oxLDL promotes EMS-induced angiogenesis by up-regulating VEGF-A expression and secretion of endometrial cells

oxLDL promotes EMS-induced angiogenesis by up-regulating VEGF-A expression and secretion of endometrial cells | 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 oxLDL promotes EMS-induced angiogenesis by up-regulating VEGF-A expression and secretion of endometrial cells Caiqi Ma, Wei Huang, Hui Wang, Wenxia Yao, Min Liang, Guifang Yu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1757587/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background Endometriosis (EMS) is a "tumor-like" gynecological disease with distant metastasis, and studies have shown that EMS can carry out distant metastasis through vascular vessels, but the driving factor and its mechanism is not clear. Methods We use EMS animal model and gene knockout technique to explore the role of EMS-induced angiogenesis in EMS metastasis in vivo and in vitro, and clarify the role and molecular mechanism of oxLDL in promoting EMS induced angiogenesis. Results We found that microvascular density (MVD) in metastasized ectopic endometrium and eutopic endometrial tissue were higher than the normal endometrial tissue, and the plasma oxLDL was positively correlated with the distant metastasis of EMS. Furthermore, we clarify that oxLDL enhanced the MVD of endometrial tissue by up-regulating the VEGF-A expression and secretion in endometrial cells. At last, we illustrated the mechanism of oxLDL promoting the VEGF-A expression through the AKT-HIF-1α signaling pathway. Conclusion oxLDL is a risk factor promoting the EMS distant metastasis by up-regulating VEGF-A expression and secretion through AKT-HIF-1α signaling. This founding may provide theoretical support and therapeutic targets for the clinical prevention and treatment of EMS. Endometriosis Vascular metastasis Angiogenesis OxLDL VEGF-A AKT-HIF-1α signaling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Endometriosis (EMS) is a gynecologic disease with high incidence and lack of radical cure(Zondervan et al. , 2020). It not only seriously affects the physical and mental health of women, but also is one of the main causes of infertility(Zondervan et al., 2018 , Evans and Decherney, 2017 ). The etiology of EMS is complex, and its pathogenesis has not been fully clarified(Horne and Saunders, 2019 ). EMS is difficult to cure for its ability to metastasis, including implant metastasis from fallopian tube, lymphatic metastasis and vascular metastasis, among them, the vascular metastasis is the predominant one (Samani et al., 2019 , Machado et al., 2008 , Keichel et al., 2011 ). And unfortunately, the specific mechanism now is still unclear. Studies have shown that the microvessels in endometrial tissue are related to the occurrence and development of EMS, and endometrial cells may metastasize remotely through blood vessels(Laschke and Menger, 2018 , Laschke et al., 2011 ). And the oxidative stress factor oxLDL is corelative with its progress(Polak et al., 2011 , Polak et al., 2013 ). But the molecular mechanism requires deeper investigation. In the process of vascular metastasis of tumor cells, tumor cells will secrete pro-angiogenesis factors to induce angiogenesis, so that they can enter the blood vessels for distant metastasis(Yang et al., 2013 ). And oxLDL can enhance the tumor-induced angiogenesis to promote the vascular metastasis of cancer cells(Han et al., 2021 ), indicating that oxLDL may promote the EMS-induced angiogenesis assisting the EMS vascular metastasis. Tumor-angiogenesis needs vascular epithelial factors like VEGF-A, VEGF-B and PDGF, to initiate the proliferation and migration of vascular epithelial cells(Claesson-Welsh and Welsh, 2013 , Saharinen et al., 2011 ). For EMS, the vascular epithelial factor which will be involved in the EMS-induced angiogenesis remains unkown. In this study, we attempted to explore the role of EMS-induced angiogenesis in EMS vascular metastasis, and clarify the molecular mechanism of oxLDL promoting EMS-induced angiogenesis. Materials And Methods Cell lines and cell culture The primary hEECs, hESCs and hUVECs were purchased from the Procell Biotech (Wuhan, China). All of them were maintained in the specific medium supplemented with 10% (v/v) FBS, 100 U/ml penicillin, and 100mg/ml streptomycin and incubated at 37℃ in a humidified incubator at 5% CO2. Tissue specimens and clinicopathological characteristics The normal endometria were obtained from the uterine curettage endometria which were tested normally, while the eutopic endometria and ectopic endometria were from the EMS patients. They were collected and histopathologically and clinically diagnosed at the Fifth Affiliated Hospital of Guangzhou Medical University between 2020 and 2021. Written informed consent was obtained from all patients prior to the study. The use of the clinical specimens for research purposes was approved by the Institutional Research Ethics Committee. Experimental animal The mature (13-15 weeks old) Wistar rats were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China). Rats were allowed to acclimate to local conditions for at least 1 week and maintained under a 12 h dark, 12 h light cycle with food and water ad libitum. Care, use, and treatment of all animals in the present study were in strict agreement with the institutionally approved protocol according to the United States Public Health Service (USPHS) Guide for the care and use of laboratory animals, as well as the guidelines set forth in the Care and Use of Laboratory Animals by the Guangzhou Medical University. The animal use protocol has been reviewed and approved by the institutional animal care and use committee of Guangzhou Medical University. The establishment of EMS rat animal model The mature (13-15 weeks old) Wistar rats were used to build the EMS animal model. After intraperitoneal anesthesia with 10% chloral hydrate (0.38 ml / 100g body weight), a longitudinal incision with a length of 2-3 cm was made at about 1 cm above the pubic symphysis, a "Y" shaped uterus was found on the dorsal side of the bladder, the morphology of the uterus was observed to keep the uterus in a relaxed position, and the diameter of the uterus was measured with a vernier caliper. At about 1.5cm from the bifurcation of the uterus, the first ligation line was ligated with silk thread, the second ligation line was ligated near the ovarian end, and the uterus about 1.5cm in between was intercepted. Cut the uterus from the first ligation line, and cut the uterine segment to be cut longitudinally, then tear the endometrial layer and serosa layer with small tweezers, and repair the serosa layer. Trim the endometrial segments with the length, width and height of about 5mm respectively. Suture the four corners of the trimmed endometrial segments with non-destructive suture on the right abdominal wall, and then close the abdomen. Part of the remaining endometrial fragments were embedded in paraffin to confirm that the transplanted tissue was endometrium and left for subsequent experiments. After the operation, the rats were kept for 15 days, and then the modeling effect was confirmed. The ectopic scar of the rats that were confirmed to be successful in modeling (obvious ectopic scar was observed) was dissected and separated. After paraffin embedded section, he staining was performed. After the ectopic endometrial tissue was confirmed by microscopic observation, IHC detected the markers of vascular endothelial cells CD34 counted their vascular density, The vascular density and lumen size of endometrium were compared between the model group and the normal control group (sham operation group). Tube formation assay Human umbilical vein endothelial cells (hUVECs) in vitro tube formation assay was performed by first pipetting 200µl Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) into each well of a 24‑well plate, which was then polymerized for 30 min at 37˚C. hUVECs (2x10 4 cells; Procell Life Technology Co., Ltd., Wuhan, China) in 200 µl conditioned medium (the culture medium from hEECs and hESCs treated with oxLDL) were added to each well and incubated at 37˚C in an atmosphere containing 5% CO2 for 12 h. Images were captured using a bright‑field with ZEISS Axio Observer Z1 (Carl Zeiss AG, Oberkochen, Germany). RNA-Seq RNA sequence was performed with Illumina platform. In brief, the first step in the workflow involved purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then had the addition of a single ‘A’ base and subsequent ligation of the adapter. The products were then purified and enriched with PCR amplification. We then quantified the PCR yield by Qubit and pooled samples together to make a single strand DNA circle (ssDNA circle), which gave the final library. DNA nanoballs (DNBs) were generated with the ssDNA circle by rolling circle replication (RCR) to enlarge the fluorescent signals at the sequencing process. The DNBs were loaded into the patterned nanoarrays and single-end read of 50 bp were read through on the Illumina platform for the following data analysis study. For this step, the Illumina platform combined the DNA nanoball-based nanoarrays and stepwise sequencing using Combinational Probe-Anchor Synthesis Sequencing Method. Tools such as bcl2fastq, FastQC, hisat2 etc. were used for the following bioinformatics analysis. In the analysis of differentially expressed genes (DEGs), the genes that had more than double fold change and the corrected P value is less than or equal to 0.05 were defined as DEGs. With DEGs, we performed KEGG pathway classification and Gene set enrichment analysis (GSEA) using R. We have upload the sequence data onto the online database SRA (Sequence Read Archieve). Our sequence data is numbered JD-KYFW-2020-874-JSFW-01. Enzyme linked immunosorbent assay The supernatant VEGF-A of hEECs treated with different concentration oxLDL was detected by VEGF-A ELISA kit (Catalog number: RAB0507, MERCK, Germany) followed the procedure from the manufacturer. The culture medium was obtained after the hEECs were treated with oxLDL for 24h. And the plasma oxLDL of the EMS patients and the normal control were tested with the freshly collected plasma from the EMS patients and normal control which were frozen and stored in liquid nitrogen before required. They were collected and histopathologically and clinically diagnosed at the Guangzhou Women and Children’s Medical Center between 2020 and 2021. H&E staining and immunohistochemistry Tissues were surgically resected and fixed in formalin and embedded in paraffin. Then 4.0 mm thick histologic sections were prepared. Before immunohistochemistry, histologic sections were stained with hematoxylin-eosin to observe the morphology. The sections were treated with endogenous peroxidase blocking solution and normal goat serum to block non-specific background. Sections were then incubated with antibodies: CD34, VEGF-A (CST, Danvers, USA). After overnight incubation at 4 ℃, sections were incubated with a biotin-conjugated second antibody at room temperature for 20 min and were incubated with enzyme conjugate (HRP-Streptavidin) at the same condition. The vessels were revealed with streptavidin-peroxidase followed by chromogenic substrate diaminobenzidine (DAB) and the sections were counterstained with hematoxylin. Images of tissue microarrays were taken by Pannoramic Viewer software and quantification of expression level was determined by gray scale scanning with Image J software. Western blotting HIF-1α, p-AKT, antibody was purchased from Abcam (Cambridge, Massachusetts, USA) and OLR1 from Santa Cruz Biotechnology (Santa Cruz, CA, USA). VEGF-A, GAPDH and β-actin antibodies were purchased from CST (Danvers, MA, USA). Cells were harvested and lysed for total protein extraction. Protein concentration was determined using BCA protein assay kit (Keygen, Nanjing, China) according to manufacturer's protocol. Aliquots of equal amounts of protein from the cell lysate were subjected to Western blot analysis. Following. Densitometry was performed using ImageJ software and normalized by β-actin levels. siRNA transfection OLR1 siRNA and a nonspecific siRNA (control) were purchased from RiboBio (Guangzhou, Guangdong, China). According to the manufacturer's instructions, transfections were performed at approximately 60% confluency using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). For each transfection reaction, 20nM OLR1 siRNA or control siRNA was used for preparation of siRNA-transfection complexes at room temperature for 20 min. Transfections were performed in 0.5 (12-well plate) or 1.5 mL (6-well plate) serum-free medium for 8 hr. After incubation, transfection complexes were removed and replaced with their corresponding media. Transfection efficiency (80%–90%) was determined by Western blotting analysis. Cells were utilized for other experiments at 24–72 hr after transfection. Immunofluorence hEECs were plated on culture slides, and incubated with siRNA and/or oxLDL (50ug/ml) for 12h. The cells were washed, and fixed in 4% paraformaldehyde. Then, cells were blocked with normal non-immune goat serum at 37℃ for 1h. After washing three times, they were incubated with rabbit phospho-Akt and HIF-1α antibodies (Abcam, Cambridge, USA) at 37℃ for 2h, then were incubated with FITC conjugated goat anti-rabbit IgG (Dako, Glostrup, Denmark) at 37℃ for 1h after three times washing. Finally, the cell nucleus was stained with 4,6-di-amino-2- phenylindole (DAPI) (Sigma). Cells were visualized under a confocal microscope (Carl Zeiss, Oberkochen, Germany). In negative-control staining, the primary antibodies were omitted. Statistical analysis Data are presented as means ± SD. Comparisons were performed by two-tailed paired Student’s t test. A value of p<0.05 was considered statistically significant. Result EMS induced angiogenesis of endometrium. The ability of distant metastasis similar to tumor cells is one of the important reasons why EMS is difficult to cure. Among the ways of metastasis, vascular metastasis is the important one, and angiogenesis is an indispensable basis for vascular metastasis. Microvessel density (MVD) is a good indicator to reflect the angiogenesis activity. We used the vascular endothelial cell specific marker CD34 to immunohistochemically mark the microvessels of endometrial tissue, and then analyzed the difference of microvessel density between normal endometrial tissue and EMS eutopic and ectopic endometrial tissues. The results showed that the endometrial microvessel density in EMS endometrial tissue was higher than that in normal control group, and the EMS ectopic endometrial tissues was higher than the eutopic ones (Fig. 1 A, C). Then, we isolated the human endometrial epithelial cells (hEECs) and endometrial stromal cells (hESCs) from normal endometrial tissue, and then incubated human umbilical vein endothelial cells (hUVECs) laid on Matrigel with their culture medium. Then observed their effects on the tube formation of hUVECs through tube formation assay. The results showed that both hEECs and hESCs could secrete factors to enhance the tube formation of hUVECs (Fig. 1 B, D). These outcomes indicated that endometrial cells can induce angiogenesis in endometrium. OxLDL enhanced the angiogenesis of EMS endometrial tissue. In order to explore the factors that cause abnormal enhancement of angiogenesis in EMS endometrial tissue, we collected the plasma of normal people and EMS patients, and detected the concentration of oxLDL with ELISA kit. It was found that the plasma concentration of oxLDL in EMS patients was higher than that in normal people (Fig. 2 A). In order to further explore whether the abnormally increased oxLDL affected the angiogenesis of endometrial tissues in EMS patients, we incubated the hEECs and hESCs with oxLDL, and then applied the supernatants to culture the hUVECs observing the affection on tube formation. The results showed that oxLDL enhanced the ability of hEECs and hESCs inducing tube formation (Fig. 2 B, C). For the deeper investigation to demonstrate the effect of oxLDL on vascular metastasis of EMS in vivo, we established an EMS rat model (Fig. 3 A). After the EMS rat models were successfully established, oxLDL and PBS were used to continuously give drugs to the two groups of rats for 30 days, and then the ectopic endometrial tissues were removed by surgery for analysis. The results showed that the endometrial tissue of the oxLDL treated group grew larger than that of the control group (Fig. 3 B). Using CD34 to mark the microvessels in the endometrial tissues, it was found that MVD in the oxLDL treated group was higher than that in the control group (Fig. 3 C, D). These results suggest that oxLDL can promote the angiogenesis in the endometrium of EMS rats. Oxldl Up-regulated The Expression And Secretion Of Vegf-a In Endometrial Cells Vascular epithelial growth factor A (VEGF-A) is an important factor in promoting angiogenesis. In order to explore the mechanism of oxLDL promoting endometrial angiogenesis, we detected the expression level of VEGF-A in endometrial cells of EMS rat model by immunohistochemical technique. It was found that the expression level of VEGF-A in endometrial cells of oxLDL treated rats was higher than the control group (Fig. 4 A), indicating that oxLDL can up-regulate the expression of VEGF-A in EMS endometrial cells. Further we detected the expression of VEGF-A in hEECs and hESCs after oxLDL treatment by Western blot, finding that the expression of VEGF-A in both hEECs and hESCs treated with oxLDL increased significantly, especially in hESCs (Fig. 4 B, C). At last, ELISA was used to detect the concentration of VEGF-A in the culture medium of hEECs and hESCs after oxLDL incubation. It also showed that the concentration of VEGF-A in the oxLDL incubation group was higher (Fig. 4 D), suggesting that oxLDL can enhance the secretion of VEGF-A in hEECs. OxLDL up-regulated the VEGF-A expression in endometrial epithelial cells via PI3K-AKT-HIF-1α signal pathway. For exploring the molecular mechanism of oxLDL up-regulating VEGF-A in hEECs, we used RNA-seq technique to detect the changes of RNA transcriptome in hEECs treated with oxLDL. Sequencing results showed that the transcription level of VEGF-A was significantly up-regulated. Combined with KEGG signal pathway analysis, it was found that the transcription level of PI3K-AKT signal pathway related genes changed significantly (Fig. 5 ), suggesting that PI3K-AKT signal pathway may participate in the regulation of VEGF-A expression by oxLDL. VEGF-A expression was reported being regulated by HIF-1α, while the activity of oxLDL often mediated by its specific receptor OLR1. Combined with the results of RNA analysis, we speculated that oxLDL may enhance HIF-1α signaling activation via OLR1-PI3K-AKT axis, and then up-regulate the expression of VEGF-A. To test the above scientific hypothesis, we treated hEECs with oxLDL after OLR1 receptor interference, and then detected p-AKT and HIF-1α expression level by Western blot and immunofluorescence techniques. The results showed that oxLDL could promote the phosphorylation level of AKT, then up-regulate HIF-1α expression (Fig. 6 A, B, C, D). For the reverse proof, we applied the AKT and HIF-1α signaling pathway inhibitors to treat the hEECs before oxLDL incubaton. Finally, we observed that the above effects of oxLDL were neutralized to some extent (Fig. 6 E, F), indicating that OLR1-PI3K-AKT-HIF-1α axis indeed mediated the up-regulation of VEGF-A expression by oxLDL (Fig. 7 ). Discussion Endometriosis (EMS) is a common gynecological disease, which is difficult to cure because its similar metastatic ability like tumors(Dahiya et al., 2021 , Kralickova et al., 2014 ). Similarly, we can also apply the idea of tumor prevention and treatment to prevent and treat it. Vascular metastasis is not only one of the main ways of tumor metastasis, but also an important pathway of EMS metastasis(Laschke and Menger, 2018 ). Therefore, anti-angiogenesis is also an effective means to prevent the progression and metastasis of EMS. Tumor cells can promote angiogenesis by paracrine VEGF-A to support its vascular metastasis(Yang et al., 2013 ). In this study, we found that endometrial cells have similar functions, especially endometrial epithelial cells. OxLDL is an important oxidative stress factor in the blood, it is also a risk factor for many diseases, including coronary atherosclerotic heart disease and cancer(Poznyak et al., 2020 , Bitorina et al., 2021 ). We found that oxLDL can enhance the risk of EMS metastasis. EMS patients with higher plasma oxLDL concentration have a greater risk of metastasis. Further studies found that oxLDL can up-regulate VEGF-A in endometrial epithelial cells and stromal cells. And VEGF-A is an important factor to promote angiogenesis(Nagy et al., 2007 ), indicating that oxLDL may enhance angiogenesis by up-regulating the expression and secretion of VEGF-A, thus promoting the distant migration of endometrial cells. ORL1 is a specific receptor of oxLDL, which mediates the most functions of oxLDL, such as NF- κ B and TNF- α signal pathway activation, promoting the formation of foam cells in the vasculature, and enhancing proliferation and metastasis of tumor cells(Pirillo et al., 2013 , Ma et al., 2019 , Poznyak et al., 2020 , Feng et al., 2014 ). The HIF-1α signal pathway activation can directly initiate the transcription and expression of VEGF-A(Palazon et al., 2017 ). Therefore, we propose a scientific hypothesis: oxLDL up-regulates the expression of VEGF-A in endometrial epithelial cells through OLR1. Finally, our experimental results verify our scientific hypothesis. Moreover, we also found that oxLDL up-regulated HIF-1α expression level to enhance HIF-1α signal pathway activation. So, how does oxLDL up-regulate HIF-1α expression through OLR1? We screened the signal pathways significantly affected by oxLDL treatment with RNA-seq. Among these pathways, AKT signal pathway was reported to regulate HIF-1α expression(Pez et al., 2011 , Zhang et al., 2018 ). Through further experimental demonstration, oxLDL does activate the AKT signaling pathway of endometrial epithelial cells. At the same time, the up-regulation of HIF-1α by oxLDL was also observed reduced after blocking the AKT signaling pathway, which proved that oxLDL did enhance HIF-1α through AKT signaling pathway, then promote the activation of HIF-1α signal pathway, and finally up-regulate the expression of VEGF-A to assist the distant metastasis of endometrial cells. Conclusion This project clarifies the molecular mechanism that oxLDL up-regulates the expression of VEGF-A in endometrial cells through AKT-HIF-1α signaling pathway, and then promotes the distant metastasis of EMS, providing new theoretical support and therapeutic targets for the prevention and treatment of EMS. Abbreviations EMS endometriosis MVD microvascular density oxLDL oxidized low-density lipoprotein OLR1 oxidized low-density lipoprotein (lectin-like) receptor 1 hEECs human endometrial epithelial cells hESCs human endometrial stromal cells hUVECs human umbilical vein endothelial cells VEGF-A Vascular epithelial growth factor A HIF-1 hypoxia inducible factor-1 FBS Fetal bovine serum. Declarations The use of the clinical specimens for research purposes was approved by the Institutional Research Ethics Committee. Ethics approval and consent to participate the animal experiment was performed according to the National Institutes of Health Guide for Care and Use of Laboratory Animals and approved by the institutional animal care and use committee of Guangzhou Medical University. Acknowledgements We would like to acknowledge the reviewers for their helpful comments on this paper. Authors’ contributions XZ, CM finished study design, CM, WH, HW, ML finished experimental studies, CM, XZ WH, HW, WY, GYfinished data analysis, CM, XZ finished manuscript editing. All authors read and approved the final manuscript. Funding This work was supported by Key Laboratory of Guangdong Higher Education Institutes (2021KSYS009); Guangzhou Science and technology planning project (202102020142) Availability of data and materials All data generated or analyzed during this study are included in this article. Consent for publication Consent for publication was obtained from the participants. Competing interests We declared that there were no conflicts of interest in this paper. Author details 1 Department of Oncology, Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong Higher Education Institutes, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510530, China. 2 Reproductive Medical Center, Affiliated Guangzhou Women and Children's Medical Center of Guangzhou Medical University, Guangzhou, 510530, China. References BITORINA AV, OLIGSCHLAEGER Y, DING L, YADATI T, WESTHEIM A, HOUBEN T, THEYS VAES,ROLDE,DS, J. & SHIRI-SVERDLOV R. (2021), "OxLDL as an Inducer of a Metabolic Shift in Cancer Cells", J Cancer , Vol. 12 No. 19, pp. 5817–5824. CLAESSON-WELSH L, WELSH M. "VEGFA and tumour angiogenesis". J Intern Med. 2013;273 No(2):114–27. DAHIYA A, THOMAS SEBASTIAN,ATHOMAS,AGEORGE,R, V. & PEEDICAYIL A. "Endometriosis and malignancy: The intriguing relationship". Int J Gynaecol Obstet. 2021;155 No(1):72–8. EVANS MB, DECHERNEY AH. "Fertility and Endometriosis". Clin Obstet Gynecol. 2017;60 No(3):497–502. FENG Y, CAI ZR, TANG Y, LU HU,G, HE J, D. & WANG S. "TLR4/NF-kappaB signaling pathway-mediated and oxLDL-induced up-regulation of LOX-1, MCP-1, and VCAM-1 expressions in human umbilical vein endothelial cells". Genet Mol Res. 2014;13 No(1):680–95. HAN L, LI D, HANG Y, ZONG X, BAI LV,J, LU X, ZHANG Y, WU PZHOU,M, Z. & HU W. (2021), "Downregulation of hsa_circ_0004543 Activates oxLDL-Induced Vascular Endothelial Cell Proliferation and Angiogenesis", Front Genet, Vol. 12632164. HORNE AW, SAUNDERS P. "SnapShot. Endometriosis" Cell. 2019;179 No(7):1677–7.e1. KEICHEL S, BARCENA DAM, SCHNEIDER REICHELT,URIEDLINGER,WF, KOHLER A, C. & MECHSNER S. "Lymphangiogenesis in deep infiltrating endometriosis". Hum Reprod. 2011;26 No(10):2713–20. KRALICKOVA M, LOSAN P, VETVICKA V. "Endometriosis and cancer". Womens Health (Lond). 2014;10 No(6):591–7. LASCHKE MW, MENGER MD. "Basic mechanisms of vascularization in endometriosis and their clinical implications". Hum Reprod Update. 2018;24 No(2):207–24. LASCHKE MW, GIEBELS C, MENGER MD. "Vasculogenesis: a new piece of the endometriosis puzzle". Hum Reprod Update. 2011;17 No(5):628–36. MA C, LUO XIE,J, YIN C, LI H, WANG R, ZHANG XXIONG,W, JIANG T, QI P, YANG WZHOU,T, WANG Z, GAO WMA,J, G. & YANG X. "OxLDL promotes lymphangiogenesis and lymphatic metastasis in gastric cancer by upregulating VEGFC expression and secretion". Int J Oncol. 2019;54 No(2):572–84. MACHADO DE, ABRAO MS, BERARDO PT, TAKIYA CM, NASCIUTTI LE. "Vascular density and distribution of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 (Flk-1) are significantly higher in patients with deeply infiltrating endometriosis affecting the rectum". Fertil Steril. 2008;90 No(1):148–55. NAGY JA, DVORAK AM, DVORAK HF. (2007), "VEGF-A and the induction of pathological angiogenesis", Annu Rev Pathol, Vol. 2251–75. PALAZON A, MACIAS TYRAKIS,PA, VELICA D, RUNDQVIST P, FITZPATRICK H, VOJNOVIC S, LOMAN NPHAN,AT, HEDENFALK N, HATSCHEK I, FOUKAKIS TLOVROT,J, T., GOLDRATH, A. W., BERGH, J. & JOHNSON RS. "An HIF-1alpha/VEGF-A Axis in Cytotoxic T Cells Regulates Tumor Progression". Cancer Cell. 2017;32 No(5):669–83.e5. PEZ F, KANIEWSKI DAYAN,FDURIVAULT,J, Le BAIMOND,G, DEUX PROVOST,GS, CLEZARDIN B, SOMMER P, P., POUYSSEGUR, J. & REYNAUD C. "The HIF-1-inducible lysyl oxidase activates HIF-1 via the Akt pathway in a positive regulation loop and synergizes with HIF-1 in promoting tumor cell growth". Cancer Res. 2011;71 No(5):1647–57. PIRILLO A, NORATA GD, CATAPANO AL. (2013), "LOX-1, OxLDL, and atherosclerosis", Mediators Inflamm, Vol. 2013152786. POLAK G, BARCZYNSKI B, KWASNIEWSKI W, BEDNAREK W, WERTEL I, DEREWIANKA-POLAK M, KOTARSKI J. (2013), "Low-density lipoproteins oxidation and endometriosis", Mediators Inflamm, Vol. 2013624540. POLAK G, MAZUREK D, NOWICKA ROGALA,E, DEREWIANKA-POLAK A, M. & KOTARSKI J. "[Increased oxidized LDL cholesterol levels in peritoneal fluid of women with advanced-stage endometriosis]". Ginekol Pol. 2011;82 No(3):191–4. POZNYAK AV, MARKIN NIKIFOROV,NG, GERASIMOVA AM,KASHIRSKIKH,DA,MYASOEDOVA,VA, E. V. & OREKHOV AN. (2020), "Overview of OxLDL and Its Impact on Cardiovascular Health: Focus on Atherosclerosis", Front Pharmacol, Vol. 11613780. SAHARINEN P, EKLUND L, PULKKI K, BONO P, ALITALO K. "VEGF and angiopoietin signaling in tumor angiogenesis and metastasis". Trends Mol Med. 2011;17 No(7):347–62. SAMANI EN, LI MAMILLAPALLI,R, MUTLU F, HUFNAGEL L, KRIKUN D, G. & TAYLOR HS. "Micrometastasis of endometriosis to distant organs in a murine model". Oncotarget. 2019;10 No(23):2282–91. YANG Y, SUN M, WANG, L. & JIAO B. "HIFs, angiogenesis, and cancer". J Cell Biochem. 2013;114 No(5):967–74. ZHANG Z, YAO L, YANG J, WANG Z, Du G. "PI3K/Akt and HIF1 signaling pathway in hypoxiaischemia (Review)". Mol Med Rep. 2018;18 No(4):3547–54. ZONDERVAN KT, BECKER, C. M. & MISSMER SA. "Endometriosis". N Engl J Med. 2020;382 No(13):1244–56. ZONDERVAN KT, MISSMER BECKER,CM,KOGA,K, TAYLOR SA, R. N. & VIGANO P. "Endometriosis". Nat Rev Dis Primers. 2018;4 No(1):9. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 27 Jun, 2022 Reviewers invited by journal 26 Jun, 2022 Editor invited by journal 20 Jun, 2022 Editor assigned by journal 20 Jun, 2022 First submitted to journal 14 Jun, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1757587","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":116476701,"identity":"1a440a9c-c7df-431e-bf13-85ba10faeef4","order_by":0,"name":"Caiqi Ma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYDACCQbGAwkgBnvjAxDFw0eEFgaIFp7DBmCKjSgtEEYyWAsDQS3m0s0HDjxsY4jmn/mY8TFvjp0MGwPzw0c38GixnHMs4UDCGYbcGbeTmQ1nbksGOozN2DgHjxaDGzkGBxIq/uc23M4/JvFxGzNQCw+bNH4t+R8OJBgw5M6/eZhNInFbPTFacoAhVsGQu+EGMxvQlsOEtVjOSDMA+2XjGbBfjvOwMRPwi7lE8sOHP9sYcucdPwwMsW3V9vzszQ8f43UYphAzHuU4tIyCUTAKRsEoQAMAlvhJC+yTvsgAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-3178-9820","institution":"Guangzhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Caiqi","middleName":"","lastName":"Ma","suffix":""},{"id":116476702,"identity":"c7b1499f-6acf-4eac-9039-52a6605f9375","order_by":1,"name":"Wei Huang","email":"","orcid":"","institution":"First Affiliated Hospital of Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Huang","suffix":""},{"id":116476703,"identity":"a424f6c5-3ecf-4a4b-9cfd-8075a96ff609","order_by":2,"name":"Hui Wang","email":"","orcid":"","institution":"Guangzhou Women and Children's Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Wang","suffix":""},{"id":116476704,"identity":"30c1154a-f462-4e2b-8146-4c47d24b4b15","order_by":3,"name":"Wenxia Yao","email":"","orcid":"","institution":"Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenxia","middleName":"","lastName":"Yao","suffix":""},{"id":116476705,"identity":"8cfae200-850c-455e-9a4d-ce944a65ff13","order_by":4,"name":"Min Liang","email":"","orcid":"","institution":"Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Liang","suffix":""},{"id":116476706,"identity":"f2b348f2-20d4-45c6-a69a-a8eb0355397b","order_by":5,"name":"Guifang Yu","email":"","orcid":"","institution":"Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guifang","middleName":"","lastName":"Yu","suffix":""},{"id":116476707,"identity":"e5ae21af-0bc0-4b5c-b4bd-380196084305","order_by":6,"name":"Xinke Zhou","email":"","orcid":"https://orcid.org/0000-0001-7840-3123","institution":"Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinke","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2022-06-14 13:35:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1757587/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1757587/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":23469589,"identity":"aa4aca3e-ec0e-4ab8-8842-73ec4b723b7c","added_by":"auto","created_at":"2022-07-05 17:04:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1746728,"visible":true,"origin":"","legend":"\u003cp\u003eEMS induced the angiogenesis in endometrial tissue\u003c/p\u003e\u003cp\u003e(A) The microvessel density in normal endometrium, eutopic endometrium and ectopic endometrium. The microvascular was stained with CD34 by immunohistochemistry. The number of each group was 8. (B) Tube formation of hUECs induced by hESCs and hEECs in co-culture system with chamber. (C) Histogram represents the microvessel density in normal endometrium, eutopic endometrium and ectopic endometrium. (D) Histogram represents the tube number in each group. * represents p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1757587/v1/27cd367445b8b72dae9c058b.png"},{"id":23469590,"identity":"c8c8d6ba-ac59-429f-9046-69075fc6a509","added_by":"auto","created_at":"2022-07-05 17:04:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":884824,"visible":true,"origin":"","legend":"\u003cp\u003eoxLDL promoted the growth of ectopic endometrium in the EMS animal models. (A) the certification of hEECs from the human endometrium. hEECs were stained by vimentin (which is expressed in hEECs) and CK-18 (which is expressed in hESCs) by immunofluorescence. (B) Three steps to build the EMS animal model. (C) the ectopic endometrium in each group of EMS animal models. The number of each group was 5. The scale bar represents 5mm.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1757587/v1/5e0f80b4989a126a3c541de0.png"},{"id":23469592,"identity":"cf0e4b2d-a286-4385-a3e1-3a1d97e32da5","added_by":"auto","created_at":"2022-07-05 17:04:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2554477,"visible":true,"origin":"","legend":"\u003cp\u003eoxLDL promoted EMS-induced angiogenesis\u003c/p\u003e\u003cp\u003e(A) The plasma oxLDL was elevated in EMS patients. the number of each patient group was 8. (B\u0026amp;C) The MVD in the ectopic endometrium from the two groups animal models. Histogram represents the MVD in each group.\u0026nbsp;(D\u0026amp;E) Tube formation induced by hESCs and hEECs treated with oxLDL and PBS. Histogram represents the tube number in each group. * represents p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1757587/v1/216334706619f6ff1d27a94b.png"},{"id":23469594,"identity":"e3b11524-3c30-4341-be43-147c21eb50a8","added_by":"auto","created_at":"2022-07-05 17:04:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1483692,"visible":true,"origin":"","legend":"\u003cp\u003eoxLDL up-regulated the expression and secretion of VEGF-A in endometrial cells. (A) The VEGF-A expression in eutopic endometrium and ectopic endometrium from the animal models treated with oxLDL and PBS. VEGF-A was stained by immunohistochemistry. (B\u0026amp;C) Western blot determined the VEGF-A expression in hESCs and hEECs treated with oxLDL and PBS. Histogram represents the gray scale of each lane from Western blot. (D) The VEGF-A secretion of hESCs and hEECs treated with oxLDL and PBS were tested by ELISA.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1757587/v1/eb9911f43f9a5bffe60d42cf.png"},{"id":23469595,"identity":"50413626-1cc2-4fd2-8153-566fb3bb764a","added_by":"auto","created_at":"2022-07-05 17:04:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":317672,"visible":true,"origin":"","legend":"\u003cp\u003eRNA-seq analysis predicted the potential mechanism of oxLDL up-regulating VEGF-A expression. (A) KEGG analysis of hEECs treated with oxLDL and PBS showed the possible signaling pathway involved in the process of oxLDL up-regulating VEGF-A. (B) Heatmap analysis predicted VEGF-A may up-regulated by oxLDL. (C) The GSEA plot shows that VEGF-A expression positively correlates with AKT-mTOR activated gene signatures.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-1757587/v1/40461eac4a0dc441d8491acd.png"},{"id":23469591,"identity":"8e72d6e1-2f32-435d-b0a1-9c23e470821e","added_by":"auto","created_at":"2022-07-05 17:04:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1117127,"visible":true,"origin":"","legend":"\u003cp\u003eoxLDL up-regulated VEGF-A expression and secretion via OLR-1/PI3K/Akt signaling. (A\u0026amp;B) p-AKT and HIF-1α expression in each group of hEECs treated with oxLDL or PBS were examined by Western blot. Histogram represents the gray scale of each lane from Western blot. (C\u0026amp;D) p-AKT and HIF-1α expression in hEECs treated with oxLDL or PBS were examined by immunofluorescence. (E\u0026amp;F) VEGF-A expression in each group of hEECs treated with oxLDL (50μg/ml), AKT inhibitor perifosine (20mM) and HIF-1α LW6 (20mM), were examined by Western blot. Histogram represents the gray scale of each lane from Western blot.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-1757587/v1/75341421c2c503d3ce0b8772.png"},{"id":23470653,"identity":"f0d74319-d5c2-4891-8d40-3aa3b03c0f1e","added_by":"auto","created_at":"2022-07-05 17:09:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":354859,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of oxLDL up-regulating VEGF-A expression.\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-1757587/v1/cafb2c16f4a8277f9c850e34.png"},{"id":23470666,"identity":"5dec3198-e3cf-48bd-803a-528709222a46","added_by":"auto","created_at":"2022-07-05 17:09:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":380001,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1757587/v1/1a58c1e6-2078-4c5f-82c1-b6beccc0aa6d.pdf"}],"financialInterests":"","formattedTitle":"oxLDL promotes EMS-induced angiogenesis by up-regulating VEGF-A expression and secretion of endometrial cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndometriosis (EMS) is a gynecologic disease with high incidence and lack of radical cure(Zondervan \u003cem\u003eet al.\u003c/em\u003e, 2020). It not only seriously affects the physical and mental health of women, but also is one of the main causes of infertility(Zondervan et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Evans and Decherney, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The etiology of EMS is complex, and its pathogenesis has not been fully clarified(Horne and Saunders, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). EMS is difficult to cure for its ability to metastasis, including implant metastasis from fallopian tube, lymphatic metastasis and vascular metastasis, among them, the vascular metastasis is the predominant one (Samani et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Machado et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Keichel et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). And unfortunately, the specific mechanism now is still unclear. Studies have shown that the microvessels in endometrial tissue are related to the occurrence and development of EMS, and endometrial cells may metastasize remotely through blood vessels(Laschke and Menger, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Laschke et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). And the oxidative stress factor oxLDL is corelative with its progress(Polak et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Polak et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). But the molecular mechanism requires deeper investigation.\u003c/p\u003e \u003cp\u003eIn the process of vascular metastasis of tumor cells, tumor cells will secrete pro-angiogenesis factors to induce angiogenesis, so that they can enter the blood vessels for distant metastasis(Yang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). And oxLDL can enhance the tumor-induced angiogenesis to promote the vascular metastasis of cancer cells(Han et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), indicating that oxLDL may promote the EMS-induced angiogenesis assisting the EMS vascular metastasis. Tumor-angiogenesis needs vascular epithelial factors like VEGF-A, VEGF-B and PDGF, to initiate the proliferation and migration of vascular epithelial cells(Claesson-Welsh and Welsh, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Saharinen et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). For EMS, the vascular epithelial factor which will be involved in the EMS-induced angiogenesis remains unkown. In this study, we attempted to explore the role of EMS-induced angiogenesis in EMS vascular metastasis, and clarify the molecular mechanism of oxLDL promoting EMS-induced angiogenesis.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eCell lines and cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe primary hEECs, hESCs and hUVECs were purchased from the Procell Biotech (Wuhan, China). All of them were maintained in the specific medium supplemented with 10% (v/v) FBS, 100 U/ml penicillin, and 100mg/ml streptomycin and incubated at 37℃ in a humidified incubator at 5% CO2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue specimens and clinicopathological characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe normal endometria were obtained from the uterine curettage endometria which were tested normally, while the eutopic endometria and ectopic endometria were from the EMS patients. They were collected and histopathologically and clinically diagnosed at the Fifth Affiliated Hospital of Guangzhou Medical University between 2020 and 2021. Written informed consent was obtained from all patients prior to the study. The use of the clinical specimens for research purposes was approved by the Institutional Research Ethics Committee.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental animal\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mature (13-15 weeks old) Wistar rats were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China). Rats were allowed to acclimate to local conditions for at least 1 week and maintained under a 12 h dark, 12 h light cycle with food and water ad libitum. Care, use, and treatment of all animals in the present study were in strict agreement with the institutionally approved protocol according to the United States Public Health Service (USPHS) Guide for the care and use of laboratory animals, as well as the guidelines set forth in the Care and Use of Laboratory Animals by the Guangzhou Medical University. The animal use protocol has been reviewed and approved by the institutional animal care and use committee of Guangzhou Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe establishment of EMS rat animal model\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mature (13-15 weeks old) Wistar rats were used to build the EMS animal model. After intraperitoneal anesthesia with 10% chloral hydrate (0.38 ml / 100g body weight), a longitudinal incision with a length of 2-3 cm was made at about 1 cm above the pubic symphysis, a \u0026quot;Y\u0026quot; shaped uterus was found on the dorsal side of the bladder, the morphology of the uterus was observed to keep the uterus in a relaxed position, and the diameter of the uterus was measured with a vernier caliper. At about 1.5cm from the bifurcation of the uterus, the first ligation line was ligated with silk thread, the second ligation line was ligated near the ovarian end, and the uterus about 1.5cm in between was intercepted. Cut the uterus from the first ligation line, and cut the uterine segment to be cut longitudinally, then tear the endometrial layer and serosa layer with small tweezers, and repair the serosa layer. Trim the endometrial segments with the length, width and height of about 5mm respectively. Suture the four corners of the trimmed endometrial segments with non-destructive suture on the right abdominal wall, and then close the abdomen. Part of the remaining endometrial fragments were embedded in paraffin to confirm that the transplanted tissue was endometrium and left for subsequent experiments. After the operation, the rats were kept for 15 days, and then the modeling effect was confirmed. The ectopic scar of the rats that were confirmed to be successful in modeling (obvious ectopic scar was observed) was dissected and separated. After paraffin embedded section, he staining was performed. After the ectopic endometrial tissue was confirmed by microscopic observation, IHC detected the markers of vascular endothelial cells CD34 counted their vascular density, The vascular density and lumen size of endometrium were compared between the model group and the normal control group (sham operation group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTube formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman umbilical vein endothelial cells (hUVECs) in vitro tube formation assay was performed by first pipetting 200\u0026micro;l Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) into each well of a 24‑well plate, which was then polymerized for 30 min at 37˚C. hUVECs (2x10\u003csup\u003e4\u003c/sup\u003e cells; Procell Life Technology Co., Ltd., Wuhan, China) in 200 \u0026micro;l conditioned medium (the culture medium from hEECs and hESCs treated with oxLDL) were added to each well and incubated at 37˚C in an atmosphere containing 5% CO2 for 12 h. Images were captured using a bright‑field with ZEISS Axio Observer Z1 (Carl Zeiss AG, Oberkochen, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-Seq\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA sequence was performed with Illumina platform. In brief, the first step in the workflow involved purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then had the addition of a single \u0026lsquo;A\u0026rsquo; base and subsequent ligation of the adapter. The products were then purified and enriched with\u003c/p\u003e\n\u003cp\u003ePCR amplification. We then quantified the PCR yield by Qubit and pooled samples together to make a single strand DNA circle (ssDNA circle), which gave the final library. DNA nanoballs (DNBs) were generated with the ssDNA circle by rolling circle replication (RCR) to enlarge the fluorescent signals at the sequencing process. The DNBs were loaded into the patterned nanoarrays and single-end read of 50 bp were read through on the Illumina platform for the following data analysis study. For this step, the Illumina platform combined the DNA nanoball-based nanoarrays and stepwise sequencing using Combinational Probe-Anchor Synthesis Sequencing Method. Tools such as bcl2fastq, FastQC, hisat2 etc. were used for the following bioinformatics analysis. In the analysis of differentially expressed genes (DEGs), the genes that had more than double fold change and the corrected P value is less than or equal to 0.05 were defined as DEGs. With DEGs, we performed KEGG pathway classification and Gene set enrichment analysis (GSEA) using R. We have upload the sequence data onto the online database SRA (Sequence Read Archieve). Our sequence data is numbered JD-KYFW-2020-874-JSFW-01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme linked immunosorbent assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe supernatant VEGF-A of hEECs treated with different concentration oxLDL was detected by VEGF-A ELISA kit (Catalog number: RAB0507, MERCK, Germany) followed the procedure from the manufacturer. The culture medium was obtained after the hEECs were treated with oxLDL for 24h. And the plasma oxLDL of the EMS patients and the normal control were tested with the freshly collected plasma from the EMS patients and normal control which were frozen and stored in liquid nitrogen before required. They were collected and histopathologically and clinically diagnosed at the Guangzhou Women and Children\u0026rsquo;s Medical Center between 2020 and 2021.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u0026amp;E staining and immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissues were surgically resected and fixed in formalin and embedded in paraffin. Then 4.0 mm thick histologic sections were prepared. Before immunohistochemistry, histologic sections were stained with hematoxylin-eosin to observe the morphology. The sections were treated with endogenous peroxidase blocking solution and normal goat serum to block non-specific background. Sections were then incubated with antibodies: CD34, VEGF-A (CST, Danvers, USA). After overnight incubation at 4 ℃, sections were incubated with a biotin-conjugated second antibody at room temperature for 20 min and were incubated with enzyme conjugate (HRP-Streptavidin) at the same condition. The vessels were revealed with streptavidin-peroxidase followed by chromogenic substrate diaminobenzidine (DAB) and the sections were counterstained with hematoxylin. Images of tissue microarrays were taken by Pannoramic Viewer software and quantification of expression level was determined by gray scale scanning with Image J software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHIF-1\u0026alpha;, p-AKT, antibody was purchased from Abcam (Cambridge, Massachusetts, USA) and OLR1 from Santa Cruz Biotechnology (Santa Cruz, CA, USA). VEGF-A, GAPDH and \u0026beta;-actin antibodies were purchased from CST (Danvers, MA, USA). Cells were harvested and lysed for total protein extraction. Protein concentration was determined using BCA protein assay kit (Keygen, Nanjing, China) according to manufacturer\u0026apos;s protocol. Aliquots of equal amounts of protein from the cell lysate were subjected to Western blot analysis. Following. Densitometry was performed using ImageJ software and normalized by \u0026beta;-actin levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003esiRNA transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOLR1 siRNA and a nonspecific siRNA (control) were purchased from RiboBio (Guangzhou, Guangdong, China). According to the manufacturer\u0026apos;s instructions, transfections were performed at approximately 60% confluency using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). For each transfection reaction, 20nM OLR1 siRNA or control siRNA was used for preparation of siRNA-transfection complexes at room temperature for 20 min. Transfections were performed in 0.5 (12-well plate) or 1.5 mL (6-well plate) serum-free medium for 8 hr. After incubation, transfection complexes were removed and replaced with their corresponding media. Transfection efficiency (80%\u0026ndash;90%) was determined by Western blotting analysis. Cells were utilized for other experiments at 24\u0026ndash;72 hr after transfection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ehEECs were plated on culture slides, and incubated with siRNA and/or oxLDL (50ug/ml) for 12h. The cells were washed, and fixed in 4% paraformaldehyde. Then, cells were blocked with normal non-immune goat serum at 37℃ for 1h. After washing three times, they were incubated with rabbit phospho-Akt and HIF-1\u0026alpha; antibodies (Abcam, Cambridge, USA) at 37℃ for 2h, then were incubated with FITC conjugated goat anti-rabbit IgG (Dako, Glostrup, Denmark) at 37℃ for 1h after three times washing. Finally, the cell nucleus was stained with 4,6-di-amino-2- phenylindole (DAPI) (Sigma). Cells were visualized under a confocal microscope (Carl Zeiss, Oberkochen, Germany). In negative-control staining, the primary antibodies were omitted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as means \u0026plusmn; SD. Comparisons were performed by two-tailed paired Student\u0026rsquo;s t test. A value of p\u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e \u003cb\u003eEMS induced angiogenesis of endometrium.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe ability of distant metastasis similar to tumor cells is one of the important reasons why EMS is difficult to cure. Among the ways of metastasis, vascular metastasis is the important one, and angiogenesis is an indispensable basis for vascular metastasis. Microvessel density (MVD) is a good indicator to reflect the angiogenesis activity. We used the vascular endothelial cell specific marker CD34 to immunohistochemically mark the microvessels of endometrial tissue, and then analyzed the difference of microvessel density between normal endometrial tissue and EMS eutopic and ectopic endometrial tissues. The results showed that the endometrial microvessel density in EMS endometrial tissue was higher than that in normal control group, and the EMS ectopic endometrial tissues was higher than the eutopic ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, C). Then, we isolated the human endometrial epithelial cells (hEECs) and endometrial stromal cells (hESCs) from normal endometrial tissue, and then incubated human umbilical vein endothelial cells (hUVECs) laid on Matrigel with their culture medium. Then observed their effects on the tube formation of hUVECs through tube formation assay. The results showed that both hEECs and hESCs could secrete factors to enhance the tube formation of hUVECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, D). These outcomes indicated that endometrial cells can induce angiogenesis in endometrium.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOxLDL enhanced the angiogenesis of EMS endometrial tissue.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn order to explore the factors that cause abnormal enhancement of angiogenesis in EMS endometrial tissue, we collected the plasma of normal people and EMS patients, and detected the concentration of oxLDL with ELISA kit. It was found that the plasma concentration of oxLDL in EMS patients was higher than that in normal people (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In order to further explore whether the abnormally increased oxLDL affected the angiogenesis of endometrial tissues in EMS patients, we incubated the hEECs and hESCs with oxLDL, and then applied the supernatants to culture the hUVECs observing the affection on tube formation. The results showed that oxLDL enhanced the ability of hEECs and hESCs inducing tube formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). For the deeper investigation to demonstrate the effect of oxLDL on vascular metastasis of EMS in vivo, we established an EMS rat model (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). After the EMS rat models were successfully established, oxLDL and PBS were used to continuously give drugs to the two groups of rats for 30 days, and then the ectopic endometrial tissues were removed by surgery for analysis. The results showed that the endometrial tissue of the oxLDL treated group grew larger than that of the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Using CD34 to mark the microvessels in the endometrial tissues, it was found that MVD in the oxLDL treated group was higher than that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). These results suggest that oxLDL can promote the angiogenesis in the endometrium of EMS rats.\u003c/p\u003e \u003cp\u003e\u003cb\u003eOxldl Up-regulated The Expression And Secretion Of Vegf-a In Endometrial Cells\u003c/b\u003e\u003c/p\u003e\n\u003cp\u003eVascular epithelial growth factor A (VEGF-A) is an important factor in promoting angiogenesis. In order to explore the mechanism of oxLDL promoting endometrial angiogenesis, we detected the expression level of VEGF-A in endometrial cells of EMS rat model by immunohistochemical technique. It was found that the expression level of VEGF-A in endometrial cells of oxLDL treated rats was higher than the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), indicating that oxLDL can up-regulate the expression of VEGF-A in EMS endometrial cells. Further we detected the expression of VEGF-A in hEECs and hESCs after oxLDL treatment by Western blot, finding that the expression of VEGF-A in both hEECs and hESCs treated with oxLDL increased significantly, especially in hESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). At last, ELISA was used to detect the concentration of VEGF-A in the culture medium of hEECs and hESCs after oxLDL incubation. It also showed that the concentration of VEGF-A in the oxLDL incubation group was higher (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), suggesting that oxLDL can enhance the secretion of VEGF-A in hEECs.\u003c/p\u003e\u003cp\u003e \u003cb\u003eOxLDL up-regulated the VEGF-A expression in endometrial epithelial cells via PI3K-AKT-HIF-1α signal pathway.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor exploring the molecular mechanism of oxLDL up-regulating VEGF-A in hEECs, we used RNA-seq technique to detect the changes of RNA transcriptome in hEECs treated with oxLDL. Sequencing results showed that the transcription level of VEGF-A was significantly up-regulated. Combined with KEGG signal pathway analysis, it was found that the transcription level of PI3K-AKT signal pathway related genes changed significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), suggesting that PI3K-AKT signal pathway may participate in the regulation of VEGF-A expression by oxLDL. VEGF-A expression was reported being regulated by HIF-1α, while the activity of oxLDL often mediated by its specific receptor OLR1. Combined with the results of RNA analysis, we speculated that oxLDL may enhance HIF-1α signaling activation via OLR1-PI3K-AKT axis, and then up-regulate the expression of VEGF-A. To test the above scientific hypothesis, we treated hEECs with oxLDL after OLR1 receptor interference, and then detected p-AKT and HIF-1α expression level by Western blot and immunofluorescence techniques. The results showed that oxLDL could promote the phosphorylation level of AKT, then up-regulate HIF-1α expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B, C, D). For the reverse proof, we applied the AKT and HIF-1α signaling pathway inhibitors to treat the hEECs before oxLDL incubaton. Finally, we observed that the above effects of oxLDL were neutralized to some extent (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F), indicating that OLR1-PI3K-AKT-HIF-1α axis indeed mediated the up-regulation of VEGF-A expression by oxLDL (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEndometriosis (EMS) is a common gynecological disease, which is difficult to cure because its similar metastatic ability like tumors(Dahiya et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Kralickova et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Similarly, we can also apply the idea of tumor prevention and treatment to prevent and treat it. Vascular metastasis is not only one of the main ways of tumor metastasis, but also an important pathway of EMS metastasis(Laschke and Menger, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Therefore, anti-angiogenesis is also an effective means to prevent the progression and metastasis of EMS. Tumor cells can promote angiogenesis by paracrine VEGF-A to support its vascular metastasis(Yang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In this study, we found that endometrial cells have similar functions, especially endometrial epithelial cells. OxLDL is an important oxidative stress factor in the blood, it is also a risk factor for many diseases, including coronary atherosclerotic heart disease and cancer(Poznyak et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Bitorina et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We found that oxLDL can enhance the risk of EMS metastasis. EMS patients with higher plasma oxLDL concentration have a greater risk of metastasis. Further studies found that oxLDL can up-regulate VEGF-A in endometrial epithelial cells and stromal cells. And VEGF-A is an important factor to promote angiogenesis(Nagy et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), indicating that oxLDL may enhance angiogenesis by up-regulating the expression and secretion of VEGF-A, thus promoting the distant migration of endometrial cells. ORL1 is a specific receptor of oxLDL, which mediates the most functions of oxLDL, such as NF- κ B and TNF- α signal pathway activation, promoting the formation of foam cells in the vasculature, and enhancing proliferation and metastasis of tumor cells(Pirillo et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Ma et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Poznyak et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Feng et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The HIF-1α signal pathway activation can directly initiate the transcription and expression of VEGF-A(Palazon et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, we propose a scientific hypothesis: oxLDL up-regulates the expression of VEGF-A in endometrial epithelial cells through OLR1. Finally, our experimental results verify our scientific hypothesis. Moreover, we also found that oxLDL up-regulated HIF-1α expression level to enhance HIF-1α signal pathway activation. So, how does oxLDL up-regulate HIF-1α expression through OLR1? We screened the signal pathways significantly affected by oxLDL treatment with RNA-seq.\u0026nbsp;Among these pathways, AKT signal pathway was reported to regulate HIF-1α expression(Pez et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Zhang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Through further experimental demonstration, oxLDL does activate the AKT signaling pathway of endometrial epithelial cells. At the same time, the up-regulation of HIF-1α by oxLDL was also observed reduced after blocking the AKT signaling pathway, which proved that oxLDL did enhance HIF-1α through AKT signaling pathway, then promote the activation of HIF-1α signal pathway, and finally up-regulate the expression of VEGF-A to assist the distant metastasis of endometrial cells.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis project clarifies the molecular mechanism that oxLDL up-regulates the expression of VEGF-A in endometrial cells through AKT-HIF-1α signaling pathway, and then promotes the distant metastasis of EMS, providing new theoretical support and therapeutic targets for the prevention and treatment of EMS.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEMS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eendometriosis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMVD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emicrovascular density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eoxLDL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoxidized low-density lipoprotein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOLR1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoxidized low-density lipoprotein (lectin-like) receptor 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ehEECs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehuman endometrial epithelial cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ehESCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehuman endometrial stromal cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ehUVECs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehuman umbilical vein endothelial cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVEGF-A\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVascular epithelial growth factor A\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\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFetal bovine serum.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe use of the clinical specimens for research purposes was approved by the Institutional Research Ethics Committee. Ethics approval and consent to participate the animal experiment was performed according to the National Institutes of Health Guide for Care and Use of Laboratory Animals and approved by the institutional animal care and use committee of Guangzhou Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge the reviewers for their helpful comments on this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXZ, CM finished study design, CM, WH, HW, ML finished experimental studies, CM, XZ WH, HW, WY, GYfinished data analysis, CM, XZ finished manuscript editing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by\u0026nbsp;Key Laboratory of Guangdong Higher Education Institutes (2021KSYS009);\u0026nbsp;Guangzhou Science and technology planning project (202102020142)\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 article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsent for publication was obtained from the participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declared that there were no conflicts of interest in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1 Department of Oncology,\u0026nbsp;Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong Higher Education Institutes, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510530, China.\u003c/p\u003e\n\u003cp\u003e2 Reproductive Medical Center, Affiliated Guangzhou Women and Children\u0026apos;s Medical Center of Guangzhou Medical University, Guangzhou, 510530, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBITORINA AV, OLIGSCHLAEGER Y, DING L, YADATI T, WESTHEIM A, HOUBEN T, THEYS VAES,ROLDE,DS, J. \u0026amp; SHIRI-SVERDLOV R. (2021), \"OxLDL as an Inducer of a Metabolic Shift in Cancer Cells\", \u003cem\u003eJ Cancer\u003c/em\u003e, Vol.\u0026nbsp;12 No. 19, pp.\u0026nbsp;5817\u0026ndash;5824.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCLAESSON-WELSH L, WELSH M. \"VEGFA and tumour angiogenesis\". J Intern Med. 2013;273 No(2):114\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDAHIYA A, THOMAS SEBASTIAN,ATHOMAS,AGEORGE,R, V. \u0026amp; PEEDICAYIL A. \"Endometriosis and malignancy: The intriguing relationship\". Int J Gynaecol Obstet. 2021;155 No(1):72\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEVANS MB, DECHERNEY AH. \"Fertility and Endometriosis\". Clin Obstet Gynecol. 2017;60 No(3):497\u0026ndash;502.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFENG Y, CAI ZR, TANG Y, LU HU,G, HE J, D. \u0026amp; WANG S. \"TLR4/NF-kappaB signaling pathway-mediated and oxLDL-induced up-regulation of LOX-1, MCP-1, and VCAM-1 expressions in human umbilical vein endothelial cells\". Genet Mol Res. 2014;13 No(1):680\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHAN L, LI D, HANG Y, ZONG X, BAI LV,J, LU X, ZHANG Y, WU PZHOU,M, Z. \u0026amp; HU W. (2021), \"Downregulation of hsa_circ_0004543 Activates oxLDL-Induced Vascular Endothelial Cell Proliferation and Angiogenesis\", Front Genet, Vol. 12632164.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHORNE AW, SAUNDERS P. \"SnapShot. Endometriosis\" Cell. 2019;179 No(7):1677\u0026ndash;7.e1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKEICHEL S, BARCENA DAM, SCHNEIDER REICHELT,URIEDLINGER,WF, KOHLER A, C. \u0026amp; MECHSNER S. \"Lymphangiogenesis in deep infiltrating endometriosis\". Hum Reprod. 2011;26 No(10):2713\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKRALICKOVA M, LOSAN P, VETVICKA V. \"Endometriosis and cancer\". Womens Health (Lond). 2014;10 No(6):591\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLASCHKE MW, MENGER MD. \"Basic mechanisms of vascularization in endometriosis and their clinical implications\". Hum Reprod Update. 2018;24 No(2):207\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLASCHKE MW, GIEBELS C, MENGER MD. \"Vasculogenesis: a new piece of the endometriosis puzzle\". Hum Reprod Update. 2011;17 No(5):628\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMA C, LUO XIE,J, YIN C, LI H, WANG R, ZHANG XXIONG,W, JIANG T, QI P, YANG WZHOU,T, WANG Z, GAO WMA,J, G. \u0026amp; YANG X. \"OxLDL promotes lymphangiogenesis and lymphatic metastasis in gastric cancer by upregulating VEGFC expression and secretion\". Int J Oncol. 2019;54 No(2):572\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMACHADO DE, ABRAO MS, BERARDO PT, TAKIYA CM, NASCIUTTI LE. \"Vascular density and distribution of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 (Flk-1) are significantly higher in patients with deeply infiltrating endometriosis affecting the rectum\". Fertil Steril. 2008;90 No(1):148\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNAGY JA, DVORAK AM, DVORAK HF. (2007), \"VEGF-A and the induction of pathological angiogenesis\", Annu Rev Pathol, Vol. 2251\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePALAZON A, MACIAS TYRAKIS,PA, VELICA D, RUNDQVIST P, FITZPATRICK H, VOJNOVIC S, LOMAN NPHAN,AT, HEDENFALK N, HATSCHEK I, FOUKAKIS TLOVROT,J, T., GOLDRATH, A. W., BERGH, J. \u0026amp; JOHNSON RS. \"An HIF-1alpha/VEGF-A Axis in Cytotoxic T Cells Regulates Tumor Progression\". Cancer Cell. 2017;32 No(5):669\u0026ndash;83.e5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePEZ F, KANIEWSKI DAYAN,FDURIVAULT,J, Le BAIMOND,G, DEUX PROVOST,GS, CLEZARDIN B, SOMMER P, P., POUYSSEGUR, J. \u0026amp; REYNAUD C. \"The HIF-1-inducible lysyl oxidase activates HIF-1 via the Akt pathway in a positive regulation loop and synergizes with HIF-1 in promoting tumor cell growth\". Cancer Res. 2011;71 No(5):1647\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePIRILLO A, NORATA GD, CATAPANO AL. (2013), \"LOX-1, OxLDL, and atherosclerosis\", Mediators Inflamm, Vol.\u0026nbsp;2013152786.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePOLAK G, BARCZYNSKI B, KWASNIEWSKI W, BEDNAREK W, WERTEL I, DEREWIANKA-POLAK M, KOTARSKI J. (2013), \"Low-density lipoproteins oxidation and endometriosis\", Mediators Inflamm, Vol. 2013624540.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePOLAK G, MAZUREK D, NOWICKA ROGALA,E, DEREWIANKA-POLAK A, M. \u0026amp; KOTARSKI J. \"[Increased oxidized LDL cholesterol levels in peritoneal fluid of women with advanced-stage endometriosis]\". Ginekol Pol. 2011;82 No(3):191\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePOZNYAK AV, MARKIN NIKIFOROV,NG, GERASIMOVA AM,KASHIRSKIKH,DA,MYASOEDOVA,VA, E. V. \u0026amp; OREKHOV AN. (2020), \"Overview of OxLDL and Its Impact on Cardiovascular Health: Focus on Atherosclerosis\", Front Pharmacol, Vol. 11613780.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSAHARINEN P, EKLUND L, PULKKI K, BONO P, ALITALO K. \"VEGF and angiopoietin signaling in tumor angiogenesis and metastasis\". Trends Mol Med. 2011;17 No(7):347\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSAMANI EN, LI MAMILLAPALLI,R, MUTLU F, HUFNAGEL L, KRIKUN D, G. \u0026amp; TAYLOR HS. \"Micrometastasis of endometriosis to distant organs in a murine model\". Oncotarget. 2019;10 No(23):2282\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYANG Y, SUN M, WANG, L. \u0026amp; JIAO B. \"HIFs, angiogenesis, and cancer\". J Cell Biochem. 2013;114 No(5):967\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZHANG Z, YAO L, YANG J, WANG Z, Du G. \"PI3K/Akt and HIF1 signaling pathway in hypoxiaischemia (Review)\". Mol Med Rep. 2018;18 No(4):3547\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZONDERVAN KT, BECKER, C. M. \u0026amp; MISSMER SA. \"Endometriosis\". N Engl J Med. 2020;382 No(13):1244\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZONDERVAN KT, MISSMER BECKER,CM,KOGA,K, TAYLOR SA, R. N. \u0026amp; VIGANO P. \"Endometriosis\". Nat Rev Dis Primers. 2018;4 No(1):9.\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mome","sideBox":"Learn more about [Molecular Medicine](https://molmed.biomedcentral.com)","snPcode":"10020","submissionUrl":"https://submission.springernature.com/new-submission/10020/3","title":"Molecular Medicine","twitterHandle":"@MolecularMedic1","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Endometriosis, Vascular metastasis, Angiogenesis, OxLDL, VEGF-A, AKT-HIF-1α signaling","lastPublishedDoi":"10.21203/rs.3.rs-1757587/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1757587/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEndometriosis (EMS) is a \"tumor-like\" gynecological disease with distant metastasis, and studies have shown that EMS can carry out distant metastasis through vascular vessels, but the driving factor and its mechanism is not clear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe use EMS animal model and gene knockout technique to explore the role of EMS-induced angiogenesis in EMS metastasis in vivo and in vitro, and clarify the role and molecular mechanism of oxLDL in promoting EMS induced angiogenesis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that microvascular density (MVD) in metastasized ectopic endometrium and eutopic endometrial tissue were higher than the normal endometrial tissue, and the plasma oxLDL was positively correlated with the distant metastasis of EMS. Furthermore, we clarify that oxLDL enhanced the MVD of endometrial tissue by up-regulating the VEGF-A expression and secretion in endometrial cells. At last, we illustrated the mechanism of oxLDL promoting the VEGF-A expression through the AKT-HIF-1α signaling pathway.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eoxLDL is a risk factor promoting the EMS distant metastasis by up-regulating VEGF-A expression and secretion through AKT-HIF-1α signaling. This founding may provide theoretical support and therapeutic targets for the clinical prevention and treatment of EMS.\u003c/p\u003e","manuscriptTitle":"oxLDL promotes EMS-induced angiogenesis by up-regulating VEGF-A expression and secretion of endometrial cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-05 17:04:22","doi":"10.21203/rs.3.rs-1757587/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2022-06-27T19:20:43+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-06-26T19:43:28+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Molecular Medicine","date":"2022-06-20T18:46:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-06-20T11:39:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Medicine","date":"2022-06-14T09:32:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mome","sideBox":"Learn more about [Molecular Medicine](https://molmed.biomedcentral.com)","snPcode":"10020","submissionUrl":"https://submission.springernature.com/new-submission/10020/3","title":"Molecular Medicine","twitterHandle":"@MolecularMedic1","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f0667851-8576-4441-935a-baae7a95026e","owner":[],"postedDate":"July 5th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-12-02T01:08:44+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-05 17:04:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1757587","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1757587","identity":"rs-1757587","version":["v1"]},"buildId":"B-jG_2CBjPDmsCi4Wdhf-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}