Biology subjects

De Kleijn, D.

Publications and source records attributed to De Kleijn, D..

4 recordsLinked to original sources

bioRxiv2025

IsoDGR-Induced Endothelial Cytoskeletal Disruption Drives Age-Related Blood-Brain Barrier Breakdown

Brain aging is characterized by progressive breakdown of the blood-brain barrier (BBB), which correlates with neuroinflammation and cognitive decline. Emerging evidence implicates degenerative modifications of the vascular proteins as a key driver of BBB dysfunction. In particular, spontaneous deamidation of Asp-Gly-Arg (NGR) motifs generates isoAsp-Gly-Arg (isoDGR) sequences that structurally mimic canonical Arg-Gly-Asp (RGD) integrin-binding ligands. Here, we show that age-associated accumulation of isoDGR in the brain cortex induces endothelial cytoskeletal collapse and tight junction disorganization, leading to BBB breakdown. Using mice lacking the L-isoaspartyl repair enzyme PCMT1 (which accelerates isoDGR accumulation) and wild type aged mice, we found markedly elevated isoDGR in brain tissues accompanied by focal microhemorrhages and increased BBB permeability. Recent whole-genome sequencing suggests that a common PCMT1 variant is linked to neurodegenerative disease risk, indicating potential clinical relevance in vascular aging. Remarkably, systemic treatment with an isoDGR-neutralizing antibody largely prevented capillary breaches and leakage, and even restored barrier integrity in aged wild-type mice. To uncover the molecular mechanism, we exposed brain endothelial cells to synthetic isoDGR-peptides, which recapitulated these effects. Unbiased RNA-sequencing reinforced these findings, revealing broad transcriptomic reprogramming of cytoskeletal, cell-cell junction, inflammatory, and stress-response pathways. Functional studies demonstrated that isoDGR triggered collapse of F-actin stress fibers, disrupted junctional ZO-1 and VE-cadherin, increased monolayer permeability to macromolecules, and impaired endothelial cell migration and proliferation. IsoDGR-treated endothelial cells exhibited increased oxidative stress, upregulation of ICAM-1/VCAM-1/CCL-2, and adopted a senescent phenotype. Our results suggest that isoDGR hijacks endothelial integrin signaling to destabilize the actin cytoskeleton and tight junctions, a process that breaches the BBB and subsequently activates inflammatory and senescence programs. In summary, we identify BBB disruption via isoDGR-induced cytoskeletal dysfunction as a central pathology of vascular aging, and demonstrate that targeting isoDGR damage preserves BBB integrity and attenuates neuroinflammation.

neuroscience↗

bioRxiv2025

Anti-isoDGR Antibody Inhibits Atherosclerosis Induced by Western Diet in ApoE-/- mice

BackgroundDegenerative protein modifications (DPMs) accumulate with aging and can alter biomolecule structure and function, including via spontaneous conversion of Asn-Gly-Arg (NGR) to isoAsp-Gly-Arg (isoDGR) motifs that can bind integrins and drive chronic inflammation. Since isoDGR-modified extracellular matrix proteins are enriched in atherosclerosis and have been associated with rupture-prone plaque characteristics, we hypothesized that antibody neutralisation can inhibit key pathological features including atherosclerotic vascular plaque formation and metabolic dysfunction. MethodsWe first examined Pcmt1-/- mice which rapidly accumulate isoDGR due to lack of the corresponding repair enzyme to assess the extent of vascular protein damage. We then treated 6-8 week old atherosclerosis-prone (ApoE-/-) mice which were fed a high-fat Western diet (WD) with weekly dose of 1mg/kg isoDGR-specific monoclonal antibody (isoDGR-mAb) or isotype-matched control (while on diet) for 2 months duration. A regular chow-fed ApoE-/- group served as baseline control. Aortic atherosclerotic burden, plaque composition, systemic inflammation, lipid profiles, hepatic steatosis, and metabolic parameters (indirect calorimetry) were assessed. ResultsPcmt1-/- mice displayed extensive isoDGR deposition and degeneration of the aortic wall, linking this DPM to vascular structural damage. In the ApoE-/- mice, WD induced large aortic root plaques with abundant isoDGR and macrophage infiltration. IsoDGR-mAb treatment decreased plaque size by [~]30% with reduced lipid and collagen content (p=0.001). Furthermore, plaques in treated mice contained significantly fewer CD68+ macrophages that also exhibited limited activation. Systemically, isoDGR-mAb modified lipoprotein profiles by decreasing atherogenic VLDL/IDL/LDL cholesterol (p=0.04) while slightly increasing HDL, accompanied by a reduction in circulating inflammatory proteins. IsoDGR-mAb also protected against hepatic lipid accumulation which was reduced by [~]60% in treated animals (p<0.001), with indirect calorimetry confirming [~]30% higher oxygen consumption and energy expenditure without change in food intake or physical activity. ConclusionWe identified isoDGR as a key pathological factor involved in the progression of atherosclerosis. Remarkably, isoDGR neutralization diminished plaque inflammation and improved atherosclerotic plaque stability. Our findings support isoDGR neutralization as a promising therapeutic strategy to mitigate both atherosclerosis and aging-associated metabolic dysfunction.

pharmacology and toxicology↗

bioRxiv2025

Kynurenine and NAD+ Pathways are Associated with Macrophage Content and Polarization in Carotid Plaques

Background and AimsMetabolism dictates macrophage function and plays a central role in atherosclerotic plaque progression. The kynurenine pathway, which metabolizes the majority of the essential amino acid tryptophan, plays a pivotal role in regulating immune responses and supporting NAD+ synthesis, essential for cellular energy metabolism. Higher circulating kynurenine levels are associated with cardiovascular disease, yet their role in atherosclerotic plaques is unclear. This study aims to investigate the underlying mechanisms driving increased kynurenine concentrations in plaques and to determine whether kynurenine serves as a mere biomarker of low-grade inflammation or reflects specific macrophage-driven metabolic alterations that could position it as a potential therapeutic target. MethodsWe used histological and transcriptomic data from two biobanks: the Athero Express Biobank (AE; n=91) and Maastricht human plaque study (MaasHPS, n= 26). Macrophages were identified through CD68 staining in AE, and M1/M2-like macrophage subtypes were distinguished by iNOS/CD68 and arginase/CD68 expression in MAASHPS. Primary human monocyte-derived cultured macrophages were polarized into M1- and M2-like phenotypes for using IFN-{gamma} and IL-4, respectively. Tryptophan, kynurenine and/or NAD+ concentrations in plaques were quantified usingliquid chromatography and metabolomics analyses. ResultsKynurenine concentrations were significantly higher in plaques with greater macrophage density (p = 0.023). Transcriptomic analysis in AE revealed upregulation of IDO2, AFMID, and KYNU in plaques with increased macrophage infiltration (p < 0.05), but not IDO1 (p = 0.16). In the MAASHPS biobank, higher IDO1, KYNU, and KMO expression correlated negatively with M2 marker positive macrophages (p < 0.001), while HAAO correlated positively (p < 0.01). In vitro, M1-like macrophages showed increased IDO1 and reduced QPRT expression compared to M2-like macrophages. We found that this disruption in kynurenine pathway gene expression led to decreased NAD+ concentrations in M1-like macrophages compared to M2-like macrophages in vitro. ConclusionHigher kynurenine levels in atherosclerotic plaques are increased by the increased presence of M1 macrophages, likely driven by both an increased IDO1 activity and reduced QPRT gene expression. This leads to decreased concentrations of NAD+, potentially determining the phenotype of the macrophages. Future studies should address whether modulation of the kynurenine pathway restores NAD+ metabolism and leads to a decrease in inflammation and an increased stable plaque phenotype.

immunology↗

bioRxiv2023

Identification of endothelial-to-mesenchymal transition gene signatures in single-cell transcriptomics of human atherosclerotic tissue

RationaleEndothelial cells can differentiate into mesenchymal-like cells via endothelial to mesenchymal transition (EndoMT). In murine models, cell transitions of EndoMT have been assessed with lineage tracing techniques. Knowledge on molecular mechanisms of EndoMT in human vascular lesions is scarce as studies in human atherosclerosis are limited by observational study designs such as histo-pathological studies. ObjectiveWe aim to identify a human EndoMT gene expression signature by combining experimentally induced in vitro EndoMT with lineage-traced pathways from atherosclerotic mice and extrapolate this to human plaque scRNA-seq data. Methods and resultsFirst, we stimulated human coronary artery endothelial cells (HCAEC) with TNF and TFG{beta} to trigger EndoMT. We executed transcriptomic analyses and defined multiple temporal patterns of gene expression changes during EndoMT. We used Cdh5-CreERT2 Rosa-eYFP apoE-/- lineage traced mouse scRNA-seq data to demonstrate that the temporal in vitro gene expression changes are reflected in EndoMT trajectories in mice plaque tissue. Finally, we constructed three candidate EndoMT lineages across multiple subpopulations of ECs and SMCs in human carotid scRNA-seq data (n=46). We examined gene expression over the course of these lineages and identified 73 markers for the presence of EndoMT such as NRG1 and DEPP1. ConclusionThis study reveals the gene expression profile of EndoMT trajectories in human atherosclerotic plaques by combining RNA-seq data from in vitro models with single-cell transcriptomic datasets. Our gene expression atlas of EndoMT in atherosclerosis could serve as a reference for future studies, providing novel inroads to study atherosclerotic mechanisms for the development of novel therapies.

molecular biology↗