Fig 1: HL3501 showed anti-glaucoma effects in a Dex-induced OHT mouse model. (A) IOP of mice over 3 weeks of treatment after topical Dex treatment for the induction of OHT; the IOP of the mouse on day 1 is shown. The mean value of the IOP was estimated. Base (basal IOP, before Dex treatment) ***P < 0.001 versus normal group; ###P < 0.001 versus vehicle-treated group. (B) ERG of mice (day 21). **P < 0.01, ***P < 0.001 versus normal group; #P < 0.05, ##P < 0.01, ###P < 0.001 versus vehicle-treated group. (C) Effect of HL3501 on fibronectin expression in the TM of murine GC-induced glaucoma. The paraffin-embedded retinal tissue was immunolabeled with fibronectin antibody (green) and SMA antibody (red) and observed under a fluorescence microscope focusing on the TM (red and yellow asterisks). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Scale bar: 100 mm. CB, ciliary body; SC, Schlemm's canal. (D) The effect of HL3501 on RGCs in murine GC-induced glaucoma. The arrows indicate the loss of RGCs. Scale bar: 200 mm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelia. (E) Effect of HL3501 on MMP-2, MMP-9, TIMP-1, and TIMP-2 protein expression in the GC-induced glaucoma model. Retinal extracts underwent western blot analysis to measure the expression of MMPs and TIMPs. Figure shows the quantification of each band normalized to a-tubulin when evaluating the amount of MMPs and TIMPs separately. Topical ocular application of HL3501 (0.04%, BID), latanoprost (0.005%, QD), or vehicle following IOP elevation induced by Dex treatment for 2 weeks. The date of measurement of the IOP of the mouse model was recorded for 21 days. Electroretinography was performed on both eyes of dark-adapted anesthetized mice on days 0 and 21. Each group had eight animals. #P < 0.05; ##P < 0.01; ###P < 0.001 versus vehicle; **P < 0.01; ***P < 0.001 versus normal.
Fig 2: Schematic showing the mechanistic role of the Hif-1a-p53 axis in cardiac rupture after myocardial infarction (MI).Hif-1a indicates hypoxia-inducible factor 1-a; Hif-1ß, hypoxia-inducible factor 1-ß; IL-1ß, interleukin-1ß; MMP/TIMP, matrix metalloproteinase/tissue inhibitor of metalloproteinase; OH, hydroxy group; and PHD, prolyl hydroxylase domain-containing protein.
Fig 3: Role of Hif-1a (hypoxia-inducible factor 1-a) in CC-8 (cleaved caspase-8), inflammation, and matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) in the myocardial infarction (MI) border zone on day 5. A, Expression of CC-8, cleaved interleukin-1ß (cIL-1ß), Mac-3, MMP-2, MMP-9, TIMP-1, and TIMP-2 in the MI border zone at day 5 after MI. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as a loading control (CTL). B, Quantification of Western blots shown in (A); n=4 in each group. Data are shown as a ratio to CTL+MI. *P<0.05, **P<0.01 vs CTL+MI, analyzed by Student t test. caHetKO indicates cardiac-specific Hif-1a hetero-knockout; and N/A indicates not applicable.
Fig 4: H3K27me3 is enriched in CDKN2A and Timp4 gene promoters. AFs in P0 were harvested for CUT&Tag assay, and AFs from P0, P1 and P2 were harvested for qRT-PCR and WB analyses. (A) Pie chart illustrating the distribution of H3K27me3 depositing sites relative to RefSeq functional categories. (B) Heatmap representing normalized H3K27me3 reads across promoters (±3 kb of TSS; Up) and averaged profiles (Bottom). TES; transcriptional end site. (C) CUT&Tag tracks show H3K27me3 potential localizations on the CDKN2a and Timp4 genes. Areas shadowed in red indicate the regions chosen for PCR validations. (D) Schematic diagram (left) showing the relative positions of the primers on the promoters of the CDKN2a (p16 and p19) and Timp4 genes. Arrows indicate the transcription start site (TSS) and direction and short black lines beneath indicate the locations for PCR. PCR bands shown on agarose gels demonstrate that H3K27me3 specifically binds to the promoter regions of CDKN2a (p16 and p19) and Timp4 genes. (E) Transcriptions of Timp1, Timp2, Timp3 and Timp4 genes were evaluated by qRT-PCR (n=3). (F) WB for analyzing Timp1~4 and three collagenase-type MMPs (n=6). Data are presented as mean ± SD. *p<0.05, **p<0.01, #p<0.001.
Fig 5: ApoA1, IL-17E, IgA, and TIMP1 increased after myocardial infarction (MI). A ApoA1 increased over the course of MI. ApoA1 increased from D0 to MI D3 in the retrospective plasma analysis (left). ApoA1 further increased from MI D3 to D7 in the prospective plasma analysis (right). B IgA, IL-17E, and TIMP-1 followed the same pattern as apoA1. The table summarizes protein expression in the plasma for both cohort 1 (D0 vs. D3) and cohort 2 (D3 vs. D7). Values are average ± SEM
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