Hyperbaric oxygen boosts long noncoding RNA MALAT1 exosome secretion to suppress microRNA-92a expression in therapeutic angiogenesis Kou-Gi Shyu, Bao-Wei.

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Hyperbaric oxygen boosts long noncoding RNA MALAT1 exosome secretion to suppress microRNA-92a expression in therapeutic angiogenesis Kou-Gi Shyu, Bao-Wei Wang, Chun-Ming Pan, Wei-Jen Fang, Chiu-Mei Lin International Journal of Cardiology Volume 274, Pages 271-278 (January 2019) DOI: 10.1016/j.ijcard.2018.09.118 Copyright © 2018 Elsevier B.V. Terms and Conditions

International Journal of Cardiology 2019 274, 271-278DOI: (10. 1016/j International Journal of Cardiology 2019 274, 271-278DOI: (10.1016/j.ijcard.2018.09.118) Copyright © 2018 Elsevier B.V. Terms and Conditions

Fig. 1 Effect of HBO on HCAEC-derived exosomal MALAT1 expression. A. Quantitative real-time PCR of HCAEC-derived exosomal MALAT1 under various degrees of HBO for 2 days. The values from treated HCAECs are expressed as a ratio of normalized values of MALAT1 mRNA in the control cells. The Tukey–Kramer comparison test was conducted for statistical analysis. **P < 0.01 vs. control; n = 4 per group. B, Quantitative real-time PCR of HCAEC-derived exosomal MALAT1 under 2.5 ATA at 1 h each day for different durations. The values from treated HCAECs are expressed as a ratio of normalized values of MALAT1 mRNA in the control cells. The Tukey–Kramer comparison test was conducted for statistical analysis. *P < 0.05 vs. control. **P < 0.01 vs. control; n = 5 per group. C, Quantitative real-time PCR of HCAEC MALAT1 under 2.5 ATA at 1 h each day for different durations. The values from treated HCAECs are expressed as a ratio of normalized values of MALAT1 mRNA in the control cells. The Tukey–Kramer comparison test was conducted for statistical analysis. *P < 0.01 vs. control; n = 4 per group. International Journal of Cardiology 2019 274, 271-278DOI: (10.1016/j.ijcard.2018.09.118) Copyright © 2018 Elsevier B.V. Terms and Conditions

Fig. 2 Effect of HBO on miR-92a and KFL2 expression in HCAEC. A, Quantitative real-time PCR of HCAEC-derived exosomal miR-92a under 2.5 ATA at 1 h each day for different durations. The values from treated HCAECs are expressed as a ratio of normalized values of miR-92a mRNA in the control cells. The Tukey–Kramer comparison test was conducted for statistical analysis. *P < 0.01 vs. control; n = 4 per group. B, Quantitative real-time PCR of HCAEC-derived exosomal miR-92a under 2.5 ATA with or without MALAT1 siRNA treatment. The values from treated HCAECs are expressed as a ratio of normalized values of miR-92a mRNA in the control cells. Scramble siRNA is a control siRNA. The Tukey–Kramer comparison test was conducted for statistical analysis. *P < 0.05 vs. control; n = 4 per group. C, Quantitative real-time PCR of HCAEC KLF2 mRNA expression under 2.5 ATA at 1 h each day for different durations. The values from treated HCAECs are expressed as a ratio of normalized values of KLF2 mRNA in the control cells. The Tukey–Kramer comparison test was conducted for statistical analysis. **P < 0.01 vs. control; n = 4 per group. D, Representative Western blot. E, Quantitative analysis of KLF2 protein expression under 2.5 ATA at 1 h each day for different durations. The values from treated HCAEC groups were normalized to match α-tubulin measurements and then expressed as a ratio of normalized values to protein in the control group. The Tukey–Kramer comparison test was conducted for statistical analysis. **P < 0.01 vs. control; n = 4 per group. International Journal of Cardiology 2019 274, 271-278DOI: (10.1016/j.ijcard.2018.09.118) Copyright © 2018 Elsevier B.V. Terms and Conditions

Fig. 3 MALAT1 siRNA and the overexpression of miR-92a inhibited both KLF2 expression in HCAECs under HBO treatment and the effect of miR-92a on KLF2 3′UTR luciferase activity under HBO treatment. A, Quantitative real-time PCR of HCAEC KLF2 mRNA expression under 2.5 ATA at 1 h each day for 5 days. The values from treated HCAECs are expressed as a ratio of normalized values of KLF2 mRNA in the control cells. The Tukey-Kramer comparison test was conducted for statistical analysis. *P < 0.01 vs. HBO; n = 4 per group. B, Representative Western blot. C, Quantitative analysis of KLF2 protein expression under 2.5 ATA at 1 h each day for 5 days. The values from treated HCAEC groups were normalized to match α-tubulin measurements and then expressed as a ratio of normalized value to protein in the control groups. The Tukey–Kramer comparison test was conducted for statistical analysis. *P < 0.01 vs. HBO; n = 4 per group. D, Sequence of the KLF2 3′UTR target site for miR-92a-3p binding, located at KLF2 3′UTR (nucleotide from 228 to 249). E, KLF2 3′UTR luciferase activity under HBO treatment with or without miR-92a-3p. The Tukey–Kramer comparison test was conducted for statistical analysis; n = 4 per group. International Journal of Cardiology 2019 274, 271-278DOI: (10.1016/j.ijcard.2018.09.118) Copyright © 2018 Elsevier B.V. Terms and Conditions

Fig. 4 Effect of HBO and HCAEC-derived exosomes with or without MALAT1 siRNA and miR-92a on the proliferation and capillary-like network formation of HCAECs as well as the effect of HCAEC-derived exosomes and HBO with or without MALAT1 siRNA and miR-92a on ratio of blood flow in hind-limb ischemia. A, Quantitative analysis of HCAEC proliferation and viability. Proliferation and viability assays were performed using the CellTiter-Glo® Luminescent Cell Viability Assay kit. The Tukey–Kramer comparison test was conducted for statistical analysis. *P < 0.001 vs. HBO-induced exosomes; n = 4 per group. B, Representative image of the tube formation of HCAECs under HBO treatment. C, Measurement of quantitative branching points. The Tukey–Kramer comparison test was conducted for statistical analysis. * P < 0.001 vs. HBO alone; n = 4 per group. D, Representative image of the tube formation of HCAECs without HBO treatment. E, Representative image of the tube formation of HCAECs. F, Representative image of Laser Doppler perfusion imaging. G, Quantitative analysis of perfusion recovery with reference to the ratio of blood flow (ischemic/normal hind-limb). The Tukey–Kramer comparison test was conducted for statistical analysis. *P < 0.01 vs. ligation; **P < 0.001 vs. ligation; n = 6 per group. International Journal of Cardiology 2019 274, 271-278DOI: (10.1016/j.ijcard.2018.09.118) Copyright © 2018 Elsevier B.V. Terms and Conditions

Fig. 4 Effect of HBO and HCAEC-derived exosomes with or without MALAT1 siRNA and miR-92a on the proliferation and capillary-like network formation of HCAECs as well as the effect of HCAEC-derived exosomes and HBO with or without MALAT1 siRNA and miR-92a on ratio of blood flow in hind-limb ischemia. A, Quantitative analysis of HCAEC proliferation and viability. Proliferation and viability assays were performed using the CellTiter-Glo® Luminescent Cell Viability Assay kit. The Tukey–Kramer comparison test was conducted for statistical analysis. *P < 0.001 vs. HBO-induced exosomes; n = 4 per group. B, Representative image of the tube formation of HCAECs under HBO treatment. C, Measurement of quantitative branching points. The Tukey–Kramer comparison test was conducted for statistical analysis. * P < 0.001 vs. HBO alone; n = 4 per group. D, Representative image of the tube formation of HCAECs without HBO treatment. E, Representative image of the tube formation of HCAECs. F, Representative image of Laser Doppler perfusion imaging. G, Quantitative analysis of perfusion recovery with reference to the ratio of blood flow (ischemic/normal hind-limb). The Tukey–Kramer comparison test was conducted for statistical analysis. *P < 0.01 vs. ligation; **P < 0.001 vs. ligation; n = 6 per group. International Journal of Cardiology 2019 274, 271-278DOI: (10.1016/j.ijcard.2018.09.118) Copyright © 2018 Elsevier B.V. Terms and Conditions

Supplemental Fig. I Electropherograms and gel-images. For visualization and better interpretation, an electropherogram and a virtual gel image were generated. A, Representative electropherogram. B, Representative gel-image. Agilent 2100 Bioanalyzer was used to identify the RNA quality of exosomes by RNA Pico Chip (Agilent Technologies, Waldbronn, Germany). International Journal of Cardiology 2019 274, 271-278DOI: (10.1016/j.ijcard.2018.09.118) Copyright © 2018 Elsevier B.V. Terms and Conditions

Supplemental Fig. II Effect of HBO derived exosome and exosome without HBO treatment on MALAT1 expression in HCAECs. Tukey-Kramer comparison test was used for statistical analysis. *P < 0.05 vs. control. **P < 0.001 vs. control. (n = 4 per group). International Journal of Cardiology 2019 274, 271-278DOI: (10.1016/j.ijcard.2018.09.118) Copyright © 2018 Elsevier B.V. Terms and Conditions

Supplemental Fig. III Quantitative real-time PCR of HCAEC-derived exosomal MALAT1 under 2.5 ATA at 1 h each day for different durations. Exosomes derived from HCAECs were isolated by Qiagen-exoEasy maxi kit. The values from treated HCAECs are expressed as a ratio of normalized values of MALAT1 mRNA in the control cells. Tukey-Kramer comparison test was used for statistical analysis. *P < 0.05 vs. control. **P < 0.01 vs. control. n = 4 per group. International Journal of Cardiology 2019 274, 271-278DOI: (10.1016/j.ijcard.2018.09.118) Copyright © 2018 Elsevier B.V. Terms and Conditions

Supplemental Fig. IV Image of SYTO RNASelect Green Fluorescent Cell Stain in human coronary artery endothelial cells (HCAECs). A, Representative image of HCAECs without exosomes treatment. B, Representative image of HCAECs with HBO-induced exosomes treatment. Green fluorescence: SYTOR RNASelect™ stain. Red fluorescence: Alexa FluorR 594 phalloidin. Blue fluorescence: DAPI. Similar results were observed in a further three independent experiments. International Journal of Cardiology 2019 274, 271-278DOI: (10.1016/j.ijcard.2018.09.118) Copyright © 2018 Elsevier B.V. Terms and Conditions

Supplemental Fig. V In situ hybridization assay detects the increased expression of MALAT1 in HBO-stimulated HCAECs. Representative microscopic images showing the presence of MALAT1 (green, arrows) in the nucleus of HCAECs. DAPI stain was used to image nuclei (blue) and rhodamine fluorescence stain was used to image HCAECs (red). Merge means image combined of detection probe. International Journal of Cardiology 2019 274, 271-278DOI: (10.1016/j.ijcard.2018.09.118) Copyright © 2018 Elsevier B.V. Terms and Conditions

Supplemental Fig. VI Effect of MALAT1 on miR-92a expression. A, Partial human MALAT1 DNA fragment (contains miR92a-3p binding site) construct. B, Cloned human MALAT1 DNA fragment contains miR-92a-3p binding sites. C, MALAT 1 wild type overexpression significantly attenuated miR-92a expression in HCAECs. Tukey-Kramer comparison test was used for statistical analysis. *P < 0.001 vs. control. (n = 4 per group). International Journal of Cardiology 2019 274, 271-278DOI: (10.1016/j.ijcard.2018.09.118) Copyright © 2018 Elsevier B.V. Terms and Conditions

Supplemental Fig. VII Presence of TSG101 in HCAEC-derived exosomes. Representative Western blot of TSG101 in HCAEC-derived exosomes. Similar findings were observed in another 2 independent experiments. International Journal of Cardiology 2019 274, 271-278DOI: (10.1016/j.ijcard.2018.09.118) Copyright © 2018 Elsevier B.V. Terms and Conditions

Supplemental Fig. VIII MALAT1 increases VEGFR2 expression in HCAECs. A, Representative Western blot and B, quantitative analysis of VEGFR2 protein expression under MALAT1-contained exosomes for different durations. The values from treated HCAEC groups have been normalized to matched α-tubulin measurement and then expressed as a ratio of normalized values to protein in the control group. Tukey-Kramer comparison test was used for statistical analysis. *P < 0.001 vs. control. N = 4 per group. International Journal of Cardiology 2019 274, 271-278DOI: (10.1016/j.ijcard.2018.09.118) Copyright © 2018 Elsevier B.V. Terms and Conditions