89ddb50ce3c54ae5ebe7ab301028e288 301740.pdf 949f871413e509e4e4561a73c6a91aa660349051 301740.pdf bc2c3766a938e0a58ca468646851a5ce8b3c32e5187e3487333fd7f97de6a134 301740.pdf Title: Contribution of Elevated Glucose and Oxidized LDL to Macrophage Inflammation: A Role for PRAS40/Akt-Dependent Shedding of Soluble CD14 Subject: Atherosclerosis, a process in which macrophages play a key role, is accelerated in diabetes. Elevated concentrations of serum-oxidized low-density lipoproteins (oxLDL) represent a common feature of both conditions. The main goal of this study was to determine the contribution of oxLDL to the inflammatory response of macrophages exposed to diabetic-mimicking conditions. THP1 cells and peripheral blood monocytes purified from non-diabetic healthy donors were cultured under normal (5 mM) or high glucose (HG) (15 mM) with oxLDL. Then, foam cell formation, expression of CD80, HLADR, CD23, CD206, and CD163, as well as toll-like receptor 4 (TLR4) and co-receptors CD36 and CD14 (both at the cell surface and soluble (sCD14)), and inflammatory mediators’ production were measured by flow cytometry, RT-qPCR, or ELISA. Additionally, serum sCD14 was determined in subjects with subclinical atherosclerosis with and without diabetes by ELISA. Our results showed that oxLDL-mediated intracellular lipid accumulation via CD36 increased under HG and that HG + oxLDL enhanced TNF, IL1B, and IL8, and decreased IL10. Moreover, TLR4 was upregulated in macrophages under HG and monocytes of subjects with diabetes and atherosclerosis. Interestingly, HG-oxLDL upregulated CD14 gene expression, although its total cellular protein abundance remained unaltered. sCD14 shedding via PRAS40/Akt-dependent mechanisms, with pro-inflammatory activity, was significantly increased in cultured macrophages and plasma from subjects with diabetes and subclinical atherosclerosis or hypercholesterolemia. Our data support an enhanced synergistic pro-inflammatory effect induced by HG and oxLDL in cultured human macrophages, possibly explained by increased sCD14 shedding. Keywords: diabetes; atherosclerosis; CD36; foam cell; monocytes; TLR4; vascular Author: Lucía Sanjurjo, Esmeralda Castelblanco, Josep Julve, Nuria Villalmanzo, Érica Téllez, Anna Ramirez-Morros, Núria Alonso, Dídac Mauricio and Maria-Rosa Sarrias Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu May 11 13:31:33 2023 CEST ModDate: Thu May 11 14:57:49 2023 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 21 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2697163 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- WEUSSB+VnURWPalladioL Type 1 Custom yes yes no 10 0 KXJEPQ+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 QJEDHD+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 HFPHIZ+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 MREUAY+VnURWPalladioL-Bold 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Destination: subsection.2.2 Item: Title: Human Samples from Subjects with and without Diabetes Destination: subsection.2.3 Item: Title: Quantification of Foam Cell Formation Destination: subsection.2.4 Item: Title: DiI-oxLDL Uptake Destination: subsection.2.5 Item: Title: Measurement of mRNA Levels by Real-Time PCR (RT-PCR) Destination: subsection.2.6 Item: Title: Western Blot Analysis of Cell Lysates and Culture Supernatants Destination: subsection.2.7 Item: Title: Silencing of CD36 Expression Destination: subsection.2.8 Item: Title: Measurement of Cytokine Secretion Destination: subsection.2.9 Item: Title: Flow Cytometry Analysis of Polarization Markers Destination: subsection.2.10 Item: Title: Immunophenotyping Destination: subsection.2.11 Item: Title: sCD14 ELISA Destination: subsection.2.12 Item: Title: Phospho-Proteome Array Destination: subsection.2.13 Item: Title: Statistical Analysis Destination: subsection.2.14 Item: Title: Results Destination: section.3 Children: Item: Title: High Glucose Potentiates Macrophage oxLDL Uptake and Foam Cell Formation through CD36 Upregulation Destination: subsection.3.1 Item: Title: Macrophage Inflammatory Responses to High Glucose Are Potentiated by oxLDL Destination: subsection.3.2 Item: Title: High Glucose and oxLDL Moderately Influenced Macrophage Polarization Destination: subsection.3.3 Item: Title: Effects of OxLDL and High Glucose on TLR4 and CD14 mRNA and Protein Expression Destination: subsection.3.4 Item: Title: High Glucose + oxLDL-Mediated Induction of CD14 and Inflammatory Responses Are PRAS40/Akt-Dependent Destination: subsection.3.5 Item: Title: TLR4 Is Elevated in a Subset of Non-Classical Monocytes from Subjects with T2D and Atherosclerosis Destination: subsection.3.6 Item: Title: sCD14 Is Elevated in T2D Subjects with Hypercholesterolemia or Atherosclerosis Destination: subsection.3.7 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.6 Info: Title: Contribution of Elevated Glucose and Oxidized LDL to Macrophage Inflammation: A Role for PRAS40/Akt-Dependent Shedding of Soluble CD14 Author: Lucía Sanjurjo, Esmeralda Castelblanco, Josep Julve, Nuria Villalmanzo, Érica Téllez, Anna Ramirez-Morros, Núria Alonso, Dídac Mauricio and Maria-Rosa Sarrias Subject: Atherosclerosis, a process in which macrophages play a key role, is accelerated in diabetes. Elevated concentrations of serum-oxidized low-density lipoproteins (oxLDL) represent a common feature of both conditions. The main goal of this study was to determine the contribution of oxLDL to the inflammatory response of macrophages exposed to diabetic-mimicking conditions. THP1 cells and peripheral blood monocytes purified from non-diabetic healthy donors were cultured under normal (5 mM) or high glucose (HG) (15 mM) with oxLDL. Then, foam cell formation, expression of CD80, HLADR, CD23, CD206, and CD163, as well as toll-like receptor 4 (TLR4) and co-receptors CD36 and CD14 (both at the cell surface and soluble (sCD14)), and inflammatory mediators’ production were measured by flow cytometry, RT-qPCR, or ELISA. Additionally, serum sCD14 was determined in subjects with subclinical atherosclerosis with and without diabetes by ELISA. Our results showed that oxLDL-mediated intracellular lipid accumulation via CD36 increased under HG and that HG + oxLDL enhanced TNF, IL1B, and IL8, and decreased IL10. Moreover, TLR4 was upregulated in macrophages under HG and monocytes of subjects with diabetes and atherosclerosis. Interestingly, HG-oxLDL upregulated CD14 gene expression, although its total cellular protein abundance remained unaltered. sCD14 shedding via PRAS40/Akt-dependent mechanisms, with pro-inflammatory activity, was significantly increased in cultured macrophages and plasma from subjects with diabetes and subclinical atherosclerosis or hypercholesterolemia. Our data support an enhanced synergistic pro-inflammatory effect induced by HG and oxLDL in cultured human macrophages, possibly explained by increased sCD14 shedding. 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