspid aortic valves (TAV) and defective signaling during the embryonic development and/or life time exposure to abnormal hemodynamic have been proposed as underlying factors. However, an explanation for the molecular mechanisms of aortopathy in BAV has not yet been provided. We combined proteomics, RNA analyses, immunohistochemistry, and electron microscopy to identify molecular differences in samples of non-dilated ascending aortas from BAV (N = 62) and TAV (N = 54) patients. Proteomic analysis was also performed for dilated aortas (N = 6 BAV and N = 5 TAV) to gain further insight into the aortopathy of BAV. Our results collectively showed the molecular signature of an endothelial/epithelial-mesenchymal (EndMT/EMT) transition-like process, associated with instability of intimal cell junctions and activation of RHOA pathway in the intima and media layers of ascending aorta in BAV patients. We propose that an improper regulation of EndMT/EMT during the spatiotemporally related embryogenesis of semilunar valves and ascending aorta in BAV individuals may result in aortic immaturity and instability prior to dilation. Exasperation of EndMT/EMT state in post embryonic life and/or exposure to non-physiological hemodynamic could lead to the aneurysm of ascending aorta in BAV individuals.


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2 Detailed Material and Methods 6 Supplementary Table 1A-C, Descriptive patient characteristics 8 Supplementary Table 2, Antibodies  9 Supplementary Table 3, Model parameter values  10 Supplementary Table 4, Significantly up and downregulated proteins, non-dilated and dilated aorta 46 Supplementary Table 5, KEGG and Hallmark analysis, non-dilated and dilated aorta 48 Supplementary Table 6, Ingenuity pathway analysis, non-dilated and dilated aorta 49 Supplementary Table 7, PubMed search for 30 top upregulated proteins in BAV-ND 51 Supplementary Table 8, PubMed search for 30 top downregulated proteins in BAV-ND 53 Supplementary Table 9, mRNA expression, non-dilated and dilated aorta 54 Figure S1, PCA and OPLS-DA of proteomic data 55 Figure S2, Protein-protein interactions (PPIs) network of proteins involved in enriched hallmark gene sets shown in Table S5b and S5d 56 Figure Figure S4, EM view of EC junctions in dilated BAV and TAV aorta 61 Figure S5, Staining of neovascularization areas as internal control 62 Figure S6, Relative distribution of PTPRB and CDH5-Y658 63 Figure S7, cell proliferation in relation to cell junction 64 Figure S8, Western blot analysis of MFNG and MYLK expression in BAV and TAV nondilated aorta 65 Figure S9, Internal thoracic arteries 66 Figure S10, Internal thoracic arteries 67 Figure S11, Internal thoracic arteries 68 Figure S12, Internal thoracic arteries 69 References (Thermo) at 60°C. Online LC-MS was performed using a hybrid Q-Exactive mass spectrometer (Thermo Scientific). FTMS master scans with 70,000 resolution (and mass range 300-1700 m/z) were followed by data-dependent MS/MS (17,500 resolution) on the top 5 ions using higher energy collision dissociation (HCD) at 30% normalized collision energy. Precursors were isolated with a 2m/z window. Automatic gain control (AGC) targets were 1e6 for MS1 and 1e5 for MS2. Maximum injection times were 100ms for MS1 and 500ms for MS2. Dynamic exclusion was used with 60s duration. Precursors with unassigned charge state or charge state 1 were excluded. An underfill ratio of 1% was used. All MS/MS spectra were searched by Sequest and processed with Percolator under the software platform Proteome Discoverer 1.4 against the human subset of Swissprot, including canonical and isoform entries, version 2015-12 (42080 sequences, from uniprot.org). Precursor mass tolerance of 10 ppm and product mass tolerance of 0.02 Da were used. The enzyme used was trypsin and up to one missed cleavage was considered. Oxidation of methionine was used as variable modification. Carbamidomethylation of cysteine, and iTRAQ8plex on peptide N-terminus and on lysine were used as fixed modifications. A cut off of 1% FDR at peptide level was used. One peptide per protein was considered sufficient for protein identification. Quantification was done using Isobaric tags for relative and absolute quantitation (iTRAQ, ABsciex). iTRAQ reporter ion quantitation and ratio calculations were done by the "Reporter ions Quantifier" module of Proteome Discoverer. The 21 samples used for proteomics were arranged in three iTRAQ8plex sets, each set containing 7 samples and one internal standard (prepared by pooling the peptides of all 21 samples), which was used as denominator in all ratio calculations. The quantitative value of each protein for each patient was calculated as the median of the PSM (peptide spectrum matches) ratios assigned to that particular protein. The obtained ratios were further normalized by median-centering of each sample. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD003702 and can be accessed at https://www.ebi.ac.uk/pride/archive/login. (Username: reviewer95031@ebi.ac.uk Password: EhAG6nMr)

Immunohistochemistry (IHC)
Localization and protein expression levels were tested by immunostaining using deparaffinized tissue sections treated with or without DIVA solution (Biocare Medical), as described previously. 4 Following antigen retrieval, sections were washed and quenched for endogenous peroxidase activity with 3% hydrogen peroxide for 5 minutes. Nonspecific binding sites were blocked with 20% normal goat or horse serum for 30 minutes and then incubated overnight at 4 o C with primary antibody (Supplementary Table 2). After washing, sections were incubated with biotinylated anti-rabbit or anti-goat IgG (Vector Laboratories) for 1 hour. Sections were then incubated with Avidin-biotin peroxidase complex (Vectastain ABC kit, Vector Laboratories) for 30 min in room temperature, followed by visualization by 3,3'-diaminobenzidine tetrahydrochloride (Dako). All sections were counterstained with Mayer's hematoxylin (Histolab Products). Quantification of percent area positive staining was performed using Image-Pro® Premier version 9.1 Software (Media Cybernetics®, Silver Spring, MD). The expression of each protein was studied in 6-10 patients from each group (BAV-ND and TAV-ND) in both aortas and internal thoracic arteries (unless otherwise stated).

Western Blot analysis
The expression level of Manic Fringe (MNFG) and MYLK (myosin light chain kinase) were analyzed by Western Blot. Protein lysates were prepared by tissue homogenization in RIPA buffer (Thermo Fisher Scientific) as described in the proteomic method section for non-dilated aorta of BAV and TAV patients. Twenty microgram of protein was re-suspended in Laemmli Sample Buffer (BioRad) containing 0.2M DTT and subjected to SDS-PAGE using 4-15% Bis-Tris Protein Gels (Invitrogen NuPAGE), or 4-20% Mini-PROTEAN TGX gels (BioRad) for 60 min at 120V. Proteins transfer was performed with Tran-Blot turbo system (Bio Rad) to nitrocellulose membranes (Bio Rad) according to standard protocols, followed by blocking in 5% dry-milk in TTBS. The blots were incubated with The array data was validated by qRT-PCR for 11 genes. 11 Differences in protein expression assessed by IHC and WB were analyzed by student's t-test (GraphPad Prism 5, La Jolla, CA). Data are expressed as mean and percent area positive staining ± SD for gene and protein expression, respectively. A Pvalue of P<0.05 was considered statistically significant. Ingenuity pathway analysis was performed on significant proteins from the OPLS-DA model. Canonical pathways are shown. KEGG pathway and Hallmark analysis within Gene Set Enrichment Analysis and the Molecular Signatures Database resource v 5.0 was used to investigate gene/protein sets. 12 Differentially expressed proteins were selected either in non-dilated or dilated samples and were mapped to protein-protein interaction database (Human Protein Reference Database. 13 The retrieved interactions are shown in Fig 1,  BMI, body mass index; hsCRP, high-sensitive C-reactive protein; ACE, Angiotensin-converting enzyme. 1 All continuous variables are presented as mean (SD) and ordinal variables are presented as n (%). P values indicate significance for differences between the two groups using a Mann-Whitney test for continuous variables and a Chi-square test for nominal variables. Non-dilated, aorta <40mm; Dilated, aorta >45mm; NS, not significant.

Supplementary Table 3. Model parameter values
Model quality parameters of PCA and OPLS-DA. No of PCs refers to the optimal number of PCs for a particular multivariate data model according to crossvalidation. RX 2 accounts for the explained variance while QY 2 accounts for the cumulative fraction of the total variation of Y that can be predicted by the model. QX 2 accounts for the cumulative fraction of the total variation of X that can be predicted by the model. RY 2 accounts for the explained variance for the Y component.

Model description No of PCs No of samples No of proteins RX 2 QX 2
Non-dilated patients only included -Supp Figure 1a     An HMG-box transcriptional repressor that was first identified in Drosophila regulating receptor tyrosine kinase (RTK) signaling. In mammals, CIC mediates RTK-dependent responses has been associated with cell proliferation, neurodegenerative diseases and cancer. 37 XIAP XIAP E3 ubiquitin-protein ligase, negative regulator of apoptosis and a potential oncogene, autophagy inhibitor, 38 overexpressed in a variety of human cancers 39 CDKN1B CDN1B Synonym with P27. Regulator of cell cycle the enhanced expression of which is associated with EMT. 40 Differential cellular localization of p27 is associated with cancer. 41 DNMT3A DNM3A Functions as de novo DNA methyltransferase. Activated in cancer. 42 Upregulated in EC exposed to disturbed flow causing increased DNA methylation of CpG islands within the promoter of the flow responsive gene KLF4 in vivo and in vitro. 43

RCN1 RCN1
Functions in secretory pathway. 44 Upegulated in EMT associated with renal fibrosis. 45 Highly upregulated in several type of cancer. 46 PSMA6 PSA6 A major component of intracellular protein degradation apparatus. PSMA6 polymorphisms associated with type2 diabetes, myocardial infarction, and coronary artery disease. 47

HGS HGS
Involved in a WNT/β-Catenin dependent cross talk in development of some types of cancer. 48 A component of ESCRT-0 complex, consisting of the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and the signal-transducing adaptor molecule (STAM) proteins, that recognizes ubiquitinylated cargo during the initial step of endosomal sorting. 49

PLXDC2 PXDC2
Type I transmembrane protein with some homology to nidogen and plexins, characterized as a mitogen for neural progenitors. 50 The expression of this protein is altered in certain types of cancer. 51 POTEI POTEI Not much information available in PubMed OTUB 1 OTUB1 A deubiquitinating enzyme overexpression of which is associated with several types of cancer via activation of RHOA and EMT. 52,53 SNRPB2 RU2B One of the 11 genes robustly and universally linked to Membrane-Type 1 Matrix Metalloproteinase (MT1-MMP). Key invasion promoting protease in multiple cancerous tumors. 54

IARS2 SYIM
Interacting partner of chaperon protein HSP90. 55 Regulation of Dynein function 56 which in turn affects endocytosis 57 and autophagy. 58 ABAT GABT An enzyme responsible for catabolism of inhibitory neurotransmitter GABA, a new function of this protein in mitochondrial metabolism has been newly discovered. 59 TCEAL3 TCAL3 Transcription elongation factor A like 3, not much information available in PubMed CNOT11 CNO11 A module of CCR4-NOT complex. This complex is a key regulator of mRNA degradation via destabilization and inhibition of translation. 60

ARSD ARSD
A metallochaperon involved in the response to arsenic environmental pollution. 61

MTHFD2 MTDC
Regulator of Vimentin expression during EMT and breast cancer cells migration and invasion. 62 Has a key role in the development of cancers. 63

ZYX ZYX
A component of focal adhesions (FAs) and stress fiber (SF). 64 Mediating the cytoskeletal and extra cellular matrix (ECM) mechanotransduction. 65 Involved in migration of mesenchymal and cancerous cells. 66 Regulates adherens junction integrity and cell motility during the EMT of lung cancer. 67 Involved in migration of the endocardial cells during EndMT of cardiac valve formation. 67

IL16 IL16
A regulator of T cell growth and has been associated with several types of cancer. 68

C2CD5 C2CD5
Characterized as a component of the MIB1 ubiquitin ligase proteomic interactome. 69

C9orf64 CI064
A somatic cell memory gene regulating the difference between induced pluripotent and embryonic stem cells differences and proposed to stabilize the cells in an intermediate stage with characteristics of both somatic and reprogrammed state. 71 Aberrant promoter methylation of this gene is associated with ovarian cancer. 72 Deleted in some cases of acute myeloid leukemia. 73

SBSPON BSPO
A gene differentially expressed in pre-eclamptic placenta, a disease characterized by angiogenic imbalance and disturbed vascular function. 74

ME1
MAOX Regulates the expression of P53. 75 Overexpressed in cancer to cover metabolic needs of cancer cells. Diminished migration and invasion were observed in ME1-repressed cancer cells. 76

SET SET
A chromatin modifying factor and histone chaperon regulating histone assembly/disassembly. 81 Involved in regulation of histone acetylation and DNA methylation thereby integrating epigenetic states of histones with DNA methylation. 82

TBC1D13 TBC13
Interacts with Rab35 which is a key regulator of membrane trafficking and endocytic recycling of numerous protein localized in plasma membrane, clathrin-coated pits and vesicles. Rab35 controls the recycling pathways for regulation of cell adhesion and migration. 83 ABHD10 ABHDA Involved in protein modification. ABHD10 is responsible for deglucuronidation of acyl glucuronides 84 which in turn covalently modifies proteins. 85

VPS25 VPS25
Subunit of Endosomal Sorting Complex required for Transport II (ESCR-II) complex involved in the regulation of endosomal biogenesis and functions. Considered to be a tumor suppress through endocytic regulation of NOTCH signaling 86 and by sorting NOTCH in Multi Vesicular Bodies (MVBs). 87 Plays a role in autophagy. 88

DLAT ODP2
Interacts with 20S proteasome component PSMA as a part of ubiquitin-independent protein degradation pathway. 89 Mutations in the gene causes neurodegenerative disease in human. 90 Supplementary Table 9 Table S5b

EM view of EC junctions in BAV-D (a-b) and TAV-D (c).
Extensive degradation at EC junction (a) and stress fiber formation adjacent to late lysosomes (b) in BAV compared to TAV patients with dilated aortas. SF, stress fibers; LL, late lysosomes. Patients: N=2 in each group.

Figure S5
Staining of neovascularization areas, as internal control. Pictures are taken from neovascularization areas of the same sections stained for localization and quantification of AJ protein, CDH5, CDH5-P, in intima, Scale bar= 50µm, Patients: N=7-9 in each group, PTPRB N=3 in each group.

Comparison between BAV-ND (a) and TAV-ND (b) patients for relative distribution of PTPRB and
CDH5-Y658, using serial sections. Scale bar= 50µm, Patients: N=3 in each group.

Figure S7
Examination of the connection between proliferative state and cell-cell junctions. We stained consecutive sections of a BAV-ND patient with high intimal proliferative activity, for CDH5, Phos-CDH5-Y658, PTPRB phosphatase, ZEB1 and KI67. The results further strengthened that the increased cell division in intimal layer, corresponded to the strong staining of anti-Phos-CDH5-Y658 and decreased PTPRB phosphatase activity, increased ZEB1 staining, implying that the higher cell proliferation, activation of EMT transcription factor and instability of the cell junctions are related and coordinated processes in BAV-ND, Scale bar= 50µm.

Figure S12
Internal thoracic arteries stained with antibodies used for aorta. NOCH pathway, Scale bar= 50µm, Patients: N=7 in each group.