C-reactive protein dissociation contributes to choroidal neovascularization in vivo: implications for age-related macular degeneration    

C-reactive protein dissociation contributes to choroidal neovascularization in vivo: implications for age-related macular degeneration | 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 C-reactive protein dissociation contributes to choroidal neovascularization in vivo: implications for age-related macular degeneration Maria Hernandez, Sara Romero-Vazquez, Sergio Recalde, Jaione Bezunartea, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5678488/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Choroidal neovascularization (CNV) and inflammation processes play an important role in retinal disease development and C-reactive protein (CRP) has been shown to contribute to development diseases like Age-related macular degeneration (AMD). Our aim was to evaluate whether monomeric and pentameric CRP (pCRP, mCRP) isoforms contribute to CNV in vivo and to characterize the mechanism of CRP dissociation in-vivo and in vitro. Both CRP isoforms were intravitreally (IVT) or intravenously (IV) injected in mice, CNV was laser-induced, retinography and fluorescein angiography were performed to evaluate edema. Lectin, mCRP, F4/80 and C5b9 localization were assessed by immunofluorescence and visualized under a confocal microscope. Intensity of fluorescence of mCRP (IF mCRP) was also quantified. To confirm pCRP dissociation in ARPE-19 cells and mice, pCRP was coupled to a fluorochrome and IVT injected. A statistical increase in CNV areas was observed in pCRP IVT injected males (p<0.05) while a statistical decrease was shown in females (p<0.05). After IV injection, pCRP males showed an increase in CNV areas only vs. mCRP injected mice (p<0.05) and in females the injection of pCRP injected mice showed higher CNV areas vs. vehicle (p<0.05) and vs. mCRP injected mice (p<0.05). Retinal edema after IVT CRP injection was observed mainly in mCRP injected mice. In females there was an IF mCRP statistical decrease in pCRP IVT injected mice vs. vehicle and a statistical increase in pCRP IV injected mice vs. vehicle (p<0.05). Mice injected with IVT isoforms showed F4/80 positive cells and C5b-9 deposition around the CNV areas. mCRP labeling was observed in the intercellular space of the endothelial cells in the angiogenic area and detected in pCRP IVT injected animals, demonstrating the dissociation of pCRP into mCRP both in vitro and in vivo in proinflammatory microenvironments. In conclusion, CRP administration increased the area of CNV and the edema observed in the subretinal space, suggesting that CRP is activated in the CNV inflammatory environment. In addition, we demonstrated that pCRP dissociates in vivo into mCRP in damaged areas close to CNV, hypothesizing that the CNV process is exacerbated by mCRP. Age macular degeneration choroidal neovascularization inflammation gender retinal pigment epithelium complement pathway immune system C-reactive protein Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Full Text Additional Declarations The authors declare no competing interests. Supplementary Files TableS1.pdf Table S1 Antibodies used in the study. FigS1.tif Fig. S1 Retinographies and lectin/mCRP immunofluorescence one week postlaser. CNV showed similar immunolabelling for mCRP (red) and lectin staining (green) in males (A, B) and females (C, D) on intact eyes (A, C) and IVT injection of vehicle (B, D). Scale bar: 100 µm. FigS2.tif Fig. S2 ZO-1 immunofluorescence in CNV areas in RPE-flatmount after IV injection. In RPE-flatmounts after vehicle (A), we observed ZO-1 staining (white) alteration in detail in RPE layer (B, 63X) due to laser induction. After pCRP injection in males (C, D) and females (E, F) similar staining was observed around the angiogenic lesion as we showed in detail in RPE layer (D, F, 63X). After mCRP injection in males (G, H) and females (I, J), similar results were observed in ZO-1 staining in detail in males (H, 40X) and females (J, 40X). DAPI (blue) label nuclei. Scale bar: 50 µm (A, C, E, G, I), 20 µm (B, D, FH, J). The yellow outlined region indicated the area magnified at 63X. Abbreviations: intravenous=IV, mCRP=monomeric CRPpCRP=pentameric CRP, retinal pigment epithelium=RPE and Zonula occludens-1=ZO-1. FigS3.tif Fig. S3 Percentage of intensity of fluorescence of mCRP (IF-mCRP) after intravitreal (IVT) and intravenous (IV) injection. The quantification was performed in total CNV lesions (A, D) and by gender in males (B, E) and females (C, F). In total samples, IF mCRP were similar in all groups. Males after two isoforms did not show changes in IF mCRP. After IVT injection of mCRP in females IF mCRP was statistically significant increased vs. pCRP (* p<0.05) and IVT injection of pCRP showed a statistical decrease vs. vehicle (* p<0.05). After IV injection of pCRP in females resulted in an increase of IF mCRP vs. vehicle and almost vs. mCRP (p=0.071). Abbreviations: intensity of immunofluorescence mCRP=IF mCRP, intravitreal=IVT, intravenous=IV, monomeric CRP=mCRP and pentameric CRP=pCRP. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions 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-5678488","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":403470913,"identity":"9c03b6c5-e609-4198-a996-590bac38ee53","order_by":0,"name":"Maria 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Navarra","correspondingAuthor":false,"prefix":"","firstName":"Idoia","middleName":"","lastName":"Belza-Zuazu","suffix":""},{"id":403470919,"identity":"90d93534-7e0a-48cd-81c2-f18088f2c290","order_by":6,"name":"Alfredo Garcia-Layana","email":"","orcid":"","institution":"Retinal Pathologies and New Therapies Group, Experimental Ophthalmology Laboratory, Department of Ophthalmology, Clínica Universidad de Navarra","correspondingAuthor":false,"prefix":"","firstName":"Alfredo","middleName":"","lastName":"Garcia-Layana","suffix":""},{"id":403470920,"identity":"ab627d93-d73e-499a-b645-a181868feba8","order_by":7,"name":"Alfredo Adan","email":"","orcid":"","institution":"Institut d’Investigacions Biomèdiques Agustí Pi i Sunyer (IDIBAPS), Ocular Inflammation Group","correspondingAuthor":false,"prefix":"","firstName":"Alfredo","middleName":"","lastName":"Adan","suffix":""},{"id":403470921,"identity":"8c64cd44-9253-4bd3-967f-6c9c8b310d09","order_by":8,"name":"Patricia Fernandez-Robredo","email":"","orcid":"","institution":"Retinal Pathologies and New Therapies Group, Experimental Ophthalmology Laboratory, Department of Ophthalmology, Clínica Universidad de Navarra","correspondingAuthor":false,"prefix":"","firstName":"Patricia","middleName":"","lastName":"Fernandez-Robredo","suffix":""},{"id":403470922,"identity":"5ece0876-02cf-4aae-9677-0d3df5e39dd8","order_by":9,"name":"Blanca Molins","email":"","orcid":"","institution":"Institut d’Investigacions Biomèdiques Agustí Pi i Sunyer (IDIBAPS), Ocular Inflammation Group","correspondingAuthor":false,"prefix":"","firstName":"Blanca","middleName":"","lastName":"Molins","suffix":""}],"badges":[],"createdAt":"2024-12-19 16:38:15","currentVersionCode":2,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5678488/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-5678488/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":74371803,"identity":"482c3264-159a-473b-899f-cd97394181a1","added_by":"auto","created_at":"2025-01-21 15:37:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":64283,"visible":true,"origin":"","legend":"\u003cp\u003eScheme of timing and experimental procedures performed in the study. Abbreviations: monomeric C-reactive protein=mCRP, pentameric C-reactive protein=pCRP, day=D, retinography=R, fluorescein angiography=FA, choroidal neovascularization=CNV, intravitreal=IVT, intravenous=IV.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5678488/v2/5321217173fac81902125f36.png"},{"id":74371411,"identity":"70d9fa0e-da09-4dc3-998c-caa783916d83","added_by":"auto","created_at":"2025-01-21 15:29:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1143215,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of CNV areas after IVT and IV mice injected with CRP isoforms. Analysis of the total area of CNV lectin staining in IVT mice (A and B), in males (A1-A3, C) and females (A4-A6, D). pCRP injected group showed a statistically significant increase in CNV areas vs. vehicle in males (*p\u0026lt;0.05, C), and females showed an increase in mCRP injected group vs. vehicle and vs. pCRP (*p\u0026lt;0.05, D). The results in IV injected mice showed a statistically significant total increase area of CNV in pCRP injected group vs. mCRP group (*p\u0026lt;0.05, F). In males this increase is similar in pCRP injected mice vs. mCRP (*p\u0026lt;0.05, E1-E3, G) and in females vs. V (*p\u0026lt;0.05) and mCRP injected mice (*p\u0026lt;0.05, E4-E6, H). Arrows indicate an example of the representative CNV area stained with lectin. Abbreviations: choroidal neovascularization=CNV, intravitreal=IVT, intravenous=IV, V=vehicle, mCRP=monomeric CRP, pCRP=pentameric CRPand optic nerve =ON. Scale bar: 500 µm.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5678488/v2/86a1758b8d756b5114079604.png"},{"id":74371802,"identity":"241802cf-9861-4845-bc97-09d06419a54c","added_by":"auto","created_at":"2025-01-21 15:37:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3265234,"visible":true,"origin":"","legend":"\u003cp\u003eMultimodal imaging of eye fundus in mice IVT and IV injected. Retinography immediately after laser induction in males (A1, B1, C1) and females (A4, B4, C4) after IVT injection and in males (D1, E1, F1) and females (D4, E4, F4) after IV injection. Retinography after 3 days from laser in males (A2, B2, C2) and females (A5, B5, C5) after IVT injection and males (D2, E2, F2) and females (A5, B5, C5) after IVT injection and retinography after 5 days from laser in males (D2, E2, F2) and females (D5, E5, F5) after IV injection, respectively. Fluorescein angiography (FA) was performed after 3 days from laser in males (A3, B3, C3) and females (A6, B6, C6) after IVT injection and 5 days after laser in males (D3, E3, F3) and females (D6, E6, F6) after IV injection respectively. Retinographies (in yellow frame) C7 and C8 showed edema in detail in males after IVT injection of mCRP (black and yellow arrows) and retinographies images C10 and C11 showed edemas in detail in females after mCRP injection (black and yellow arrows). FA (in yellow frame) C9 showed edema after fluorescein injection in males injected with mCRP IVT and FA images C12 showed edema after fluorescein injection in females injected with mCRP IVT. Abbreviations: intravitreal=IVT, intravenous=IV, monomeric CRP=mCRP, pentameric CRP=pCRP. Asterisk showed the group statistically significant in lectin areas quantification.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5678488/v2/4b5ab44fb41482680ef1b260.png"},{"id":74371443,"identity":"07d0f3cf-13c2-43ba-8bd1-842b3e91bfa4","added_by":"auto","created_at":"2025-01-21 15:29:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":12197401,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence of mCRP and lectin staining in CNV areas after IVT injection. RPE-flatmount stained with lectin, in green, indicates the CNV areas and mCRP labeling is shown in red. RPE-flatmounts after vehicle injection in four areas performed (A1-A4, 10X) and in detail in CNV area number 2 (B1-B4, 40X). A total mCRP immunofluorescence in mice injected with vehicle is similar to the two isoforms. In males (C, D, G, H) we observed a similar immunolabelling in four areas of CNV injected with pCRP (C1-C4, 10X) and in detail in CNV area 3 (D1-D4, 40X) vs. vehicle and vs. mCRP injected in four areas (G1-G4, 10X) and in detail in CNV area 2 (H1-H4, 40X). In females injected with pCRP (E, F) there was a decrease in labelling mCRP in four areas (E1, E4, 10X) and in detail in CNV area 1 (F1-F4, 40X) vs. pCRP injected mice in four areas (I1-I4, 10X) and in detail in CNV area 1 (J1-J4, 40X). The outlined regions indicate the areas of CNV around the optic nerve (ON). Hoechst (blue) label nuclei. Scale bar: 100 µm (A, C, E, G, I), 20 µm (B, D, F, H, J). Abbreviations: choroidal neovascularization=CNV, intravitreal=IVT, optic nerve=ON, monomeric CRP=mCRP and pentameric CRP=pCRP.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5678488/v2/c5970e5595c2ae05b788f721.png"},{"id":74371452,"identity":"4d1d4925-7c4c-4f8d-8e82-2e63715592a6","added_by":"auto","created_at":"2025-01-21 15:29:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":25987557,"visible":true,"origin":"","legend":"\u003cp\u003eMagnification images of immunofluorescence of mCRPand lectin staining in CNV areas after IVT injection. We observed in detail a specific localization of the mCRP immunofluorescence (red) that did not colocalize with endothelial cells labelled with lectin (green) in vehicles (A). After pCRP injection (B, C) and mCRP injection (F, G) in males, and females respectively (D, E) and images (H, I), we observed at higher magnification (40-63X) in orthogonal projection (C, E and G, I) the similar localization of the mCRP immunolabeling, between endothelial cells. Hoechst (blue) label nuclei. Scale bar: 20 µm. Abbreviations: intravitreal=IVT, monomeric CRP=mCRP and pentameric CRP=pCRP.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5678488/v2/a0296ee9d708b8d85b6ecb7b.png"},{"id":74371425,"identity":"8745673f-8c0e-46b6-bb68-29ff0b114e77","added_by":"auto","created_at":"2025-01-21 15:29:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13013004,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence of mCRP and lectin staining in CNV areas after IV injection. RPE-flatmount stained with lectin, in green, indicates the CNV areas and mCRP labeling is shown in red. RPE-flatmounts after vehicle injection in four areas (A1-A4, 10X) and in detail in CNV area number 1 (B1-B4, 40X). A total mCRP immunofluorescence in mice injected with vehicles is similar to the two isoforms. In males (C, D, G, H) we observed a similar mCRP labelling in pCRP injected mice in four areas of CNV (C1-C4, 10X) and in detail in CNV area 1 (D1-D4, 40X), and in mCRP injected mice in four areas of CNV (G1-G4, 10X) and in detail in CNV area1 (H1-H4, 40X). In females injected with pCRP (E, F) in four areas of CNV (E1-E4, 10X) and in detail in CNV area 1 (F1-F4, 40X), we showed a statistical increase of mCRP labelling in pCRP injected mice vs. and vs mCRP injected mice in four areas of CNV (I1-I4) and in detail in CNV area 3 (J1-J4, 40X). The outlined regions indicate the areas of CNV around the optic nerve (ON). Hoechst (blue) label nuclei. Scale bar: 100 µm (A, C, E, G, I). Abbreviations: Choroidal neovascularization=CNV, intravenous=IV, optic nerve=ON, monomeric CRP=mCRP and pentameric CRP=pCRP\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5678488/v2/592ac33ae1a266adb7860f2d.png"},{"id":74371457,"identity":"b82e12cc-b9d4-4a0d-919b-2ecbbfaef27c","added_by":"auto","created_at":"2025-01-21 15:29:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":14495255,"visible":true,"origin":"","legend":"\u003cp\u003eMagnification images of immunofluorescence of mCRP and lectin staining in CNV areas after IV injection. The results were similar to IVT injection of the two CRP isoforms. We observed in detail a specific localization of the mCRP labelling (red) that did not colocalize with endothelial cells labelled with lectin (green) in vehicles (A). After pCRP injection (B, C) and mCRP injection (F, G) in males, and females respectively (D, E) and (H, I), we observed at higher magnification (40-63X) in orthogonal projection (C, G and E, I) the similar localization of the mCRP immunolabeling, between endothelial cells. Hoechst (blue) label nuclei. Scale bar: 20 µm. Abbreviations: intravenous=IV, monomeric CRP=mCRP and pentameric CRP=pCRP.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-5678488/v2/b6e7c993c65dd4e7bc76d2c0.png"},{"id":74371407,"identity":"9e5867b6-c933-41b8-bda2-9aaba98b5a50","added_by":"auto","created_at":"2025-01-21 15:29:56","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":927526,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro porcine RPE cells incubated with pCRP-555. RPE cells, control (A, no LPS) and LPS incubated (B) in transwell inserts were incubated with pCRP-555 (red) (C, G) and immunolabeled with mCRP (green) (B, F) and a pCRP-555 fluorochrome (red). Two isoforms were observed only in LPS-RPE cells. DAPI (blue) (D, H) label nuclei. Scale bar: 20 µm. Abbreviations: Lipopolysaccharide=LPS, monomeric CRP=mCRP, pentameric CRP=pCRPand retinal pigment epithelium=RPE.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-5678488/v2/2c25e3e6da1d6a72ff4c0b6e.png"},{"id":74371807,"identity":"b61af696-126d-4439-a57e-90317f8b47fc","added_by":"auto","created_at":"2025-01-21 15:37:57","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":11988329,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo laser CNV mice model IVT injected with pCRP-555. RPE-flatmonts were immunolabeled with mCRP (green) and visualized the pCRP (red) in CNV area (A-D) and close to CNV in RPE and ONL (E-O). Super-resolution images showed the two isoforms dissociated in RPE (E-H) and in RPE-ONL layer (I-O). No colocalization between the two isoforms was observed in orthogonal projection (D, H, O). In Figure D we showed the different retinal layers in orthogonal projection. The outlined regions indicate the area magnified in detail. Hoechst (blue) label nuclei. Scale bar: 20µm (A-D), 10 µm (L), 2 µm (I-K, M-O). Abbreviations: choroidal neovascularization=CNV, retinal pigment epithelium=RPE, intravitreal=IVT, monomeric CRP=mCRP, pentameric CRP=pCRP, outer nuclear layer=ONL, inner nuclear layer=INL, ganglion cell layer=GCL.\u003c/p\u003e","description":"","filename":"Fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-5678488/v2/561bfe97939fca8c8280ea71.png"},{"id":74371813,"identity":"449e0390-787f-4fb4-b9f9-d4f1e688f0a1","added_by":"auto","created_at":"2025-01-21 15:37:58","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":24903652,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of mCRP and F4/80 immunofluorescence in a representative lectin-CNV area after IVT injection. In RPE-flatmounts an increase of mCRP (red) and F4/80 (white) labeling was observed in mice injected with vehicle (A-I) as we observed in an example of a CNV area in detail in border of the CNV area with the different markers (B-H, 40X) and in orthogonal projection (I, 63X). After pCRP (J-Q) and mCRP (R-Z) injection in mice, increase in mCRP labeling (O, X) and a strong F4/80 immunolabeling (Q, Z) were observed compared to vehicles (F and G respectively). The outlined region in image A was shown in detail in B-H images. DAPI (blue) label nuclei. Scale bar: 20 µm. Abbreviations: choroidal neovascularization=CNV, intravitreal=IVT, monomeric CRP=mCRP and pentameric CRP=pCRP.\u003c/p\u003e","description":"","filename":"Fig10.png","url":"https://assets-eu.researchsquare.com/files/rs-5678488/v2/0f6649541bb791b2f56ffbbf.png"},{"id":74371444,"identity":"b66bef15-a81e-4487-a3de-dc308445f503","added_by":"auto","created_at":"2025-01-21 15:29:57","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":4497112,"visible":true,"origin":"","legend":"\u003cp\u003eC5b9 immunofluorescence images in CNV areas in mice injected with IVT pCRP-555. In In RPE layer, a light pCRP (red) labelling was observed in CNV areas (A) and mCRP (green) was close to C5b9 (white) (C), in super-resolution image at 63X (D-F) and in orthogonal projection image (G). In retinal ganglion cell layer (RGCL) (H-N), C5b9 was observed in cells around the mCRP positive vessels (L-N) with super-resolution images (H-M) and orthogonal projection images (N). Hoechst (blue) label nuclei. Scale bar: 50 µm (A-C, H, I), 10 µm (D-F) and 20 µm (G, J-N). Abbreviations: choroidal neovascularization=CNV, intravitreal=IVT, monomeric CRP=mCRP, pentameric CRP=pCRP, retinal pigment epithelium=RPE, retinal ganglion cell layer=RGCL.\u003c/p\u003e","description":"","filename":"Fig11.png","url":"https://assets-eu.researchsquare.com/files/rs-5678488/v2/5dfd1b1f9c983be3209e5445.png"},{"id":74372322,"identity":"90318c2a-0e50-4066-9756-5123bc069cdc","added_by":"auto","created_at":"2025-01-21 15:54:39","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7817337,"visible":true,"origin":"","legend":"","description":"","filename":"Hernandezetal2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5678488/v2_covered_9eb08d54-18e3-4b2b-94ba-54066e2ed756.pdf"},{"id":74371406,"identity":"2be5b565-f9cd-4695-96d4-6eeaaef5997b","added_by":"auto","created_at":"2025-01-21 15:29:56","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":111122,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1 Antibodies used in the study.\u003c/p\u003e","description":"","filename":"TableS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5678488/v2/7908569447d8de90900b3b17.pdf"},{"id":74371805,"identity":"662ca044-7460-43fd-9d06-d2123a933b13","added_by":"auto","created_at":"2025-01-21 15:37:56","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":699254,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S1 Retinographies and lectin/mCRP immunofluorescence one week postlaser. CNV showed similar immunolabelling for mCRP (red) and lectin staining (green) in males (A, B) and females (C, D) on intact eyes (A, C) and IVT injection of vehicle (B, D). Scale bar: 100 µm.\u003c/p\u003e","description":"","filename":"FigS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5678488/v2/376313922b0be365c0087f66.tif"},{"id":74371806,"identity":"9df20eef-9f5b-48ca-bd15-3da539abd446","added_by":"auto","created_at":"2025-01-21 15:37:56","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":6722068,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S2 ZO-1 immunofluorescence in CNV areas in RPE-flatmount after IV injection. In RPE-flatmounts after vehicle (A), we observed ZO-1 staining (white) alteration in detail in RPE layer (B, 63X) due to laser induction. After pCRP injection in males (C, D) and females (E, F) similar staining was observed around the angiogenic lesion as we showed in detail in RPE layer (D, F, 63X). After mCRP injection in males (G, H) and females (I, J), similar results were observed in ZO-1 staining in detail in males (H, 40X) and females (J, 40X). DAPI (blue) label nuclei. Scale bar: 50 µm (A, C, E, G, I), 20 µm (B, D, FH, J). The yellow outlined region indicated the area magnified at 63X. Abbreviations: intravenous=IV, mCRP=monomeric CRPpCRP=pentameric CRP, retinal pigment epithelium=RPE and Zonula occludens-1=ZO-1.\u003c/p\u003e","description":"","filename":"FigS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-5678488/v2/f1b49d318ef55a25c6a9829d.tif"},{"id":74371414,"identity":"4b788706-f874-4ac6-8eb0-bd235f7b97bc","added_by":"auto","created_at":"2025-01-21 15:29:56","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1093648,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S3 Percentage of intensity of fluorescence of mCRP (IF-mCRP) after intravitreal (IVT) and intravenous (IV) injection. The quantification was performed in total CNV lesions (A, D) and by gender in males (B, E) and females (C, F). In total samples, IF mCRP were similar in all groups. Males after two isoforms did not show changes in IF mCRP. After IVT injection of mCRP in females IF mCRP was statistically significant increased vs. pCRP (* p\u0026lt;0.05) and IVT injection of pCRP showed a statistical decrease vs. vehicle (* p\u0026lt;0.05). After IV injection of pCRP in females resulted in an increase of IF mCRP vs. vehicle and almost vs. mCRP (p=0.071). Abbreviations: intensity of immunofluorescence mCRP=IF mCRP, intravitreal=IVT, intravenous=IV, monomeric CRP=mCRP and pentameric CRP=pCRP.\u003c/p\u003e","description":"","filename":"FigS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-5678488/v2/5c1b6c3c2045dc39a87d2c31.tif"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"C-reactive protein dissociation contributes to choroidal neovascularization in vivo: implications for age-related macular degeneration ","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"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":"Age macular degeneration, choroidal neovascularization, inflammation, gender, retinal pigment epithelium, complement pathway, immune system, C-reactive protein","lastPublishedDoi":"10.21203/rs.3.rs-5678488/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5678488/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChoroidal neovascularization (CNV) and inflammation processes play an important role in retinal disease development and C-reactive protein (CRP) has been shown to contribute to development diseases like Age-related macular degeneration (AMD). Our aim was to evaluate whether monomeric and pentameric CRP (pCRP, mCRP) isoforms contribute to CNV in vivo and to characterize the mechanism of CRP dissociation in-vivo and in vitro. Both CRP isoforms were intravitreally (IVT) or intravenously (IV) injected in mice, CNV was laser-induced, retinography and fluorescein angiography were performed to evaluate edema. Lectin, mCRP, F4/80 and C5b9 localization were assessed by immunofluorescence and visualized under a confocal microscope. Intensity of fluorescence of mCRP (IF mCRP) was also quantified. To confirm pCRP dissociation in ARPE-19 cells and mice, pCRP was coupled to a fluorochrome and IVT injected. A statistical increase in CNV areas was observed in pCRP IVT injected males (p\u0026lt;0.05) while a statistical decrease was shown in females (p\u0026lt;0.05). After IV injection, pCRP males showed an increase in CNV areas only vs. mCRP injected mice (p\u0026lt;0.05) and in females the injection of pCRP injected mice showed higher CNV areas vs. vehicle (p\u0026lt;0.05) and vs. mCRP injected mice (p\u0026lt;0.05). Retinal edema after IVT CRP injection was observed mainly in mCRP injected mice. In females there was an IF mCRP statistical decrease in pCRP IVT injected mice vs. vehicle and a statistical increase in pCRP IV injected mice vs. vehicle (p\u0026lt;0.05). Mice injected with IVT isoforms showed F4/80 positive cells and C5b-9 deposition around the CNV areas. mCRP labeling was observed in the intercellular space of the endothelial cells in the angiogenic area and detected in pCRP IVT injected animals, demonstrating the dissociation of pCRP into mCRP both in vitro and in vivo in proinflammatory microenvironments. In conclusion, CRP administration increased the area of CNV and the edema observed in the subretinal space, suggesting that CRP is activated in the CNV inflammatory environment. In addition, we demonstrated that pCRP dissociates in vivo into mCRP in damaged areas close to CNV, hypothesizing that the CNV process is exacerbated by mCRP.\u003c/p\u003e","manuscriptTitle":"C-reactive protein dissociation contributes to choroidal neovascularization in vivo: implications for age-related macular degeneration ","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2025-01-21 15:29:51","doi":"10.21203/rs.3.rs-5678488/v2","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}},{"code":1,"date":"2025-01-17 10:57:27","doi":"10.21203/rs.3.rs-5678488/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":"0b358dc6-a597-4473-ab45-f60c44ec2adf","owner":[],"postedDate":"January 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-22T07:23:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-21 15:29:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-5678488","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5678488","identity":"rs-5678488","version":["v2"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}