Multi-Level Analysis of Adipose Tissue Reveals the Relevance of Perivascular Subpopulations and an Increased Endothelial Permeability in Early-Stage Lipedema.

Lipedema is a chronic, progressive disease of adipose tissue with unknown etiology. Based on the relevance of the stromal vascular fraction (SVF) cell population in lipedema, we performed a thorough characterization of subcutaneous adipose tissue, SVF isolated thereof and the sorted populations of endothelial cells (EC), pericytes and cultured adipose-derived stromal/stem cells (ASC) of early-stage lipedema patients. We employed histological and gene expression analysis and investigated the endothelial barrier by immunofluorescence and analysis of endothelial permeability in vitro. Although there were no significant differences in histological stainings, we found altered gene expression of factors relevant for local estrogen metabolism (aromatase), preadipocyte commitment (ZNF423) and immune cell infiltration (CD11c) in lipedema on the tissue level, as well as in distinct cellular subpopulations. Machine learning analysis of immunofluorescence images of CD31 and ZO-1 revealed a morphological difference in the cellular junctions of EC cultures derived from healthy and lipedema individuals. Furthermore, the secretome of lipedema-derived SVF cells was sufficient to significantly increase leakiness of healthy human primary EC, which was also reflected by decreased mRNA expression of VE-cadherin. Here, we showed for the first time that the secretome of SVF cells creates an environment that triggers endothelial barrier dysfunction in early-stage lipedema. Moreover, since alterations in gene expression were detected on the cellular and/or tissue level, the choice of sample material is of high importance in elucidating this complex disease.

CRISPR/Cas9 Genome Editing vs. Over-Expression for Fluorescent Extracellular Vesicle-Labeling: A Quantitative Analysis.

Over-expression of fluorescently-labeled markers for extracellular vesicles is frequently used to visualize vesicle up-take and transport. EVs that are labeled by over-expression show considerable heterogeneity regarding the number of fluorophores on single particles, which could potentially bias tracking and up-take studies in favor of more strongly-labeled particles. To avoid the potential artefacts that are caused by over-expression, we developed a genome editing approach for the fluorescent labeling of the extracellular vesicle marker CD63 with green fluorescent protein using the CRISPR/Cas9 technology. Using single-molecule sensitive fluorescence microscopy, we quantitatively compared the degree of labeling of secreted small extracellular vesicles from conventional over-expression and the CRISPR/Cas9 approach with true single-particle measurements. With our analysis, we can demonstrate a larger fraction of single-GFP-labeled EVs in the EVs that were isolated from CRISPR/Cas9-modified cells (83%) compared to EVs that were isolated from GFP-CD63 over-expressing cells (36%). Despite only single-GFP-labeling, CRISPR-EVs can be detected and discriminated from auto-fluorescence after their up-take into cells. To demonstrate the flexibility of the CRISPR/Cas9 genome editing method, we fluorescently labeled EVs using the HaloTag® with lipid membrane permeable dye, JaneliaFluor® 646, which allowed us to perform 3D-localization microscopy of single EVs taken up by the cultured cells.

SVF-derived extracellular vesicles carry characteristic miRNAs in lipedema.

Lipedema is a chronic, progressive disease of adipose tissue with lack of consistent diagnostic criteria. The aim of this study was a thorough comparative characterization of extracellular microRNAs (miRNAs) from the stromal vascular fraction (SVF) of healthy and lipedema adipose tissue. For this, we analyzed 187 extracellular miRNAs in concentrated conditioned medium (cCM) and specifically in small extracellular vesicles (sEVs) enriched thereof by size exclusion chromatography. No significant difference in median particle size and concentration was observed between sEV fractions in healthy and lipedema. We found the majority of miRNAs located predominantly in cCM compared to sEV enriched fraction. Surprisingly, hierarchical clustering of the most variant miRNAs showed that only sEVmiRNA profiles - but not cCMmiRNAs - were impacted by lipedema. Seven sEVmiRNAs (miR-16-5p, miR-29a-3p, miR-24-3p, miR-454-p, miR-144-5p, miR-130a-3p, let-7c-5p) were differently regulated in lipedema and healthy individuals, whereas only one cCMmiRNA (miR-188-5p) was significantly downregulated in lipedema. Comparing SVF from healthy and lipedema patients, we identified sEVs as the lipedema relevant miRNA fraction. This study contributes to identify the potential role of SVF secreted miRNAs in lipedema.

Peptide-based vaccination against OPN integrin binding sites does not improve cardio-metabolic disease in mice.

Obesity causes insulin resistance via a chronic low-grade inflammation. This inflammation is characterized by elevated pro-inflammatory markers and macrophage accumulation in the adipose tissue (AT). AT inflammation is a key factor causing insulin resistance and thus type 2 diabetes, both linked to atherosclerotic cardiovascular disease. Osteopontin (OPN), a well-known inflammatory cytokine, is involved in obesity-linked complications including AT inflammation, insulin resistance, atherosclerosis and CVD. During inflammation, OPN is proteolytically cleaved by matrix metalloproteinases or thrombin leading to increased OPN activity. Therefore, OPN provides a new interesting target for immunological prevention and treatment of obesity-associated diseases. The aim of our study was to evaluate peptide-based vaccines against integrin binding sites of OPN and to examine whether these active immunotherapies are functional in reducing metabolic tissue inflammation, insulin resistance, and atherosclerosis in a cardio-metabolic (Ldlr-/- mice) and a diet-induced obesity model (WT mice). However, atherosclerosis, insulin resistance and AT inflammation were not diminished after treatment with OPN-derived peptides in murine models. Lack of efficacy was based on a failure to induce antibodies capable to bind epitopes in the context of functional OPN protein. In conclusion, our data point to unexpected challenges in the immunotherapeutic targeting of adhesive motives, such as RGD containing sequences, on endogenous proteins.

A humanized osteopontin mouse model and its application in immunometabolic obesity studies.

Osteopontin (OPN) is a multifunctional protein involved in several inflammatory processes and pathogeneses including obesity-related disorders and cancer. OPN binds to a variety of integrin receptors and CD44 resulting in a proinflammatory stimulus. Therefore, OPN constitutes a novel interesting target to develop new therapeutic strategies, which counteract OPN's proinflammatory properties. We established a humanized SPP1 (hSPP1) mouse model and evaluated its suitability as a model for obesity and insulin resistance. Unchallenged hSPP1 animals did not significantly differ in body weight and gross behavioral properties compared to wild-type (WT) animals. High-fat diet-challenged hSPP1 similarly developed obesity and inflammation, whereas insulin resistance was markedly changed. However, OPN expression profile in tissues was significantly altered in hSPP1 compared to WT depending on the diet. In conclusion, we developed a versatile humanized model to study the action of OPN in vivo and to develop strategies that target human OPN in a variety of pathologies.