Bieback, K. | bioRxiv Science

bioRxiv2022

Secreted long non-coding RNAs Gadlor1 and Gadlor2 affect multiple cardiac cell types and aggravate cardiac remodeling during pressure overload

BackgroundPathological overload triggers maladaptive myocardial remodeling that leads to heart failure. Recent studies have shown that long non-coding RNAs (lncRNAs) regulate cardiac remodeling. This study investigates two recently discovered, secreted lncRNAs, Gadlor1 and Gadlor2 (Gadlor 1/2). MethodsWe generated compound Gadlor1/2 knock-out (KO) mice and compared their response to pressure overload by transverse aortic constriction (TAC) to that of wild-type (WT) littermates. Endothelial cells, fibroblasts and cardiomyocytes were isolated from the hearts of both genotypes after TAC and their transcriptome was investigated by RNA sequencing. Gadlor target proteins were identified by RNA antisense purification coupled with mass spectrometry (RAP-MS) in cardiomyocytes. In addition, we investigated the effects of cardiac overexpression of Gadlor1/2. ResultsGadlor1/2 are jointly upregulated in failing mouse hearts as well as in the myocardium of heart failure patients. Cardiac overexpression of Gadlor1 and Gadlor2 aggravated myocardial dysfunction and enhanced hypertrophic and fibrotic remodeling in mice exposed to pressure overload. Compound Gadlor1/2 KO mice, in turn, exerted markedly reduced myocardial hypertrophy, fibrosis and dysfunction, but more angiogenesis during short and long-standing pressure overload. Paradoxically, Gadlor1/2 KO mice suffered from sudden death during prolonged overload, possibly due to cardiac arrhythmia. Gadlor1 and Gadlor2, which are mainly expressed in endothelial cells (ECs) in the heart, where they inhibit pro-angiogenic gene-expression, are strongly secreted within extracellular vesicles (EVs). These EVs transfer Gadlor lncRNAs to cardiomyocytes, where they bind and activate calmodulin-dependent kinase II, induce pro-hypertrophic gene-expression and enhance calcium re-uptake into the sarcoplasmic reticulum. ConclusionGadlor1 and Gadlor2 are lncRNAs that are mainly enriched in EC-derived EVs and are jointly upregulated in mouse and human hearts during pathological overload. We reveal a crucial endothelial cell-cardiomyocyte crosstalk, which aims at restoring calcium homeostasis in cardiomyocytes during overload at the cost of aggravated hypertrophy and fibrosis.

systems biology↗

bioRxiv2022

Multicentre comparison of biological and functional properties of mesenchymal stromal cells from different sources cultivated using a harmonised manufacturing workflow

BackgroundMesenchymal stromal cells (MSCs), commonly sourced from adipose tissue, bone marrow and umbilical cord, have been widely used in many medical conditions due to their therapeutic potential. Yet, the still limited understanding of the underlying mechanisms of action hampers clinical translation. Clinical potency can vary considerably depending on tissue source, donor attributes, but importantly, also culture conditions. Lack of standard procedures hinders inter-study comparability and delays the progression of the field. The aim of this study was A-to assess the impact on MSC characteristics when different laboratories performed analysis on the same MSC material using harmonised culture conditions and B-to understand source-specific differences. MethodsThree independent institutions performed a head-to-head comparison of human-derived adipose (A-), bone marrow (BM-), and umbilical cord (UC-) MSCs using harmonised culture conditions. In each centre, cells from one specific tissue source were isolated and later distributed across the network to assess their biological properties, including cell expansion, immune phenotype, and tri-lineage differentiation (part A). To assess tissue specific function, angiogenic and immunomodulatory properties and the in vivo biodistribution were compared in one expert lab (part B). ResultsBy implementing a harmonised manufacturing workflow, we obtained largely reproducible results across three independent laboratories in part A of our study. Unique growth patterns and differentiation potential were observed for each tissue source, with similar trends observed between centres. Immune phenotyping verified expression of typical MSC surface markers and absence of contaminating surface markers. Depending on the established protocols in the different laboratories, quantitative data varied slightly. Functional experiments in part B concluded that conditioned media from BM-MSCs significantly enhanced tubulogenesis and endothelial migration in vitro. In contrast, immunomodulatory studies reported superior immunosuppressive abilities for A-MSCs. Biodistribution studies in healthy mice showed lung entrapment after administration of all three types of MSCs, with a significantly faster clearance of BM-MSCs. ConclusionThese results show the heterogeneous behaviour and regenerative properties of MSCs as a reflection of intrinsic tissue-origin properties while providing evidence that the use of standardised culture procedures can reduce but not eliminate inter-lab and operator differences. HighlightsIn this study, we have: - Provided a harmonised manufacturing workflow that has demonstrated reproducible results across three independent laboratories when expanding MSCs. - Defined a multi-assay matrix capable of identifying functional differences in terms of angiogenesis, wound healing abilities and immunosuppressive properties. - Demonstrated similar in vivo biodistribution properties regardless of cell origin.

cell biology↗