
==== Front
iScience
iScience
iScience
2589-0042
Elsevier

S2589-0042(24)01661-4
10.1016/j.isci.2024.110436
110436
Article
SOX6 expression and aneurysms of the thoracic and abdominal aorta
Carmona-Berrio David 1214
Adarve-Rengifo Isabel 1214
Marshall Andrea G. 3
Vue Zer 3
Hall Duane D. 4
Miller-Fleming Tyne W. 5
Actkins Ky’Era V. 5
Beasley Heather K. 3
Almonacid Paula M. 6
Barturen-Larrea Pierina 12
Wells Quinn S. 7
Lopez Marcos G. 8
Garza-Lopez Edgar 9
Dai Dao-Fu 10
Shao Jianqiang 11
Neikirk Kit 3
Billings Frederic T. IV 8
Curci John A. 12
Cox Nancy J. 5
Gama Vivian 12
Hinton Antentor Jr. antentor.o.hinton.jr@vanderbilt.edu
3∗
Gomez Jose A. jagoc502@gmil.com
1315∗∗
1 Vanderbilt University, Cell and Developmental Biology, Nashville, TN 37232, USA
2 Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA
3 Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, USA
4 Department of Internal Medicine, Abboud Cardiovascular Research Center, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA
5 Vanderbilt Genetics Institute, Vanderbilt University Medical Center, Nashville, TN 37232, USA
6 Department of Economics, EAFIT University, Medellín, Antioquia, Columbia
7 Department of Medicine, Division of Cardiovascular Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA
8 Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
9 Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA
10 Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA
11 Central Microscopy Research Facility, University of Iowa, Iowa City, IA 52242, USA
12 Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
13 Department of Medicine / Clinical Pharmacology Division. Vanderbilt University Medical Center, Nashville, TN 37232, USA
∗ Corresponding author antentor.o.hinton.jr@vanderbilt.edu
∗∗ Corresponding author jagoc502@gmil.com
14 These authors contributed equally

15 Lead contact

24 7 2024
20 9 2024
24 7 2024
27 9 11043629 8 2022
31 1 2024
28 6 2024
© 2024 Published by Elsevier Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Abdominal and thoracic aortic aneurysms (AAAs, TAAs) remain a major cause of deaths worldwide, in part due to the lack of reliable prognostic markers or early warning signs. Sox6 has been found to regulate renin controlling blood pressure. We hypothesized that Sox6 may serve as an important regulator of the mechanisms contributing to hypertension-induced aortic aneurysms. Phenotype and laboratory-wide association scans in a clinical cohort found that SOX6 gene expression is associated with aortic aneurysm in subjects of European ancestry. Sox6 and tumor necrosis factor alpha (TNF-α) expression were upregulated in aortic tissues from patients affected by either AAA or TAA. In Sox6 knockout mice with angiotensin-II-induced AAA, we found that Sox6 plays critical role in the development and progression of AAA. Our data support a regulatory role of SOX6 in the development of hypertension-induced AAA, suggesting that Sox6 may be a therapeutic target for the treatment of aortic aneurysms.

Graphical abstract

Highlights

• Transcription factor SOX6 is associated with abdominal and thoracic aortic aneurysm

• LabWAS provides clinical measurements associated with aortic aneurysm diagnosis

• Knocking out Sox6 attenuated hypertension-induced abdominal aortic aneurysm

Medical science; Cardiovascular medicine; Molecular Genetics

Subject areas

Medical science
Cardiovascular medicine
Molecular Genetics
Published: July 24, 2024
==== Body
pmcIntroduction

Abdominal aortic aneurysm (AAA) is a devastating disease that can lead to rupture of the aorta and is estimated to cause up to 200,000 yearly deaths worldwide.1,2 An estimated 2.78 per 100,000 people suffer from an aortic aneurysm or dissection worldwide.2,3 In the United States, aortic aneurysms are more common in men and present in 4%–8% of those undergoing screening.4,5 There are no prognostic markers for AAA development, and warning signs for impending aortic rupture are lacking. While AAAs are the most common, aneurysms may also appear in the aorta of other tissues. For instance, while AAA occurs in the infrarenal abdominal aorta, thoracic aortic aneurysms (TAAs) occur in the ascending and descending aorta in the thoracic cavity. Compared to AAAs, TAAs are less common but more heritable. For example, several connective tissue diseases are associated with the development of TAAs including Marfan syndrome (MFS), Loeys-Dietz syndrome (LDS), and Ehlers-Danlos syndrome (EDS).6 Marfan syndrome is characterized for the presence of TAA caused by a mutation in fibrillin-1.6 Preclinical models of TAA have identified transforming growth factor β (TGF-β) signaling as a key molecule in the development of aneurysm. In these models, angiotensin II (Ang II) is responsible for increased TGF-β signaling.7 AAAs and TAAs develop when the vessel wall dilates at specific points. This process increases the risk of aorta dissection, during which the vessel intima is damaged. In the event of aorta dissection, blood enters the underlying layers of the vessel wall and creates a false lumen where coagulation, immune cell infiltration, and further inflammation occur. In the case of rupture, aortic aneurysms and dissections can be fatal.

TAAs and AAAs have similar pathogenic characteristics, with smooth muscle loss and elastic tissue degeneration as the most common features in both diseases.8 However, there are several differences, including genetic origin. In TAA, about 20% of patients have an autosomal dominant inheritance resulting from a mutation in a single gene.8 Genes associated with TAA do not confer risk for AAA. AAA is a chronic disease whose risk factors are shared with atherosclerosis.9,10 Genes associated with atherosclerosis have also been found to be associated with AAA.11 Hypertension is a risk factor for AAA development. TAA high-risk genes are known as the Heritable risk for Thoracic Aortic Disease (HTAD) group and include genes involved in smooth muscle contraction (SMC), TGF-β signaling, and extracellular matrix (ECM) among others.8

Hypertension is a major risk factor for AAA, and risk of rupture is increased by 30% for each 10-mmHg increase in blood pressure in humans.12 While there is compelling evidence to support the role of hypertension in AAA development,3,13 the pathophysiology linking them remains poorly understood. Hypertension, in combination with smoking and age, are the main risk factors for AAA development. Hypertension is associated with overactivation of the renin-angiotensin-aldosterone system (RAAS), which facilitates oxidative stress and degradation of the extracellular matrix. This leads to smooth muscle cell death within the aorta, which has been postulated to contribute to the development of aortic aneurysms.14 In RAAS, the main effector molecule is the vasoconstrictor Ang II, which raises blood pressure primarily through activation of type 1 angiotensin receptors (AT1R).15 Ang II regulates endothelial permeability and serves as a growth factor for new blood vessels. Ang II is furthermore linked to hypertension, age-induced vascular stiffening, atherosclerosis, AAA, and dissection development.16,17

In this manuscript, we seek to further identify the relationship linking hypertension to the development of aortic aneurysms so that additional diagnostic markers and therapeutic targets may be developed to decrease mortality and morbidity from AAA and TAA. We have previously found that Sox6 functions as a regulator of renin expression, controlling the rate-limiting step in RAAS.18,19 Sox6 depletion in Ren1d+ cells inhibited renal-artery-stenosis-induced hypertension and kidney injury.18 Sox6 is a transcription factor member of the D-subfamily of the sex-determining region. It is in the y-related family of transcription factors, characterized by a conserved DNA-binding domain known as the high-mobility group box and an ability to bind the minor groove of DNA. Sox6 is associated with numerous disease states including cancer, cardiomyopathy, and diabetic nephropathy.20 Sox6 has previously been reported as a specific regulator of smooth muscle cells in a single-cell RNA sequencing (RNA-seq) study of murine abdominal aortic aneurysm.21 Thus, we hypothesized that Sox6 may serve to regulate the mechanisms contributing to aortic aneurysms, and here we present human and mouse data indicating that Sox6 expression plays a role in the development of abdominal and thoracic aortic aneurysms.

Using a large, clinical biobank population, we found that gene expression of SOX6 is associated with aortic aneurysm in humans of European ancestry. Moreover, analysis of tissue from patients affected by abdominal and thoracic aortic aneurysm Sox6 expression was upregulated. To determine if Sox6 expression was necessary for the development of aortic aneurysm in mice, the gene was specifically knocked out in smooth muscle cells. In mice, knocking out Sox6 expression in smooth muscle cells inhibited the development of abdominal aortic aneurysm in mice induced by hypertension. The data presented here suggest that the transcription factor Sox6 has a previously unreported function in the development of abdominal aortic aneurysms and that SOX6 may be a molecular marker of human AAA and TAA.

Results

Based on our previous findings,18,19 we sought to test the hypothesis that SOX6 contributes to the etiology of aortic aneurysm. To determine the relationship between SOX6 gene expression and aortic aneurysms in humans, we used a large clinical biobank at Vanderbilt (BioVU) to identify participants with aortic aneurysm. Individuals diagnosed with aortic aneurysm (n = 1,209) were identified using phecodes mapped from ICD9/10 billing codes of de-identified electronic health records (EHRs) (see STAR Methods). The biobank we used contains genotype information of 70,439 individuals of European ancestry and 15,174 individuals of African ancestry. To assess SOX6 gene expression in the biobank participants, we used a previously established method of imputing gene expression from genotype data to model the genetically regulated gene expression (GREX) of SOX6 (Figure 1).22,23 The relationship between SOX6 GREX and the aortic aneurysm diagnosis was then evaluated by logistic regression models, correcting for the age, sex, and genetic ancestry of each individual. In the group of individuals of European ancestry, we found that SOX6 GREX was significantly associated with aortic aneurysm diagnosis (n = 1,208; p = 0.0265; Table 1). However, there was no significant association between SOX6 GREX and aortic aneurysm in individuals of African ancestry. This was perhaps due to the small sample size (n = 98; p = 0.1452; Table 1).Figure 1 Modeling SOX6 genetically regulated gene expression (GREX) and aortic aneurysm in a medical biobank

We leveraged a large, clinical biobank at Vanderbilt (BioVU) that contains deidentified electronic health record (EHR) data linked to genotype data for 85,613 participants. We sought to test whether imputed gene expression of SOX6 and SOX6-regulated genes is associated with an aortic aneurysm diagnosis. To identify aortic aneurysm (AA) cases and controls, we required cases to have at least two instances of the aortic aneurysm phecode/diagnosis code, while controls had no instances of these codes (see STAR Methods, n cases = 1,306 and n controls = 71,748, top left panel). Genetically regulated gene expression for SOX6 and SOX6-regulated genes were calculated in BioVU participants using models built from the GTEx version 8 data (top right panel), which contains genotype data matched to RNA-seq data from 838 donors across 49 tissues. These models capture the impact of genetic variation on gene expression, allowing us to calculate how much each genetic variant is contributing to gene expression. We used these models to predict or impute gene expression in the BioVU population, which only have genotype data. Imputed gene expression was calculated in BioVU and assessed for association in AA cases versus controls using logistic regression models (bottom panel), accounting for genetic ancestry (principal components/PC 1–10), sex, age, number of medical center visits, and genotyping batch.

Table 1 Phenotype association results for SOX-6 GREX

Genetic Ancestry Group	Phenotype	GREX	p	n_models	n_samples	p_best	best_tissue	
European	AorticAneurysm	SOX6	0.0265	14	59307	0.0326	SkinSunExposed	
African	AorticAneurysm	SOX6	0.1452	14	13649	0.0300	ColonSigmoid	

Next, we analyzed the expression patterns of SOX6 in humans with aortic aneurysms. We collected tissue from patients affected by AAA and TAA during aneurysm repair surgeries. Non-aneurysmal aortic tissue samples from ascending aorta punch sites of saphenous vein grafts from patients undergoing coronary artery bypass surgery were collected as controls. The burden of AAA increases with age and is more prevalent in men than women.3 Table S1 (Tables S1 and S2) describes patient details and clinical characteristics. Elastin staining analysis showed significant elastic fragmentation and cystic medial degeneration in aneurysmal tissue from both AAA and TAA patients (Figure S1). Furthermore, there was loss of smooth muscle cells, increased collagen deposition, and fibrosis (Figures S2 and S3).

To better understand the transcriptional pathways activated in disease states, we analyzed gene expression via RNA-seq. RNA-seq was applied to RNA from TAA tissue and non-aneurysmal aortic tissue. Analysis showed that SOX6 expression is upregulated in TAA (Figures 2A and 2B). Other genes upregulated in TAA tissue included Gremlin 1 (GREM1), C-C motif chemokine ligand 21 (CCL21), and FYN binding protein 1 (ADIPOQ) (Figures 2A and 2B). GREM1 is a bone morphogenic protein (BMP) antagonist and is upregulated in aortic tissue from Loeys-Dietz syndrome.24 CCL21 is present in the conditioned medium isolated from AAA split arterial layer.25 Adiponectin (ADIPOQ) has an inhibitory function in AAA development induced by Ang II and β-aminopropionitrile.26 We performed ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA) on significant differentially regulated genes to identify the signaling pathways, associated biological functions, and key upstream regulators that are likely altered and contribute to the TAA phenotype (Figures 2C–2E). IPA diseases and functions enrichment showed that there was a downregulation of several functional pathways, including endocytosis (Figure 2C). GSEA:KEGG (Gene Set Enrichment Analysis: Kyoto Encyclopedia of Genes and Genomes) analyses indicated that in TAA, peroxisome-proliferator-activated receptor (PPAR) signaling and cytokine-cytokine receptor interactions are upregulated (Figure 2D). This aligns with previous studies that have found that PPARγ agonist decreases inflammation in patients with aortic aneurysm and has a function in AAA development.27 IPA Causal Networks annotations pointed to PRKAA2, SGK3, and GNGA3 as activated upstream regulators contributing to the significantly altered gene dataset in the TAA samples. PRKAA2 (protein kinase AMP-activated catalytic subunit alpha 2) is a gene essential in energy-sensing enzyme that monitors cellular energy status. SGK3 (serum/glucocorticoid regulated kinase family member 3) has a role in neutral amino acid transport and activation of potassium and chloride channels. Finally, CNGA3 (cyclic nucleotide gated channel subunit alpha 3) is believed to be important for vision and olfactory signal transduction. We found that these genes are likely to have increased activity in human TAA samples (Figure 2E).Figure 2 RNA sequence analysis of human TAA samples

RNA-seq analysis indicates a modest change in growth-related pathway in thoracic aortic aneurysm (TAA) samples.

(A) Heatmap of 55 significant differentially expressed genes (DEGs) found in TAA samples relative to control (n = 4 per group).

(B) Expression of significant (black circles) versus non-significant (gray circles) DEGs displayed as a volcano plot. FDR, false discovery rate.

(C) Ingenuity pathway analysis (IPA) results of DEGs for enriched diseases and function annotations.

(D) Enriched KEGG pathway terms from gene set enrichment analysis (GSEA).

(E) Enriched IPA causal networks.

Gene transcriptional differences between AAA tissue and non-aneurysmal aortic tissue were also determined via RNA-seq (Figure 3A). The immune-related gene JCHAIN (joining chain of multimeric IgA and IgM) and the guanine nucleotide exchange factor DENND1C (DENN domain containing 1C) were found to have the highest fold increase in AAA (Figure 3B). IPA showed that several canonical pathways previously associated with AAA development28,29,30 were upregulated in AAA samples, including T cell (Th2 and Th1) pathways, PTEN signaling, and oxidative stress in macrophages (Figure 3C). Most of the upregulated functions identified by IPA are related to immune responses known to play a role in AAA28,29,30 (Figures 3D and S4). Furthermore, IPA analysis of causal networks suggested that CoREST, SOX11, and ACE2 may have increased activity in AAA (Figure 3E). CoREST determines neural cell differentiation, SOX11 is important for regulation of embryonic development and in the determination of the cell fate and is consistent with our data. ACE2 was previously reported as a modulator of Ang II induction of AAA.31Figure 3 RNA sequence analysis of human AAA samples

RNA sequencing analysis suggests a robust immune response occurs in abdominal aortic aneurysm (AAA).

(A) Heatmap of 1,022 significant differentially expressed genes (DEGs) found in AAA samples relative to control (n = 4 per group).

(B) Expression of significant (black circles) versus non-significant (gray circles) DEGs displayed as a volcano plot. Log2 FC, log2 fold change in control vs. AAA expression; FDR, false discovery rate.

(C–E) Ingenuity pathway analysis (IPA) results of DEGs for enriched canonical pathway (C), diseases and functions annotation (D), and causal network (E) terms. Example enriched terms are indicated.

Next, we use RT-qPCR to validate the expression of genes found in the RNA-seq analysis by RT-qPCR. We focused on those genes matching two criteria: (1) were common to both AAA and TAA (Figure S5; Table S3); (2) possessed promoters containing SOX6 binding sites (Figure S6; Tables S4 and S5). Genes that matched these criteria were COTL1, CCL21, and PKD1. COTL1 and CCL21 were upregulated in both TAA and AAA, whereas PKD1 was downregulated in both TAA and AAA. In silico analysis indicated that SOX6 has binding sites in the promoter of those three genes, suggesting a regulatory function (Figure S7). JCHAIN expression was upregulated in AAA samples based on our RNA-seq analysis. The promoter of this gene has several binding sites for SOX6, and the mRNA was upregulated in AAA samples (Figure S6B). SOX6 transcripts were elevated in about half of AAA and TAA samples (not significant due to small sample size (Figure S6B). Likewise, PKD1 and CCL21 were also detected in the samples (Tables S4 and S5). There were no statistically significant differences.

Next, protein expression in human aortic samples was examined by western blot. SOX6 protein expression was significantly upregulated in AAA and TAA aortic tissue compared to control tissue (Figures 4A–4F). Tumor necrosis factor alpha (TNF-α) has a known function in the development of both TAA32 and AAA33 and was used as a positive control. The expression of TNF-α was upregulated in both AAA and TAA (Figures 4A–4F). In silico analysis of the TNF-α promoter shows that there are several SOX6 binding sites suggesting a possible function of SOX6 in the control of TNF-α expression during both AAA and TAA development. Moreover, Sox6 was identified as part of a regulon in murine aortic smooth muscle cells in a mouse model of AAA.21 Transforming growth factor β (TGF-β) and its molecular pathway are associated with both TAA and AAA in humans.34,35 In the TAA human samples, TGF-β expression was significantly upregulated (Figure S8), but not in the human AAA samples (data not shown). SMAD2 and its phosphorylated form were found to be significantly increased in the human AAA samples when compared to control samples (Figure S8). These results suggest that SOX6 is upregulated in AAA and TAA, indicating a likely role in the etiology of these diseases.Figure 4 Human abdominal and thoracic aortic aneurysm Sox6 expression western blot analysis

Human aorta samples were collected from tissue discarded after AAA or TAA or coronary artery bypass surgery (control). Samples were flash freeze in liquid nitrogen and stored at −80°C. Expression of SOX6 was analyzed by western blot using a specific antibody.

(A) Western blot images showing levels of SOX6 and TNF-α expression in human TAA.

(B) Densitometric analysis of SOX6 and (C) TNF-α protein bands are shown to the right of western blots.

(D) Western blot images showing levels of SOX6 and TNF-α expression in human AAA and control samples. Beta-actin was used as a loading control.

(E) Densitometric analysis of SOX6 and (F) TNF-α protein bands are shown to the right of western blots. TAA N = 4, AAA N = 3, Control N = 3–5. Data are presented as the mean ± SD. p value calculated with unpaired t test. ∗p < 0.05.

We then sought to evaluate the clinical implications of genes upregulated in the TAA and AAA tissues by leveraging the large medical biobank, BioVU. The GREX for the genes identified by TAA and AAA tissue RNA-seq analysis was calculated in BioVU participants (Tables 2 and 3 show Patient Demographics). Additionally, clinical laboratory measurements were extracted for 70,337 individuals of European ancestry and 15,123 individuals of African ancestry (323 and 241 lab tests, respectively) using the Quality Lab pipeline (Figure S9).5 We tested the relationship between AAA and TAA gene GREX (ADIPOQ, APOC1, COTL1, CST7, DENND1C, FABP4, FLT4, GREM1, HLADQA1, IL7R, MYO1G, PKD1, PLIN1, PYHIN1, RSPO3, TGFB1, TLR9) and lab measurements using linear regression models, correcting for age, sex, and genetic ancestry across all patients. We found significant associations between APOC1 (apolipoprotein C1) GREX with triglyceride in serum or plasma (n = 32,852; p = 2.11 x 10−14) and HLA-DQA1 (major histocompatibility complex, class II, DQ alpha 1) GREX with glycated hemoglobin A1c (n = 23,162; p = 1.31 x 10−13), glucose (n = 66,124; p = 8.19 x 10−8), and tau protein in body fluid (n = 47,235; p = 3.29 x 10−6) across individuals of European ancestry (Figure S10; Table S6). APOC1 was reported as a possible molecular marker for AAA.36 A locus in the HLA-DQA1 gene was identified as a possible risk factor of AAA development.37 We did not note any significant associations between GREX and lab values in the much smaller sample of individuals of African ancestry (Figure S11; Table S6).Table 2 Demographics of patients with atrioventricular block

Demographics	Case	Control	p	
N	3,812	427,662		
Mean age (SD)	68.92 (15.11)	46.02 (16.96)	<0.001	
Mean BMI (SD)	29.01 (6.18)	28.84 (6.82)	0.114	
Race (%)			<0.001	
 White	3,439 (90.2)	346,245 (81.0)		
 Black	285 (7.5)	47,912 (11.2)		
 Asian	29 (0.8)	8,254 (1.9)		
 Other	59 (1.5)	25,251 (5.9)		
Sex (%)			<0.001	
 Female	1,609 (42.2)	270,107 (63.2)		
 Male	2,203 (57.8)	157,549 (36.8)		
 Unknown	0 (0.0)	6 (0.0)		
Ethnicity (%)			<0.001	
 Hispanic	20 (0.5)	11,240 (2.6)		
 Non-Hispanic	3,752 (98.4)	395,374 (92.5)		
 Unknown	40 (1.0)	21,048 (4.9)		
Other includes individuals with an EHR-reported race of Asian, Native American, Pacific Islander, Indian, other, mixed race, unknown, and did not report. BMI, body mass index.

Table 3 Demographics of patients with chronic vascular insufficiency of intestine

Demographics	Case	Control	p	
N	3,812	427,662		
Mean age (SD)	59.88 (17.42)	47.17 (17.40)	<0.001	
Mean BMI (SD)	25.44 (5.55)	28.97 (6.90)	<0.001	
Race (%)			0.04	
 White	283 (86.8)	435,313 (81.4)		
 Black	29 (8.9)	60,824 (11.4)		
 Asian	6 (1.8)	9,648 (1.8)		
 Other	8 (2.5)	29,144 (5.4)		
Sex (%)			0.14	
 Female	221 (67.8)	333,960 (62.4)		
 Male	105 (32.2)	200,962 (37.6)		
 Unknown	0 (0.0)	7 (0.0)		
Ethnicity (%)			0.02	
 Hispanic	5 (1.5)	13,330 (2.5)		
 Non-Hispanic	316 (96.9)	497,857 (93.1)		
 Unknown	5 (1.5)	23,742 (4.4)		
Other includes individuals with an EHR-reported race of Asian, Native American, Pacific Islander, Indian, other, mixed race, unknown, and did not report. BMI, body mass index.

In follow-up analyses, we tested the association between the AAA and TAA genes identified by RNA-seq analysis with aortic aneurysm diagnosis in the BioVU patient population (Figures 2 and 3). We modeled gene expression in the multiple genes identified by RNA-seq analysis (ADIPOQ, APOC1, COTL1, CST7, DENND1C, FABP4, FLT4, GREM1, HLADQA1, IL7R, MYO1G, PKD1, PLIN1, PYHIN1, RSPO3, TGFB1, TLR9) and found a nominal association with ADIPOQ GREX in individuals of European ancestry (p = 0.0455) with aortic aneurysm diagnosis (Table S8). Adiponectin (ADIPOQ) has an inhibitory function in abdominal aortic aneurysm development induced by Ang II and β-aminopropionitrile.26

As described earlier, SOX6 expression was upregulated in AAA and TAA human samples, suggesting a function in the etiology of these diseases. To perform a proof of principle animal experiment and to determine if Sox6 plays a key role in the development of hypertension-induced AAA, we generated a smooth-muscle-cell-specific Sox6-specific knockout mouse model using a tamoxifen inducible Cre. These mice are identified as Myh11CreERT2/Sox6fl/fl (Sox6 KO) mice. Myh11CreERT2/Sox6w/w (Sox6 WT) littermate mice were used as controls.38 Cre expression was induced via intraperitoneal injections of tamoxifen for five consecutive days to ablate Sox6 expression prior to Ang II infusion. Sox6 WT and Sox6 KO adult mice received continuous infusion of Ang II 1000 ng/kg/min for 28 days via a subcutaneous Alzet 2004 pump. Tamoxifen did not affect blood pressure or induce AAA. The baseline SBP were similar across all groups (102.7 ± 10.1 mmHg in Sox6 WT and 108.1 ± 10.5 mmHg in Sox6 KO): Ang II increased systolic blood pressure by about 35 mmHg (139.4 ± 15.5 in Sox6 WT and 139.2 ± 26.8 in Sox6 KO) (Figure 5A). Kaplan Meier curves demonstrate that 44% of the Sox6 WT mice died of AAA rupture, most within the initial 11 days of Ang II infusion. In contrast, we only observed 6% mortality in Sox6 KO mouse (log rank [Mantel-Cox] test; p < 0.001; Figures 5B and S12). AAA did not develop in control-vehicle-treated mice (Figure 5B). Figure S12 shows representative images of Ang-II-induced fatal rupture of AAA in Sox6 WT mice. Using ultrasound measurement, we showed that Ang-II-induced dilation of thoracic aortas was significantly higher in Sox6 WT than those in Sox6 KO (Figure S12). This finding suggests that Sox6 ablation prevented Ang-II-induced AAA (Figures 5 and S12). Histological analysis of the abdominal aortas sections of the surviving mice displayed abdominal aorta cystic medial degeneration, which led to AAA in Sox6 WT-Ang II (Figures 5C, 5D, and 6). Significant elastin fragmentation and degradation leading to cystic medial degeneration was also observed in Sox6 WT Ang-II-treated mice (Figure 6). In vehicle-treated controls, as well as Sox6 KO Ang-II-treated samples, cystic medial degeneration was similar, suggesting that Ang-II-induced elastic fragmentation, cystic medial degeneration, leading to aortic dilation and AAA development in Sox6 WT mice, was inhibited by knocking out Sox6 expression in smooth muscle cells (Figure 6). Taken together, the in vivo data suggest that Sox6 controls a smooth muscle cell transcriptional network important in the development of AAA induced by hypertension and that knocking out Sox6 in smooth muscle cells may decouple hypertension from AAA development.Figure 5 Knocking out Sox6 in smooth muscle cells inhibits hypertension-induced abdominal aortic changes in mice

C57Bl6 Myh11CreERT2/Sox6fl/fl (Sox6 KO) and Myh11CreERT2/Sox6w/w (Sox6 WT) mice were intraperitoneally injected with 2.5 mg of tamoxifen for five consecutive days. Then osmotic mini pumps were implanted subcutaneously for infusion of Ang II (1000 ng/kg/min x 28 days). Knocking out Sox6 in SMCs inhibits degradation of the ECM.

(A) Blood pressure changes were measured using tail cuff method (n = 12–14). p values calculated using a two-way ANNOVA. ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001 when comparing Sox6 KO Ang-II-treated group to both vehicles. ###p < 0.001 and #### p < 0.0001 when comparing Sox6 WT Ang-II-treated group to both vehicles.

(B) Ang-II-induced abdominal aortic aneurysm in Sox WT mice (n = 18). p values calculated using log rank (Mantel-Cox) test. Sox6 WT vehicle (n = 14), Sox6 KO vehicle (n = 13), and Sox6 KO Ang II (n = 18). ∗∗∗∗p < 0.0001.

(C) Trichrome blue showed areas of elastic fragmentation in both Sox6 WT-Ang II and Sox6 KO-Ang II (arrows).

(D) Hematoxylin and eosin.

Figure 6 Knocking out Sox6 in smooth muscle cells inhibits hypertension-induced abdominal aorta cystic medial degeneration in mice

C57Bl6 Myh11CreERT2/Sox6fl/fl (Sox6 KO) and Myh11CreERT2/Sox6w/w (Sox6 WT) mice were intraperitoneally injected with 2.5 mg of tamoxifen for five consecutive days. Then osmotic mini pumps were implanted subcutaneously for infusion of angiotensin II (1,000 ng/kg/min x 28 days). Knocking out Sox6 in SMCs inhibits degradation of ECM.

(A) Representative Van Gieson Stain images for elastin show cystic medial degeneration in Sox6 WT-Ang II (arrows). 20× magnification with 100 microns scale bars.

(B) Quantification of hypertension-induced abdominal aorta cystic medial degeneration. Percentage of total cystic area. Sox6 WT vehicle (n = 3), Sox6 KO vehicle (n = 3), Sox6 WT Ang II (n = 6), and Sox6 KO Ang II (n = 6). p values were calculated using one-way ANOVA. ∗p < 0.05 and ∗∗p < 0.01.

Discussion

Here, we utilize multiple approaches to evaluate the relationship between SOX6 and aortic aneurysm, including inquiries into human clinical health records linked with genotype data, evaluation of mRNA, and protein expression in human abdominal and thoracic aortic aneurysm patient samples. We also tested an Ang-II-induced hypertension model of abdominal aortic aneurysm in Sox6 knockout mice. Analysis of human clinical data suggests that SOX6 is associated with both abdominal and thoracic aortic aneurysms. Using a Sox6-specific knockout in smooth muscle cells, we established that the transcription factor Sox6 has a previously unidentified function in the development of hypertension-induced abdominal aortic aneurysm.

EHRs linked to patient genotype data aid in the discovery of new markers of human diseases and in the development of personalized medicine approaches and therapies. Using the institutional resource BioVU, we found that imputed expression of SOX6 is associated with aortic aneurysm diagnosis. Moreover, expression of this transcription factor was upregulated in both abdominal and thoracic aortic aneurysm tissue samples, indicating a direct transcriptional effect of SOX6 in these diseases. Furthermore, in TAA samples, upregulation of genes involved PPAR signaling, and cytokine receptor interaction was identified in TAA samples, indicating a key role for the immune system in the disease of this stage (Figures 2C and 2D). Previous reports indicate that PPARγ has a function in AAA development.27

RNA-seq analysis of aneurysmal aortic tissue from patients with AAA suggests the involvement of canonical pathways regulating the immune system, oxidative damage, macrophage production of NO and reactive oxygen species (ROS), natural killer cell signaling, and the T cell pathway (Figure 3C). The disease pathways upregulated were immune system driven such as leukocyte migration and quantity, leukopoiesis, and lymphopoiesis (Figure 3D). There are differences in the AAA and TAA RNA-seq that may reflect molecular distinctions in the development of both diseases. These results confirm the key role the immune system plays in the development of AAA.28,29,30,39 The AAA tissue samples were compared to non-aneurysmal aortic samples collected from patients undergoing coronary artery bypass surgery, and as such, our findings may reflect a partial assessment of the genes upregulated in the AAA samples. In Sox6 WT mice, Ang II induces cystic medial degradation in the abdominal aorta, while knocking out Sox6 inhibited this process (Figure 6). The abdominal aorta of Sox6 WT mice is disarranged, with an increase in fibrosis, as seen in the human samples, but this was not present in the Sox6 KO mice (Figures 5, 6, and S1–S3).

AAA accounts for more than 175,000 deaths globally, with AAA rupture causing 1% of deaths in men older than 65 years.12 Transabdominal ultrasound is used to diagnose AAA patients 65 years or older with a history of smoking, and they should be screened with at least one ultrasound at 65 years old, and the detection of rupture is not common. The disease is usually detected upon rupture, carrying an 80% mortality rate.4,40 TAA symptoms are rare, making it difficult to diagnose,41 with a high percentage of TAA patients dying without medical attention.41 Laboratory tests performed at hospitals and clinics are widespread practices to diagnose diseases, and these lab results are reported in the EHR. One of the challenges of the treatment for thoracic and abdominal aortic aneurysm is the asymptomatic nature of both diseases. TAA is harder to diagnose,41 requiring cross-sectional imaging (computed tomography [CT] or magnetic resonance [MR]), and there is a lack of molecular markers for predicting disease progression; therefore, it is important to develop molecular markers and clinical tests to better diagnose TAA and AAA. BioVU links genomic and clinical data from a large sample of patients and provides the opportunity to identify clinical data that are associated with these diseases. To address this, we performed a laboratory-wide association study (LabWAS), using the BioVU resource to identify the laboratory measurements associated with an aortic aneurysm diagnosis (Figure S13; Table S10). Aortic aneurysm diagnosis was significantly associated with multiple biomarkers and measurements of cardiovascular and metabolic health, including cholesterol, glucose, calcium, hemoglobin A1C, triglycerides, QRS duration, and T-wave axis from electrocardiograms. Additionally, we also saw significant associations with creatinine, immune system markers (white blood cells, eosinophils, lymphocytes), and red blood cell width and distribution.

LabWAS results (Figure S13; Table S10) indicate that changes in erythrocyte distribution width was associated with aortic aneurysms. This finding has been previously reported42 and based on our findings, may be a good clinical laboratory test to detect aortic aneurysm. The clinical cardiac parameters QRS duration and ST segment were associated with aortic aneurysm in our analysis. Two reports suggest that QRS may serve as an additional risk assessment of aortic dissection.43 Moreover, ST segment depression, concomitant with T-wave inversion, had been used to diagnose type A aortic dissection in a hypertensive patient.44 Decrease in creatinine clearance, associated with increase in serum creatinine after endovascular aneurysm repair, was also reported after a meta-analysis.45 Our LabWAS found creatinine as a parameter associated with aortic aneurysms. High-density lipoprotein (HDL) and a calcium clinical test were also strongly associated with aortic aneurysms. Low HDL is considered a risk factor for AAA.46 Other clinical parameters (Figure S12; Table S10) may also serve as clinicals test to detect aortic aneurysm, aiding in the diagnosis of AAA and TAA.

Additionally, we performed a LabWAS within the BioVU population to discern associations between genetic variation across SOX6 and SOX6-regulated genes. Significant associations were found between APOC1 GREX with triglyceride in serum or plasma and HLA-DQA1 GREX with glycated hemoglobin A1c, glucose, and tau protein in individuals of European ancestry (Figure S9; Table S6). APOC1 and HLA-DQA1 were reported as possible molecular markers of AAA.36,37 Our data and previous reports24,26 indicate that adiponectin may serve as a molecular marker for aortic aneurysm in patients of European ancestry. No significant results were detected in patients of African ancestry likely due to the smaller sample size (Figure S11; Table S7). Analysis within BioVU suggests that SOX6 may represent a molecular marker for AAA and TAA. The use of clinical laboratory measurements in the EHR may benefit translational research; however, the utility of this type of analysis is yet to be determined,47 and no conclusive results have been reported. However, our findings provide multiple clinical laboratory values associated with aortic aneurysm diagnosis, providing a more comprehensive list of potential biomarkers for the disease. These results may promote the development of improved patient risk stratification for aortic aneurysms and may lead to more informed referral for imaging for definitive diagnosis of aortic aneurysms.

There is compelling evidence to support the role of hypertension in AAA development.3,12,13 However, current antihypertensive therapies do not typically work against aortic aneurysm growth and rupture,48 with success only observed in patients suffering aortic aneurysm due to Marfan syndrome.49 Our data indicate that knocking out Sox6 in smooth muscle cells (Sox6 KO) decouples hypertension and AAA development (Figure 5). Typically, Sox6 WT mice that developed AAA rupture died soon after treatment with Ang II. This was not observed in Sox6 knockout mice. The ECM organization of the abdominal aorta in the Sox6 KO mice was preserved from hypertension-induced damage (Figures 5 and 6), indicating that smooth muscle cells are key in the Ang-II-induced ECM damage with Sox6 playing a key function. Additionally, we find that SOX6 GREX is significantly associated with hypertension diagnosis in BioVU and within the UK Biobank dataset (Table S9). Gene centric arrays and genome wide association studies (GWAS) indicate a strong association of Sox6 with hypertension (reviewed in ref.20). In combination, these data suggest that Sox6 has an unidentified function in the development of aortic aneurysms and may serve as a promising target.

In consensus with other reports, TNF-α protein expression is upregulated in human TAA tissue,32,50 and its expression is also increased in human AAA tissue (Figure 4). Concomitant with TNF-α, the expression of SOX6 was significantly upregulated in both human thoracic and abdominal aortic aneurysm tissue, suggesting a function for SOX6 in the transcription of genes associated with these diseases. The human tissue use in our study was collected from patients affected by AAA and TAA of thoracic and abdominal aorta once the aneurysm is developed, indicating that SOX6 plays a role in the advanced disease stage in humans.

Limitations of the study

We used the PheWAS method and identified the association of SOX6 with aortic aneurysm in humans. We are limited by the number of patients in the Vanderbilt University Medical Center biobank and human genomes included in the system. LabWAS is a new approach to uncover relevant clinical information to diagnose human diseases; one limitation is that the measurements are affected by the medications given to patients. We use a Bonferroni correction in the statistical analysis, which is a conservative approach. The Ang II induction of aortic aneurysm in mice model recapitulates dissection and aneurysm formation. However, Ang II does not reproduce all the features of aortic aneurysm in humans. Despite this limitation, this mouse model continues to be a reliable research animal model.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Antibodies	
	
Sox6	abcam	ab30455	
TNF alpha	abcam	ab183218	
Beta actin	abcam	ab178787	
TGF beta	Sigma-Aldrich	A1978	
SMAD2	abcam	ab40855	
pSMAD2	abcam	ab280888	
Cre	Novus	NB100-56133F	
Alpha smooth muscle actin	abcam	ab21027	
RNA isolation Quick-RNA MiniPrep kit	ZYMO RESEARCH	R1055	
Massons tricome	Fisher	NC9900705	
H&E	Fisher	NC1231542	
Elastin	Fisher	M1005910500	
cDNA SuperScript VILO MasterMix	Thermofisher	11755050	
	
Oligonucleotides	
	
GAPDH Taqman primer	Thermofisher	4331182 Hs02786624_g1	
COTL1 taqman primer	Thermofisher	4331182 Hs00332755_m1	
JCHAIN Taqman primer	Thermofisher	4331182 Hs00376160_m1	
SOX6 Taqman primer	Thermofisher	4331182 Hs00264525_m1	
PKD1 Taqman primer	Thermofisher	4331182 Hs00947377_m1	
GREM1 Taqman primer	Thermofisher	4331182 Hs01879841_s1	
CCL21 Taqman primer	Thermofisher	4331182 Hs00171076_m1	
ATP6AP2 Taqman primer	Thermofisher	4331182 Hs00997145_m1	
TNF Alpha Taqman primer	Thermofisher	4331182 Hs00174128_m1	
TGFB1 Taqman primer	Thermofisher	4331182 Hs00998133_m1	
MMP9 Taqman primer	Thermofisher	4331182 Hs00957562_m1	
MMP2 Taqman primer	Thermofisher	4331182 Hs01548727_m1	
EIF1B2 Taqman primer	Thermofisher	4331182 Hs00426752_m1	
	
Deposited data	
	
GEO data base accession GSE202267			

Resource availability

Lead contact

Further information and resources and reagents request should be directed to the lead contact, Dr Jose A. Gomez (jagoc502@gmail.com).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• RNA sequence data have been deposited into the GEO database and are publicly available as of the date of publication. Accession number is listed in the key resources table.

• This paper does not report original code.

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Experimental model and study participant details

Human studies

Aortic Aneurysm Aorta (AAA), Thoracic Aorta Aneurysm (TAA) and Control Aorta (Control) samples were collected from the OR at Vanderbilt University Medical Center, flash freeze in liquid nitrogen, and stored at -80°C. Full informed consent was obtained for all tissue samples.

Institutional Review Board Statement: All studies involving human samples was IRB Board approved (Perioperative vascular reactivity; IRB Number: 191351).

Study participants details

This study used electronic health record from Vanderbilt University Medical Center. All the participants details are presented in Tables 2 and 3.

Animals

Mice were housed and cared for at the Vanderbilt University Medical Center (VUMC) Division of Animal Care following the National Institutes of Health (NIH) guidelines and the Guide for the Care and Use of Laboratory Animals, US Department of Health and Human Services. All animal procedures were approved by the VUMC Institutional Animal Care and Use Committee prior to starting the experiments (protocol number M1900022-01). All the animals used in the in vivo studies, were maintained on a 12-hour light / 12-hour dark cycle at an ambient temperature of 24°C and 60% humidity. Six-week-old Myh11CreERT2/Sox6fl/fl (Sox6-KO) and Myh11CreERT2/Sox6wt/wt (Sox6-WT) control littermate mice were used in this study. The precise number of the animals for Sox6 KO and WT littermates are provided in the figure legends. The sequences of the primers for genotyping are: Sox6fl/fl, Forward Primer: 5’-GTC ACT CAG AGG TTA CTA TGG TG-3’; Reverse Primer: 5’-TTG GAG GCT TTA GCA GCT CTC-3’.18 Myh11 CreERT2 38,52-54., Transgene Forward 5’-TGA CCC CAT CTC TTC ACT CC-3’, Transgene Reverse 5’-AGT CCC TCA CAT CCT CAG GTT-3’, Internal positive controls Forward 5’- CAG CCA ACT TTA CGC CTA GC-3’, and Internal positive control Reverse 5’- TCT CAA GAT GGA CCT AAT ACG G-3’. Band to each of the PCR products obtained are presented in Figure S13A. The expression of Cre in aortic smooth muscle cells was determined using IHC with a protocol previously reported18,19 and presented in Figure S13B. IHC was performed by following the previously published protocol of our lab.18,19 Briefly, aortas were fixed with 10% neutral buffered formalin solution, dehydrated in graduated ethanol series, and embedded in paraffin. Aorta sections were cut at 10 microns thickness. Histo-Clear solution (catalog no. HS-202, National Diagnostics) was used to deparaffinized the sections and permeabilized with 0.2% Triton X-100 at room temperature (RT). Thereafter, sections were blocked with 5% BSA-PBS at RT and incubated with primary antibodies prepared in 1% BSA-PBS overnight at 4°C. Cre (Novus cat # NB100-56133F) and alpha smooth muscle actin (abcam cat # ab21027). Next morning, sections were washed with PBS (3X5 min). After three washes, sections were incubated with fluorochrome-conjugated secondary antibodies for 1 h at RT. The secondary antibodies were prepared in 1% BSA-PBS (1/500) and were chosen based on the primary antibodies and Alexa fluor fluorophores (ThermoFisher). DAPI was used to counterstain the nuclei. The tissue sections images were acquired with Nikon Eclipse Ti, (Software NIS-Elements AR 4.40.00 64-bit).

Method details

Calculating gene expression (GREX) in BioVU

Vanderbilt University Medical Center curates a biorepository of genotype data matched to de-identified electronic health records (EHR) for over 259,000 individuals. Genotype data for 85,613 BioVU individuals was generated using the Illumina MultiEthnic Genotype Array (MEGAEX) and imputed to the HRC reference panel using the Michigan imputation server 55,56. Gene expression was calculated in BioVU individuals from models built using the genotype-tissue expression (GTEx) project data 57. Details on the BioVU program, including oversight, patient engagement and ethical considerations have been previously discussed 51. This opt-in program collects leftover blood samples from patient visits at clinics across Tennessee. BioVU initiated sample collection in 2007 and is ongoing. Data from the 1000 Genome Project were used to define genetic ancestry with principal component analysis (PCA) 58,59. Genetically regulated gene expression was calculated in BioVU individuals from models built using the genotype-tissue expression (GTEx) project data 57. GTEx version 8 includes genotype data matched to RNA sequencing for over 838 donors across 49 distinct tissues. The best performing GREX models based on the highest r2 values from PrediXcan, UTMOST, and JTI approaches were used to model gene expression for SOX6 and additional genes from BioVU genotype data 22,23,60.

GREX Phenotype association study in BioVU

Aortic aneurysm cases and controls were identified in BioVU by at least two mentions of the aortic aneurysm phecode (442.1) mapped from ICD9/10 diagnosis billing codes (International Classification of Diseases, 9th and 10th editions), including: aortic aneurysm and dissection, dissecting aneurysm of aorta, dissecting aneurysm of aorta- unspecified site, dissection of aorta thoracic, dissecting aneurysm of abdominal aorta, dissecting aneurysm of thoracoabdominal aorta, thoracic aortic aneurysm- ruptured, thoracic aneurysm without rupture, aortic aneurysm of unspecified site- ruptured, thoracoabdominal aortic aneurysm- ruptured, thoracoabdominal aortic aneurysm- ruptured, thoracoabdominal aortic aneurysm- without mention of rupture, aortic aneurysm- not otherwise specified, aneurysm of aorta in diseases classified elsewhere. Controls included individuals without any mention of the aortic aneurysm phecode and excluded individuals with any mention of the similar, yet distinct phecodes: diseases of arteries, arterioles, and capillaries identified using the PheWAS package in R (version 0.99.5-2 and 3.6.0, respectively) 61,62. MultiXcan was used to combine the GREX of each gene across all tissues 63. Quantification and statistical analysis: we identified 1,208 individuals of European ancestry and 98 individuals of African ancestry as cases for aortic aneurysm in BioVU (controls = 58,099 and 13,551, respectively). Logistic regression was used to examine the relationship between SOX6 GREX or AAA/TAA gene GREX and the aortic aneurysm disease outcome. Age, sex, median age of medical record, principal components 1-10, genotype batch, and number of medical center visits were used as covariates in the analysis. More details in Supplemental Methods.

GREX laboratory-wide association scan (LabWAS) in BioVU

We evaluated all labs with measurements from at least 100 individuals, which resulted in testing 323 labs across 70,337 individuals of European ancestry and 241 labs across 15,123 individuals of African ancestry. We examined the relationship between the GREX of SOX6 and additional AAA/TAA genes with all available clinical lab measurements in linear regression models. Quantification and statistical analysis: Age, sex, principal components 1-10, genotype batch, median age of medical record, and number of medical center visits were used as covariates in the analysis. We used a Bonferroni-corrected threshold to account for the number of GREX-lab pairs present in the associations tested (i.e. 0.05/(number of labs∗number of unique gene-tissue pairs)). Laboratory values were extracted from the Vanderbilt University Medical Center de-identified EHR database and cleaned using the QualityLab pipeline as previously described.47

Aortic aneurysm diagnosis LabWAS in the Vanderbilt synthetic derivative

The Vanderbilt Synthetic Derivative includes insurance billing codes (International Classification of Diseases, 9th and 10th editions/ICD-9 and ICD-10 codes), laboratory measurements, and clinician notes. We used the Quality Labs pipeline to extract lab measurements in individuals classified as cases or controls for aortic aneurysm based on the phecode 442.1 for aortic aneurysm. Quantification and statistical analysis: we evaluated 335 labs across 2,273 cases and 1,268,204 controls for aortic aneurysm. Age, sex, median age of medical record, EHR-reported race and ethnicity, and number of medical center visits were used as covariates in the analysis. We used a Bonferroni-corrected threshold to account for the number of labs tested (i.e. 0.05/(number of labs tested)). The Vanderbilt Synthetic Derivative is a de-identified database of the electronic health records for over 3 million patients at Vanderbilt, dating back more than 30 years 64.

Osmotic mini pump implantation

Mice were anesthetized with isoflurane for the duration of the procedure. Adult male (8 to 12 weeks old) Myh11CreERT2/Sox6fl/fl (Sox6 KO) and Myh11CreERT2/Sox6wt/wt (Sox6 WT) were intraperitoneally injected with 2.5 mg of Tamoxifen for five consecutive days to induced Sox6 knock-out (Figure S14). One week later, mice were anesthetized using isoflurane, and osmotic mini-pumps (alzet model 2004) implanted subcutaneously for continuous infusion of Ang II (1000 ng/kg/min x 28 days) or its vehicle. A subcutaneous (SC) injection of ketoprofen (Dose: 5mg/kg BW) was given before procedure and 24 h as post-operative analgesia.

Blood pressure measurement

Blood pressure measurements were performed using a tail-cuff method following previously reported recommendation for precise measurements previously reported.18 Blood pressure was also measured 4 weeks after Ang II infusion.

RT-qPCR

Preparation of template cDNA. Aortic Aneurysm Aorta (AAA), Thoracic Aorta Aneurysm (TAA) and Control Aorta (CA) samples were collected from the OR at Vanderbilt University Medical Center, flash freeze in liquid nitrogen, and stored at -80°C. The RNA was extracted from a portion of each sample by homogenizing two times during 30 seconds with Tissue-Tearor (Model 985370, BioSpec Products, Inc.) resuspended in 600 uL RNA Lysis Buffer from Quick-RNA MiniPrep kit (ZYMO RESEARCH) RNA was extracted following the manufacturer's instructions. The amount of RNA extracted was quantified on NanoDrop One (Thermo Scientific). The cDNA was obtained from the RNA extracted from the samples using SuperScript VILO MasterMix (Invitrogen, ThermoFisher Scientific) following manufacturer's instructions. The amount, quality and purity of the cDNA was measured using the NanoDrop One (Thermo Scientific). Real-time PCR was performed in the QuantStudio 3 Real-Time PCR System (Applied Biosystems) using TaqMan Fast Advanced Master Mix (Applied Biosystems, containing AmpliTaq Fast DNA Polymerase, uracil-N-glycosylase, dNTPs with dUTP, ROX dye as passive reference, and optimized buffer components). The total reaction volume (20 μL) consisted of the following: 1 μL cDNA (100 ng), 10 μL TaqMan Fast Advanced Master Mix, 1 μL of each TaqMan Gene Expression Assay, and 8 μL ultrapure DNase-free water. The thermal cycle parameters were as follows: UNG incubation at 50°C for 2 min, polymerase activation at 95°C for 20 s, denaturation at 95°C for 1 s and then annealing and extension at 60°C for 20 s. We used Thermofisher Taqman proves for each gene assay. The gene assays used in this study were polycystin 1 (PKD1), gremlin 1 (GREM1), coactosin like F-actin binding protein 1 (COTL1), joining chain of multimeric IgA and IgM (JCHAIN), C-C motif chemokine ligand 21 (CCL21), ATPase H+ transporting accessory protein 2 (ATPGAP2), tumor necrosis factor (TNF), transforming growth factor beta (TGFβ1), SRY-box transcription factor 6 (SOX6), matrix metallopeptidase 9 (MMP9), matrix metalloproteinase (MMP2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and eukaryotic translation initiation factor 2B subunit alpha (EIF2B1) were used as housekeeping genes. Real-Time qPCR data normalization and analysis. The samples included in the study allowed to see how normally the gene assays are expressed under tissue normal conditions, in the other hand the pathological conditions allowed us to see if it has any difference in terms of expression over those candidate genes. The comparative CT method was used to calculate the relative expression of the transcripts in all the samples and genes were normalized according to the housekeeping genes.

Western blot

Preparation of BCA Standard Curve and getting protein concentration from the samples. The BCA standard curve was made on a microplate and constructed using the Pierce™ BCA Protein Assay Kit (Thermo Scientific) following manufacturer's instructions mixing the reagent A and reagent B (50:1) with the BCA and dilutions, and with the samples (AAA, TAA, and CA) from protein extraction procedure. Incubation was performed during 30 minutes at 37°C, let the plate cool down and measure the absorbance at 562nm on a plate reader. With the results, the standard curve was constructed and calculated the original concentration of the samples to define how much amount of protein is going to be added to the electrophoresis gel. SDS-PAGE Electrophoresis Gel. To perform the electrophoresis the Mini-PROTEAN TGX Precast Gels (BIO-RAD, Cat. #456-1096) were used once the running buffer 25 mM Tris, 190 mM Glycine, and 0.1% SDS) was added to the electrophoresis chamber Mini-PROTEAN Tetra System (BIO-RAD). The samples were prepared with 10 μL 4x Laemmli Sample Buffer (BIO-RAD, Cat. #1610747) and 10 μL of sample and Nuclease free water. The samples were heated during 5 min at 95°C and each well was loaded with 30 ug of protein with 10 μL of Precision Plus Protein Standards (BIO-RAD) ladder. The conditions to run the gels were stablished for 5 min at 50V and then for 70 min at 100 V. The sponges and filter papers were incubated in cold transfer buffer (25 mM Tris, 190 mM Glycine, and 20% methanol) for 15 min at 4°C. The PVDF membrane was activated in methanol for 30 seconds, put between the electrodes and the filter papers along with the polyacrylamide gel to run it for 120 min at 100 V in a 4°C room. Once the electro transfer is completed, we can proceed with the antibodies looking for the protein of interest. Immunoblotting. The PVDF membrane is transferred directly in fresh blocking buffer (milk 5% in TBS 1X-T 0.1%) for 1-2 hours at room temperature, later the primary antibody is diluted (1:100) in milk 5% with TBS 1X-T 0.1% and it’s added to the PVDF membrane overnight at 4°C, then washed 3x with TBS 1X-T 0.1% incubated for 10 min at room temperature. The second antibody is added (1:1000) diluted in milk 5% with TBS 1X-T 0.1%, let incubate it for 1 hour at room temperature and washed 3x with TBS 1X-T 0.1% incubated for 10 min at room temperature. Now revealed with Clarity Western ECL Substrate (BIO-RAD) following software’s instructions. For stripping the blot Restore Western Blot Stripping Buffer (Thermo Scientific) was used and the immunoblotting was repeated if necessary, according to the different antibodies needed. Protein bands were quantified and normalized with the house-keeping gene beta actin (Sigma-Aldrich cat # A1978) using software integrated with the image station.

RNA sequencing

RNA was isolated from Aortic Aneurysm Aorta (AAA), Thoracic Aorta Aneurysm (TAA) and Control Aorta (CA). Total RNA was isolated using a PicoPure Arcturus kit (Invitrogen) according to the manufacturer’s instructions. Complimentary cDNA was generated with an Ovation Pico WTA System V2 kit (NuGEN) which maintains the stoichiometry of the original RNA population. library construction was performed NEBNext® Ultra™ II for DNA Library Prep. All samples are sequenced and performed at multiplex Paired-End 150 bp on the Illumina NovaSeq 6000 by VANTAGE core at VUMC. Raw sequencing data was uploaded and analyzed using the Illumina BaseSpace platform. Sequences (151 bp paired end) were trimmed from both ends to 125 bp using the FastQ Toolkit app. Transcripts were aligned to the human hg19 genome and quantified with the RNA Express app. Differentially expressed genes (DEGs) were determined using a false discovery rate (FDR) threshold of 0.05 and expression mean count >10 for any group. Data have been deposited into the GEO database (accession GSE202267). Expression heatmaps were generated with Morpheus (Broad Institute) via hierarchical clustering using average linkage and Spearman’s correlation. Enriched pathway predictions were performed with Ingenuity Pathway Analysis (IPA, QIAGEN) and through gene set enrichment analysis (GSEA, WebGestalt.org). An absolute activation Z-score of >2.0 and a p-value of <0.05 were deemed as significant for enriched terms.

Human and murine tissue staining

Abdominal aortas of anesthetized (isoflurane) mice 4 weeks after Ang II fusion were harvested and fixed overnight at 4°C in 4%v/v neutral buffered formalin, followed by incubation in 30%w/v sucrose for a further 24hrs at 4°C. The aortas were paraffin embedded. For each section, 5 10-micron slices were taken and mounted onto a glass slide. Masson’s trichrome, Hematoxylin and Eosin, and Van Geison staining was then performed according to standard techniques in the Pathology core at VUMC. Human samples were fixed overnight at 4°C in 4%v/v neutral buffered formalin, followed by incubation in 30%w/v sucrose for a further 24hrs at 4°C. The aortas were paraffin embedded. For each section, 5 10-micron slices were taken and mounted onto a glass slide. Masson’s trichrome, Hematoxylin and Eosin, and Van Geison staining was then performed according to standard techniques in the Pathology core at VUMC.

Quantification and statistical analysis

All statistical analysis were performed using GraphPad Prism 8.2. Data are presented as mean ± SEM unless otherwise indicated. Two-way ANOVA was used for experiments with three or more conditions followed by Tukey’s tests for comparisons between individual groups. One way ANOVA was used for experiments with three conditions. Student t-test was used to compare the mRNA expression values between control aortic sample and TAA or control aortic sample and AAA. A p-value equal or less than 0.05 was considered significant. We used a Bonferroni-corrected threshold to account for the number of GREX-lab pairs present in the associations tested (i.e. 0.05 / (number of labs∗number of unique gene-tissue pairs)). Logistic regression was used to examine the relationship between SOX6 GREX or AAA/TAA gene GREX and the aortic aneurysm disease outcome. We used a Bonferroni-corrected threshold to account for the number of labs tested (i.e. 0.05/(number of labs tested)). Data analysis was performed blinded. Animals were divided by genotyped and randomized to the procedure. N values or sample numbers and p values are presented in each figure and legends.

Supplemental information

Document S1. Figures S1–S14 and Tables S1, S2, S4, S5, S8, and S9

Table S3. RNA sequence data

Table S6. LabWAS results for individuals of European ancestry (n = 70,337, 323 labs)

Table S7. LabWAS results for individuals of African ancestry (n = 15,123, 241 labs)

Table S10. LabWAS results for aortic aneurysm diagnosis (n = 2,273 cases/1,268,204 controls, 335 labs)

Acknowledgments

We want to thank Dr. Lea Davis for her technical assistance with the BioVU data set analysis. The Synthetic Derivative and BioVU projects at Vanderbilt University Medical Center are supported by numerous sources: institutional funding, private agencies, and federal grants, including the 10.13039/100000002 NIH -funded Shared Instrumentation grants S10OD017985 and S10RR025141 and 10.13039/100016220 CTSA grants UL1TR002243 , UL1TR000445 , and UL1RR024975 from the 10.13039/100006108 National Center for Advancing Translational Sciences . Its contents are solely the responsibility of the authors and do not necessarily represent official views of the 10.13039/100006108 National Center for Advancing Translational Sciences or the 10.13039/100000002 National Institutes of Health . Genomic data are also supported by investigator-led projects that include U01HG004798 , R01NS032830 , RC2GM092618 , P50GM115305 , U01HG006378 , U19HL065962 , and R01HD074711 and additional funding sources listed at https://victr.vumc.org/biovu-funding/. 10.13039/100021144 UNCF /BMS EE United Negro College Fund/Bristol-Myers Squibb E.E. Just Postgraduate Fellowship in the Life Sciences Fellowship to H.K.B., The 10.13039/100021144 United Negro College Fund /Bristol-Myers Squibb E.E. Just Faculty Fund, 10.13039/100000861 Burroughs Wellcome Fund Career Awards at the Scientific Interface Award, 10.13039/100000861 Burroughs Wellcome Fund Ad-hoc Award, National Institutes of Health Small Research Pilot Subaward to 5R25HL106365-12 from the 10.13039/100000002 National Institutes of Health PRIDE Program, DK020593 , Vanderbilt Diabetes and Research Training Center for DRTC Alzheimer’s Disease Pilot & Feasibility Program to A.H.J. Research was supported by NHLBI Research Scientist Development Grant (1K01HL135461-01 ) to J.A.G. Small Research Project (SRP) Subaward from PRIDE-CVD to J.A.G.; SRPs are funded by an 10.13039/100000050 NHLBI grant #R25 HL105446 to Dr. Mohamed Boutjdir.

Author contributions

J.A.G. conceived the original idea. D.C., I.A., A.H.J., and J.A.G. designed research. D.D.H. and A.G.M. performed RNA-seq data analysis. T.W.M., K.V.A., P.M.A., Q.S.W., H.K.B., D.F.D., and N.J.C. provided access to BioVU data and performed the analysis. D.C., I.A., P.B.L., and J.A.G. performed experiments. Z.V. and A.G.M. collected mouse samples. Human samples were collected by M.G.L., F.T.B., and J.A.C. The first draft of the manuscript was written by D.C., I.A., T.W.M., D.D.H., and J.A.G. All the authors listed revised, edited, and approved the manuscript.

Declaration of interests

The authors declare no conflicts of interest, financial, or otherwise.

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2024.110436.
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