Supplementary MaterialsFigure S1: (A) Consultant western blot images of proteins extracted from AC10 cell cultures in normoxia or hypoxia (2% O2). images taken after 6 h culture. Results are expressed in arbitrary units (= 3, * 0.05). Image_2.tif (158K) GUID:?F14334E2-E8A9-448E-BF21-33C5B1A86851 Physique S3: Cell proliferation assay of AC10, fibroblasts and human umbilical cord vein cells (HUVEC). Representative example of cell proliferation measured by CCK-8 assay after 48 h of culture in presence of Nx and Hx CM-EVs. Results are presented in arbitrary units (= 3). Image_3.tif (35K) GUID:?E2BD7B94-C586-4BA2-A282-3BE450C0EB42 Table S1: Protein identification in cardiomyocyte-derived extracellular vesicles in normoxia. Table_1.DOCX (24K) GUID:?FFD3E4EA-FEFB-499C-A107-9F410D9B0215 Table S2: Protein identification in cardiomyocyte-derived extracellular vesicles in hypoxia. Table_2.DOCX (32K) GUID:?31917C7E-ABBE-4C81-AB0C-C1B82C6AA959 Table S3: Gene ontology biological processes for proteins identified in extracellular vesicles derived in normoxia. Table_3.DOCX (25K) GUID:?B3CD570B-6F61-4BE6-B53F-6110BB5938D7 Table S4: Gene ontology biological processes for proteins identified in extracellular vesicles derived in hypoxia. Table_4.DOCX (28K) GUID:?AD4CCC3C-EE30-4752-84AD-C3A590488895 Abstract Extracellular vesicles (EVs) are small membrane vesicles secreted by most cell types with important roles in cell-to-cell communication. To assess their relevance in the context of heart ischemia, EVs isolated from the AC10 ventricular cardiomyocyte cell line (CM-EVs), exposed to normoxia (Nx) or hypoxia (Hx), were incubated with fibroblasts (Fb) and Rabbit Polyclonal to B3GALT4 endothelial cells (EC). CM-EVs were studied using electron microscopy, nanoparticle tracking analysis (NTA), western blotting and proteomic analysis. Results showed that EVs had a strong preference to be internalized by EC over fibroblasts, suggesting an active exosome-based communication mechanism between CM and EC in the heart. In Matrigel tube-formation assays, Hx CM-EVs were inferior to Nx CM-EVs in angiogenesis. By contrast, in a wound-healing Lamivudine assay, wound closure was faster in fibroblasts treated with Hx CM-EVs than with Nx CM-EVs, supporting a pro-fibrotic effect of Hx CM-EVs. Overall, these observations were consistent with the different protein cargoes detected by proteomic analysis under Nx and Hx conditions and the biological pathways identified. The paracrine crosstalk between CM-EVs, Fb, and EC in different physiological conditions could account for the contribution of CM-EVs to cardiac remodeling after an ischemic insult. for 16 h. The induction of Hx in cell cultures was monitored by stabilization of hypoxia inducible factor-1 alpha (HIF-1) expression and cell viability reduction (Physique S1). Extracellular vesicle purification We used ultracentrifugation without sucrose-gradient centrifugation step to isolate EVs from cardiomyocytes. Accordingly, we refer to the isolated pool of vesicles as CM-EVs and not CM-exosomes. Approximately 150 mL of culture media was collected and EVs were isolated by several ultracentrifugation actions as explained (9). Briefly, supernatants were centrifuged first at 2,000 for 20 min (Eppendorf 5804 benchtop centrifuge, A-4-62 rotor), 10,000 for 70 min (Hitachi CP100NX centrifuge, Beckman Coulter 50.2 Ti rotor) and subsequently filtered manually through a 0.22 m filter to eliminate cell debris using a syringe. Then, EVs were pelleted by ultracentrifugation at 110,000 for 120 min (Hitachi CP100NX centrifuge, Beckman Coulter 50.2 Ti rotor), filtered through a 0.22 m filter to maintain sterility and ultracentrifuged again at 110,000 for 120 min (Hitachi CP100NX centrifuge, Beckman Coulter 50.2 Ti rotor). The manipulation of EV extracts was performed in a laminar circulation hood to preserve sterility. EV protein concentration was decided with the Pierce BCA Protein Assay Kit (ThermoFisher Scientific) Lamivudine to make sure equal levels of proteins samples. EVs had been suspended in RIPA buffer [1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate in Tris-buffered saline (TBS), Sigma-Aldrich] for western blotting or phosphate buffered saline (PBS) for nanoparticle monitoring, electron microscopy, stream cytometry, functional and proteomic analysis. Extracellualar vesicle incorporation tests Capture of tagged EVs by fibroblasts and EC was performed using techniques modified from prior reviews (10). EVs had been fluorescently stained with carboxyfluorescein diacetate succinimidyl diester (CFSE; 5 M) (ThermoFisher Scientific) for 15 min at 37C, and unincorporated dye was taken out ultracentrifugation. Lamivudine As a poor control to normalize, the same quantity of PBS was put into CFSE to monitor unincorporated dye transported over following the staining guidelines (PBS control). Cells had been seeded at 50% confluency in 24-well plates and incubated with CFSE-labeled EVs at 2 g/mL (5.00 E + 08 contaminants/mL). After 3 h of incubation, cells had been cleaned in frosty PBS double, trypsinized and examined by stream cytometry utilizing a FACS Canto II on the Cytomics Device from the Instituto de Investigacin Sanitaria, La Fe. Traditional western blot evaluation EVs or cells had been lysed in 100 L of RIPA buffer formulated with protease (Comprehensive, Sigma-Aldrich) and phosphatase (PhosSTOP, Sigma-Aldrich) inhibitors..