Involvement of HIF-1/Trf/Fe2+ signaling pathway in the Pathogenesis of Stanford Type A Aortic Dissection

Involvement of HIF-1/Trf/Fe2+ signaling pathway in the Pathogenesis of Stanford Type A Aortic Dissection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Involvement of HIF-1/Trf/Fe2+ signaling pathway in the Pathogenesis of Stanford Type A Aortic Dissection Li Li, Yangchao Zhao, Yanyu Lu, Tao Zhang, Bin Lin, Yupeng Liu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7845557/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 16 You are reading this latest preprint version Abstract Background Stanford type A aortic dissection (TAAD), as one of the most lethal cardiovascular disease, may be mediated by cellular ferroptosis, which exacerbates structural modification and dysfunction of endothelial and smooth muscle cells. Hypoxia inducible factor-1 (HIF-1), as a transcription factor has shown participate in the course of ferroptosis. However, the mechanism involved has not been uncovered clearly. Methods We collected clinical basic information and image data of TAAD patients received surgical operation for aortic replacement and non- TAAD patients who suffered from other surgeries including cardiac transplantation or aortic valve replacement. Then aortic tissues being replaced from each group was analyzed through RNA-sequencing to reveal underlying molecular mechanism participated in TAAD. Then, according to genes and signaling pathway selected, qRT-PCR and western blot were further implemented to verify the expression quantity of proteins. Results Baseline characteristics including age, gender, and other variables were non-differential between two groups. Computed tomography angiography (CTA) showed that linear low-density structure referring to tearing intima was floated in the full-course of aortic lumen which was separated into the true lumen and the false lumen. Disordered cells arrangement and fractured fibrous tissue were displayed in the H&E staining and Masson staining. RNA-seq found that more than 5000 differential genes were expressed in TAAD patients if |log 2 FC|≥1 and P value ≤ 0.5. Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEEG) analysis revealed that genes related cellular ferroptosis and HIF-1 signaling pathway were expressed differentially and significantly during the development of TAAD. Moreover, the proteins of SLC7A11 and GPX4 which related ferroptosis were up-regulated, as well as the HIF-1 protein and its downstream protein of transferrin receptor 1(Trf1), which were in line with the results of RT-PCR. The amount of Fe 2+ was greater in aorta from TAAD patients than that from non-TAAD patients. Conclusion Cellular feroptosis plays a vital role during the development of TAAD, and the HIF-1/Trf/Fe 2+ pathway was involved in the regulation of cellular ferroptosis. ferroptosis TAAD HIF-1 signaling pathway RNA-seq Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction As a fatal cardiovascular disease, aortic dissection is characterized by the formation of a false lumen of aorta rooted in blood flow into the medial layer from the tearing of intimal layer 1 . Stanford type A aortic dissection (TAAD) is extremely dangerous with a mortality rate of around 50% within two days 2 .Though imaging techniques such as computed tomography angiography (CTA) and surgical interventions including aortic replacement decreased the in-hospital mortality rate 3 , it remains a challenge for diagnosis and treatment. Pathological mechanisms including loss of smooth muscle cells, extracellular matrix (ECM) degradation and inflammatory cell infiltration are widely accepted in the development of AD 4 . The functional change, structural injury and even death of constituent cells of the aortic wall,such as vascular smooth muscle cells (VSMCs), endothelial cells and immune cells including macrophages, can directly lead to the degeneration of aortic wall and the imbalance of microenvironment homeostasis, which is an important cause of the onset and continuous progression of aortic dissection 5 . Multiple regulatory cell death patterns are involved in the loss of aortic wall cells, and ferroptosis are among. A large number of studies have validated that ferroptosis plays a key role in promoting vascular diseases including aortic dissection, abdominal aortic aneurysm and atherosclerosis 2 , 6 – 8 . Unlike apoptosis, autophagy or cell necrosis, ferroptosis, as a form of iron-dependent regulatory cell death, are mainly characterized by iron metabolism disorders and lipid peroxidation 9 . Scholars found that the ferroptosis enrichment score in aortic dissection tissues was significantly higher than that in normal aortic tissues through the analysis of human TAAD data in the high-throughput Gene Expression Omnibus (GEO) database 6 . Moreover, the enrichment score of ferroptosis was higher than that of copper death, pyroptosis, immunogenic cell death, necroptosis and autophagy 10 . The expression of ferroptosis driver genes in the aorta in mice treated with β-aminopropionitrile would be significantly upregulated with the degeneration of the aorta, along with the increase of lipid peroxidation products 8 , 11 . It is suggested that ferroptosis is closely related to aortic dissection and affects various cell types located in the inner and middle layers of the aorta. However, multiple factors could regulate ferroptosis and the specific pathway is unknown. Herein, to determine whether ferroptosis contribute to the development of TAAD, and to reveal the underline signal pathway, we collected aortic tissues from 18 TAAD patients and 10 non-TAAD patients to carry out transcriptome sequencing, screened out the relevant signal pathways through enrichment analysis, and the related gene and protein expressions were further detected by RT-PCR and Western blotting based on the results of mRNA seq. In our study, hypoxia inducible factor-1 (HIF-1) signal pathway was differently expressed in TAAD patients and the downstream protein of Trf was also upregulated, which may provide a new intervening measure for TAAD therapy. Methods Materials Hematoxylin and Eosin Staining kit, Masson’s Trichrome Staining Kit and Lipid Peroxidation Assay Kit with BODIPY 581/591 C11 were acquired from Beyotime (Shanghai, China). Antibodies against GPX4, CD71 (Trf1), HIF-1ɑ, SLC7A11 used for western blot were acquired from Abcam (Cambridge, UK). Ferrous ion content detection kit was acquired from Solarbio® life science (Beijing, China). Human specimens The aortic tissues were obtained from TAAD patients (n = 18) undergoing aortic replacement surgery at cardiac surgery department of the First Affiliated hospital of Zhengzhou university between January 2023 and December 2023.The tissues of non-TAAD patients with aortic valve disease were obtained after aortic valve replacement surgery.In this study, all TAAD was diagnosed by computed tomography angiography (CTA). All patients or their families signed the written informed consent form according to the declaration of Helsinki, and this study protocol was approved by the medical ethic committee of the First Affiliated Hospital of Zhengzhou University. Computed tomography scan imaging Patients suspected TAAD were received computed tomography angiography (CTA) imaging after excluding absolute contraindications. Immediately after administration of contrast agent through median cubital vein, CTA imaging was obtained using CT-imaging system with respiratory synchronization at the indicated points. Three-dimension images of aortic tissues were built to facilitate the visualization of the vasculacture. Patients received CTA were told to drink plenty of water after examination in order to promote the removal of contrast agent. Immunohistochemical staining Tissues were fixed in 4% formaldehyde solution, embedded in paraffin and cut into 5 µm cross-section. Then, the slices suffered dewaxed, hydrated and sequentially incubated in hematoxylin and eosin solution for about 3 min. In the end, the slices were dehydrated before scanning with anoptical microscope (Olympus, Tokyo, Japan). Representative histological pictures were displayed. Masson’s trichrome staining was carried out according to the manufacturer’s instruction to visualize the elastic fiber integrity of abnormal aorta Immunofluorescent staining Aortic sections were dewaxed and rehydrated respectively in xylene and a series of gradient ethanols. Antigen retrieval by microwave treatment were performed in citrate buffer (pH 6.0).Then, aortic sections were blocked with donkey serum for 30 minutes in order to reduce nonspecific staining. Sections were incubated with the BODIPY 581/591 C11 working solution which was prepared according to the manufacturer’s instruction at room temperature for 2 hours. At last, sections were washed with PBS containing Tween-20. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China). The aortic sections were examined using an Olympus fluorescence microscope (Shanghai Nikon Instrument Co., LTD). RNA sequencing Aortic tissues obtained from TAAD or non-TAAD patients were adopted for RNA extraction, library construction, and sequencing. Total RNA was extracted with Trizol reagent (Beyotime, Shanghai, China) according to the manufacturer’s instruction. The integrity and purity of RNA were detected by agarose gel electrophoresis and Nanodrop 2000 (Thermo Fisher Science, Shanghai, China). Then, cDNA libraries were constructed and sequenced by Wuhan Servicebio technology Co., LTD.(Wuhan, China). Real-time quantitative PCR (qRT-PCR) Total RNA was extracted by using Trizol reagent mixed with aortic tissues in a grinder. 20 µL reaction system was reverse-transcripted to cDNA under specific temperature procedures. Then, reaction system containing 2×SYBR Green qPCR mix, gene primer, and reverse transcription product was amplified on a fluorescence quantitative PCR instrument after forty cycles of 95℃ for 15s, 60℃ for 30s. Primer sequences for targeted genes are listed in Supplementary material online, Table S1 . Western Blot : Human aortas were lysed in RIPA buffer to extracted the whole protein. Before loaded onto 10% SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes, the lysates were qualified to detect the concentration using Bradford protein assay. Subsequently, the membranes were blocked in 5% non-fat milk at room temperature for 1 h and probed with primary antibodies against GPX4 (1:1000, Abcam, Cambridge, UK), SMA (1:1000, Abcam, Cambridge, UK), SLC7A11 (1:1000, Abcam, Cambridge, UK), CD71 (1:1000, Abcam, Cambridge, UK), and HIF (1:1000, Abcam, Cambridge, UK). After washing, the membranes were incubated with species appropriate secondary antibodies at room temperature for 1 h. The luminescence reaction is displayed using Pierce ECL luminescence kit (Affinity Biosciences, Cincinnati, OH, USA) and β-actin was used as an internal control. The protein strips were analyzed with the gel image processing system (Image-pro Plus 6.0). Ferrous ion detection Equal amounts of human aortic tissues were homogenized on ice until dissolved completely. Then, the homogenate was centrifuged at 4℃ for 10 minutes, 3000 r/min. According to the manufacture’s instruction, the standard substances and the samples to be tested were prepared before measuring the absorbance on a spectrophotometer. Calculate the content of ferrous ions based on the results above. Statistical analysis The SPSS 22.0 software (SPSS, Inc, Chicago, IL, USA) was used for our statistical analysis. Normality of the data was examined using the Shapiro–Wilk test. For normally distributed data, the equality of variances was examined using the Levene test and presented as mean ± SD. For non-normally distributed data, median (IQR) was used. For two group comparisons, Student t-test was used. One-way ANOVA was utilized to compare data among multiple groups. P < 0.05 was considered statistically significant. Results The basic characteristics of clinical data The clinical characteristics of each group were summarized in Table 1 including age, gender, body weight, medical history, basic vital signs and laboratory findings. As a result,The TAAD group showed significant differences from the control group in terms of age, gender, prevalence of hypertension, D-dimer levels and FDP levels. In order to better understand the occurrence of type A aortic dissection, typical CTA images of TAAD patients were presented in Fig. 1 b. As we can see, the rupture of the vascular intima is located in the ascending aorta, involving the entire aorta, and even extended to aorta abdominalis and iliac artery. While, for patients of control group, the aorta was intact and smooth, and no rupture was found throughout the whole course (Fig. 1 a). Further, the H&E stain and Masson stain of aorta in TAAD group showed that the aortic vascular wall tissue was significantly damaged, the morphology of smooth muscle cells was irregular, and the elastic fibers were incomplete, severely fractured, and fragmented. In contrast, the aortic wall structure was intact, the smooth muscle cells were closely and orderly arranged, and the elastic fibers were intact and continuous in control group (Fig. 1 c,d). Table 1 Baseline characteristics of all participants Characteristic AD(n = 18) Control (n = 9) P value Age, Mean ± SD 52.94 ± 12.82 60.88 ± 10.3 0.169 ≥ 50, n (%) 10(55.56) 8 (88.89) 0.205 <50, n (%) 8(44.44) 1 (11.11) 0.012 Gender, n (%) Male 13 (72.22) 8 (88.89) 0.049 Female 5 (27.78) 1 (11.11) 0.062 Body weight 80.11 (65, 92.5) 77.38 (61.02, 90.64) 0.375 Alcohol, n (%) 5 (27.78) 3(33.33) 0.250 Smoking, n (%) 9 (50) 5(55.56) 0.999 RR 18.99 ± 1.21 19.67 ± 1.03 0.999 HR 80.17 ± 4.68 76.67 ± 5.01 0.989 SBP 145.18 ± 18.96 131.17 ± 18.15 0.051 DBP 88.74 ± 14.05 83.5 ± 13.79 0.768 Diseases, n (%) Hypertension 16(88.89) 5 (55.56) 0.000 Diabetes 3 (16.67) 1 (11.11) 0.441 Cardiovascular disease 5 (27.78) 2 (22.22) 0.874 Others 7 (38.89) 4 (44.44) 0.250 Laboratory test, ALT (mean ± SD) 44.28 ± 23.12 34.22 ± 11.58 0.193 AST (mean ± SD) 52.64 ± 27.57 32.22 ± 10.24 0.085 TBIL (mean ± SD) 14.63 ± 2.32 11.24 ± 1.46 0.999 Cr, median (IQR) 88.61(72.25,104.75) 76.12 (59.62,100.33) 0.045 eGFR, median (IQR) 83.26 (69, 99.25) 89.88 (71,105.61) 0.068 T-CHO 5.98 ± 1.02 3.47 ± 0.79 0.049 TG 2.77 ± 0.85 1.31 ± 0.66 0.661 L-LDL 2.01 ± 0.63 1.97 ± 0.6 0.999 H-LDL 1.03 ± 0.27 1.06 ± 0.33 0.999 Blood sugar 5.42 ± 0.86 5.06 ± 0.61 0.812 HbA1c 5.41 ± 1.36 5.0 ± 1.38 0.999 WBC, median (IQR) 12.36 (10.65, 14.13) 5.28 (3.77, 6.79) 0.023 Hb, median (IQR) 131.94 (117, 145) 122.85 (105.1, 134.65) 0.060 PLT (mean ± SD) 163.89 ± 57.52 160.22 ± 54.14 0.078 N% (mean ± SD) 86.03 ± 8.66 85.9 ± 8.14 0.125 PT (mean ± SD) 12.28 ± 0.51 10.84 ± 0.81 0.250 D-dimer (mean ± SD) 15.51 ± 6.67 1.94 ± 0.79 0.000 FDP (mean ± SD) 91.33 ± 20.72 34.95 ± 9.82 0.004 Annotation: RR: respiratory rate; HR: heart rate; SBP: systolic blood pressure; DBP: diastolic blood pressure; ALT: glutamic pyruvic transaminase; AST: glutamic oxaloacetic transaminase; TBIL: total bilirubin; Cr: creatinine; eGFR: estimated glomerular filtration rate; TG: triglyceride; T-CHO: total cholesterol; L-LDL: low density lipoprotein cholesterol; H-LDL: high density lipoprotein cholesterol; HbA1c: glycosylate hemoglobin type A1c; WBC: white blood cell; Hb: hemoglobin; PLT: platelet; N%: neutrophile percentage; PT: prothrombin; FDP:fibrin degradation products. The genes of patients in aortic dissection group expressed differentially In order to reveal the underlying pathogenesis of aortic dissection, we performed high-throughput sequencing on the aortic tissues of patients in the dissection group and the control group. Before screening for differentially expressed genes, principal component analysis (PCA) of all samples was implemented to identify the degree of similarity among samples. As Fig. 2 a displayed, the samples of dissection group were close to each other, so as the control group, suggesting a good genetic similarity among each group. The heat map showed conspicuous differences of encoding genes between the dissection group and control group (Fig. 2 b). There totally existed 5109 differentially expressed genes, among which 1904 up-expressed genes and 3205 down-expressed genes if |log 2 FC|≥1and P value ≤ 0.5 (Fig. 2 c). Subsequently, the differential genes were annotated according to three categories including biological process, cellular component and molecular function which exhibited with Gene ontology (GO) analysis (Fig. 2 d). The GO bubble chart described top 20 significantly expressed genes based on enrich factor, most of which participate in the biological process. The KEGG pathway annotation showed that among the top 20 differential genes, almost all were related to immune inflammation (Fig. 2 e). From this, we can see that certain biological processes have occurred in patients with aortic dissection and may be regulated by inflammation-related signaling pathways. Ferroptosis was involved in development process of TAAD In order to have a comprehensive understanding of the functions of differentially expressed genes, in-depth KEGG annotation was performed. The result demonstrated that multiple signal pathways were directly or indirectly associated with the biological process of ferroptosis, including p53 signaling pathway, JAK-STAT signaling pathway, PI3K-Akt signaling pathway and ferroptosis (Fig. 3 a). Figure 3 b listed the ferroptosis-related differentially expressed genes. Then, we conducted immunofluorescence staining and western blot experiments to verify the above analysis results.The C11-BODIPY 581/591 stain indicating lipid peroxidation showed that the aortic tissue of dissection group had a stronger green fluorescence compared to control group (Fig. 3 c,d). In addition, the result of western blot also revealed that the expression quantity of SLC7A11 protein (a protein which generally marked ferroptosis) of aortic tissue in TAAD patients (n = 7) were significantly down-regulated along with ɑ-SMA (ɑ-smooth muscle actin, which usually decreased in AD) protein compared with the amount of proteins in patients of control group (n = 7). These results were displayed in Fig. 3 e,f. To sum up, cell ferroptosis played an important role in the pathogenesis of TAAD. HIF-1 signaling pathway regulated the ferroptosis process Based on the results of Fig. 2 e and Fig. 3 , we observed that HIF-1 signaling pathway which closely related to the inflammatory response was significantly enriched during the process of functional annotation (Fig. 4 a). To further ascertain whether the HIF-1 signaling pathway take part in regulating ferroptosis during the process of aortic dissection, RT-PCR and western blot were carried out for detection of symbolic factors. As a result, the mRNA expression of HIF-1 and CD71 (also named transferrin receptor 1,Trf1) in aortic tissues of TAAD patients were dramatically up regulated in comparison with expression in control group (Fig. 4 b,c). While, the mRNA expression of glutathione peroxidase 4 (GPX4, an important regulatory factor of ferroptosis) was down regulated in TAAD patients (Fig. 4 d), which was along with and opposite to the expression of HIF-1 and CD7. The trend of proteins expression of HIF-1, CD71 and GPX4 was in line with mRNA expression (Fig. 4 e,f), suggesting HIF-1 signal pathway may influence the cellular ferroptosis process during the development of TAAD. Finally, we tested the Fe 2+ content of aortic tissues in both groups to support the speculation. As Fig. 4 g shown, the concentration of Fe 2 + in TAAD group was significantly higher than that in control group, which may facilitate and enhance the iron-dependent programmed cell death form-cellular ferroptosis. Discussion TAAD is an acute and devastating cardiovascular disease with high mortality in short time if the surgical repair operation is not carried out immediately 12 . Although technical intervention significantly improved survival rate of TAAD patients, operative mortality still remains high 13 . Further researches are warrant to comprehensively understand the pathogenesis of TAAD for prompt diagnosis and management. Due to a tear in the intimal layer of the ascending aorta, lumenal blood enters the arterial wall, and the intima and media of the aorta are stripped in rapid sequence, resulting in the formation of false lumen. It has been reported that several risk factors are related to aortic dissection, such as age, hypertension, and dyslipidemia 14 , 15 . In this study, we found that the more than half of TAAD patients whose aorta was tore from ascending to aortaventrails even iliac artery were over 50 years old, and 77.2% were males. Importantly, almost 90% TAAD patients had suffered from hypertension for a long time. The proportion that had a dyslipidemia was approximately 70%, which was in accordance with previous study 16 . As a new type of regulatory cell death, ferroptosis has been found to be involved in in many diseases, especially in the field of cancer since its proposal in 2012 17 . In recent years, researches have shown that ferroptosis also plays important role in the development of various cardiovascular diseases, including doxorubicin-induced cardiotoxicity, ischemia/reperfusion cardiomyopathy, heart failure and coronary heart disease 18 – 21 . Such being the case, Standford type A aortic dissection, one of the most common and critical cardiovascular diseases, whether affected by ferroptosis or not on earth and how? In order to answer this question, we sequenced the transcriptome of the abandoned aortic tissues of TAAD patients undergoing aortic replacement surgery. The results in Fig. 2 showed that thousands of genes were differentially expressed in comparison with non-TAAD patients, which was as expected. Further analysis found that multiple genes were reached in ferroptosis pathway or ferroptosis-related pathway, heavily suggesting ferroptosis took place in the course of AD. Subsequently, we performed C11-BODIPY 581/591 staining on the obtained aortic tissues.This lipophilic dye can quickly enter the membrane and be used as a fluorescent probe to detect lipid peroxidation and antioxidant capacity in living cells. After oxidation by lipid hydrogen peroxide substances, the maximum excitation and emission wavelengths of the dye shifted and mainly presents green fluorescence 22 . In our study, the intensity of green fluorescence in TAAD patients’ tissue was significantly greater than that in control group, demonstrating that lipid peroxidation occured in aortic dissection. Interestingly, lipid peroxidation of unsaturated fatty acids on the cell membrane is the main mechanism of ferroptosis, which process is catalyzed under the action of divalent iron or ester oxygenase, thereby inducing cell death 17 , 23 . Moreover, the protein of SLC7A11 separated by Western Blot also hint that ferroptosis participated in TAAD. SLC7A11, solute carrier family 7 member 11, also named xCT, belongs to the cystine/glutamic acid reverse transporter protein 24 . Multiple studies have shown that the down-regulation of SLC7A11 can decrease intracellular levels of cysteine, exhaust the biosynthesis of GSH, and indirectly inhibit the activity of GPX4 25, 26 , ultimately inducing ferroptosis in cells for the accumulation of lipid peroxides. In this study, the expression amount of SLC7A11 in TAAD was significantly down-regulated compared to control group, which was consistent with the trend of ɑ-SMA expression. Hereto, we have confirmed that ferroptosis is involved in the occurrence and development of aortic dissection at the genetic, mRNA and protein levels. Considering that ferroptosis is regulated by many pathways, we conducted further enrichment analysis on the differentially expressed genes. As a result, HIF-1 signaling pathway drew our attention. As a key factor in responding to hypoxic stress, HIF-1 can regulate cell growth, proliferation, migration and apoptosis, being related to various physiological and pathological processes 27 . It participates in multiple signal transduction pathways and plays an important role in diseases such as inflammation and cardiovascular diseases 28 , 29 . Previous study showed that transferrin receptor was involved in HIF-1 signaling pathway in cervical cancer. Inspired by the results of this study and combined with the relationship between transferrin receptors (Tfr) and ferroptosis 30 , we speculate that HIF-1 may involved in cellular ferroptosis in the development of TAAD mediated by Tfr. The sequent RT-PCR results verified that the relative mRNA expression amount of HIF-1, Tfr1 and GPX4 in aortic tissue were significantly higher in TAAD patients that that in control group, which was in line with the western blot results. Tfr1 (i.e.CD71) is an important transmembrane protein that mediate the uptake of iron by cells. While intracellular iron metabolism is closely related to ferroptosis 31 , the iron content within the cells was also detected. As expect, the iron content in aortic tissue of TAAD patients was obviously exceeded the gross of control group, reminding that iron metabolism has changed in the process of aortic dissection. Integrating these experimental results together, we inferred that Tfr1-regulated iron metabolism probably interceded HIF-1 signal pathway to involve in the pathological mechanism of aortic dissection through mediating cellular feroptosis. For all this, the design of this study is relatively rudimentary, and more experiments are needed to clarify the specific role of ferroptosis in the progression of aortic dissection. For instance, the gender ratio of the patients included in this study was unbalanced,which would impact the results in a certain. it’s still unknown that which type of aortic cells have undergone ferroptosis. Is it vascular smooth muscle cells, endothelial cells, immune cells which include monocytes, macrophages, lymphocytes? Or more than one type cells involved? Additionally, more elaborate and rigorous experimental designs are requisite to confirm the causality of Tfr1 and HIF-1 signaling pathway, which could introduce genetically knocked-out or overexpressed mice. Besides, it is necessary to elucidate the upstream and downstream genes of the HIF-1 signaling pathway. Conclusion To sum up, we analyzed case data of TAAD patients and those with other related aortic diseases from clinical practice, as well as discarded aortic tissues after surgery. Transcriptome sequencing revealed that ferroptosis was involved in the pathogenesis of the disease, which was supported by fluorescence staining and related protein electrophoresis. And, HIF-1 signaling pathway may influence cellular ferroptosis through Tfr1-mediated iron metabolism. Abbreviations TAAD type A aortic dissection HIF-1 hypoxia inducible factor-1 RT-qPCR real-time quantitative polymerase chain reaction CTA computed tomography angiography RNA-seq ribonucleic acid-sequence GO gene ontology KEEG Kyoto Encyclopedia of Genes and Genomes ECM extracellular matrix GEO gene expression omnibus JAK-STAT Janus kinase-signal transducer and activator of transcription PI3K-Akt phosphoinositide-3 kinase protein kinase B RIPA radio immunoprecipitation assay SDS sodium dodecyl sulfate FDP Fibrin degradation products. Declarations Ethics approval and consent to participate All patients or their families signed the written informed consent form according to the declaration of Helsinki, and this study protocol was approved by the medical ethic committee of the First Affiliated Hospital of Zhengzhou University. Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. Author’s contributions L. L and JZ.D participated in the designed of the study, L.L, YY. L and T.Z conducted the human samples, YC.Z and B.L carried out experiments, YP.L performed statistical analysis, L.L and ZF.L drafted and revised the manuscript, JZ.D was the principal investigator of the laboratory, participated in design and help to revise the manuscript. Conflict of interest All of the authors declare no conflict of interest. Funding This study was supported by the Joint Construction Project for Medical Science and Technology Research of Henan Province (NO. LHGJ20230258 and NO.LHGJ20230290). References Zhu, Y.; Lingala, B.; Baiocchi, M.; Tao, J. J.; Toro Arana, V.; Khoo, J. W.; Williams, K. M.; Traboulsi, A. A.; Hammond, H. C.; Lee, A. M.; et al. Type A Aortic Dissection-Experience Over 5 Decades: JACC Historical Breakthroughs in Perspective. J Am Coll Cardiol 2020, 76 (14), 1703–1713. 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1","display":"","copyAsset":false,"role":"figure","size":269265,"visible":true,"origin":"","legend":"\u003cp\u003eThe typical images and pathological staining of TAAD.(a)The CTA images of aorta in control group; (b) The typical CTA images of aorta in type A aortic dissection; (c) The H\u0026amp;E stain of aortic tissue from TAAD patients and control patients; (d) The Masson stain of aortic tissue from TAAD patients and control patients. All scar bar were 200 μm.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7845557/v1/75779549be139f5eaec882ae.jpeg"},{"id":97706069,"identity":"a7747b92-6c22-4c6a-b638-baa50ea4f774","added_by":"auto","created_at":"2025-12-08 12:54:38","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":134109,"visible":true,"origin":"","legend":"\u003cp\u003eThe mRNA transcriptome sequencing results of aortic tissues from clinical patients. (a)The principal component analysis (PCA) of aortic tissues from TAAD patients and controls (case=TAAD group n=11, Control=control group n=7). (b) The heatmap of differently expressed genes between TAAD group and control group (G1=TAAD group, G2=control group). (c)The volcano plot of differently expressed genes between TAAD group and control group (purple plots represent down-regulated genes, blue plots represent up-regulated genes). (d) Top 20 of Gene Ontology (GO) annotation from TAAD group as compared with control group. (e) Top 20 of KEGG enrichment analysis from TAAD group as compared with control group.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7845557/v1/4800c1a59c6b2f13a5777357.jpeg"},{"id":97895087,"identity":"7c3d9af6-c6bc-48a6-8231-9699b28519c1","added_by":"auto","created_at":"2025-12-10 15:33:30","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":145171,"visible":true,"origin":"","legend":"\u003cp\u003eFerroptosis was found to involve in the development of TAAD. (a)The histogram of KEGG analysis according to the -log(\u003cem\u003eP\u003c/em\u003e value), red frames represent signal pathways that related to ferroptosis; (b) The heatmap of genes related to ferroptosis which were significantly expressed in TAAD patients as compared to control group (G1=Control group, G2=TAAD group, red meant up-expressed, while blue meant down-expressed); (c) The representative images of C11-BODIPY\u003csup\u003e581/591 \u003c/sup\u003estain of aortic tissues from TAAD patients and control patients (Scar bar: 100 μm);(d) The intensity of green fluorescence of control group and TAAD group; (f) The proteins expression amount of SLC7A11 and ɑ-SMA in western blot results of aortic tissue (n=7); (e) The gray value of proteins from both groups (n=7).\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7845557/v1/9ae606fde7997e17804136e3.jpeg"},{"id":97706046,"identity":"aee71a1c-8ccb-4c8e-86fe-2f9a29870130","added_by":"auto","created_at":"2025-12-08 12:54:37","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":99863,"visible":true,"origin":"","legend":"\u003cp\u003eHIF-1 signaling pathway influenced ferroptosis via Tfr1. (a) The bubble diagram of KEGG enrichment analysis showed HIF-1 signaling pathway was significantly enriched; (b) The relative mRNA expression of HIF-1; (c)The relative mRNA expression of CD71 (Tfr1); (d) The relative mRNA expression of GPX4; (e) The gray value of proteins including GPX4, CD71, and HIF-1 from both groups(n=7); (f) The proteins expression amount of GPX4, CD71, and HIF-1 in western blot results of aortic tissue (n=7); (g) The concentration of Fe\u003csup\u003e2+\u003c/sup\u003e in aortic tissue from both groups.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7845557/v1/080bef99fcc33a13e9d03aa7.jpeg"},{"id":97902631,"identity":"ff0551d2-f7d3-47b8-aa8d-4acd95527bc2","added_by":"auto","created_at":"2025-12-10 15:53:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1460375,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7845557/v1/73cd108d-1ae3-49b7-a96f-58ac440f86fb.pdf"},{"id":97706041,"identity":"79d79379-070f-48db-8e75-815a43853937","added_by":"auto","created_at":"2025-12-08 12:54:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":712656,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7845557/v1/8510100c51a5708ea8970107.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Involvement of HIF-1/Trf/Fe2+ signaling pathway in the Pathogenesis of Stanford Type A Aortic Dissection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs a fatal cardiovascular disease, aortic dissection is characterized by the formation of a false lumen of aorta rooted in blood flow into the medial layer from the tearing of intimal layer\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Stanford type A aortic dissection (TAAD) is extremely dangerous with a mortality rate of around 50% within two days\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.Though imaging techniques such as computed tomography angiography (CTA) and surgical interventions including aortic replacement decreased the in-hospital mortality rate\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, it remains a challenge for diagnosis and treatment.\u003c/p\u003e\u003cp\u003ePathological mechanisms including loss of smooth muscle cells, extracellular matrix (ECM) degradation and inflammatory cell infiltration are widely accepted in the development of AD\u003csup\u003e4\u003c/sup\u003e. The functional change, structural injury and even death of constituent cells of the aortic wall,such as vascular smooth muscle cells (VSMCs), endothelial cells and immune cells including macrophages, can directly lead to the degeneration of aortic wall and the imbalance of microenvironment homeostasis, which is an important cause of the onset and continuous progression of aortic dissection\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Multiple regulatory cell death patterns are involved in the loss of aortic wall cells, and ferroptosis are among. A large number of studies have validated that ferroptosis plays a key role in promoting vascular diseases including aortic dissection, abdominal aortic aneurysm and atherosclerosis\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eUnlike apoptosis, autophagy or cell necrosis, ferroptosis, as a form of iron-dependent regulatory cell death, are mainly characterized by iron metabolism disorders and lipid peroxidation\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Scholars found that the ferroptosis enrichment score in aortic dissection tissues was significantly higher than that in normal aortic tissues through the analysis of human TAAD data in the high-throughput Gene Expression Omnibus (GEO) database\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Moreover, the enrichment score of ferroptosis was higher than that of copper death, pyroptosis, immunogenic cell death, necroptosis and autophagy\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The expression of ferroptosis driver genes in the aorta in mice treated with β-aminopropionitrile would be significantly upregulated with the degeneration of the aorta, along with the increase of lipid peroxidation products\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, 11\u003c/sup\u003e. It is suggested that ferroptosis is closely related to aortic dissection and affects various cell types located in the inner and middle layers of the aorta. However, multiple factors could regulate ferroptosis and the specific pathway is unknown.\u003c/p\u003e\u003cp\u003eHerein, to determine whether ferroptosis contribute to the development of TAAD, and to reveal the underline signal pathway, we collected aortic tissues from 18 TAAD patients and 10 non-TAAD patients to carry out transcriptome sequencing, screened out the relevant signal pathways through enrichment analysis, and the related gene and protein expressions were further detected by RT-PCR and Western blotting based on the results of mRNA seq.\u0026nbsp;In our study, hypoxia inducible factor-1 (HIF-1) signal pathway was differently expressed in TAAD patients and the downstream protein of Trf was also upregulated, which may provide a new intervening measure for TAAD therapy.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003cp\u003eHematoxylin and Eosin Staining kit, Masson\u0026rsquo;s Trichrome Staining Kit and Lipid Peroxidation Assay Kit with BODIPY 581/591 C11 were acquired from Beyotime (Shanghai, China). Antibodies against GPX4, CD71 (Trf1), HIF-1ɑ, SLC7A11 used for western blot were acquired from Abcam (Cambridge, UK). Ferrous ion content detection kit was acquired from Solarbio\u0026reg; life science (Beijing, China).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eHuman specimens\u003c/strong\u003e\u003cp\u003eThe aortic tissues were obtained from TAAD patients (n\u0026thinsp;=\u0026thinsp;18) undergoing aortic replacement surgery at cardiac surgery department of the First Affiliated hospital of Zhengzhou university between January 2023 and December 2023.The tissues of non-TAAD patients with aortic valve disease were obtained after aortic valve replacement surgery.In this study, all TAAD was diagnosed by computed tomography angiography (CTA). All patients or their families signed the written informed consent form according to the declaration of Helsinki, and this study protocol was approved by the medical ethic committee of the First Affiliated Hospital of Zhengzhou University.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eComputed tomography scan imaging\u003c/strong\u003e\u003cp\u003ePatients suspected TAAD were received computed tomography angiography (CTA) imaging after excluding absolute contraindications. Immediately after administration of contrast agent through median cubital vein, CTA imaging was obtained using CT-imaging system with respiratory synchronization at the indicated points. Three-dimension images of aortic tissues were built to facilitate the visualization of the vasculacture. Patients received CTA were told to drink plenty of water after examination in order to promote the removal of contrast agent.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eImmunohistochemical staining\u003c/strong\u003e\u003cp\u003eTissues were fixed in 4% formaldehyde solution, embedded in paraffin and cut into 5 \u0026micro;m cross-section. Then, the slices suffered dewaxed, hydrated and sequentially incubated in hematoxylin and eosin solution for about 3 min. In the end, the slices were dehydrated before scanning with anoptical microscope (Olympus, Tokyo, Japan). Representative histological pictures were displayed. Masson\u0026rsquo;s trichrome staining was carried out according to the manufacturer\u0026rsquo;s instruction to visualize the elastic fiber integrity of abnormal aorta\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eImmunofluorescent staining\u003c/strong\u003e\u003cp\u003eAortic sections were dewaxed and rehydrated respectively in xylene and a series of gradient ethanols. Antigen retrieval by microwave treatment were performed in citrate buffer (pH 6.0).Then, aortic sections were blocked with donkey serum for 30 minutes in order to reduce nonspecific staining. Sections were incubated with the BODIPY 581/591 C11 working solution which was prepared according to the manufacturer\u0026rsquo;s instruction at room temperature for 2 hours. At last, sections were washed with PBS containing Tween-20. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China). The aortic sections were examined using an Olympus fluorescence microscope (Shanghai Nikon Instrument Co., LTD).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eRNA sequencing\u003c/strong\u003e\u003cp\u003eAortic tissues obtained from TAAD or non-TAAD patients were adopted for RNA extraction, library construction, and sequencing. Total RNA was extracted with Trizol reagent (Beyotime, Shanghai, China) according to the manufacturer\u0026rsquo;s instruction. The integrity and purity of RNA were detected by agarose gel electrophoresis and Nanodrop 2000 (Thermo Fisher Science, Shanghai, China). Then, cDNA libraries were constructed and sequenced by Wuhan Servicebio technology Co., LTD.(Wuhan, China).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eReal-time quantitative PCR (qRT-PCR)\u003c/strong\u003e\u003cp\u003eTotal RNA was extracted by using Trizol reagent mixed with aortic tissues in a grinder. 20 \u0026micro;L reaction system was reverse-transcripted to cDNA under specific temperature procedures. Then, reaction system containing 2\u0026times;SYBR Green qPCR mix, gene primer, and reverse transcription product was amplified on a fluorescence quantitative PCR instrument after forty cycles of 95℃ for 15s, 60℃ for 30s. Primer sequences for targeted genes are listed in Supplementary material online, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern Blot\u003c/b\u003e: Human aortas were lysed in RIPA buffer to extracted the whole protein. Before loaded onto 10% SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes, the lysates were qualified to detect the concentration using Bradford protein assay. Subsequently, the membranes were blocked in 5% non-fat milk at room temperature for 1 h and probed with primary antibodies against GPX4 (1:1000, Abcam, Cambridge, UK), SMA (1:1000, Abcam, Cambridge, UK), SLC7A11 (1:1000, Abcam, Cambridge, UK), CD71 (1:1000, Abcam, Cambridge, UK), and HIF (1:1000, Abcam, Cambridge, UK). After washing, the membranes were incubated with species appropriate secondary antibodies at room temperature for 1 h. The luminescence reaction is displayed using Pierce ECL luminescence kit (Affinity Biosciences, Cincinnati, OH, USA) and β-actin was used as an internal control. The protein strips were analyzed with the gel image processing system (Image-pro Plus 6.0).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFerrous ion detection\u003c/strong\u003e\u003cp\u003eEqual amounts of human aortic tissues were homogenized on ice until dissolved completely. Then, the homogenate was centrifuged at 4℃ for 10 minutes, 3000 r/min. According to the manufacture\u0026rsquo;s instruction, the standard substances and the samples to be tested were prepared before measuring the absorbance on a spectrophotometer. Calculate the content of ferrous ions based on the results above.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003cp\u003eThe SPSS 22.0 software (SPSS, Inc, Chicago, IL, USA) was used for our statistical analysis. Normality of the data was examined using the Shapiro\u0026ndash;Wilk test. For normally distributed data, the equality of variances was examined using the Levene test and presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. For non-normally distributed data, median (IQR) was used. For two group comparisons, Student t-test was used. One-way ANOVA was utilized to compare data among multiple groups. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eThe basic characteristics of clinical data\u003c/strong\u003e\u003cp\u003eThe clinical characteristics of each group were summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e including age, gender, body weight, medical history, basic vital signs and laboratory findings. As a result,The TAAD group showed significant differences from the control group in terms of age, gender, prevalence of hypertension, D-dimer levels and FDP levels. In order to better understand the occurrence of type A aortic dissection, typical CTA images of TAAD patients were presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb. As we can see, the rupture of the vascular intima is located in the ascending aorta, involving the entire aorta, and even extended to aorta abdominalis and iliac artery. While, for patients of control group, the aorta was intact and smooth, and no rupture was found throughout the whole course (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Further, the H\u0026amp;E stain and Masson stain of aorta in TAAD group showed that the aortic vascular wall tissue was significantly damaged, the morphology of smooth muscle cells was irregular, and the elastic fibers were incomplete, severely fractured, and fragmented. In contrast, the aortic wall structure was intact, the smooth muscle cells were closely and orderly arranged, and the elastic fibers were intact and continuous in control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec,d).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBaseline characteristics of all participants\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCharacteristic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAD(n\u0026thinsp;=\u0026thinsp;18)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge, Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e52.94\u0026thinsp;\u0026plusmn;\u0026thinsp;12.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e60.88\u0026thinsp;\u0026plusmn;\u0026thinsp;10.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.169\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;50, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10(55.56)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8 (88.89)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.205\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026lt;50, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8(44.44)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (11.11)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.012\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGender, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13 (72.22)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8 (88.89)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.049\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5 (27.78)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (11.11)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.062\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBody weight\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e80.11 (65, 92.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e77.38 (61.02, 90.64)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.375\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlcohol, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5 (27.78)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3(33.33)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.250\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSmoking, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9 (50)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5(55.56)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.999\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.999\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e80.17\u0026thinsp;\u0026plusmn;\u0026thinsp;4.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e76.67\u0026thinsp;\u0026plusmn;\u0026thinsp;5.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.989\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSBP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e145.18\u0026thinsp;\u0026plusmn;\u0026thinsp;18.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e131.17\u0026thinsp;\u0026plusmn;\u0026thinsp;18.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.051\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDBP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e88.74\u0026thinsp;\u0026plusmn;\u0026thinsp;14.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e83.5\u0026thinsp;\u0026plusmn;\u0026thinsp;13.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.768\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDiseases, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHypertension\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16(88.89)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5 (55.56)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDiabetes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3 (16.67)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (11.11)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.441\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCardiovascular disease\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5 (27.78)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (22.22)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.874\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOthers\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7 (38.89)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4 (44.44)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.250\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLaboratory test,\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eALT (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e44.28\u0026thinsp;\u0026plusmn;\u0026thinsp;23.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34.22\u0026thinsp;\u0026plusmn;\u0026thinsp;11.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.193\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAST (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e52.64\u0026thinsp;\u0026plusmn;\u0026thinsp;27.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e32.22\u0026thinsp;\u0026plusmn;\u0026thinsp;10.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.085\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTBIL (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14.63\u0026thinsp;\u0026plusmn;\u0026thinsp;2.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.999\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCr, median (IQR)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e88.61(72.25,104.75)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e76.12 (59.62,100.33)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.045\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eeGFR, median (IQR)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e83.26 (69, 99.25)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e89.88 (71,105.61)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.068\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT-CHO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.049\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.661\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eL-LDL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.999\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH-LDL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.999\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBlood sugar\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.812\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHbA1c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.999\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWBC, median (IQR)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12.36 (10.65, 14.13)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.28 (3.77, 6.79)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.023\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHb, median (IQR)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e131.94 (117, 145)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e122.85 (105.1, 134.65)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.060\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePLT (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e163.89\u0026thinsp;\u0026plusmn;\u0026thinsp;57.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e160.22\u0026thinsp;\u0026plusmn;\u0026thinsp;54.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.078\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN% (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e86.03\u0026thinsp;\u0026plusmn;\u0026thinsp;8.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e85.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.125\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePT (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.250\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD-dimer (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.51\u0026thinsp;\u0026plusmn;\u0026thinsp;6.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFDP (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e91.33\u0026thinsp;\u0026plusmn;\u0026thinsp;20.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34.95\u0026thinsp;\u0026plusmn;\u0026thinsp;9.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.004\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAnnotation: RR: respiratory rate; HR: heart rate; SBP: systolic blood pressure; DBP: diastolic blood pressure; ALT: glutamic pyruvic transaminase; AST: glutamic oxaloacetic transaminase; TBIL: total bilirubin; Cr: creatinine; eGFR: estimated glomerular filtration rate; TG: triglyceride; T-CHO: total cholesterol; L-LDL: low density lipoprotein cholesterol; H-LDL: high density lipoprotein cholesterol; HbA1c: glycosylate hemoglobin type A1c; WBC: white blood cell; Hb: hemoglobin; PLT: platelet; N%: neutrophile percentage; PT: prothrombin; FDP:fibrin degradation products.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eThe genes of patients in aortic dissection group expressed differentially\u003c/strong\u003e\u003cp\u003eIn order to reveal the underlying pathogenesis of aortic dissection, we performed high-throughput sequencing on the aortic tissues of patients in the dissection group and the control group. Before screening for differentially expressed genes, principal component analysis (PCA) of all samples was implemented to identify the degree of similarity among samples. As Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea displayed, the samples of dissection group were close to each other, so as the control group, suggesting a good genetic similarity among each group. The heat map showed conspicuous differences of encoding genes between the dissection group and control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). There totally existed 5109 differentially expressed genes, among which 1904 up-expressed genes and 3205 down-expressed genes if |log\u003csub\u003e2\u003c/sub\u003eFC|\u0026ge;1and \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026le;\u0026thinsp;0.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Subsequently, the differential genes were annotated according to three categories including biological process, cellular component and molecular function which exhibited with Gene ontology (GO) analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The GO bubble chart described top 20 significantly expressed genes based on enrich factor, most of which participate in the biological process. The KEGG pathway annotation showed that among the top 20 differential genes, almost all were related to immune inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). From this, we can see that certain biological processes have occurred in patients with aortic dissection and may be regulated by inflammation-related signaling pathways.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFerroptosis was involved in development process of TAAD\u003c/strong\u003e\u003cp\u003eIn order to have a comprehensive understanding of the functions of differentially expressed genes, in-depth KEGG annotation was performed. The result demonstrated that multiple signal pathways were directly or indirectly associated with the biological process of ferroptosis, including p53 signaling pathway, JAK-STAT signaling pathway, PI3K-Akt signaling pathway and ferroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eb listed the ferroptosis-related differentially expressed genes. Then, we conducted immunofluorescence staining and western blot experiments to verify the above analysis results.The C11-BODIPY\u003csup\u003e581/591\u003c/sup\u003e stain indicating lipid peroxidation showed that the aortic tissue of dissection group had a stronger green fluorescence compared to control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ec,d). In addition, the result of western blot also revealed that the expression quantity of SLC7A11 protein (a protein which generally marked ferroptosis) of aortic tissue in TAAD patients (n\u0026thinsp;=\u0026thinsp;7) were significantly down-regulated along with ɑ-SMA (ɑ-smooth muscle actin, which usually decreased in AD) protein compared with the amount of proteins in patients of control group (n\u0026thinsp;=\u0026thinsp;7). These results were displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ee,f. To sum up, cell ferroptosis played an important role in the pathogenesis of TAAD.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eHIF-1 signaling pathway regulated the ferroptosis process\u003c/strong\u003e\u003cp\u003eBased on the results of Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ee and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e, we observed that HIF-1 signaling pathway which closely related to the inflammatory response was significantly enriched during the process of functional annotation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). To further ascertain whether the HIF-1 signaling pathway take part in regulating ferroptosis during the process of aortic dissection, RT-PCR and western blot were carried out for detection of symbolic factors. As a result, the mRNA expression of HIF-1 and CD71 (also named transferrin receptor 1,Trf1) in aortic tissues of TAAD patients were dramatically up regulated in comparison with expression in control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eb,c). While, the mRNA expression of glutathione peroxidase 4 (GPX4, an important regulatory factor of ferroptosis) was down regulated in TAAD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), which was along with and opposite to the expression of HIF-1 and CD7. The trend of proteins expression of HIF-1, CD71 and GPX4 was in line with mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ee,f), suggesting HIF-1 signal pathway may influence the cellular ferroptosis process during the development of TAAD. Finally, we tested the Fe\u003csup\u003e2+\u003c/sup\u003e content of aortic tissues in both groups to support the speculation. As Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eg shown, the concentration of Fe\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;in TAAD group was significantly higher than that in control group, which may facilitate and enhance the iron-dependent programmed cell death form-cellular ferroptosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTAAD is an acute and devastating cardiovascular disease with high mortality in short time if the surgical repair operation is not carried out immediately\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Although technical intervention significantly improved survival rate of TAAD patients, operative mortality still remains high\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Further researches are warrant to comprehensively understand the pathogenesis of TAAD for prompt diagnosis and management.\u003c/p\u003e\u003cp\u003eDue to a tear in the intimal layer of the ascending aorta, lumenal blood enters the arterial wall, and the intima and media of the aorta are stripped in rapid sequence, resulting in the formation of false lumen. It has been reported that several risk factors are related to aortic dissection, such as age, hypertension, and dyslipidemia\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In this study, we found that the more than half of TAAD patients whose aorta was tore from ascending to aortaventrails even iliac artery were over 50 years old, and 77.2% were males. Importantly, almost 90% TAAD patients had suffered from hypertension for a long time. The proportion that had a dyslipidemia was approximately 70%, which was in accordance with previous study\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAs a new type of regulatory cell death, ferroptosis has been found to be involved in in many diseases, especially in the field of cancer since its proposal in 2012\u003csup\u003e17\u003c/sup\u003e. In recent years, researches have shown that ferroptosis also plays important role in the development of various cardiovascular diseases, including doxorubicin-induced cardiotoxicity, ischemia/reperfusion cardiomyopathy, heart failure and coronary heart disease\u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Such being the case, Standford type A aortic dissection, one of the most common and critical cardiovascular diseases, whether affected by ferroptosis or not on earth and how? In order to answer this question, we sequenced the transcriptome of the abandoned aortic tissues of TAAD patients undergoing aortic replacement surgery. The results in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e showed that thousands of genes were differentially expressed in comparison with non-TAAD patients, which was as expected. Further analysis found that multiple genes were reached in ferroptosis pathway or ferroptosis-related pathway, heavily suggesting ferroptosis took place in the course of AD. Subsequently, we performed C11-BODIPY\u003csup\u003e581/591\u003c/sup\u003e staining on the obtained aortic tissues.This lipophilic dye can quickly enter the membrane and be used as a fluorescent probe to detect lipid peroxidation and antioxidant capacity in living cells. After oxidation by lipid hydrogen peroxide substances, the maximum excitation and emission wavelengths of the dye shifted and mainly presents green fluorescence\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In our study, the intensity of green fluorescence in TAAD patients\u0026rsquo; tissue was significantly greater than that in control group, demonstrating that lipid peroxidation occured in aortic dissection. Interestingly, lipid peroxidation of unsaturated fatty acids on the cell membrane is the main mechanism of ferroptosis, which process is catalyzed under the action of divalent iron or ester oxygenase, thereby inducing cell death\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Moreover, the protein of SLC7A11 separated by Western Blot also hint that ferroptosis participated in TAAD. SLC7A11, solute carrier family 7 member 11, also named xCT, belongs to the cystine/glutamic acid reverse transporter protein\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Multiple studies have shown that the down-regulation of SLC7A11 can decrease intracellular levels of cysteine, exhaust the biosynthesis of GSH, and indirectly inhibit the activity of GPX4\u003csup\u003e25, 26\u003c/sup\u003e, ultimately inducing ferroptosis in cells for the accumulation of lipid peroxides. In this study, the expression amount of SLC7A11 in TAAD was significantly down-regulated compared to control group, which was consistent with the trend of ɑ-SMA expression. Hereto, we have confirmed that ferroptosis is involved in the occurrence and development of aortic dissection at the genetic, mRNA and protein levels.\u003c/p\u003e\u003cp\u003eConsidering that ferroptosis is regulated by many pathways, we conducted further enrichment analysis on the differentially expressed genes. As a result, HIF-1 signaling pathway drew our attention. As a key factor in responding to hypoxic stress, HIF-1 can regulate cell growth, proliferation, migration and apoptosis, being related to various physiological and pathological processes\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. It participates in multiple signal transduction pathways and plays an important role in diseases such as inflammation and cardiovascular diseases\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Previous study showed that transferrin receptor was involved in HIF-1 signaling pathway in cervical cancer. Inspired by the results of this study and combined with the relationship between transferrin receptors (Tfr) and ferroptosis\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, we speculate that HIF-1 may involved in cellular ferroptosis in the development of TAAD mediated by Tfr. The sequent RT-PCR results verified that the relative mRNA expression amount of HIF-1, Tfr1 and GPX4 in aortic tissue were significantly higher in TAAD patients that that in control group, which was in line with the western blot results. Tfr1 (i.e.CD71) is an important transmembrane protein that mediate the uptake of iron by cells. While intracellular iron metabolism is closely related to ferroptosis\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, the iron content within the cells was also detected. As expect, the iron content in aortic tissue of TAAD patients was obviously exceeded the gross of control group, reminding that iron metabolism has changed in the process of aortic dissection. Integrating these experimental results together, we inferred that Tfr1-regulated iron metabolism probably interceded HIF-1 signal pathway to involve in the pathological mechanism of aortic dissection through mediating cellular feroptosis.\u003c/p\u003e\u003cp\u003eFor all this, the design of this study is relatively rudimentary, and more experiments are needed to clarify the specific role of ferroptosis in the progression of aortic dissection. For instance, the gender ratio of the patients included in this study was unbalanced,which would impact the results in a certain. it\u0026rsquo;s still unknown that which type of aortic cells have undergone ferroptosis. Is it vascular smooth muscle cells, endothelial cells, immune cells which include monocytes, macrophages, lymphocytes? Or more than one type cells involved? Additionally, more elaborate and rigorous experimental designs are requisite to confirm the causality of Tfr1 and HIF-1 signaling pathway, which could introduce genetically knocked-out or overexpressed mice. Besides, it is necessary to elucidate the upstream and downstream genes of the HIF-1 signaling pathway.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTo sum up, we analyzed case data of TAAD patients and those with other related aortic diseases from clinical practice, as well as discarded aortic tissues after surgery. Transcriptome sequencing revealed that ferroptosis was involved in the pathogenesis of the disease, which was supported by fluorescence staining and related protein electrophoresis. And, HIF-1 signaling pathway may influence cellular ferroptosis through Tfr1-mediated iron metabolism.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTAAD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etype A aortic dissection\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHIF-1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehypoxia inducible factor-1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRT-qPCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ereal-time quantitative polymerase chain reaction\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCTA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecomputed tomography angiography\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRNA-seq\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eribonucleic acid-sequence\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003egene ontology\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKEEG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eECM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eextracellular matrix\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGEO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003egene expression omnibus\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eJAK-STAT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eJanus kinase-signal transducer and activator of transcription\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePI3K-Akt\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ephosphoinositide-3 kinase protein kinase B\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRIPA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eradio immunoprecipitation assay\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSDS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003esodium dodecyl sulfate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFDP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFibrin degradation products.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients or their families signed the written informed consent form according to the declaration of Helsinki, and this study protocol was approved by the medical ethic committee of the First Affiliated Hospital of Zhengzhou University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;L. L and JZ.D participated in the designed of the study, L.L, YY. L and T.Z conducted the human samples, YC.Z and B.L carried out experiments, YP.L performed statistical analysis, L.L and ZF.L drafted and revised the manuscript, JZ.D was the principal investigator of the laboratory, participated in design and help to revise the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll of the authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Joint Construction Project for Medical Science and Technology Research of Henan Province (NO. LHGJ20230258 and \u0026nbsp; NO.LHGJ20230290).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhu, Y.; Lingala, B.; Baiocchi, M.; Tao, J. J.; Toro Arana, V.; Khoo, J. W.; Williams, K. M.; Traboulsi, A. A.; Hammond, H. 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DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jep.2023.116289\u003c/span\u003e\u003cspan address=\"10.1016/j.jep.2023.116289\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTang, L. J.; Zhou, Y. J.; Xiong, X. M.; Li, N. S.; Zhang, J. J.; Luo, X. J.; Peng, J. Ubiquitin-specific protease 7 promotes ferroptosis via activation of the p53/TfR1 pathway in the rat hearts after ischemia/reperfusion. \u003cem\u003eFree Radic Biol Med\u003c/em\u003e 2021, \u003cem\u003e162\u003c/em\u003e, 339\u0026ndash;352. 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DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/nu15030591\u003c/span\u003e\u003cspan address=\"10.3390/nu15030591\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-medical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejmr","sideBox":"Learn more about [European Journal of Medical Research](http://eurjmedres.biomedcentral.com)","snPcode":"40001","submissionUrl":"https://submission.nature.com/new-submission/40001/3","title":"European Journal of Medical Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ferroptosis, TAAD, HIF-1, signaling pathway, RNA-seq","lastPublishedDoi":"10.21203/rs.3.rs-7845557/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7845557/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eStanford type A aortic dissection (TAAD), as one of the most lethal cardiovascular disease, may be mediated by cellular ferroptosis, which exacerbates structural modification and dysfunction of endothelial and smooth muscle cells. Hypoxia inducible factor-1 (HIF-1), as a transcription factor has shown participate in the course of ferroptosis. However, the mechanism involved has not been uncovered clearly.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe collected clinical basic information and image data of TAAD patients received surgical operation for aortic replacement and non- TAAD patients who suffered from other surgeries including cardiac transplantation or aortic valve replacement. Then aortic tissues being replaced from each group was analyzed through RNA-sequencing to reveal underlying molecular mechanism participated in TAAD. Then, according to genes and signaling pathway selected, qRT-PCR and western blot were further implemented to verify the expression quantity of proteins.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eBaseline characteristics including age, gender, and other variables were non-differential between two groups. Computed tomography angiography (CTA) showed that linear low-density structure referring to tearing intima was floated in the full-course of aortic lumen which was separated into the true lumen and the false lumen. Disordered cells arrangement and fractured fibrous tissue were displayed in the H\u0026amp;E staining and Masson staining. RNA-seq found that more than 5000 differential genes were expressed in TAAD patients if |log\u003csub\u003e2\u003c/sub\u003eFC|\u0026ge;1 and \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026le;\u0026thinsp;0.5. Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEEG) analysis revealed that genes related cellular ferroptosis and HIF-1 signaling pathway were expressed differentially and significantly during the development of TAAD. Moreover, the proteins of SLC7A11 and GPX4 which related ferroptosis were up-regulated, as well as the HIF-1 protein and its downstream protein of transferrin receptor 1(Trf1), which were in line with the results of RT-PCR. The amount of Fe\u003csup\u003e2+\u003c/sup\u003e was greater in aorta from TAAD patients than that from non-TAAD patients.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eCellular feroptosis plays a vital role during the development of TAAD, and the HIF-1/Trf/Fe\u003csup\u003e2+\u003c/sup\u003e pathway was involved in the regulation of cellular ferroptosis.\u003c/p\u003e","manuscriptTitle":"Involvement of HIF-1/Trf/Fe2+ signaling pathway in the Pathogenesis of Stanford Type A Aortic Dissection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 12:54:16","doi":"10.21203/rs.3.rs-7845557/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-22T08:32:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-22T05:26:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9916139840731537566413028707106039289","date":"2026-01-10T12:22:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-06T21:46:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"12012712563145231995530603714192538234","date":"2026-01-06T19:25:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"339372094022686357661504260429668727926","date":"2026-01-06T15:09:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-06T11:04:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"93992494965020039898140661438527183766","date":"2026-01-06T10:54:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-05T09:10:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66874358548072548509143425673774824906","date":"2026-01-05T08:58:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-30T22:18:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272725685084985010311418435455907715974","date":"2025-12-05T02:42:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-04T23:21:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-10T05:40:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-09T11:21:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Medical Research","date":"2025-11-08T02:10:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-medical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejmr","sideBox":"Learn more about [European Journal of Medical Research](http://eurjmedres.biomedcentral.com)","snPcode":"40001","submissionUrl":"https://submission.nature.com/new-submission/40001/3","title":"European Journal of Medical Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4e5a5765-e25d-49ff-9717-adaf9dc1dcc4","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-01-22T08:41:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-08 12:54:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7845557","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7845557","identity":"rs-7845557","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}