Background
Thoracic aortic dissection (TAD) is a fatal cardiovascular disease with rapid progression, high mortality, poor prognosis, and increasing incidence rate [
1]. TAD is characterized by the rapid development of the intimal flap, which can extend both forward and backward from the site where the intima tears up [
1]. Aortic remodeling, which involves alterations in the structure and function of blood vessels, is a histological characteristic of TAD that exhibits regional and heterogeneous variations [
1‐
4]. The spatial expression distribution and function of genes associated with aortic remodeling are crucial for understanding the pathophysiology of TAD [
5]. Studies have suggested that the spatial distribution of inflammatory-related markers tends to gradually increase from the intact area to the border area and the tear area (TA) [
6]. Intimal tear has been suggested as the initiating event in TAD [
1], but the underlying molecular changes of the site of the tearing up remain poorly characterized. It is crucial to elucidate the initiating mechanisms that trigger the tearing or rupture of the aorta.
Previous studies have consistently relied on tissues extracted from various individuals, resulting in discernible individual variations. Meanwhile, traditional transcriptomes from whole-tissue homogenates mix varying levels of lesion information and lack the spatial resolution to accurately reflect the true situation [
7,
8]. Tomography RNA sequencing (Tomo-seq), a spatial resolution transcription method proposed in 2014, provides continuous changes in gene expression from diseased areas to relatively normal areas based on continuous cryosections of specific regions [
9‐
12]. Our histology examination confirmed the pathological differences along the tear and remote area in TAD. We performed Tomo-seq to identify the spatial gene expression signatures along the tear and remote area in TAD. For the first time, spatial distribution patterns in gene expression along the tear and remote area have been observed.
NINJ1, encoding Ninjurin 1, had expression peaks in the TA. NINJ1 is a homophilic transmembrane adhesion molecule involved in various processes of tissue remodeling such as inflammation and cell death [
13‐
15]. In our study, NINJ1 exhibited a similar trend to the genes associated with inflammatory responses. Moreover, we also noticed that NINJ1 co-localized with various cell death markers, including GSDMD, HMGB1, and TUNEL. Inhibition of NINJ1 by short hairpin RNA (shRNA) and neutralizing antibody delayed the development of beta-aminopropionitrile (BAPN)-induced TAD and reduced overall mortality. Our results indicated that NINJ1 may be associated with the formation of TAD. Tomo-seq could be used to increase our understanding of the mechanisms of vascular remodeling, which could help facilitate the development of effective therapeutics for TAD.
Methods
Patient specimens collection
The aorta tissues of TAD were obtained from eight patients diagnosed with sporadic TAD who underwent the aortic repair surgery operated within 24 h of onset. The study was approved by the Human Ethics Committee of Guangzhou First People’s Hospital (K-2019–156-02). Written informed consent was obtained from each patient. Patients with aortic disease triggered by gene mutation, such as Marfan’s syndrome, or associated with bicuspid aortic valves were excluded based on clinical diagnosis. All samples were collected within 30 min after aorta excision. Samples were repeatedly flushed with saline at 4 °C to remove blood and mural thrombus adhering to the aortic wall, and photographs recorded the tear boundary. Afterward, these samples were cut according to the needs of the study (Additional file
1: Fig. S1).
Tomo-seq
Tomo-seq is a spatial resolution transcription method based on continuous sections of the target area of frozen tissues, followed by RNA extraction and sequencing from individual sections [
9,
10,
16]. In short, 4-mm wide portions of human TAD tissue spanning from the tear to the remote area were embedded in a tissue-freezing medium, frozen on dry ice, and cryosectioned into 20 continuous slices of 5 μm thickness into an Eppendorf tube, 30 tubes in total. After the total RNA extraction from continuous cryosections in an individual Eppendorf tube and DNase treatment, magnetic beads with Oligo (dT) were used to isolate mRNA. The mRNA was fragmented into short fragments. Then cDNA is synthesized using the mRNA fragments as templates. Short fragments were purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments were connected with adapters. During the QC steps, Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System were used to quantify and qualify the sample library. At last, the library could be sequenced using BGISEQ-500. Tomo-seq data analysis was referred to as reported by Wu et al. [
11]. Fastq data were aligned to human genome hg38 using STAR (v2.5.3a), and then gene expression TPM (transcript per million) was calculated using RSEM (v1.2.31). We used GENCODE v31 as gene annotation. Genes expressed in at least three sections were selected for downstream analysis. We performed Tomo-seq data analysis using R and Bioconductor package tomoda (
https://doi.org/doi:10.18129/B9.bioc.tomoda). The procedures of analysis were adapted from Wu et al. [
16]. In brief, we scale the TPM of all genes across sections to obtain
Z scores (i.e., the TPM of a gene in all sections minus the mean value divided by the standard deviation). For correlation analysis, Pearson correlation coefficients between any two sections were calculated based on the
Z score of all genes. Next, we performed hierarchical clustering of sections using the TPM of all genes to find the borders of different zones. We found highly expressed (
Z score > 1) genes in at least four consecutive sections and calculated the statistical significance using permutation tests to identify locally expressed genes. Almost all (337 out of 339) identified locally expressed genes are significant at
p-value < 0.05. Then we analyzed the expression pattern in sections of identified locally expressed genes. The similarity of expression patterns among genes was measured using both dimensional reductions with t-SNE and correlation analysis. Both methods showed four groups of genes that were locally expressed in different sections. The plots of gene TPM changes across sections were locally smoothed with LOESS.
Statistical analysis
Values are presented as mean ± standard error of the mean (SEM). Statistical analyses between two groups were conducted using the two-tailed unpaired or paired Student’s t-test. Comparison among > two groups was performed using one-way ANOVA analysis. For classified data, the chi-square test was used. Pearson’s correlation coefficients were used to calculate gene pair correlation based on gene expression in human samples. Gene ontology (GO)-term analysis on ranked lists was performed using the online database Metascape. P value < 0.05 was interpreted to denote statistical significance. All the statistical analysis methods were indicated in the corresponding figure legends. GraphPad Prism 8.0.1 software (San Diego, CA, USA) was used for statistical analyses.
Histological analysis
The aortas were fixed in 10% formalin, embedded in paraffin, and cut into 5-μm-thick sections. The hematoxylin and eosin (H&E), Verhoeff’s Van Gieson (EVG) staining (G1597, Solarbio), and Masson’s trichrome staining (G1340, Solarbio) were performed on the paraffin sections of the aorta according to the kit instructions and finally observed with a pathologic scanner (Zeiss, Oberkochen, Germany). The elastin degradation was graded on a scale of 1–4, where 1 for < 25% degradation, 2 for 25–50% degradation, 3 for 50–75% degradation, and 4 for > 75% degradation, dissection, or rupture [
17]. Quantitative analysis of related positive area and number of cells in tissues (the ratio of a total number of positive points /the area of the entire section) using Image-Fiji.
Immunohistochemistry (IHC)
Antigen retrieval was performed on the paraffin sections using ethylenediaminetetraacetic acid (EDTA) solution (pH 9.0, ZLI-9068, ZSBG-BIO, China). The endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 15 min after being washed in PBS, and nonspecific binding was blocked using goat serum (ZLI-9056, ZSBG-BIO, China) for 45 min at room temperature. Sections were stained with the primary antibodies overnight at 4 °C. After washing 3 times with PBS, the slides were incubated with appropriate HRP-labeled secondary antibodies for 20 min at room temperature and then performed chromogenic DAB staining. The positive area (%) of NINJ1 was determined utilizing the Image J IHC Toolbox plugin. In short, the overall area of NINJ1-positive pixels is computed and divided by the overall area of the tissue contained within the section to acquire the NINJ1 area ratio.
Multiple immunostaining
The Opal 7 multiplexed assay (PerkinElmer, MA, USA) was used to generate multiple immunostainings. The best concentration of antibodies was determined before multiple immunostainings, according to the instructions. The primary antibodies used were as follows: anti-CD45 (1:200, ab10558, Abcam), anti-α-SMA (1:2000, ab12964, Abcam), anti-VIM (1:150, ab8978, Abcam), anti-CD31 (1:50,ab9498,Abcam), anti-NINJ1 (1:200, GTX31596, GeneTex), anti-TPPP3 (1:200, GTX33554, GeneTex), anti-CD3 (1:200, 17,617–1-P,Thermo), anti-MMP9 (1:1000, ab74003,Abcam), anti-CD11b (1:3000, ab133357, Abcam), anti-CD31 (1:50, ab28364,Abcam), anti-GSDMD (1:800, 36,425, Cell signaling), anti-HMGB1(1:500, ab79823, Abcam). The stained slides were analyzed by Vectra Polaris Quantitative Pathology Imaging Systems (PerkinElmer).
TUNEL apoptosis assay
Paraffin-embedded aortic sections were stained with the One Step TUNEL Apoptosis Assay Kit A (C1090, Beyotime) and counterstained with hematoxylin according to the manufacturer’s recommendation.
Mouse TAA/TAD model
Wildtype male C57BL/6J were used for this study. Animals were purchased from the Charles River Laboratories (Beijing, China) and approved by the Animal Ethics Committee at Fuwai Hospital (FW-2022–0015). Animal experiments were designed according to The ARRIVE guidelines 2.0 (Additional file
2) [
18]. All mice were maintained in IVCs at the density of 3–5 mice per cage in an SPF animal room with temperature-controlled (23 ± 2 °C), humidity of 50 ± 5%, and a dark/light cycle of 12 h. Age- and weight-matched mice were randomized into different groups by random number table method. Three-week-old male C57BL/6 mice were fed a normal diet and administered freshly prepared BAPN (Sigma-Aldrich, USA) solution dissolved in the drinking water (1g/kg/day) for consecutive 4 weeks to establish the TAA (Thoracic aortic aneurysm)/TAD model as previously described [
19‐
21]. The same-aged mice fed with a regular diet and drinking water were served as controls. All mice that died before the expected end time of the experiment were autopsied immediately to confirm whether they died of aortic rupture, we excluded mice that did not die from TAD. The aorta of mice was examined by ultrasonic examination at 3 weeks after BAPN induction. Mice that survived for 4 weeks were sacrificed using an overdose of sodium pentobarbital, and their thoracic aortic tissue was collected for further analysis. TAA is defined as arterial dilation to more than 50% of the normal diameter. TAD is characterized by the formation of a false lumen with the blood in the medial layer, or massive blood clots in the thoracic cavity. Mouse feeding, conduction of the TAA/TAD model, and phenotype identification are handled by different individuals, and the person responsible for phenotype identification is not aware of the group information in advance. The minimum number of mice in each group was not less than six.
Adeno-associated virus (AAV) and NINJ1 knockdown
Ninj1 shRNA fragment was cloned into adeno-associated virus 9 (AAV9) vector (pAV-U6-shRNA-CMV-GFP) (WZ Biosciences Inc.) to construct AAV9-shNinj1. Three-week-old male C57BL/6 mice were injected with 20 μl of either 5.0 × 1011 v.g AAV-shNinj1(Ninj1-shRNA group, n = 10) and with AAV-NC (Vehicle group, n = 10) via retrobulbar vein. Seven days after the pretreatment, we constructed the BAPN-induced TAA/TAD mice model. Mice given the same dose of BPAN alone at the same time were used as the model group (n = 12) and the same aged mice fed with regular diet and drinking water for 4 weeks were served as controls (n = 6). The above experiments were all conducted on mice of the same age and subjected to different treatments during the same period.
NINJ1-neutralization antibody treatment
Three-week-old male C57BL/6 mice were administered NINJ1-neutralizing antibody (2.5 μg/20 μL, BD Biosciences) (anti-NINJ1 group, n = 12) or with mouse IgG antibody (IgG group, n = 10) as control by retrobulbar vein injection at 4 days before the onset of TAA/TAD induction. Mice given the same dose of BPAN alone at the same time were used as the model group (n = 12) and the same aged mice fed with regular diet and drinking water for 4 weeks were served as controls (n = 6). The above experiments were all conducted on mice of the same age and subjected to different treatments during the same period.
Phenyl-β-d-glucopyranoside (PDG) treatment
PDG (292,710, Sigma; CAS number: 24857607) was purchased from Merck. Forty-three-week-old male C57BL/6 mice were randomized into three groups: the normal group (n = 6), the model group (n = 24) received freshly prepared BAPN (Sigma-Aldrich, USA) solution dissolved in the drinking water (1 g/kg/day) for 4 weeks, and the PDG group (n = 10) that received BAPN with the same way as the model mice and daily injected intraperitoneally with PDG (100 mg/kg/day) for 3 weeks. The above experiments were all conducted on mice of the same age and subjected to different treatments during the same period.
Quantitative RT-PCR
Total RNA was extracted from tissues using TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA), followed by isopropyl alcohol precipitation. Quantitative RT-PCR was performed using the Q5 Real-Time PCR System (Applied Biosystems, Foster City, CA) with SYBR Green Master Mix (Takara). The expression levels of the target genes were normalized against GAPDH by 2^(-△△Ct) method.
Western blot (WB)
Aorta tissue was ground into fine powder under liquid nitrogen freezing, and proteins were extracted by RPIA containing PMSF. Primary antibodies against NINJ1 (1:200, sc-136295, Santa Cruz) and GAPDH (1:10,000, KC5G5, Aksomics) were utilized and left to incubate overnight at 4°C. Bands were observed using Pierce™ ECL peroxidase substrate (Thermo Scientific, 32,209), and then images were taken using the Tanon chemiluminescent imaging system.
Discussion
Our study has demonstrated the following: (1) Significant neointima formation, tissue fibrosis, media degeneration, and inflammatory infiltration were observed in the TA. (2) Tomo-seq analysis revealed a spatial partitioning in gene expression between the TA and the RA. Furthermore, it identified NINJ1 as a key molecule involved in inflammation and tissue remodeling of TAD. (3) In the human aorta, NINJ1 co-localized with macrophages, T cells, and CD31
+α-SMA
+ cells, primarily showing clustering at the TA. (4) Inhibition of NINJ1 significantly reduced the morbidity and mortality associated with BAPN-induced TAD in mice. Previous studies found lesions of TAD are regional and transmural specific, with the outer media of the ascending aorta being a susceptible site [
26]. The present study demonstrated that neointima formation accompanied by inflammatory infiltration and fibrosis was also an essential feature of TAD, especially in the TA.
We utilized Tomo-seq to capture the continuous change trajectory of genes in human TAD tissue, spanning from the TA to the RA, and accurately identified genes associated with TAD remodeling. Following the Tomo-seq protocol [
12,
16,
33], we employed a single-cryosection thickness of ≤ 10 μm and consecutive mixed slices to analyze specific localized information for sequencing. GO-term analysis of the genes expressed locally emphasized the typical steps in the complex process of TAD: inflammation and extracellular matrix organization [
34]. Searching for genes that exhibit similar transcription patterns to these classic markers holds the promise of discovering novel key genes closely associated with TAD. Cluster I was at the edge of the TA and contained the well-known T cell marker CD3E and was enriched in the immune system process. T-cell activation plays a vital role in neointima formation in response to arterial injury [
35]. Some studies have reached the conclusion that Th1 immune responses are positively correlated with vascular remodeling and intimal expansion of TAD [
35,
36]. Our data showed novel genes associated with intimal inflammation in cluster I, including NINJ1 and TPPP3. Recent studies have shown that NINJ1 regulated the tissue remodeling and inflammation in vascular [
15,
37]. Immunostaining analyses demonstrated that the co-location of NINJ1 with T cells and macrophages trended toward aggregation at the TA in human tissue. TPPP3, a member of tubulin polymerization-promoting proteins, which was involved in palmitic acid-induced endothelial oxidative injury [
38,
39]. We verified that TPPP3 was also abundantly enriched in the neointima of TA.
NINJ1 was involved in multiple diseases and played various roles. Ninj1 could stimulate the inflammatory response of macrophages and thus promote the development of pulmonary fibrosis [
40]. Leukocyte upregulation of Ninj1 could increase cell adhesion and transport in the inflammation of the central nervous system [
37,
41]. NINJ1 was involved in endothelial dysfunction and inhibition of NINJ1 expression was a potential therapeutic strategy to prevent endothelial dysfunction in diabetes [
42]. NINJ1 also could mediate plasma membrane rupture during lytic cell death [
13]. The role of NINJ1 in TAD has not been explored. In our study, we observed that NINJ1 was enriched at the TA of TAD and co-localized with cell death markers, such as GSDMD and Tunel, and inflammation marker HMGB1. Therefore, we hypothesized that NINJ1 is closely associated with TAD.
We then utilized shRNA to downregulate NINJ1 expression in the BAPN-induced TAD model, which resulted in a notable reduction in TAD formation. Moreover, this approach also led to diminished infiltration of inflammatory cells and a decrease in the number of CD31
+α-SMA
+ cells. The NINJ1-neutralizing antibody also demonstrated comparable therapeutic effects and can effectively impede the formation of TAD. Inflammation can induce the generation of EndMT, and EndMT can in turn exacerbate inflammation, forming a vicious cycle [
23,
43]. Our results showed inhibition of NINJ1 could reduce inflammation and EndMT, which is the potentially effective approach for treating TAD. On the other hand, we used PDG to treat BAPN-induced TAD models and found that it has an effect of delaying the development of TAD.
Our study also suggested that Tomo-seq would be suitable for various cardiovascular diseases. For example, Studies have shown that plaques associated with acute coronary syndrome exhibit more significant longitudinal heterogeneity. Therefore, at every single point in time, various parts of the same plaque may show different stages and trajectories [
44,
45]. Tomo-seq can potentially facilitate the analysis of arterial remodeling mechanisms in plaque development. In valvular heart disease, continuous calcium deposition is the primary cause that leads to the gradual narrowing of the aortic valve [
46]. Stenotic aortic valves existed in non-calcified regions and calcified regions [
47]. Tomo-seq will help to understand the pathological progress of vascular diseases.
The limitation of this study is that we did not knock down NINJ1 in specific types of cells. The main reason for this is that we observed NINJ1 was expressed in multiple cell types associated with TAD progression, such as macrophages, T cells, and CD31+α-SMA+ cells. As a result, we chose to employ a broad NINJ1 inhibition to investigate the association between NINJ1 and TAD.
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