Abstract 1458 Extracellular Vesicles-Incorporated GAS5 Ameliorates in Patients with Coronary Artery Disease and Regulates Function of Endothelial Cells via Cellular Crosstalk

Accumulating evidence indicates that long noncoding RNAs (lncRNAs) are playing a crucial role in cardiovascular diseases (CVDs) such as myocardial infarction (MI), ischemia, coronary artery disease (CAD), and he art failure. Intercellular transfer of extracellular vesicles (EVs) transmits ncRNAs and regulates the function of recipient cells via diverse mechanisms in CVD. However, the specific role of EV-lncRNA in recipient endothelial cells (ECs) and the molecular mechanism of shuttling of EV-lncRNA into recipient cells were poorly investigated.

A human lncRNA array revealed that certain EV-lncRNAs are significantly dysregulated in CAD patients. Circulating EVs from patients with (n=30) or without (n=30) CAD were used to quantify GAS5, MALAT1, PUNISHER, and H19 RNA levels. GAS5 (p=0.02) were significantly increased in patients with CAD, compared to non-CAD patients. RNA interference in ECs revealed that GAS5 is an important regulator EC function which decreases viability, cell proliferation, increased apoptosis, and cytotoxicity, which is quantified by using an MTT assay, Brd-U incorporation assay, and Caspase 3/7 activity. Loss of GAS5 decreases endothelial network formation and migration capacity. In vitro, atherosclerotic conditions augment GAS5 in EC and corresponding EVs. EV-RNA degradation and fluorescent labeling of EV by PKH67 experiments demonstrated that GAS5 mainly encapsulated into EVs to facilitate its suttling to recipient cells. In vitro functional experiments revealed tube formation, angiogenic sprouting, migration, proliferation; apoptosis is impaired in recipient cells upon treatment with GAS5 silenced EV compared to controls suggesting that GAS5 is an important regulator of target EC function. To unveil the underlying molecular mechanism, mass spectrometry idenfied RNA-binding proteins in EVs as hnRNPU, hnRNPK, hnRNPA2B1. RNA immunoprecipitation and RNA pulldown experiments confirmed that GAS5 interacts with hnRNPU. Loss of hnRNPU in ECs phenocopied effects of GAS5. Pathway analysis was performed via Cignal 45 combined with dual-luciferase assays revealed that NF-kB activity and apoptotic markers (e.g. caspases) are upregulated upon GAS5 silencing. Immunoblotting and qPCR revealed the upregulation of NF-kB and its cognate genes. These data indicate that GAS5 exerts its function via an NF-kB-dependent mechanism. In vivo endothelial regeneration murine model and zebrafish experiments demonstrated that EV-GAS5 is an important regulator of target EC function, in particular angiogenic response via vesicular shuttling.

Our data indicate that EV-incorporated GAS5 has an anti-apoptotic role via large vesicular shuttling, sensing the angiogenic function of EC. GAS5 regulates its function via interaction with hnRNPU to shuttle into recipient cells via an NF-kB-dependent mechanism. Our study indicates that GAS5 may be useful for targeted therapeutics in CVD.

Central figure. LncRNA expression in circulating EVs from patients with or without CAD and analysis of lncRNA GAS5. (A) Volcano plot showing differentially regulated human lncRNAs in EVs derived from patient plasma by using a PCR-based human lncRNA array. Thresholds (dotted lines) of a two-fold change and P-values (FDR-adjusted)<0.05 were set to distinguish lncRNAs of interest. n=3 for NCAD, n=3 for CAD.

Clin Res Cardiol (2023). https://doi.org/10.1007/s00392-023-02180-w
Extracellular Vesicles-Incorporated GAS5 Ameliorates in Patients with Coronary Artery Disease and Regulates Function of Endothelial Cells via Cellular Crosstalk
M. Baumschabl¹, K. Maus², K. Maus¹, L. Zhou¹, P. Düsing¹, A. Zietzer¹, P. R. Goody¹, R. Chennupati³, S. Uchida⁴, F. Jansen¹, G. Nickenig¹, M. R. Hosen¹
¹Medizinische Klinik und Poliklinik II, Universitätsklinikum Bonn, Bonn; ²Molekulare Kardiologie // Geb. 370, Universitätsklinikum Bonn, Bonn; ³Klinik für Kardiologie, Pneumologie und Angiologie, Universitätsklinikum Düsseldorf, 40225 Düsseldorf; ⁴Center for RNA Medicine, Aalborg University, Department of Clinical Medicine,, Copenhagen, DK;
Background: Accumulating evidence indicates that long noncoding RNAs (lncRNAs) are playing a crucial role in cardiovascular diseases (CVDs) such as myocardial infarction (MI), ischemia, coronary artery disease (CAD), and he art failure. Intercellular transfer of extracellular vesicles (EVs) transmits ncRNAs and regulates the function of recipient cells via diverse mechanisms in CVD. However, the specific role of EV-lncRNA in recipient endothelial cells (ECs) and the molecular mechanism of shuttling of EV-lncRNA into recipient cells were poorly investigated. Methods and Results: A human lncRNA array revealed that certain EV-lncRNAs are significantly dysregulated in CAD patients. Circulating EVs from patients with (n=30) or without (n=30) CAD were used to quantify GAS5, MALAT1, PUNISHER, and H19 RNA levels. GAS5 (p=0.02) were significantly increased in patients with CAD, compared to non-CAD patients. RNA interference in ECs revealed that GAS5 is an important regulator EC function which decreases viability, cell proliferation, increased apoptosis, and cytotoxicity, which is quantified by using an MTT assay, Brd-U incorporation assay, and Caspase 3/7 activity. Loss of GAS5 decreases endothelial network formation and migration capacity. In vitro, atherosclerotic conditions augment GAS5 in EC and corresponding EVs. EV-RNA degradation and fluorescent labeling of EV by PKH67 experiments demonstrated that GAS5 mainly encapsulated into EVs to facilitate its suttling to recipient cells. In vitro functional experiments revealed tube formation, angiogenic sprouting, migration, proliferation; apoptosis is impaired in recipient cells upon treatment with GAS5 silenced EV compared to controls suggesting that GAS5 is an important regulator of target EC function. To unveil the underlying molecular mechanism, mass spectrometry idenfied RNA-binding proteins in EVs as hnRNPU, hnRNPK, hnRNPA2B1. RNA immunoprecipitation and RNA pulldown experiments confirmed that GAS5 interacts with hnRNPU. Loss of hnRNPU in ECs phenocopied effects of GAS5. Pathway analysis was performed via Cignal 45 combined with dual-luciferase assays revealed that NF-kB activity and apoptotic markers (e.g. caspases) are upregulated upon GAS5 silencing. Immunoblotting and qPCR revealed the upregulation of NF-kB and its cognate genes. These data indicate that GAS5 exerts its function via an NF-kB-dependent mechanism. In vivo endothelial regeneration murine model and zebrafish experiments demonstrated that EV-GAS5 is an important regulator of target EC function, in particular angiogenic response via vesicular shuttling. Conclusion and future perspectives: Our data indicate that EV-incorporated GAS5 has an anti-apoptotic role via large vesicular shuttling, sensing the angiogenic function of EC. GAS5 regulates its function via interaction with hnRNPU to shuttle into recipient cells via an NF-kB-dependent mechanism. Our study indicates that GAS5 may be useful for targeted therapeutics in CVD. Central figure. LncRNA expression in circulating EVs from patients with or without CAD and analysis of lncRNA GAS5. (A) Volcano plot showing differentially regulated human lncRNAs in EVs derived from patient plasma by using a PCR-based human lncRNA array. Thresholds (dotted lines) of a two-fold change and P-values (FDR-adjusted)<0.05 were set to distinguish lncRNAs of interest. n=3 for NCAD, n=3 for CAD.
https://dgk.org/kongress_programme/jt2023/aP2244.html