Macrophage M2 polarization with down-regulated NOX2 promotes placental angiogenesis

Macrophage M2 polarization with down-regulated NOX2 promotes placental angiogenesis | 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 Macrophage M2 polarization with down-regulated NOX2 promotes placental angiogenesis Ruonan Zhang, Lu Zhang, Ruixue Wang, Ayinisa Wubulikasimu, Mengtian Wei, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5419879/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The polarised state of placental Hofbauer cells (HBCs) is frequently accompanied by alterations in inflammatory factors and marker proteins, which are crucial in normal pregnancy. NOX2, as one of NADPH oxidase, is commonly expressed in phagocytes and mediates oxidative stress. A recent paper reported that the upregulation of NOX2 in porcine placenta led to reduced angiogenesis through increased oxidative stress, which attracted our interesting. To investigate whether NOX2 expression in human placenta is the cause of reduced vascularity in placentas of Fetal growth restriction (FGR), we examined the expression and localisation of NOX2 in placental villous and analysed the expression of NOX2 in different macrophages subtypes as well as its effect on vascular endothelial function. Our results showed that NOX2 was predominantly expressed in HBCs of placental villi. Moreover M2 polarisation of macrophages were inversely associated with NOX2 expression and promoted angiogenesis. The expression of NOX2 was significantly elevated in the placenta of FGR compared with normal placentas, which might be related to the reduction of placental vasculature in FGR. These results suggested that NOX2 can be used as a marker of HBCs and as a result of M2 polarization, and is also deeply involved in the functional regulation of placental vascular endothelial cells. These evidences providing a potential target for the diagnosis and treatment of FGR. NOX2 Hofbauer cells fetal growth restriction M2-polarized angiogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Fetal growth restriction(FGR), also known as intrauterine growth restriction(IUGR), is a prevalent complication during pregnancy that determinant the global morbidity and mortality rates. The pathogenesis of FGR is not fully understood, and its etiology may included maternal medical conditions, infectious diseases of fetal, placental disorders and umbilical cord abnormalities [ 1 ] . Placental-related fetal growth restriction arises primarily due to deficient remodeling of the uterine spiral arteries supplying the placenta and resultant malperfusion [ 2 ] . Therefore, one of the principal features of the placenta in cases of FGR is the reduction in volume, surface area, and vascularization of the intermediate and terminal villi that mediate maternal-fetal exchange [ 3 – 5 ] . The process of angiogenesis is balanced by numerous factors: secreted soluble molecules, hypoxia, shear stress, components of the extracellular matrix (ECM) and direct cell–cell interaction with distinct cell types, such as pericytes, fibroblasts and tissue resident macrophages [ 6 – 8 ] . NOX2, also known as gp91phox, is the prototype NADPH oxidase. The NOX family NADPH oxidases are proteins with the capability of transferring electrons across biofilms. These enzymes all possess the ability to transfer electrons through the plasma membrane and generate superoxides as well as other downstream reactive oxygen species (ROS) [ 9 ] . NOX2 was initially identified in neutrophils and macrophages, and it widely distributed among a large number of tissues including placenta [ 10 ] . The macrophages in the placenta were mainly Hofbauer cells (HBCs), which are abundant in the villous stroma [ 11 , 12 ] . These macrophages exhibit considerable plasticity and heterogeneity throughout pregnancy. Some reports suggest that in normal pregnancy, these Hofbauer cells demonstrate an M2-like phenotype and expressed some specific marker protein of M2 macrophages [ 13 ] . However, in the context of placental disorders, the balance between M1 and M2 may be disrupted. The role of HBCs in the placenta is not yet clearly understood. However, given their location and paracrine capabilities, they are believed to be involved in early placental development, placental immunology, and the development and maturation of placental mesenchyme throughout pregnancy [ 14 – 18 ] . There is emerging evidence that Hofbauer cells may serve important roles in both normal and abnormal pregnancy. To enhance our understanding of Hofbauer cell biology and their functions in health and disease, it is essential to recognize their heterogeneous nature [ [ 19 ] . Recently, the expression of NOX2 in porcine placenta was reported. Using porcine vascular endothelial cells (PVECs), increases placental oxidative stress and decreases placental angiogenesis possibly through the upregulation of NOX2 [ 20 ] . However, the exact location of NOX2 in human placental tissue and the function to angiogenesis still unknown. We thus predict that M1:M2 ratio of macrophage is associated with the expression of NOX2 which as an alter gene of FGR, eventually affects angiogenesis in the placenta. We initially discovered, through immunofluorescence staining, that NOX2 is mainly expressed on HBCs, which might be related to the reduction in blood vessel density in the placenta of FGR. Since the pathogenesis of FGR involves the disturbance of placental angiogenesis, we analyzed from the perspective of M1/M2 and found that M2 macrophages expressed lower levels of NOX2, which may be the reason for the increased expression of NOX2 in the placental of FGR. Our results provide insights into the biomarkers of HBCs and the relationship between NOX2 with angiogenesis. NOX2 can be used as a differential gene of FGR, which is also valuable for disease prediction and diagnosis and treatment. Materials and Methods Study population and sample collection All participants in the study were pregnant women who underwent natural delivery in Shanghai First Maternity and Infants Hospital from January 2022 to June 2023. The patients in this study were divided into normal late pregnancy (n = 4, 37–39 weeks), and Intrauterine growth restriction (n = 6, 33–39 weeks), as shown in Table 1 . In this study, the placental tissues of all patients were obtained from the operating room with the approval of the Scientific Ethics Committee of Shanghai First Maternity and Infants Hospital affiliated with Tongji University. After the blood clots were cleaned on ice with PBS, they were cut into 1 cm \(\:\times\:\) 1 cm tissue blocks and stored in liquid nitrogen in frozen storage tubes for further use. Table 1 The characteristics of women from normal and FGR pregnancies. Parameter Normal (n = 4) FGR (n = 4) Maternal age (yr) 28 ± 0.707 29 ± 2.345 Gestational age (wk) 38.425 ± 1.064 38.25 ± 1.126 Gravidity 1 ± 0.000 1.25 ± 0.433 Parity 1 ± 0.000 1 ± 0.000 Birth weight (grams) 3077.5 ± 202.993 2837.5 ± 243.323 Placental weight (grams) 550 ± 72.572 512.5 ± 90.381 Supplementary Table 1. Western Blot Antibodies Western blot analysis The concentration of proteins was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. Proteins were separated by 10% SDS PAGE gels and transferred to PVDF membranes by gel electrophoresis and electroblotting. After blocking with 5% BSA, the blots were probed with primary antibodies at 4°C overnight. Then, the membranes were washed and incubated with secondary antibodies. Ultimately, proteins were visualized using enhanced chemiluminescence reagents (Thermo Fisher Scientific). The antibodies used are listed in Table S1 . The relative protein expression levels were analyzed by densitometry using ImageJ imaging analysis software (NIH). Immunofluorescence staining Store frozen slices in -80°C. After three washes with ice-cold PBS, the tissue were fixed with 4% paraformaldehyde (PFA, Dingguo, China) for 30 minutes and then permeabilized with 0.1% Triton X-100 (Dingguo) for 15 minutes. Subsequently, the tissue were blocked with 5% bovine serum albumin (Thermo Fisher Scientific) for 1 hour at room temperature and incubated with an appropriate concentration of primary antibodies against NOX2, CD163 or CD31 overnight at 4°C. Then, the tissue were incubated with Alexa Fluor® 488-conjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies for 1 hour. The stained tissue were observed under a fluorescence confocal microscope. The antibodies used are listed in Table S2 . RNA isolation and quantitative RT-PCR Total RNA was extracted using TRIzol (Thermo Fisher Scientific), and RNA was then reverse transcribed using the SuperScript First Strand cDNA System (Takara) according to the manufacturer's instructions. Quantitative RT-PCR (qRT-PCR) was performed using the SYBR Green PCR master mix (Takara) and the StepOnePlus PCR system (Thermo Fisher Scientific) according to the manufacturer's instructions. The housekeeping gene GAPDH was used as an endogenous control. The primer sequences are shown in Table S3 . Cell culture and establishment THP-1 and 293T cells obtained from ATCC (Rockville, MD, USA) were maintained at 37°C in a humidified atmosphere with 5% CO 2 and 95% air. Cells were cultured in RPMI 1640 medium (HyClone, Logan, UT, USA) or DMEM-high glucose medium (HyClone) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin. Human umbilical vein endothelial cells (HUVECs) were isolated using 0.1% collagenase II. Immediately after isolation, cells were cultured and steadily expanded in Endothelial Cell Medium (ECM, ScienCell, San Diego, CA, USA) containing 5% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% endothelial cell growth supplement (ECGS, ScienCell) under 37°C in a humidified atmosphere with 5% CO 2 . Cell proliferation assay To test the effect of macrophage polarization on HUVECs proliferation, 2 \(\:\times\:\) 10 3 cm cells per well were plated in 96-well culture plates in 100 µL of FBS-containing ECM medium. The medium was replaced with RPMI 1640 medium from M0/M1/M2 macrophages after 16 hours. After 48 hours, relative cell numbers were determined using MTS (Promega, Madison, WI, USA) reagent in a 96-well plate reader at 490 nm. Cell migration assay The migration of HUVECs was determined using a modified system according to the manufacturer’s instructions (BD Bioscience, Heidelberg, Germany). In this assay, cell migration was monitored using a 24-well transwell plate with inserts containing 8 µm pores (Costar, Corning, NY, USA). In brief, 1.5 \(\:\times\:\) 10 4 cells were seeded on the upper side of the transwell inserts in serum-free medium. RPMI 1640 medium which cultured macrophages was added to the companion plate. Following incubation at 37°C with 5% CO 2 and 95% air for 16 hours, the fluorescent stain calcein-AM (Thermo Fisher Scientific) was added to each chamber and incubated for 30 minutes. The numbers of migrated cells were determined by fluorescence image analysis (Nikon, Tokyo, Japan). Tube formation assay The formation of capillary-like structures was assessed in a 48-well plate using Growth Factor Reduced Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). HUVECs (1.5 \(\:\times\:\) 10 3 cells/well) were resuspended in 500 uL ECM medium then plated on top of solidified Matrigel (200 µL/well) following incubation at 37°C for 1 h, and then replaced with RPMI 1640 medium from macrophages and ECM medium(500 uL and 200 uL respectively). They were viewed per hour under fluorescence microscopy (Nikon Eclipse Ti). PMA exposure and polarization THP-1 cells were counted about 1×10 6 cells/ml, and seeded at 6mm dish in 1 mL of RPMI medium with PMA(100 ng/ml), maintained at 37°C, 5% CO 2 in a humidified tissue culture incubator. After 48 hours, THP-1 cells gradually adhere to the dish bottom and differentiation into M0 macrophages. RPMI medium with PMA were removed and M0 macrophages were exposured to polarizing cytokines. The medium was replaced with fresh medium containing either IFNγ + LPS (20 ng/mL and 100 ng/mL respectively) or IL-4 (100 ng/mL). The cells were maintained in medium with cytokines for 48 hours. Medium with cytokines were removed and replaced with RPMI 1640 medium containing 0.1% FBS for 24 hours, then collect macrophages’ supernatant for co-cultured with HUVECs. Statistical analyses Data are expressed as the means ± SD and were analyzed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was analyzed by unpaired Student's t tests or one-way ANOVA. A p value < 0.05 was considered statistically significant. RESULTS Location of NOX2 in human placental tissues To explore the functional role of NOX2 in human placental angiogenesis, we first observed NOX2 distribution in human placenta by confocal detection. HBCs exhibit M2 macrophage phenotype so we used the CD163 protein to label the HBCs, our data showed that NOX2 was mainly expressed in the Hofbauer cells (HBCs) (Fig. 1 A). Using CD31 to represent vascular endothelial cell in the placenta, we observed less angiogenesis in the placenta with FGR (Fig. 1 B). These findings indicate that NOX2 co-localises with HBC's known marker which is associated with reduced angiogenesis in the placenta of FGR. Inducing macrophages with M2 characteristics We attempted to induce THP-1 cells to M2-polarized macrophages to mimic HBCs. First, we established the process and method of inducing macrophages [ 21 ] . We analyzed the expression of markers for different macrophage phenotypes, CCR-7, IL-6 and CXCL-10 for M1-polarized macrophages (Fig. 2 A) and CCL-17, Arg-1 and CD-206 for M2-polarized macrophages (Fig. 2 B) using rt-qPCR. Our experimental results show that we successfully induced M2-polarized macrophages. Effect of M2-polarized macrophages on HUVECs angiogenesis We speculate that the number of NOX2-expressing HBCs colocalized with endothelial cells is related to the blood vessels in the placenta. Therefore, we analyzed the impact of macrophages CM on network formation of primary HUVECs. Indeed, CM of M2-polarized macrophages induced the migration (Fig. 3 A), network formation (Fig. 3 B) and proliferation (Fig. 3 C) of HUVECs, as compared with M0/M1-polarized macrophages’ medium. The different polarization states of macrophages affect the formation of peripheral blood vessels, which was consistent with results described in other reports [ 22 , 23 ] . Expression of NOX2 in FGR’ placental tissues and M2-polarized macrophages We compared NOX2 in placental expression of FGR with normal. The placenta from FGR contained higher protein levels of NOX2 than that from healthy pregnancies, as shown by Western blot detection (Fig. 4 A). Since NOX2 is mainly expressed in macrophages, we further analyzed the relationship between different macrophages and the expression of NOX2. We found that M2-polarized macrophages expressed less NOX2 (Fig. 4 B). The upregulation of NOX2 expression in the FGR placenta may be due to the downregulation of the proportion of M2-polarized macrophages, which leads to impaired placental angiogenesis. DISCUSSION NOX2 can be expressed in porcine placental vascular endothelial cells [ 20 , 24 ] , but its distribution in human placental tissues is not clear. It’s seems that the abnormal expression of NOX2 may be associated with pregnancy-related diseases [ 25 , 26 ] . HBCs of the chorionic villi are usually adjacent to endothelial cells and trophoblasts, and their polarisation state will change in response to environmental stimuli [ 27 ] . However, whether HBCs regulate placental angiogenesis and their relationship with abnormal placentas have seldom been reported. For the first time, we have observed that NOX2 is expressed in HBCs within human placental tissue. This is evidenced by the co-localisation of NOX2 with a known marker protein (CD163) for HBCs. Other papers have reported that NOX2 is expressed in trophoblasts or endothelial cells [ 28 , 29 ] , but we witnessed a different phenomenon. By measuring CD31 in normal and FGR placentas, we found that the vascular density of FGR placentas was significantly lower than normal, which is consistent with other reports [ 30 ] . To investigate the effect of NOX2-expressing HBCs on endothelial cell function, we induced M2-polarised of THP1 cells. We demonstrated that macrophages expressing NOX2 decreased after M2-polarisation and could promote tube formation of placental vascular endothelial cells. Another study showed that NOX2 is involved in the regulation of macrophage adhesion to endothelial cells [ 28 , 31 ] . These results are fully supporting the role for macrophages in relation to vascular endothelial cells. This confirms our speculation that upregulation of placental NOX2 expression may inhibit angiogenesis in FGR placentas as a consequence of a reduction in M2-type macrophages. Consistent with an existing paper, the protective role of M2’ HBCs in FGR is indirectly demonstrated [ 32 ] . At the same time, the above findings strongly imply a role for NOX2 as an HBCs marker, in predicting FGR and reduced placental vascularity. Nevertheless, there are also some puzzling points in this topic, which will be improved in the subsequent research. NOX2 expression had no significant regulatory effect on the tube formation assay of HUVECs. This is inconsistent with what has been reported in the literature [ 24 ] , perhaps because of the differential regulatory role of NOX2 in different species. We did not isolate HBCs from placental villous progenitors, nor did we use macrophages for transfection of NOX2. So it can only be said that down-regulation of NOX2 expression by M2 macrophages correlates with angiogenesis, but not that NOX2 down-regulation promotes angiogenesis. The role of HBCs in promoting angiogenesis and chorionic villus growth needs to be further demonstrated. Conditioned medium of HBCs promotes vascular growth, which may be related to the ability of HBCs to secrete proangiogenic factors. The available literature [ 31 ] , including our conclusions, only proves that NOX2 does have a regulatory effect on placental vasculature. However, the specific mechanism of NOX2 regulation of endothelial cell function still needs to be further investigated. In summary, our data indicated that NOX2 was expressed in HBCs of the human placenta throughout pregnancy. Notably, NOX2 expression was increased in FGR, which may be a result of M2 polarisation concurrently inhibiting angiogenesis. Indirect proof the role of HBCs in placental vascular development. Therefore, this study provides a better understanding of the role of NOX2 in placental development and highlights that NOX2 serves as a potential therapeutic target for placental disorders. In the future, it may be possible in the future to treat placental vascular diseases by influencing HBCs polarity or altering NOX2 expression(Fig. 5 ). Declarations Ethics approval and Consent to participate This study was approved by the Scientific and Ethical Committee of the Shanghai First Maternity and Infant Hospital affiliated with Tongji University. Consent to participate was obtained from all participants, and informed consent obtained was written. Ethical number: KS212332. Consent for Publication All authors are consent the manuscript being published in BMC Pregnancy and Childbirth . Competing Interests The authors declared that they have no conflicts of interest to this work. Funding: This work was supported by the National Natural Science Foundation of China (grant number 82071663 to T Duan) and the National Key Research and Development Program of China (grant number 2022YFC2704703 to K Wang). Author Contribution RN.Z., L.Z., K.W. and T.D. conceived the study; RN.Z. performed the experiments; analyzed the data; RN.Z. wrote the original draft and K.W. wrote the review & editing; T.D. and K.W. approved the manuscript. Other work have done by RN.Z. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgements: None Data Availability All data generated or analysed during this study are included in this published article [and its supplementary information files] References American College of O. Gynecologists' Committee on Practice B-O, and the Society f-F. ACOG Practice Bulletin 204: Fetal Growth Restriction [J]. Obstet Gynecol. 2019;133(2):e97–109. Burton GJ, Jauniaux E. Pathophysiology of placental-derived fetal growth restriction [J]. Am J Obstet Gynecol. 2018;218(2S):S745–61. Teasdale F. Idiopathic intrauterine growth retardation: histomorphometry of the human placenta [J]. Placenta. 1984;5(1):83–92. 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Wiad Lek. 2020;73(2):215–9. Lim R, Acharya R, Delpachitra P et al. Activin and NADPH-oxidase in preeclampsia: insights from in vitro and murine studies [J]. Am J Obstet Gynecol, 2015, 212(1): 86 e1-12. Gaw SL, Hromatka BS, Ngeleza S et al. Differential Activation of Fetal Hofbauer Cells in Primigravidas Is Associated with Decreased Birth Weight in Symptomatic Placental Malaria [J]. Malar Res Treat, 2019, 2019: 1378174. Additional Declarations No competing interests reported. Supplementary Files SF1.tif Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-5419879","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":388217212,"identity":"31096789-d3e7-4635-9480-c896fee73f8d","order_by":0,"name":"Ruonan Zhang","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ruonan","middleName":"","lastName":"Zhang","suffix":""},{"id":388217213,"identity":"f6cb658b-3cad-4f40-90fc-6681970095f7","order_by":1,"name":"Lu Zhang","email":"","orcid":"","institution":"Shanghai First Maternity and Infant 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Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xuanchen","middleName":"","lastName":"Wei","suffix":""},{"id":388217224,"identity":"f9c02e41-736f-481d-a2b8-18580967cc2f","order_by":8,"name":"Xinyu Deng","email":"","orcid":"","institution":"Shanghai First Maternity and Infant Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Deng","suffix":""},{"id":388217225,"identity":"321f7c12-4e24-43e0-b9f2-a492a0eb2ea4","order_by":9,"name":"Ruomeng Bi","email":"","orcid":"","institution":"Shanghai First Maternity and Infant Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ruomeng","middleName":"","lastName":"Bi","suffix":""},{"id":388217226,"identity":"b4661e25-8192-4674-a993-542593d14f35","order_by":10,"name":"Tao Duan","email":"","orcid":"","institution":"Shanghai First Maternity and Infant Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Duan","suffix":""},{"id":388217227,"identity":"fe7e3898-135f-4ac3-9261-aa457cc6c1c9","order_by":11,"name":"Kai Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYBACCQbGBgiLvbHxQUIFAw8JWngOHzb4cIYoLXBWWprkzDYiHCbZfrhN4ueOWnlzhhwDad55dTLm7AcYP3zMwa1FmiexTbL3zHHDnQ1nDIx5tx3msexJYJacuQ23FjmGxLYbvG3HEgwO9hgk8247wGNwIIGNmRefFv6HbTf/grQc5jE4zDunjsfg/AP8WqQlEttu87bVJBgcY0tsnNnAzGNwg4AtkjMetv+WbTtguOEM82GGD8eAdt142IzXLxLn0x8bvm2rkze4/7D9R0JNnb3B+eSDHz7i0QIFh5E5sMjFD+qIUTQKRsEoGAUjFQAAEHhXm76UCLoAAAAASUVORK5CYII=","orcid":"","institution":"Shanghai First Maternity and Infant Hospital","correspondingAuthor":true,"prefix":"","firstName":"Kai","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-11-09 05:23:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5419879/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5419879/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71168756,"identity":"0168a5bd-bd69-433f-88b9-aaf5f138d748","added_by":"auto","created_at":"2024-12-11 18:27:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2337612,"visible":true,"origin":"","legend":"\u003cp\u003eLocation of NOX2 in human placental tissues. Immunolocalization of NOX2 in human placental villi from normal pregnancies or FGR was assessed using confocal microscopy (A. Bar, 200 μm). Green indicates positive staining for NOX2, golden and red indicates CD163 and CD31 respective, and blue indicates DAPI. (B) Statistical assay of blood vessel density in placenta of normal pregnancies and FGR. *p\u0026lt;0.05 indicates that the difference between the two groups was statistically significant.\u003c/p\u003e","description":"","filename":"F1.png","url":"https://assets-eu.researchsquare.com/files/rs-5419879/v1/6a4de77fa6cb9822707d0072.png"},{"id":71168941,"identity":"1bc8a419-f7ab-4f42-83ef-0b8059d08140","added_by":"auto","created_at":"2024-12-11 18:35:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":351795,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification the polarization state of macrophages. The mRNA levels of CCR-7, IL-6 and CXCL-10 for M1-polarized macrophages (A) and CCL-17, Arg-1 and CD-206 for M2-polarized macrophages (B) using rt-qPCR. ***p\u0026lt;0.001 indicates that the difference between M0 and M1/2-polarized macrophages was statistically significant.\u003c/p\u003e","description":"","filename":"F2.png","url":"https://assets-eu.researchsquare.com/files/rs-5419879/v1/7dbdc2435dba29856ab04a5c.png"},{"id":71168761,"identity":"31d0cbd0-3657-492a-8702-cd0355b6da3a","added_by":"auto","created_at":"2024-12-11 18:27:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7593757,"visible":true,"origin":"","legend":"\u003cp\u003eM2-polarized macrophages promotes angiogenesis of HUVECs. (A) Effects of M2-polarized macrophages on the migration of HUVECs. (B) Effects of M2-polarized macrophages on the Tubule formation of HUVECs. Each hole had 4 fields of view, and the number of cells in the field of view was calculated. The cell number is relative to three independent experiments. (***P\u0026lt;0.001) Bar, 200 μm. (C) Effects of M2-polarized macrophages on the proliferation of HUVECs.\u003c/p\u003e","description":"","filename":"F3.png","url":"https://assets-eu.researchsquare.com/files/rs-5419879/v1/6c0b6c14e672b89d3955340e.png"},{"id":71168942,"identity":"e2e6eb76-e5f3-4f4b-8e0e-92fd2d690fb0","added_by":"auto","created_at":"2024-12-11 18:35:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":597636,"visible":true,"origin":"","legend":"\u003cp\u003eThe relationship between NOX2 with FGR’ placental tissues and M2-polarized macrophages. (A) The protein levels of NOX2 from normal pregnancies and FGR, representative Western blot images are shown for NOX2 and β-actin. (B) The mRNA levels of NOX2 from M0 and M2-polarized macrophages detected by rt-qPCR (****P\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"F4.png","url":"https://assets-eu.researchsquare.com/files/rs-5419879/v1/76e107c9ce2a0c982d34f663.png"},{"id":71168757,"identity":"55962f94-9d7d-4de4-aea6-ee3ba537e092","added_by":"auto","created_at":"2024-12-11 18:27:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":98244,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstracts (Created in BioRender.com, thanks BioRender Support for science). The placenta of FGR may have fewer M2 macrophages and therefore express more NOX2. By affecting the proliferation, migration and tubulation of vascular endothelial cells, it ultimately affects the formation of placental blood vessels.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5419879/v1/5c3524295d87b2685136d087.png"},{"id":73925856,"identity":"bb61d81c-bb31-414f-8ede-ff6f6808ca8d","added_by":"auto","created_at":"2025-01-16 04:46:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9800438,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5419879/v1/3d998315-f104-4565-b071-8dbb0880d6d8.pdf"},{"id":71168759,"identity":"23e9b65f-7e3d-42ce-ad97-c77a66c6e49a","added_by":"auto","created_at":"2024-12-11 18:27:59","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3045072,"visible":true,"origin":"","legend":"","description":"","filename":"SF1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5419879/v1/77e390a4857db3f61ef759f6.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Macrophage M2 polarization with down-regulated NOX2 promotes placental angiogenesis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eFetal growth restriction(FGR), also known as intrauterine growth restriction(IUGR), is a prevalent complication during pregnancy that determinant the global morbidity and mortality rates. The pathogenesis of FGR is not fully understood, and its etiology may included maternal medical conditions, infectious diseases of fetal, placental disorders and umbilical cord abnormalities\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Placental-related fetal growth restriction arises primarily due to deficient remodeling of the uterine spiral arteries supplying the placenta and resultant malperfusion\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Therefore, one of the principal features of the placenta in cases of FGR is the reduction in volume, surface area, and vascularization of the intermediate and terminal villi that mediate maternal-fetal exchange\u003csup\u003e[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. The process of angiogenesis is balanced by numerous factors: secreted soluble molecules, hypoxia, shear stress, components of the extracellular matrix (ECM) and direct cell\u0026ndash;cell interaction with distinct cell types, such as pericytes, fibroblasts and tissue resident macrophages\u003csup\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\u003eNOX2, also known as gp91phox, is the prototype NADPH oxidase. The NOX family NADPH oxidases are proteins with the capability of transferring electrons across biofilms. These enzymes all possess the ability to transfer electrons through the plasma membrane and generate superoxides as well as other downstream reactive oxygen species (ROS)\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. NOX2 was initially identified in neutrophils and macrophages, and it widely distributed among a large number of tissues including placenta\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe macrophages in the placenta were mainly Hofbauer cells (HBCs), which are abundant in the villous stroma\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. These macrophages exhibit considerable plasticity and heterogeneity throughout pregnancy. Some reports suggest that in normal pregnancy, these Hofbauer cells demonstrate an M2-like phenotype and expressed some specific marker protein of M2 macrophages\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. However, in the context of placental disorders, the balance between M1 and M2 may be disrupted.\u003c/p\u003e \u003cp\u003eThe role of HBCs in the placenta is not yet clearly understood. However, given their location and paracrine capabilities, they are believed to be involved in early placental development, placental immunology, and the development and maturation of placental mesenchyme throughout pregnancy\u003csup\u003e[\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. There is emerging evidence that Hofbauer cells may serve important roles in both normal and abnormal pregnancy. To enhance our understanding of Hofbauer cell biology and their functions in health and disease, it is essential to recognize their heterogeneous nature [\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecently, the expression of NOX2 in porcine placenta was reported. Using porcine vascular endothelial cells (PVECs), increases placental oxidative stress and decreases placental angiogenesis possibly through the upregulation of NOX2\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. However, the exact location of NOX2 in human placental tissue and the function to angiogenesis still unknown.\u003c/p\u003e \u003cp\u003eWe thus predict that M1:M2 ratio of macrophage is associated with the expression of NOX2 which as an alter gene of FGR, eventually affects angiogenesis in the placenta. We initially discovered, through immunofluorescence staining, that NOX2 is mainly expressed on HBCs, which might be related to the reduction in blood vessel density in the placenta of FGR. Since the pathogenesis of FGR involves the disturbance of placental angiogenesis, we analyzed from the perspective of M1/M2 and found that M2 macrophages expressed lower levels of NOX2, which may be the reason for the increased expression of NOX2 in the placental of FGR. Our results provide insights into the biomarkers of HBCs and the relationship between NOX2 with angiogenesis. NOX2 can be used as a differential gene of FGR, which is also valuable for disease prediction and diagnosis and treatment.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy population and sample collection\u003c/h2\u003e \u003cp\u003eAll participants in the study were pregnant women who underwent natural delivery in Shanghai First Maternity and Infants Hospital from January 2022 to June 2023. The patients in this study were divided into normal late pregnancy (n\u0026thinsp;=\u0026thinsp;4, 37\u0026ndash;39 weeks), and Intrauterine growth restriction (n\u0026thinsp;=\u0026thinsp;6, 33\u0026ndash;39 weeks), as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In this study, the placental tissues of all patients were obtained from the operating room with the approval of the Scientific Ethics Committee of Shanghai First Maternity and Infants Hospital affiliated with Tongji University. After the blood clots were cleaned on ice with PBS, they were cut into 1 cm\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e1 cm tissue blocks and stored in liquid nitrogen in frozen storage tubes for further use.\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\u003eThe characteristics of women from normal and FGR pregnancies.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNormal (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFGR (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaternal age (yr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.707\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e29\u0026thinsp;\u0026plusmn;\u0026thinsp;2.345\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGestational age (wk)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e38.425\u0026thinsp;\u0026plusmn;\u0026thinsp;1.064\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e38.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.126\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGravidity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.433\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBirth weight (grams)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3077.5\u0026thinsp;\u0026plusmn;\u0026thinsp;202.993\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2837.5\u0026thinsp;\u0026plusmn;\u0026thinsp;243.323\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlacental weight (grams)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e550\u0026thinsp;\u0026plusmn;\u0026thinsp;72.572\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e512.5\u0026thinsp;\u0026plusmn;\u0026thinsp;90.381\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eSupplementary Table\u0026nbsp;1. Western Blot Antibodies\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eThe concentration of proteins was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. Proteins were separated by 10% SDS PAGE gels and transferred to PVDF membranes by gel electrophoresis and electroblotting. After blocking with 5% BSA, the blots were probed with primary antibodies at 4\u0026deg;C overnight. Then, the membranes were washed and incubated with secondary antibodies. Ultimately, proteins were visualized using enhanced chemiluminescence reagents (Thermo Fisher Scientific). The antibodies used are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The relative protein expression levels were analyzed by densitometry using ImageJ imaging analysis software (NIH).\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence staining\u003c/h3\u003e\n\u003cp\u003eStore frozen slices in -80\u0026deg;C. After three washes with ice-cold PBS, the tissue were fixed with 4% paraformaldehyde (PFA, Dingguo, China) for 30 minutes and then permeabilized with 0.1% Triton X-100 (Dingguo) for 15 minutes. Subsequently, the tissue were blocked with 5% bovine serum albumin (Thermo Fisher Scientific) for 1 hour at room temperature and incubated with an appropriate concentration of primary antibodies against NOX2, CD163 or CD31 overnight at 4\u0026deg;C. Then, the tissue were incubated with Alexa Fluor\u0026reg; 488-conjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies for 1 hour. The stained tissue were observed under a fluorescence confocal microscope. The antibodies used are listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eRNA isolation and quantitative RT-PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted using TRIzol (Thermo Fisher Scientific), and RNA was then reverse transcribed using the SuperScript First Strand cDNA System (Takara) according to the manufacturer's instructions. Quantitative RT-PCR (qRT-PCR) was performed using the SYBR Green PCR master mix (Takara) and the StepOnePlus PCR system (Thermo Fisher Scientific) according to the manufacturer's instructions. The housekeeping gene GAPDH was used as an endogenous control. The primer sequences are shown in Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eCell culture and establishment\u003c/h3\u003e\n\u003cp\u003eTHP-1 and 293T cells obtained from ATCC (Rockville, MD, USA) were maintained at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% air. Cells were cultured in RPMI 1640 medium (HyClone, Logan, UT, USA) or DMEM-high glucose medium (HyClone) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin.\u003c/p\u003e \u003cp\u003eHuman umbilical vein endothelial cells (HUVECs) were isolated using 0.1% collagenase II. Immediately after isolation, cells were cultured and steadily expanded in Endothelial Cell Medium (ECM, ScienCell, San Diego, CA, USA) containing 5% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% endothelial cell growth supplement (ECGS, ScienCell) under 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation assay\u003c/h2\u003e \u003cp\u003eTo test the effect of macrophage polarization on HUVECs proliferation, 2\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e3\u003c/sup\u003ecm cells per well were plated in 96-well culture plates in 100 \u0026micro;L of FBS-containing ECM medium. The medium was replaced with RPMI 1640 medium from M0/M1/M2 macrophages after 16 hours. After 48 hours, relative cell numbers were determined using MTS (Promega, Madison, WI, USA) reagent in a 96-well plate reader at 490 nm.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell migration assay\u003c/h3\u003e\n\u003cp\u003eThe migration of HUVECs was determined using a modified system according to the manufacturer\u0026rsquo;s instructions (BD Bioscience, Heidelberg, Germany). In this assay, cell migration was monitored using a 24-well transwell plate with inserts containing 8 \u0026micro;m pores (Costar, Corning, NY, USA). In brief, 1.5\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e4\u003c/sup\u003e cells were seeded on the upper side of the transwell inserts in serum-free medium. RPMI 1640 medium which cultured macrophages was added to the companion plate. Following incubation at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% air for 16 hours, the fluorescent stain calcein-AM (Thermo Fisher Scientific) was added to each chamber and incubated for 30 minutes. The numbers of migrated cells were determined by fluorescence image analysis (Nikon, Tokyo, Japan).\u003c/p\u003e\n\u003ch3\u003eTube formation assay\u003c/h3\u003e\n\u003cp\u003eThe formation of capillary-like structures was assessed in a 48-well plate using Growth Factor Reduced Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). HUVECs (1.5\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e3\u003c/sup\u003e cells/well) were resuspended in 500 uL ECM medium then plated on top of solidified Matrigel (200 \u0026micro;L/well) following incubation at 37\u0026deg;C for 1 h, and then replaced with RPMI 1640 medium from macrophages and ECM medium(500 uL and 200 uL respectively). They were viewed per hour under fluorescence microscopy (Nikon Eclipse Ti).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePMA exposure and polarization\u003c/h2\u003e \u003cp\u003eTHP-1 cells were counted about 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/ml, and seeded at 6mm dish in 1 mL of RPMI medium with PMA(100 ng/ml), maintained at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified tissue culture incubator. After 48 hours, THP-1 cells gradually adhere to the dish bottom and differentiation into M0 macrophages.\u003c/p\u003e \u003cp\u003eRPMI medium with PMA were removed and M0 macrophages were exposured to polarizing cytokines. The medium was replaced with fresh medium containing either IFNγ\u0026thinsp;+\u0026thinsp;LPS (20 ng/mL and 100 ng/mL respectively) or IL-4 (100 ng/mL). The cells were maintained in medium with cytokines for 48 hours.\u003c/p\u003e \u003cp\u003eMedium with cytokines were removed and replaced with RPMI 1640 medium containing 0.1% FBS for 24 hours, then collect macrophages\u0026rsquo; supernatant for co-cultured with HUVECs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eData are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and were analyzed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was analyzed by unpaired Student's \u003cem\u003et\u003c/em\u003e tests or one-way ANOVA. A p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLocation of NOX2 in human placental tissues\u003c/h2\u003e \u003cp\u003eTo explore the functional role of NOX2 in human placental angiogenesis, we first observed NOX2 distribution in human placenta by confocal detection. HBCs exhibit M2 macrophage phenotype so we used the CD163 protein to label the HBCs, our data showed that NOX2 was mainly expressed in the Hofbauer cells (HBCs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Using CD31 to represent vascular endothelial cell in the placenta, we observed less angiogenesis in the placenta with FGR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These findings indicate that NOX2 co-localises with HBC's known marker which is associated with reduced angiogenesis in the placenta of FGR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eInducing macrophages with M2 characteristics\u003c/h2\u003e \u003cp\u003eWe attempted to induce THP-1 cells to M2-polarized macrophages to mimic HBCs. First, we established the process and method of inducing macrophages\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. We analyzed the expression of markers for different macrophage phenotypes, CCR-7, IL-6 and CXCL-10 for M1-polarized macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and CCL-17, Arg-1 and CD-206 for M2-polarized macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) using rt-qPCR. Our experimental results show that we successfully induced M2-polarized macrophages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffect of M2-polarized macrophages on HUVECs angiogenesis\u003c/h2\u003e \u003cp\u003eWe speculate that the number of NOX2-expressing HBCs colocalized with endothelial cells is related to the blood vessels in the placenta. Therefore, we analyzed the impact of macrophages CM on network formation of primary HUVECs. Indeed, CM of M2-polarized macrophages induced the migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), network formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) of HUVECs, as compared with M0/M1-polarized macrophages\u0026rsquo; medium. The different polarization states of macrophages affect the formation of peripheral blood vessels, which was consistent with results described in other reports\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eExpression of NOX2 in FGR\u0026rsquo; placental tissues and M2-polarized macrophages\u003c/h2\u003e \u003cp\u003eWe compared NOX2 in placental expression of FGR with normal. The placenta from FGR contained higher protein levels of NOX2 than that from healthy pregnancies, as shown by Western blot detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Since NOX2 is mainly expressed in macrophages, we further analyzed the relationship between different macrophages and the expression of NOX2. We found that M2-polarized macrophages expressed less NOX2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The upregulation of NOX2 expression in the FGR placenta may be due to the downregulation of the proportion of M2-polarized macrophages, which leads to impaired placental angiogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eNOX2 can be expressed in porcine placental vascular endothelial cells\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, but its distribution in human placental tissues is not clear. It\u0026rsquo;s seems that the abnormal expression of NOX2 may be associated with pregnancy-related diseases \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. HBCs of the chorionic villi are usually adjacent to endothelial cells and trophoblasts, and their polarisation state will change in response to environmental stimuli\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. However, whether HBCs regulate placental angiogenesis and their relationship with abnormal placentas have seldom been reported.\u003c/p\u003e \u003cp\u003eFor the first time, we have observed that NOX2 is expressed in HBCs within human placental tissue. This is evidenced by the co-localisation of NOX2 with a known marker protein (CD163) for HBCs. Other papers have reported that NOX2 is expressed in trophoblasts or endothelial cells\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, but we witnessed a different phenomenon. By measuring CD31 in normal and FGR placentas, we found that the vascular density of FGR placentas was significantly lower than normal, which is consistent with other reports\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. To investigate the effect of NOX2-expressing HBCs on endothelial cell function, we induced M2-polarised of THP1 cells. We demonstrated that macrophages expressing NOX2 decreased after M2-polarisation and could promote tube formation of placental vascular endothelial cells. Another study showed that NOX2 is involved in the regulation of macrophage adhesion to endothelial cells\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. These results are fully supporting the role for macrophages in relation to vascular endothelial cells. This confirms our speculation that upregulation of placental NOX2 expression may inhibit angiogenesis in FGR placentas as a consequence of a reduction in M2-type macrophages. Consistent with an existing paper, the protective role of M2\u0026rsquo; HBCs in FGR is indirectly demonstrated\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. At the same time, the above findings strongly imply a role for NOX2 as an HBCs marker, in predicting FGR and reduced placental vascularity.\u003c/p\u003e \u003cp\u003eNevertheless, there are also some puzzling points in this topic, which will be improved in the subsequent research. NOX2 expression had no significant regulatory effect on the tube formation assay of HUVECs. This is inconsistent with what has been reported in the literature\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, perhaps because of the differential regulatory role of NOX2 in different species. We did not isolate HBCs from placental villous progenitors, nor did we use macrophages for transfection of NOX2. So it can only be said that down-regulation of NOX2 expression by M2 macrophages correlates with angiogenesis, but not that NOX2 down-regulation promotes angiogenesis. The role of HBCs in promoting angiogenesis and chorionic villus growth needs to be further demonstrated. Conditioned medium of HBCs promotes vascular growth, which may be related to the ability of HBCs to secrete proangiogenic factors. The available literature\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, including our conclusions, only proves that NOX2 does have a regulatory effect on placental vasculature. However, the specific mechanism of NOX2 regulation of endothelial cell function still needs to be further investigated.\u003c/p\u003e \u003cp\u003eIn summary, our data indicated that NOX2 was expressed in HBCs of the human placenta throughout pregnancy. Notably, NOX2 expression was increased in FGR, which may be a result of M2 polarisation concurrently inhibiting angiogenesis. Indirect proof the role of HBCs in placental vascular development. Therefore, this study provides a better understanding of the role of NOX2 in placental development and highlights that NOX2 serves as a potential therapeutic target for placental disorders. In the future, it may be possible in the future to treat placental vascular diseases by influencing HBCs polarity or altering NOX2 expression(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and Consent to participate\u003c/strong\u003e \u003cp\u003e This study was approved by the Scientific and Ethical Committee of the Shanghai First Maternity and Infant Hospital affiliated with Tongji University. Consent to participate was obtained from all participants, and informed consent obtained was written. Ethical number: KS212332.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for Publication\u003c/strong\u003e \u003cp\u003eAll authors are consent the manuscript being published in \u003cem\u003eBMC Pregnancy and Childbirth\u003c/em\u003e.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declared that they have no conflicts of interest to this work.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (grant number 82071663 to T Duan) and the National Key Research and Development Program of China (grant number 2022YFC2704703 to K Wang).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRN.Z., L.Z., K.W. and T.D. conceived the study; RN.Z. performed the experiments; analyzed the data; RN.Z. wrote the original draft and K.W. wrote the review \u0026amp; editing; T.D. and K.W. approved the manuscript. Other work have done by RN.Z. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eNone\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmerican College of O. Gynecologists' Committee on Practice B-O, and the Society f-F. ACOG Practice Bulletin 204: Fetal Growth Restriction [J]. Obstet Gynecol. 2019;133(2):e97\u0026ndash;109.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurton GJ, Jauniaux E. Pathophysiology of placental-derived fetal growth restriction [J]. Am J Obstet Gynecol. 2018;218(2S):S745\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeasdale F. 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Caffeic acid phenethyl ester suppresses monocyte adhesion to the endothelium by inhibiting NF-kappaB/NOX2-derived ROS signaling [J]. J Clin Biochem Nutr. 2016;58(3):174\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSavchenko SE, Dyadyk OO, Chaika KV, et al. Pathomorphological characteristics and immunohistochemical features of placentae from hiv-positive pregnant women with fetal growth retardation [J]. Wiad Lek. 2020;73(2):215\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLim R, Acharya R, Delpachitra P et al. Activin and NADPH-oxidase in preeclampsia: insights from in vitro and murine studies [J]. Am J Obstet Gynecol, 2015, 212(1): 86 e1-12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGaw SL, Hromatka BS, Ngeleza S et al. Differential Activation of Fetal Hofbauer Cells in Primigravidas Is Associated with Decreased Birth Weight in Symptomatic Placental Malaria [J]. Malar Res Treat, 2019, 2019: 1378174.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"NOX2, Hofbauer cells, fetal growth restriction, M2-polarized, angiogenesis","lastPublishedDoi":"10.21203/rs.3.rs-5419879/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5419879/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe polarised state of placental Hofbauer cells (HBCs) is frequently accompanied by alterations in inflammatory factors and marker proteins, which are crucial in normal pregnancy. NOX2, as one of NADPH oxidase, is commonly expressed in phagocytes and mediates oxidative stress. A recent paper reported that the upregulation of NOX2 in porcine placenta led to reduced angiogenesis through increased oxidative stress, which attracted our interesting. To investigate whether NOX2 expression in human placenta is the cause of reduced vascularity in placentas of Fetal growth restriction (FGR), we examined the expression and localisation of NOX2 in placental villous and analysed the expression of NOX2 in different macrophages subtypes as well as its effect on vascular endothelial function. Our results showed that NOX2 was predominantly expressed in HBCs of placental villi. Moreover M2 polarisation of macrophages were inversely associated with NOX2 expression and promoted angiogenesis. The expression of NOX2 was significantly elevated in the placenta of FGR compared with normal placentas, which might be related to the reduction of placental vasculature in FGR. These results suggested that NOX2 can be used as a marker of HBCs and as a result of M2 polarization, and is also deeply involved in the functional regulation of placental vascular endothelial cells. These evidences providing a potential target for the diagnosis and treatment of FGR.\u003c/p\u003e","manuscriptTitle":"Macrophage M2 polarization with down-regulated NOX2 promotes placental angiogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-11 18:27:54","doi":"10.21203/rs.3.rs-5419879/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"553f0b18-70bd-4f55-a61b-3175af93aa8b","owner":[],"postedDate":"December 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-06T09:08:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-11 18:27:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5419879","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5419879","identity":"rs-5419879","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}