c4a3179a321be85ee961a65bcdeae45c 282304.pdf 6ea59deb8cea4399a51b4fbd412377fbde54c036 282304.pdf 3989a25de4933c5301e53e7a562602f7767458788872fadf0ba11e7abcdcce77 282304.pdf Title: Retinal Pigment Epithelium-Secreted VEGF-A Induces Alpha-2-Macroglobulin Expression in Endothelial Cells Subject: Alpha-2-macroglobulin (A2M) is a protease inhibitor that regulates extracellular matrix (ECM) stability and turnover. Here, we show that A2M is expressed by endothelial cells (ECs) from human eye choroid. We demonstrate that retinal pigment epithelium (RPE)-conditioned medium induces A2M expression specifically in ECs. Experiments using chemical inhibitors, blocking antibodies, and recombinant proteins revealed a key role of VEGF-A in RPE-mediated A2M induction in ECs. Furthermore, incubation of ECs with RPE-conditioned medium reduces matrix metalloproteinase-2 gelatinase activity of culture supernatants, which is partially restored after A2M knockdown in ECs. We propose that dysfunctional RPE or choroidal blood vessels, as observed in retinal diseases such as age-related macular degeneration, may disrupt the crosstalk mechanism we describe here leading to alterations in the homeostasis of choroidal ECM, Bruch’s membrane and visual function. Keywords: Alpha-2-macroglobulin (A2M); retinal pigment epithelium (RPE); vascular endothelial growth factor-A (VEGF-A); endothelial cells (ECs); extracellular matrix (ECM); matrix metalloproteinase-2 (MMP-2); protease inhibitor; age-related macular degeneration (AMD); Bruch’s membrane Author: Guillermo L. Lehmann, Michael Ginsberg, Daniel J. Nolan, Cristina Rodríguez, José Martínez-González, Shemin Zeng, Andrew P. Voigt, Robert F. Mullins, Shahin Rafii, Enrique Rodriguez-Boulan and Ignacio Benedicto Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sat Sep 24 09:27:30 2022 CEST ModDate: Sat Sep 24 09:32:11 2022 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1466518 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- TLGRFU+VnURWPalladioL Type 1 Custom yes yes no 10 0 PQVHWF+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 DFNCPT+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 IIQDJD+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 IOJCNK+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 PFKOHU+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 46 0 VIWIOF+CMSY10 Type 1 Builtin yes yes no 71 0 ROWTUD+PazoMath Type 1 Builtin yes yes no 75 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 79 0 KHEHMI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 99 0 KHEHMK+PalatinoLinotype TrueType WinAnsi yes yes no 102 0 KHEHMM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 105 0 KHEHMI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 119 0 KHEHMJ+PalatinoLinotype CID TrueType Identity-H yes yes yes 122 0 KHEHMK+PalatinoLinotype TrueType WinAnsi yes yes no 128 0 KHEHMM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 131 0 KHEHMI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 149 0 KHEHMJ+PalatinoLinotype CID TrueType Identity-H yes yes yes 152 0 KHEHMK+PalatinoLinotype TrueType WinAnsi yes yes no 158 0 KHEHMM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 161 0 KHEHMI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 183 0 KHEHMK+PalatinoLinotype TrueType WinAnsi yes yes no 186 0 KHEHMM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 189 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-10-07 02:27:15 CEST RepresentationInformation: 282304.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-10-06 09:55:55 CEST Size: 1466518 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 509 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Children: Item: Title: Human RPE/Choroid scRNAseq Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Cell Culture Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Conditioned Medium and A2M Induction Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Real-Time PCR Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Western Blot Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: ELISA Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Cell Viability Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Transient Transfection and Luciferase Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Zymography Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Lentiviral Production and shRNA Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Children: Item: Title: A2M Expression Is Enriched in Human Choroidal and Retinal ECs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: RPE Conditioned Medium Induces A2M Expression in ECs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: RPE-Secreted VEGF-A Induces A2M Expression in ECs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: RPE-Induced A2M Expression in ECs Reduces MMP-2 Activity in Culture Supernatants Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Info: Title: Retinal Pigment Epithelium-Secreted VEGF-A Induces Alpha-2-Macroglobulin Expression in Endothelial Cells Author: Guillermo L. Lehmann, Michael Ginsberg, Daniel J. Nolan, Cristina Rodríguez, José Martínez-González, Shemin Zeng, Andrew P. Voigt, Robert F. Mullins, Shahin Rafii, Enrique Rodriguez-Boulan and Ignacio Benedicto Subject: Alpha-2-macroglobulin (A2M) is a protease inhibitor that regulates extracellular matrix (ECM) stability and turnover. Here, we show that A2M is expressed by endothelial cells (ECs) from human eye choroid. We demonstrate that retinal pigment epithelium (RPE)-conditioned medium induces A2M expression specifically in ECs. Experiments using chemical inhibitors, blocking antibodies, and recombinant proteins revealed a key role of VEGF-A in RPE-mediated A2M induction in ECs. Furthermore, incubation of ECs with RPE-conditioned medium reduces matrix metalloproteinase-2 gelatinase activity of culture supernatants, which is partially restored after A2M knockdown in ECs. We propose that dysfunctional RPE or choroidal blood vessels, as observed in retinal diseases such as age-related macular degeneration, may disrupt the crosstalk mechanism we describe here leading to alterations in the homeostasis of choroidal ECM, Bruch’s membrane and visual function. Keywords: Alpha-2-macroglobulin (A2M); retinal pigment epithelium (RPE); vascular endothelial growth factor-A (VEGF-A); endothelial cells (ECs); extracellular matrix (ECM); matrix metalloproteinase-2 (MMP-2); protease inhibitor; age-related macular degeneration (AMD); Bruch’s membrane Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sat Sep 24 09:27:30 CEST 2022 ModDate: Sat Sep 24 09:32:11 CEST 2022 ID: 0xb2a51471ddd1525e533f554d5ab6b6a3, 0xb2a51471ddd1525e533f554d5ab6b6a3 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: DFNCPT+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 239 FontDescriptor: FontName: DFNCPT+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: VIWIOF+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: VIWIOF+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: 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254, 192, 291, 203 Annotation: Subtype: Link Rect: 59, 159, 96, 169 Annotation: Subtype: Link Rect: 279, 135, 317, 146 Annotation: Subtype: Link Rect: 323, 135, 359, 146 Annotation: Subtype: Link Rect: 169, 113, 207, 123 Annotation: Subtype: Link Rect: 471, 89, 508, 100 Annotation: Subtype: Link Rect: 515, 89, 551, 100 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 117, 711, 155, 721 Annotation: Subtype: Link Rect: 239, 676, 276, 687 Annotation: Subtype: Link Rect: 307, 653, 345, 664 Annotation: Subtype: Link Rect: 95, 630, 133, 640 Annotation: Subtype: Link Rect: 104, 607, 141, 617 Annotation: Subtype: Link Rect: 424, 583, 461, 594 Annotation: Subtype: Link Rect: 59, 550, 96, 560 Annotation: Subtype: Link Rect: 102, 550, 138, 560 Annotation: Subtype: Link Rect: 250, 526, 288, 537 Annotation: Subtype: Link Rect: 294, 526, 330, 537 Checksum: 4819cc72 Type: CRC32 Checksum: c4a3179a321be85ee961a65bcdeae45c Type: MD5 Checksum: 6ea59deb8cea4399a51b4fbd412377fbde54c036 Type: SHA-1