7e0e11519be752b867318d858784270a 298685.pdf 42067dbe7831e53f6c77ee7004ed8a0e11ed6098 298685.pdf 1f61ca7fa16f32dca2847d9b2c3a0248dd37db489c678137d2f6f7c6dc898015 298685.pdf Title: Differentiation of Adipose Tissue Mesenchymal Stem Cells into Endothelial Cells Depends on Fat Depot Conditions: Regulation by miRNA Subject: The development of obesity is associated with substantial modulation of adipose tissue (AT) structure. The plasticity of the AT is reflected by its remarkable ability to expand or reduce in size throughout the adult lifespan, which is linked to the development of its vasculature. This increase in AT vasculature could be mediated by the differentiation of adipose tissue-derived stem cells (ASCs) into endothelial cells (ECs) and form new microvasculature. We have already shown that microRNA (miRNA)-145 regulates the differentiation of ASCs into EC-like (ECL) cells. Here, we investigated whether ASCs-differentiation into ECs is governed by a miRNAs signature that depends on fat depot location and /or the metabolic condition produced by obesity. Human ASCs, which were obtained from white AT by surgical procedures from lean and obese patients, were induced to differentiate into ECL cells. We have identified that miRNA-29b-3p in both subcutaneous (s)ASCs and visceral ASCs and miRNA-424-5p and miRNA-378a-3p in subcutaneous (s)ASCs are involved in differentiation into EC-like cells. These miRNAs modulate their pro-angiogenic effects on ASCs by targeting FGFR1, NRP2, MAPK1, and TGF-2, and the MAPK signaling pathway. We show for the first time that miRNA-29b-3p upregulation contributes to ASCs’ differentiation into ECL cells by directly targeting TGFB2 in both sASCs and visceral ASCs. Moreover, our results reveal that, independent of sASCs’ origin (obese/lean), the upregulation of miRNA-378a-3p and the downregulation of miRNA-424-5p inhibit MAPK1 and overexpress FGFR1 and NRP2, respectively. In summary, both the adipose depot location and obesity affect the differentiation of resident ASCs through the expression of specific miRNAs. Keywords: adipose tissue; miRNAs; angiogenesis; ASCs; endothelial cells Author: Gemma Arderiu, Anna Civit-Urgell, Alberto Díez-Caballero, Fabrizio Moscatiello, Carlos Ballesta and Lina Badimon Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Fri Mar 15 08:28:34 2024 CET ModDate: Fri Mar 15 08:33:27 2024 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 19 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 5608242 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- WLENKM+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 VPMMBP+URWPalladioL-Bold Type 1 Custom yes yes yes 16 0 GNPVEY+URWPalladioL-Roma Type 1 Custom yes yes yes 22 0 ZCWCYZ+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 CRJHCU+PazoMath-Italic Type 1 Builtin yes yes yes 32 0 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TrueType WinAnsi yes yes yes 298 0 YNXJDR+PalatinoLinotype-Bold TrueType WinAnsi yes yes yes 302 0 FIHUXD+PalatinoLinotype-Roman TrueType WinAnsi yes yes yes 306 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-08-07 02:36:02 CEST RepresentationInformation: 298685.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-08-05 09:47:29 CEST Size: 5608242 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 566 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: ASC Isolation and Characterization Destination: subsection.2.1 Item: Title: MTS Viability/Proliferation Assay Destination: subsection.2.2 Item: Title: Induction of Endothelial Differentiation and Characterization Destination: subsection.2.3 Item: Title: miRNA Isolation and Quantification Destination: subsection.2.4 Item: Title: miRNA 4.0 Affymetrix Library Preparation and Sequencing Destination: subsection.2.5 Item: Title: miRNA Data Analysis Destination: subsection.2.6 Item: Title: qRT-PCR Analysis of miRNA Expression Destination: subsection.2.7 Item: Title: Integrated Target Prediction of Differentially Expressed miRNAs and Enrichment Analysis Destination: subsection.2.8 Item: Title: miRNA or Anti-miRNA Transfection Destination: subsection.2.9 Item: Title: RNA Isolation and qRT-PCR of Target Genes Destination: subsection.2.10 Item: Title: Constructs and Plasmids Destination: subsection.2.11 Item: Title: Luciferase Reporter Assay Destination: subsection.2.12 Item: Title: Statistical Analysis Destination: subsection.2.13 Item: Title: Results Destination: section.3 Children: Item: Title: bFGF Induces Subcutaneous ASCs and Visceral ASCs to Differentiate into ECL Cells Destination: subsection.3.1 Item: Title: Differential miRNA Profile in ASCs from Different Fat Depots and Metabolic Conditions after Their Differentiation into ECL Cells Destination: subsection.3.2 Item: Title: MiRNA-424-5p, miRNA-378a-3p, and miRNA-29b-3p Regulate ASCs’ Angiogenic Differentiation Destination: subsection.3.3 Item: Title: In Silico Analysis: miRNAs Target Angiogenesis-Associated Genes FGFR1, NRP2, MAPK1, and TGF-2 in Differentiated ASCs into ECL Cells Destination: subsection.3.4 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: appendix.A. Info: Title: Differentiation of Adipose Tissue Mesenchymal Stem Cells into Endothelial Cells Depends on Fat Depot Conditions: Regulation by miRNA Author: Gemma Arderiu, Anna Civit-Urgell, Alberto Díez-Caballero, Fabrizio Moscatiello, Carlos Ballesta and Lina Badimon Subject: The development of obesity is associated with substantial modulation of adipose tissue (AT) structure. The plasticity of the AT is reflected by its remarkable ability to expand or reduce in size throughout the adult lifespan, which is linked to the development of its vasculature. This increase in AT vasculature could be mediated by the differentiation of adipose tissue-derived stem cells (ASCs) into endothelial cells (ECs) and form new microvasculature. We have already shown that microRNA (miRNA)-145 regulates the differentiation of ASCs into EC-like (ECL) cells. Here, we investigated whether ASCs-differentiation into ECs is governed by a miRNAs signature that depends on fat depot location and /or the metabolic condition produced by obesity. Human ASCs, which were obtained from white AT by surgical procedures from lean and obese patients, were induced to differentiate into ECL cells. We have identified that miRNA-29b-3p in both subcutaneous (s)ASCs and visceral ASCs and miRNA-424-5p and miRNA-378a-3p in subcutaneous (s)ASCs are involved in differentiation into EC-like cells. These miRNAs modulate their pro-angiogenic effects on ASCs by targeting FGFR1, NRP2, MAPK1, and TGF-2, and the MAPK signaling pathway. We show for the first time that miRNA-29b-3p upregulation contributes to ASCs’ differentiation into ECL cells by directly targeting TGFB2 in both sASCs and visceral ASCs. Moreover, our results reveal that, independent of sASCs’ origin (obese/lean), the upregulation of miRNA-378a-3p and the downregulation of miRNA-424-5p inhibit MAPK1 and overexpress FGFR1 and NRP2, respectively. In summary, both the adipose depot location and obesity affect the differentiation of resident ASCs through the expression of specific miRNAs. 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