Fig 2: EC-SMC cross talk via exosome-delivered miR-483-3p. (a) A representative electron microscopy image of HUVEC-derived exosomes. Scale bar, 100 nm. (b) A nanoparticle tracking analysis of the size distribution of exosomes. (c) Immunofluorescence images of HASMCs stained with a-SMA (red) and DAPI (blue). HASMCs were treated with HUVEC-derived exosomes (PKH67-label, green) for 0 or 24 hours. Laser scanning confocal images are shown. Scale bar, 20 µm. (d) The qPCR analysis of miR-483-3p levels in HUVECs transfected with pre-miR-Ctrl or pre-miR-483-3p for 48 hours. (e) Exosomes isolated from HUVECs were identified by CD81 and CD63 immunostaining. (f) miR-483-3p levels in exosomes isolated from HUVECs transfected with pre-miR-Ctrl or pre-miR-483-3p. (g–i) HASMCs were incubated with HUVEC-derived exosomes for 24 hours. The qPCR analysis of (g) miR-483-3p and ACE1, TGF-ß, and (h) CTGF in HASMCs. (i) Western blotting analysis of these proteins in HASMCs. Data are shown as mean ± SEM. Student's two-tailed t test was used for statistical analysis of data. *P < 0.05; ***P < 0.001.

Fig 3: Knockdown of Dot1L alleviates cardiac fibrosis and improves cardiac function during MI. Lentivirus carrying Dot1L shRNA (shDot1L) or Control shRNA (shCtrl) was injected into myocardium of mice undergoing LAD ligation for 14 days to specially knock down the cardiac Dot1L. A The expressions of Dot1L, CTGF, FN, Col 1, and MMP9 in MI mice were analyzed by western blot. B The level of H3K79me3 was analyzed by western blot. C Representative images of immunohistochemistry staining of Dot1L and Col 3 of cross sections of whole heart from MI mice. D Cardiac functions were measured by echocardiography and EF%, FS% were also statistically analyzed. E Representative images of immunofluorescence double staining of Dot1L and MMP9 of cross sections of whole heart. All data are represented as means ± SD. ANOVA, ***p < 0.001 versus Sham group, ##p < 0.01, ###p < 0.001 versus MI treated with shCtrl, n = 6/per group

Fig 4: A negative effect of HOXA4 on VSMC phenotypic switching is mediated by YAP/TEAD ARepresentative Western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding shRNAs of HOXA4 and TEAD1/3/4. Quantification of alpha-smooth muscle actin and calponin protein levels is expressed as the means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, by ANOVA with Sidak's correction.BProliferation assay in VSMCs transduced with indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then, cell viability was analyzed by Cell Counting Kit-8 at indicated days at each time point as indicated. *P < 0.05 versus shCtrl, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.CRepresentative immunoblotting images and quantification of the interaction between endogenous TEAD1 and HOXA4 or YAP using co-IP experiments in human VSMCs with knockdown of HOXA4. *P < 0.05 versus shCtrl, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.DChIP assays of CTGF, CYR61, and a-SMA promoters using anti-FLAG-tag, anti-Myc-tag, and anti-TEAD1 antibodies in human VSMCs transfected with plasmids expressing Myc-tagged YAP-5SA and/or FLAG-tagged HOXA4. *P < 0.05, **P < 0.01 versus FLAG-GFP, by unpaired two-tailed Student's t-test. Data are expressed as the means ± SEM of three independent experiments.

Fig 5: miR-133a blunted cardiac fibroblast proliferation and activation. (a) PVFs were treated with or without Ang II for 24 h. miR-133a level in PVFs was quantified by RT-PCR. Data are mean ± SEM (n = 5 independent experiments); *p < 0.05, unpaired 2-tail t-test. (b–e) PVFs were transfected with miR-133a mimics (133a-m) or negative control (m-NC) and inhibitors (133a-i) or negative control (i-NC) for 24 h. (b) The proliferation rate of PVFs in each group was detected by the CCK-8 assay. (c) CTGF mRNA levels in each group were quantified by RT-PCR. (d) The protein levels of CTGF, Col 1, and Col 3 were detected by western blot analysis. (e) Quantification of CTGF, Col 1, and Col 3 in (d). Data are mean ± SEM (n = 5 independent experiments); *p < 0.05, one-way ANOVA with the Bonferroni posttest. (f–k) PVFs were transfected with 133a-m or 133a-i for 24 h and treated with Ang II for 24 h. (f–g) CTGF mRNA levels in each group were quantified by RT-PCR. (h, j) The protein levels of CTGF, Col 1, and Col 3 were detected by western blot analysis. (i, k) Quantification of CTGF, Col 1, and Col 3 in (h) and (j). Data are mean ± SEM (n = 5 independent experiments); *p < 0.05, two-way ANOVA with the Bonferroni posttest.