ERBB4 as a treatment target in aortic dissection and its role in cell therapies for blood vessel repair

Aortic tissue samples were obtained from three patients undergoing Stanford type A dissection surgery, while control aortic tissue (Control) was collected from the aortas of coronary artery bypass patients (n ═ 3). Each sample was divided into two parts: one portion was stored at −80 ^∘C for western blot analysis, and the other was fixed in a 4% paraformaldehyde solution for further examination. This study adhered to the principles of the Declaration of Helsinki and was approved by the Ethics Committee of the First Affiliated Hospital of Bengbu Medical University.

IHC analysis was performed on aortic tissue. Samples were treated with 3% hydrogen peroxide for 10 min, rinsed with phosphate-buffered saline (PBS), and blocked with 10% normal goat serum in PBS for 1 h at 37 ^∘C. Sections were then incubated overnight at 4 ^∘C with primary antibodies, followed by the appropriate secondary antibody and DAB staining (Beyotime, Shanghai, China). Finally, the sections were sealed with gum.

RNA sequencing was conducted by Seqhealth Technology Co., Ltd. (Wuhan, China). Briefly, total RNA was extracted from human aortic tissue samples using TRIzol Reagent (Invitrogen, Cat. No. 15596026, USA). Stranded RNA sequencing libraries were prepared from 2 µg of total RNA using the KCTM Stranded mRNA Library Prep Kit for Illumina^® (Wuhan Seqhealth Co., Ltd., China), following the manufacturer’s instructions. PCR products were enriched, quantified, and sequenced on a DNBSEQ-T7 sequencer (MGI Tech Co., Ltd., China) using the PE150 model. DEGs were identified using the edgeR package (version 3.12.1), with a fold-change cutoff of 2 and a P value threshold of 0.05 for statistical significance. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses for DEGs were performed using KOBAS software (version 2.1.1).

Human aortic smooth muscle cells (HASMCs) were obtained from the American Type Culture Collection (ATCC, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were maintained at 37 ^∘C with 5% CO₂. In the model group, HASMCs were cultured in high-glucose DMEM. Three siRNAs were designed by Ribobio (Guangzhou, China) and transfected into HASMCs using a siRNA transfection kit (Thermo Fisher Scientific, USA), following the manufacturer’s instructions.

After transfection, the cells were evenly seeded into a 96-well plate at a density of 2000 cells per well and cultured for 72 h. The culture medium was then discarded, and CCK-8 detection reagent was added. The cells were incubated at 37 ^∘C for 1 h, and the optical density at 450 nm was measured using a microplate reader.

HASMCs were seeded into a six-well plate and cultured until reaching approximately 80%–90% confluency. A linear wound was then created in the center of each well using a sterile 200 µL pipette tip. Debris was removed by washing the wells twice with PBS, after which the cells were incubated in DMEM containing 1% FBS to minimize proliferation. Treatments—including the vehicle control, growth factors, or pharmacological inhibitors—were added as indicated. Images of the wound area were captured at 0, 24, and 48 h using an inverted microscope equipped with a camera. The distance between the advancing wound edges was measured using ImageJ software (USA), and data were expressed as the percentage of wound closure relative to the initial wound area.

The transfected cells were collected, resuspended in serum-free medium, and added to the upper chamber. DMEM high-glucose medium containing 20% FBS (Hyclone, USA) was added to the lower chamber. After 24 h of incubation, the medium was discarded, and the cells were fixed with methanol for 30 min. The cells were then stained with 0.1% crystal violet (MedChemExpress, USA) for 20 min. Non-migrated cells on the upper chamber were gently removed with a cotton swab. Finally, the migrated cells were observed and photographed under an optical microscope.

Following a 48-h transfection period, cells from each experimental group were harvested, fixed in absolute ethanol, and resuspended in centrifuge tubes. A total of 2.0 × 10⁵ cells were collected and subsequently treated with 500 µL of propidium iodide (PI) staining solution (Abcam, UK) for 30 min in the dark. Flow cytometry (BD, USA) was then used to analyze the cell cycle.

The formation of capillary-like structures in vitro was assessed using the standard Matrigel assay. HASMCs were seeded at a density of 2 × 10⁴ cells per well in 24-well plates with different treatments in serum-free media. The plates were pre-coated with growth factor-reduced Matrigel matrix (BD Biosciences, USA) and incubated at 37 ^∘C for 12 h. Tube formation was quantified by analyzing four random fields at 200× magnification per well and comparing the results to untreated control wells.

A total of 10 male Sprague-Dawley (SD) rats (three weeks old) were purchased from The Shanghai Slake Laboratory Animal Co., Ltd (China). After a seven-day adaptive feeding period, an aortic dissection model was induced by administering β-aminopropionitrile via gavage at a dosage of 1 g/kg/day for six weeks, until the formation of aortic dissection. Rats that did not successfully develop the model were excluded from the study after experimentation. Successful modeling criteria included survival during drug administration, followed by anesthesia, euthanasia via cervical dislocation, and extraction of the aorta for pathological analysis. The animals were then divided into two groups: shCtrl and shERBB4. Each rat received a 200 µL injection of adenovirus (designed by Ribobio) via the tail vein. The adenovirus was administered two days before β-aminopropionitrile treatment and then once every two weeks. The occurrence of aortic dissection was confirmed by the presence of a significant false lumen containing numerous red blood cells.

Aortic specimens were fixed overnight in 4% paraformaldehyde, then dehydrated, embedded in paraffin, and sectioned into 5 µm slices. After deparaffinization and rehydration, the sections were stained with H&E (Solarbio, Beijing, China), dehydrated with alcohol, cleared with xylene, cover-slipped, and examined under an optical microscope. Images were captured and recorded.

The aortic tissue samples were deparaffinized and stained using Masson’s trichrome (Beyotime, Shanghai, China). Subsequently, images of five microscopic fields were captured at 200× magnification using an optical microscope (Olympus, Japan).

The aortic tissue was cut into 5 µm sections, then dewaxed and hydrated. EVG staining was applied for 30 min, followed by background differentiation and counterstaining with EVG (Solarbio, Beijing, China). After rinsing with distilled water, the sections were mounted and examined under a microscope for image acquisition and analysis.

Trizol reagent (Thermo Fisher Scientific, USA) was added to the collected cells to extract total RNA. The extracted RNA was then reverse transcribed into cDNA using a reverse transcription kit. qPCR was performed using cDNA as a template and a SYBR qPCR kit (Beyotime, Shanghai, China). GAPDH served as an internal reference, and gene expression levels were calculated using the 2^−ΔΔCT method.

Cells from each experimental group were harvested and lysed using Radioimmunoprecipitation Assay (RIPA) buffer (Beyotime, Shanghai, China) to extract total cellular protein. Protein concentration was determined using the bicinchoninic acid (BCA) assay (Beyotime, Shanghai, China). Equalized protein samples were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). To prevent non-specific binding, membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature, followed by overnight incubation at 4 ^∘C with primary antibodies against ERBB4 (1:1000, Proteintech, Wuhan, China), α-actin (1:1000, Proteintech), Transgelin (TAGLN) (1:1000, Elabsience, Shanghai, China), SPP1 (1:1000, ZENBIO, Chengdu, China), epiregulin (1:1000, Biorbyt, UK), CD151 (1:1000, Proteintech), ITGAE (1:1000, ab224202, Abcam, UK), ITGB5 (1:1000, Proteintech), p-IκBα (1:5000, Proteintech), IκBα (1:5000, Proteintech), p-NF-κB-65 (1:2000, Proteintech), NF-κB-65 (1:1000, Proteintech), and GAPDH (1:50000, Proteintech) as a loading control. After incubation, membranes were washed three times with TBST and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000, Absin, Shanghai, China) for 1 h at room temperature. Following another three TBST washes, protein bands were detected using an enhanced chemiluminescence (ECL) system, and their intensities were quantified by densitometry.

All experiments performed in this study adhered to the ethical guidelines of the Declaration of Helsinki and were approved by the Ethics Committees of Anhui Medical University (No. 2024253). No patients participated in this study.

Data from multiple experimental repeats were collected and statistically analyzed using GraphPad Prism 8.0. Differences between experimental groups were compared using Student’s t-test.

IHC results revealed that α-actin and TAGLN expression were reduced in the thoracic aortic dissection (TAD) group. Western blot analysis further confirmed these findings. Additionally, western blot results showed that the expression levels of SPP1 and epiregulin were increased in the TAD group compared to the control group (Figure 1, P < 0.05).

RNA sequencing results showed 1878 upregulated genes and 3440 downregulated genes (Figure 2A and 2B). GO enrichment analysis revealed that the DEGs) were primarily enriched in leukocyte migration (GO:0050900), viral transcription (GO:0019083), SRP-dependent cotranslational protein targeting to the membrane (GO:0006614), integrin binding (GO:0005178), focal adhesion (GO:0005925), and lamellipodium formation (GO:0030032) (Figure 2C). Similarly, KEGG enrichment analysis indicated that the DEGs were mainly associated with pathways, such as Phagosome (hsa04145), Amoebiasis (hsa05146), Dilated cardiomyopathy (hsa05414), Fc gamma R-mediated phagocytosis (hsa04666), and the NF-kappa B signaling pathway (hsa04064) (Figure 2D).

We analyzed the mRNA expression levels of KRT16, VCAN, ITGA10, ERBB4, and CCN3. Among them, KRT16, Versican (VCAN), and ERBB4 were significantly upregulated in the TAD group compared to the Control group (Figure 3A–3E, P < 0.05). Based on these results, we selected ERBB4 for further experiments. To suppress ERBB4 expression, we designed three siRNAs. qRT-PCR and Western blot analyses confirmed a significant reduction in ERBB4 expression following siRNA transfection. Among the three, siRNA-2 exhibited the highest knockdown efficiency. Therefore, we used siRNA-2 for all subsequent experiments (Figure 3F–3H, P < 0.05).

CCK-8 results revealed that cell viability was inhibited in the siERBB4 group compared to the siCtrl group (Figure 4A, P < 0.05). Colony formation assay results showed that cell proliferation was suppressed in the siERBB4 group relative to the siCtrl group (Figure 4B, P < 0.05). Cell cycle analysis indicated a decrease in the number of cells in the S phase and cell cycle arrest at the G1/S or G2/M checkpoints in the siERBB4 group. These findings suggest that ERBB4 knockdown suppresses cell proliferation (Figure 4C, P < 0.05).

The Transwell and wound scratch healing assays indicated that cell migration was diminished in the siERBB4 group compared to the siCtrl group (Figure 5A, B; P < 0.05). Tube formation capacity in HASMCs was evaluated based on the number of tubes and branch length. The results showed that HASMCs in the siCtrl group exhibited typical tube formation, whereas tube formation was significantly inhibited in the siERBB4 group (Figure 5C; P < 0.05).

HE staining of aortic tissue revealed a large false lumen filled with numerous red blood cells, confirming the presence of an aortic dissection. In the shCtrl group, aortic wall inflammation was significantly increased, whereas inflammatory cell infiltration was reduced in the shERBB4 group (Figure 6A). Masson staining showed a significant increase in the contractile strength of collagen fibers in the shERBB4 group compared to the shCtrl group (Figure 6B). Additionally, in the shCtrl group, elastic fibers were increased but sparsely arranged and locally broken. In contrast, the shERBB4 group exhibited dense, regularly arranged elastic fibers, although some remained locally broken (Figure 6C).

According to GO enrichment analysis (GO:000517 and GO:00018, P < 0.05), we examined the protein expression levels of integrin-binding molecules (CD151, ITGAE, ITGB5) and key components of the NF-κB signaling pathway (p-IκBα, IκBα, p-NF-κB-65, NF-κB-65) in tissue samples. Western blot results showed that the expression levels of CD151, ITGAE, ITGB5, p-IκBα, and p-NF-κB-65 were significantly reduced in the shERBB4 group (P < 0.05; Figure 6D).

The data used to support the findings of this study are available from the corresponding author upon request.