A functional variant of SMAD4 enhances macrophage recruitment and inflammatory response via TGF-β signal activation in Thoracic aortic aneurysm and dissection

Genetic association studies

The demographic characteristics and clinical features of the study participants in screening and validation cohort were listed in Supplementary Table 1. Data of 400 healthy controls were documented in our previous study [19]. The candidate 20 SNPs were genotyped in 202 TAAD patients and 400 healthy controls using MALDI-TOF MS (Fig. 1A). Large vessels CT angiography and 3D scanning images for thoracic aortic aneurysm and dissection patients were shown in Fig. 1B.

Figure 1. The MALDI-TOF MS spectrum of candidate SNPs and large vessels CT angiography for clinical features of TAAD patients. (A) The MALDI-TOF MS spectrum of candidate SNPs. Genotypes of SNPs are determined by plotting peak intensity (y-axis) against mass (Da) (x-axis). The spectrum of rs12455792 is indicated by a circle. (B) Large vessels CT angiography and 3D scanning images for clinical features of thoracic aortic aneurysm (a, c) and dissection (b, d) patients.

The discriminations of genotype distribution for 20 candidate SNPs between cases and healthy controls were statistical analyzed using multiple regression (Supplementary Table 2). Genotype frequencies of all the SNPs in healthy controls were conformed to Hardy-Weinberg equilibrium (HWE) (P>0.05). Of the 20 SNPs, rs12913975, rs10757278, rs10770612, rs10733710 and rs12455792 were significantly associated with increased TAAD susceptibility, with ORs of 1.489 (P=0.012), 2.006 (P=0.005), 1.767 (P=0.003), 1.412 (P=0.005), 1.585 (P=0.011) adjusting for age and gender, respectively. Among them, the functional SNP rs12455792 C>T variant was correlated with increased aortic diameter of TAAD patients (r=0.164, P=0.020, positive correlation in screening data), yielding the most significant results (Supplementary Table 3). rs12455792 was bioinformatical predicted as a proximal transcription regulatory loci, i.e. it was a functional SNP. And we have proved that rs1245792 variant reduced the expression of SMAD4 [19]. Collectively, we further investigated the relationship between rs12455792 and pathological progression of TAAD.

rs12455792 C>T variant enhanced both canonical and non-canonical TGF-β signaling transduction in HASMCs

To determine the TGF-β signaling changes with rs12455792 C>T variant in HASMCs, we isolated normal human thoracic aortic SMCs and cultured them to passage 3 in SMC special medium. Since we have proved that rs12455792 variant suppressed SMAD4 expression, siRNA-SMAD4 were transfected into HASMCs to mimic the impact of rs12455792 variant genotype. Real-time PCR was used to detect the transcription of canonical ligands and downstream targets in TGF-β signaling in HASMCs transfected with siRNA-NC or siRNA-SMAD4. We found there was no change for the expression of TGFB1, TGFB2, TGFB3 and ID-1 between control and SMAD4 low-expression group. But the expression of PAI-1 was obviously reduced in SMAD4 low-expression group. PAI-1 was a direct target of canonical TGF-β pathway. Compared to control, we also found an obvious increase for the expression of CTGF in SMAD4 low-expression group (Fig. 2A). This increase was offset with addition of canonical and non-canonical TGF-β signaling mediator inhibitors (Supplementary Figure 2). CTGF, an essential regulator in fibronectin assembly and vascular remodeling, is a well-known factor in TGF-β responsiveness.

Figure 2. Enhanced canonical and non-canonical TGF-β signaling in SMAD4-interfered HASMCs. (A) Real-time PCR assay for the expression of canonical and non-canonical TGF-β signaling molecules in different HASMCs. (B) Western blot assay for the expression of canonical and non-canonical TGF-β signaling mediators in HASMCs transfected with siRNA-NC or siRNA-SMAD4. (C) Western blot assay for the expression of canonical and non-canonical TGF-β signaling mediators in HASMCs carrying rs12455792 CC, CT or TT genotypes. (D, E) Quantitative analysis for B and C, respectively. (F) Real-time PCR assay for the expression of essential chemotactic factors in different HASMCs. The experiments were repeated three times. Data were presented as mean ± SD. *P<0.05, **P<0.01, ns: not significant.

Furthermore, we examined the activation of intracellular mediators in canonical or non-canonical TGF-β signaling. The results revealed an enhanced activation of SMAD2, SMAD3, JNK and ERK in SMAD4 low-expression group (Fig. 2B, D). We also demonstrated the increased activation of SMAD2, and JNK in HASMCs carrying rs12455792 variant genotypes without gene dosage effect, compared to that with wild type genotype (Fig. 2C, E). These data confirmed that both canonical and non-canonical TGF-β signaling responsiveness elevated by SMAD4 low-expression or rs12455792 variant in HASMCs.

rs12455792 C>T variant leads to MØ recruitment accompanied by the increased expression of chemotactic factors

Next, we investigated whether TGF-β signal interfering in HASMCs could directly trigger MØ recruitment. First, we checked the alterations for chemotactic factors in HASMCs transfected with siRNA-SMAD4. The real-time PCR data declared a significant upregulation of numerous chemokines, including CCL2, CXCL3, CXCL9, CXCL10 and CXCL12 in siRNA-SMAD4 group (Fig. 2F). Among these CCL and CXCL family members, CCL2 and CXCL12 have been previously reported to facilitate monocyte chemotactic activity and be involved in vascular inflammation [20]. Therefore, we further detected MØ chemotaxis status with SMAD4-low expression and rs12455792 variant. Before functional assay, HASMCs were identified with over 95% SM22α immunostained and MØ was successfully induced from THP-1 human monocyte cell line by 100ng/ml PMA. The transwell migration assays verified an evident stimulating role of the SMAD4-silenced HASMCs on MØ chemotaxis, in compliance with the excessive CCL2 and CXCL12 secretion from the SMAD4-silenced HASMCs (Fig. 3A-B). Similarly, we observed rs12455792 C>T variant strikingly promoted MØ migrating to HASMCs (Fig. 3C-D).

Figure 3. rs12455792 C>T variant promote the chemotaxis of MØ to HASMCs. (A) Representative fluorescence images of MØ migrating to HASMCs transfected with siRNA-NC or siRNA-SMAD4 in transwell assay. (B) The quantitative analysis of MØ cell numbers in different groups, n=5/group. (C) Representative fluorescence images of MØ migrating to HASMCs carrying rs12455792 CC, CT or TT genotypes in transwell assay. (D) The quantitative analysis of MØ cell numbers in different groups, n=5/group. Red: Dil-prestained MØ; Green: Dio-prestained HASMCs; Blue: DAPI. Data are means ± SD. **P<0.01, ns: not significant. Scale bar: 50μm.

rs12455792 C>T variant facilitates MØ infiltration in human TAAD specimens

To evaluate the actual phenomena of MØ infiltration in human thoracic aorta specimens from TAAD patients with different genotype, we examined immunofluorescence staining for CD68 with frozen sections of specimens under a laser scanning confocal microscopy. The number of CD68+ cells in rs12455792 CT and TT group was significantly higher than that in CC group (Fig. 4A-B). This suggested that rs12455792 C>T variant notably facilitated MØ infiltration in human TAAD specimens. Furthermore, we detected the expression of pro-inflammatory marker TNF-α (M1 type MØ) and anti-inflammatory marker CD206 (M2 type MØ) in human TAAD specimens with different genotype. The average integral optical density of TNF-α in CT or TT group was markedly increased, compared to that in CC group (Fig. 5A-B). rs12455792 C>T variant elevated M1 type MØ marker expression, indicating that the variant led to an activated inflammatory circumstance initiated via MØ polarization. But there was no significant change for CD206 expression with rs12455792 variant.

Figure 4. rs12455792 C>T variant promotes MØ infiltration in human thoracic aorta specimens. (A) Representative confocal-microscopy graphs of immunostaining for CD86 in specimens from thoracic aortic aneurysm patients with rs12455792 CC, CT or TT genotypes. Red: CD68; Blue: DAPI. (B) The quantitative analysis of MØ cell numbers in different groups, n=8/group. Data are means ± SD. **P<0.01, ns: not significant. Scale bar: 100μm.

Figure 5. rs12455792 C>T variant enhances TNF-α but not CD206 expression in human thoracic aorta specimens. (A) Immunofluorescence staining for TNF-α and CD206 in specimens from thoracic aortic aneurysm patients with rs12455792 CC, CT or TT genotypes. Red: TNF-α; Green: CD206; Blue: DAPI. (B) The plot of average integral optical density for TNF-α and CD206 expression in different groups, n=8/group. Data are means ± SD. *P<0.05, **P<0.01, ns: not significant. Scale bar: 100μm.

SMAD4 knockdown facilitates the progression of TAAD in animal model

To mimic the impact of rs12455792 - SMAD4 low expression generally, we constructed the SMAD4 KD mouse and detected the associated pathological progress of TAAD. Animal model was constructed with peritoneal injection of 15mg/kg.d AngII for 4w. The procedure was shown in Fig. 6A. The SMAD4 KD mouse was a heterozygote with one chromosome inserted by red fluorescence protein (RFP) flag and piggyBac (PB) transposon in exon 8 of SMAD4 (Fig. 6B). It was identified by electrophoresis of mouse tail DNA (Fig. 6C). Applying ultraviolet transmission, the red fluorescence of inserted RFP flag was observed in the naked skin of SMAD4 KD mice (Fig. 7A). With AngII administration for 4w, we found SMAD4 knockdown strikingly contributed to the progressive dilatation of both thoracic aortas and abdominal aortas using echocardiography (from 1w to 4w) (Fig. 7B-D). Moreover, we separated the aortas in different groups and stained them by oil-red (Fig. 8A). As the aneurysm developed, SMAD4 KD mice displayed typical pathological performances of the inherited predispose to TAAD, including aortic diameter increase, plaque accumulation, aortic wall thickening, monocytes infiltration and medial degeneration characterized by destructive vascular matrix remodeling (Fig. 8B-E). Notably, histological analysis of the thoracic aortic wall in SMAD4 KD mice revealed that aortic dissection rapidly developed, accompanied with erythrocyte extravasation into the media lesion region and intense inflammatory cell infiltration (Fig. 8D-E).

Figure 6. The construction of TAA animal model. (A) The flow diagram of modeling process for TAA mouse in Wild Type and SMAD4 KD group; (B) The schematic diagram for generation of SMAD4 KD mice; (C) The DNA electrophoretogram of identification for Wild Type mice and SMAD4 KD mice.

Figure 7. Repression of SMAD4 leads to dilatation of both thoracic aorta and abdominal aorta. (A) Representative holistic view of Wild Type and SMAD4 KD mice under ultraviolet. The red fluorescence is observed in the naked skin of SMAD4 KD mice. (B) The polygram for time-dependent aorta dilatation in different groups; (C) The quantitative analysis for diameter of aortas among different groups at 4W after modeling; (D) Representative echocardiography images of aorta diameter in Wild Type and SMAD4 KD group at 2W or 4W, n=5/group. *: P<0.05. The data were presented as mean ± SD.

Figure 8. Repression of SMAD4 promotes the formation of TAAD. (A) The gross view and profile view of aortas from Wild Type and SMAD4 KD mouse models. Quantitative analysis of maximal aortic diameter (B), plaques area (C) and lesion area (D) between different groups, n=5/group; (E) Representative graphs of HE staining for thoracic aortas from Wild Type and SMAD4 KD mouse models. Data are means ± SD. *P<0.05, **P<0.01. Scale bars: 100μm.

Subjects

All experiments referring human or animal samples were approved by Institutional Review Board (IRB), the first affiliated hospital of Soochow University (Jan. 2010 - Dec. 2016). All subjects were chosen from Han Chinese population of eastern China. 202 cases of screening cohort, 227 cases of validation cohort and 400 healthy controls were included in this study. They have signed the informed consent. The inclusion criteria of sporadic TAAD patients was described in previous report [19]. Controls were frequency matched via age and gender to cases. The aorta status of patients were measured by echocardiography, angiography, CT or MRI. The demographic and clinicopathological information were documented in patients' interview. Fresh thoracic aorta tissues around focus were collected from 39 TAAD patients receiving the Bentall procedures in Department of Cardiovascular Surgery, the first affiliated hospital of Soochow University.

SNP selecting and genotyping

We selected 30 SNPs essential in pathogenesis of TAAD by analyzing GWAS-based TAAD reports [16-18], OMIM database (http://www.omim.org/) and Han Chinese data from 1000 Genome Project resources (http://www.1000genomes.org). Among them, 10 SNPs were difficult to detected using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and excluded, by virtue of homologous interference, hairpin structure, short intervals and so on. All the candidate SNPs meet the following criteria: (1) the minor allele frequency (MAF)>0.05; (2) r²>0.80 for each paired SNPs. Functinal SNPs were bioinformatical analyzed using online tools- rsnp (http://rsnp.psych.ac.cn/) and snpinfo (http://snpinfo.niehs.nih.gov/cgi-bin/snpinfo/snpfunc.cgi).

Candidate SNPs were genotyped using MALDI-TOF MS as previously described [19]. Briefly, The PCR product was dispensed into a 384-format SpectroCHIP and the MALDI-TOF MS assay was performed on a MassARRAY Compact Analyzer (Sequenom, San Diego, CA, USA). Amplification and single-base extension primers in multiple PCR were synthesized by Benegene (Benegene Biotechnology, Shanghai, China) and all the sequences were listed in Supplementary Table 4. Genotype calling was carried out with MassARRAY RT package 3.0. Genotyping quality was evaluated by Sanger sequencing for ~15% randomly chosen samples, finally generating a 100% concordance. The success rate of SNP genotyping was >99%.

Animal models

The SMAD4 knock-down (KD) mouse were designed and constructed by Biogle (Biogle Inc., Hangzhou, China). All mice were maintained with C57BL/6J genetic background. Experiments regarding to animal model were performed according to institutional guidelines for laboratory animals. To induce aneurysm formation of TAA model, we chose 10-12w old mice and intraperitoneal injected with Angiotensin II (Ang II) (Sangon Inc., Shanghai, China) (15mg/kg.d, n =10 per group). All experiments were conducted using both male and female mice. Controls were sex-matched littermates. Four weeks later, mice were sacrificed, and thoracic aortas were histochemistry stained and evaluated.

Echocardiography

Mice were anesthetized with 5% isoflurane by Small Animal Anesthesia Ventilator System (RWD Life Science Inc,. Shenzhen, China). The thoracic aortic diameter was evaluated using a Vevo2100 cardiovascular ultrasound system with MS400 (30mHz) microprobe (VisualSonics Inc., Toronto, Canada). Echocardiographic detection was performed in B mode. Each experiment was repeated at three times from 10 mice per group.

Cell culture

Primary patients human aortic smooth muscle cells (HASMCs) were isolated from thoracic aorta of TAAD patients who received Bentall procedures. Normal HASMCs was obtained from aorta from patients during aortic valve replacement. In brief, the media of fresh thoracic aortas were tore off and cut into 1mm² pieces, then cultured in 10cm dishes with human SMC medium (Sciencell, San Diego, CA, USA). Three days after primary culture, HASMCs were spread out from aortic tissues. With growth to 80% confluence, cells were digested by 0.25% trypsin-EDTA and passaged. In the experiments, HASMCs were used in passage 3-8 with at least 95% purity.

Real-time PCR and western blotting

Real-time PCR was performed on an ABI StepOnePlus™ Real-Time PCR System (ABI, Carlsbad CA, USA). The primers used for amplification of chemotactic factors and TGF-β molecules were listed in Supplementary Table 5. In western blotting assay, proteins were extracted from HASMCs or human thoracic aorta tissues and subjected to immunoblotting with rabbit polyclonal antibodies against p-ERK、p-JNK、p-GSK, mouse monoclonal antibodies against pSMAD2、pSMAD3 (1:1000, Santacruz, California, USA). GAPDH was used as internal control. Gel-Pro analyzer 4.0 software package was used to analysis the grey scales of target proteins, which were standardized against that of GAPDH (Media Cybernetics, Silver Spring, MD, USA).

Chemotatic assay

MØ was derived from THP-1 monocytes with 100ng/ml PMA induction for 48h. Cell chemotaxis were measured using 24-well Transwell® units (8.0 μm pore, Costar Corning, NY, USA). One hour before experiment, MØ was stained with Dil (red, Beyotime, Shanghai, China), a bioactive fluorescence probe. HASMCs were stained with Dio (green, Beyotime, Shanghai, China). Then, MØ (5×10³) in 100 μl medium was added into each upper insert, and HASMCs in different group (5×10⁴) in 500 μl medium were added into lower chamber. The plate were incubated at 37 °C, 5% CO₂ for 24h. To examine MØ chemotaxic-migrating to lower chamber, cells was fixed by methanol for 10 minutes, washed, and stained with DAPI. Finally, the images of chmotaxic-migrated MØ were captured with an inversion fluorescence microscope (Olympus IX51, Tokyo, Japan). MØ was quantified by counting 10 independent visual fields per group via Image Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA). Each assay was performed in triplicate.

Quantification of aortic lesions

The aortic tissue of mouse was removed from the ascending aorta to the iliac artery branch and fixed with 4% paraformaldehyde overnight at 4°C. After fixation, the aortic intimal surface was exposed by a longitudinal cut through the aortic arch that extended down the whole length of the aortic tree. Then the aortic tissues were stained using Oil Red-O and images were captured. To quantify the phanerous lesions in intimal surface, mean lesion areas was analyzed by Image Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).

Histological and immunocytochemical characterization of TAAD

To evaluate the pathological performances of TAAD, murine aortas were separated and fixed in the 4% paraformaldehyde after 4w of treatment. Serial paraffin cross section (5 μm) of murine thoracic aortas were prepared for histological analysis. Aorta sections were stained with eosin and hematoxylin for morphological assessment. Immunostaining of CD86 and polarization markers (TNF-α, CD206) (1:1000, Santacruz, California, USA) was performed to detect MØ infiltration in frozen sections of human thoracic aortas. The images of immunofluorescence were digitally captured on a Carl Zeiss LSM880 laser scanning confocal microscope (Carl zeiss, Jena, Germany). CD68+ Cell numbers or average integral optical density of CD206 and TNF-α in the aortic wall were quantitative analyzed from 10 samples per group using Image Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).

Statistical analysis

Differences of the demographic and clinical features, and frequencies of genotypes in case-control study were tested by Student's t test (for continuous variables) or Chi-square test (for categorical variables). The comparisons of cell numbers, mean intergral optical density, lesion areas and so on were using Student's t test (between 2 groups) or One way ANOVA (between more than 2 groups). Hardy–Weinberg equilibrium (HWE) was evaluated using online analytical tools. For analyzing main effect of candidate SNPs, univariate or multivariate logistic regression models were performed to generate ORs and corresponding 95%CIs with adjustment by possible confounders. All statistical tests were two tailed and conducted using Statistical Program for Social Sciences (SPSS 18.0, Chicago, IL, USA) or R software(http://www.r-project.org/). P value <0.05 was considered statistically significant.