Identification of the delivery potential to proangiogenic genetics exhibited by hypoxic cardiomyocyte-derived exosomes via paracrine

Experimental Design and Cell GroupingThis study aimed to identify the potential of CMs to activate angiogenic pathways under hypoxic stress and to achieve intercellular communication through the delivery of angiogenic markers via CM-Exos by characterizing the phenotype of an in vitro hypoxia-induced CMs model and CM-Exos. H9C2 cells (Pricella, CL-0089) were cultured in DMEM (Sigma-Aldrich, PM150210) supplemented with 10% fetal bovine serum (Procell, 164210-50) and 1% penicillin/streptomycin (Procell, PB180120) under normoxic conditions (21% O₂, 5% CO₂, 37°C). Upon reaching 80–90% confluence, H9C2 cells were seeded at a density of 1×10⁵ cells/mL into culture dishes. For Exos identification experiments, H9C2 cells were cultured in DMEM without serum and GFs. The study was conducted in two stages: (1) determination of optimal hypoxic conditions for exosome secretion from cardiomyocytes; and (2) evaluation of the angiogenic potential for the release of angiogenic regulators and markers. Phase Ⅰ: H9C2 cells were randomly divided into five groups based on hypoxia exposure time: 0 h, 24 h, 48 h, 72 h, and 96 h, with the 0 h group serving as the control. Phase Ⅱ: H9C2 cells were divided into two groups: (1) control and (2) hypoxia, using the optimal conditions determined in Phase Ⅰ.Establishment of a CM hypoxic model in vitroExperiments were initiated using H9C2 cells at passage to the third generation. The culture medium consisted of DMEM supplemented with 10% fetal bovine serum (Procell, 164210-50) and 1% penicillin/streptomycin (Procell, PB180120). H9C2 cells were treated with 0, 200, 400, 600, 800, and 1000 μM CoCl₂ for 0, 24, 48, 72, or 96 hours at 37°C, as previously described. Cell lysates were collected for downstream analyses. Hypoxia induction was assessed by measuring cell proliferation and apoptosis levels.Cell counting kit-8 assay (CCK-8)The enhanced Cell Counting Kit-8 (Elabscience, E-CK-A362) was used following the manufacturer’s protocol. Briefly, treated H9C2 cells were seeded into 96-well plates at 8×10³ cells/well in serum-free DMEM and incubated at 37°C in a CO₂ incubator. H9C2 cells were incubated for 0, 24, 48, 72, or 96 hours. Subsequently, a 10% CCK-8 working solution was prepared in basal culture medium and added to per well (100 µL), followed by 2 hours of incubation at 37°C with 5% CO₂. Finally, absorbance at 450 nm was measured using a microplate reader.Flow cytometryThe Annexin V-PE/7-AAD apoptosis detection kit (Elabscience, E-CK-A216) was used following the manufacturer's instruction. H9C2 cells were digested with trypsin and centrifuged at 300 ×g for 5 minutes to collect cells. The cell pellet was resuspended in 50 μL of 1×Binding Buffer. A total of 5 μL of Annexin V-PE and 5 μL of 7-AAD were added, and cells were incubated in the dark at room temperature for 15 minutes. After incubation, 400 μL of 1×Binding Buffer was added to per tube, followed by filtration to prepare single-cell suspensions. To quantify apoptosis rates by fluorescence intensity, BD FACSCalibur flow cytometer was used to analyzing the stained samples.Purification and characterization of Exos from H9C2 cellsExosome Extraction and Purification Kit (Umibio, UR52121) was used following the instructions. As previously described, H9C2 cells from each group were transferred to centrifuge tubes and centrifuged at 3,000 g for 10 min to remove cellular debris from the samples. After centrifugation again, the supernatant was transferred to fresh centrifuge tubes. Exosome Concentration Solution (ECS) was added at a sample-to-ECS ratio of 4:1. The mixture was vortexed for 1 min and incubated for 2 h. After centrifugation at 10,000 g for 60 min, the supernatant was discarded. After resuspending 100 μL of 1× PBS, the pellet was centrifuged at 12,000 g for 2 min with the supernatant being retained. The pellet contained exosome-enriched fractions (primary exosome preparation). The sample was transferred to the upper chamber of an Exosome Purification Filter (EPF) column and centrifuged at 3,000 g for 10 min. The liquid collected at the bottom of the EPF column contained purified exosome particles ready for subsequent analysis. All procedures were performed at 4°C.Transmission electron microscopy assay (TEM)Firstly, 10 μL of Exos suspension was loaded onto a copper grid and left to stand for 1 minute. The sample was fixed in 50 μL of 1% glutaraldehyde solution for 5 minutes and rinsed 8 times with ddH₂O. Then, samples were stained with 1% phosphotungstic acid solution for 2 minutes. Excess liquid was removed using filter paper, and the copper grid was air-dried at room temperature. Lastly, images were acquired using TEM (Hitachi HT7800, Japan) at 100 kV.Nanoparticle tracking analysis (NTA)Nanoparticle tracking analysis (NTA) was performed using a NanoSight system (NS300, Malvern Panalytical, UK) to assess particle size and concentration. As previously described. Briefly, purified exosome samples were diluted in 1×PBS at room temperature. Final particle concentrations were adjusted to 5×10⁸–2×10⁹ particles/mL. Analysis was conducted using NanoSight software (version 3.0).Western blotting analysisCells were collected and washed with cold PBS, lysed in RIPA buffer (Servicebio, G2002), and centrifuged to collect lysates. Total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and quantified using a BCA kit (Beyotime, P0009). Add 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg/mL of standard solution, all in 20 μL volume. A standard curve was constructed after diluting the sample 20 times, add 20 μl of diluted sample to make 3 duplicate wells, then add 200 μl of BCA working solution (BCA reagent A and B 50:1), place at 37℃ for 30 minutes, with measure the absorbance at 562 nm. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, K2MA8350E) and incubated overnight at 4°C with primary antibodies against CD63, Hsp70, and TSG101 (Proteintech, 1:10000). The PVDF membranes were incubated with secondary antibody, HRP-labeled goat anti-rabbit IgG (Invitrogen, AB_228338, 1:3000), and bands were visualized using enhanced chemiluminescence (Affinity, KF8003). TSG101 was used as housekeeping protein.Quantitative reverse transcription real‐time polymerase chain reaction (RT‐qPCR)Total RNA was extracted from H9C2 cells and exosomes separately by using TRIzol (Invitrogen) and reverse-transcribed into cDNA using Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix (Yeasen, 11141ES60). Then, RT-qPCR analysis was performed using 2×Universal Blue SYBR® Green qPCR Master Mix Kit (Servicebio, G3326-05), and the gene expression was calculated via the 2-∆∆Ct method. Immunofluorescence assayH9C2 cells were seeded into 24-well plates at 1×10⁵ cells/well, and fixed with 4% paraformaldehyde at room temperature overnight. Cells were permeabilized with pepsin retrieval solution at room temperature for 30 min and add 3% H₂O₂ for 20 min in the dark to block the endogenous peroxidase activity. Cells were blocked with 5% bovine serum albumin (BSA) for 30 minutes at 37°C. Cells were incubated with primary antibodies (1:100 dilution with 2% BSA) targeting VEGF, ET-1, NOS, vWF, Klotho, FGF23, MALAT1, and Ang-II (green), and CD81 (red) were applied overnight in a humidified chamber at 4˚C. Secondary antibodies (Alexa Fluor® 488/555 conjugated Goat pAb to Rb lgG H&L) were incubated at room temperature for 30 minutes. Finally, cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole AS1075; ASPEN Biotechnology) for 5 minutes at room temperature in the dark. Exos co-localized markers (CD81), angiogenesis regulatory indicators (MALAT1, Klotho, FGF23) and downstream angiogenesis markers (VEGF, ET-1, NO, vWF, Ang-Ⅱ) were visualized under confocal laser scanning microscopy.Statistical analysisThe data in this study were expressed as mean ± standard deviation (mean ± SD). Student’s t-test was used for comparisons between two groups, while one-way ANOVA followed by Bonferroni’s post hoc test was performed for multiple comparisons. Statistical analyses and visualizations were performed using GraphPad Prism v10.1 software (Inc., San Diego, CA, USA.), and Adobe Illustrator v2023 (Adobe, San Jose, USA) was employed to design and compose graphical figures. A P-value < 0.05 was considered statistically significant.