Journal: Molecular Therapy. Nucleic Acids

Article Title: Inducible miR-1224 silences cerebrovascular Serpine1 and restores blood flow to the stroke-affected site of the brain

doi: 10.1016/j.omtn.2022.12.019

Figure Lengend Snippet: Serpine1 is a target of miR-1224 (A) Angiogenic protein array showing the difference in control mimic- and miR-1224 mimic-transfected bEnd.3 cells. The Proteome Profiler Mouse Angiogenesis Array Kit (cat. no. ARY015) was used to simultaneously assess the relative levels of 53 mouse angiogenesis-related proteins. (B) SERPINE1 protein (n = 9) and (C) Serpine1 transcript abundance in miR-1224 mimic-delivered cells (n = 6). (D) Binding site to mouse transcript ENSMUST00000041388-3′ UTR position 652–674 to miR-1224-5p (MIMAT0005460); binding energy −30.5 kcal/mol. Proposed binding sites for miR-1224 were identified in the SERPINE1 3′ UTR that was inserted into the Gaussia luciferase plasmid vector (wild type). The pEZX-MT05 vector also contains the secreted Gaussia luciferase (GLuc) open reading frame, driven by the SV40 promoter, as a reporter of the 3′ UTR expression, and a secreted alkaline phosphatase (SEAP) reporter, driven by a CMV promoter, as an internal control. (E) Quantification of luciferase reporter assay (n = 12) of wild-type and mutated versions of the miR-1224 binding site on the SERPINE1 3′ UTR. GLuc activity and alkaline phosphatase activity were assayed after 48 h of transfection using GeneCopoeia’s Secrete-Pair dual luminescence assay kit. Percentage change in GLuc activity was calculated after normalizing to alkaline phosphatase activity. Data are shown as mean ± SEM.

Article Snippet: SERPINE1 levels were measured using a commercially available ELISA kit (DY3828-05; R&D Systems, Minneapolis, MN) following the manufacturer’s protocol as previously described.

Techniques: Protein Array, Control, Transfection, Binding Assay, Luciferase, Plasmid Preparation, Expressing, Reporter Assay, Activity Assay, Luminescence Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inducible miR-1224 silences cerebrovascular Serpine1 and restores blood flow to the stroke-affected site of the brain

doi: 10.1016/j.omtn.2022.12.019

Figure Lengend Snippet: Downregulation of Serpine1 induced angiogenic response in mouse primary brain microvascular endothelial cells Matrigel tube formation was visualized by phase-contrast microscopy at 18–24 h in control or SERPINE1 siRNA-transfected cells. pMBMECs transfected with nontargeting siRNA (control siRNA) or SERPINE1 siRNA were plated on Matrigel 72 h following transfection. (A) Tube formation in control and SERPINE1 siRNA-transfected pMBMECs. Scale bars, 100 μm. (B) SERPINE1 protein (ELISA) after transfection of SERPINE1 siRNA (n = 12). Standard angiogenic parameters were quantified (n = 7) using ImageJ and the Angiogenesis Analyzer plug-in tool. (C) Total tube length, (D) number of nodes, (E) number of junctions, and (F) number of meshes. Data shown as mean ± SEM. (G–K) Alternative studies were conducted with the SERPINE1 inhibitor TM5441. (G) Tube formation in control and TM5441 (10 μM, 24 h)-treated pMBMECs. Scale bars, 100 μm. Angiogenic parameters were quantified (n = 8) using ImageJ and the Angiogenesis Analyzer plug-in tool. (H) Total tube length, (I) number of nodes, (J) number of junctions, and (K) number of meshes. Data are shown as mean ± SEM.

Article Snippet: SERPINE1 levels were measured using a commercially available ELISA kit (DY3828-05; R&D Systems, Minneapolis, MN) following the manufacturer’s protocol as previously described.

Techniques: Microscopy, Control, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inducible miR-1224 silences cerebrovascular Serpine1 and restores blood flow to the stroke-affected site of the brain

doi: 10.1016/j.omtn.2022.12.019

Figure Lengend Snippet: Small-molecule SERPINE1 inhibition increased cerebrovascular blood flow at the stroke-affected site and protected against stroke (A) Timeline of TM5441 treatment, MCAO, LSI, MRI, and brain harvest. (B) Real-time, noninvasive, cerebrovascular perfusion images of the dorsal surface of the brain were acquired in mice during occlusion and at 48 h post-stroke. (C) Perfusion quantification in TM5441-treated mice showed significantly higher perfusion in stroke-affected brain (n = 7 and 8). The SERPINE1 inhibitor TM5441 improved FITC-lectin perfusion to the S1 cortex at the stroke-affected site at 48 h post-stroke. (D) Fluorescence micrographs of stroke-affected S1 cortex of control and TM5541-treated mice. The inset images (indicated by white dashed lines) show a zoomed-in view of the selected region on the right. Scale bars, 50 μm. (E) Quantification of lectin-perfused vessel length (μm 2 area, n = 7). (F) Representative 9.4 T MRI (48 h post-stroke) images and (G) percentage hemisphere lesion volume calculated based on T2-weighted MRI at 48 h post-stroke (n = 7). (H) Representative mobility track plots from baseline and 48 h post-stroke of control or TM5441-treated mice. Tracks start at blue dots and end at red dots. (I) TM5441 treatment significantly improved 48 h post-stroke distance traveled, mean speed, and time mobile (n = 6 and 8) compared with control. Data are shown as mean ± SEM.

Article Snippet: SERPINE1 levels were measured using a commercially available ELISA kit (DY3828-05; R&D Systems, Minneapolis, MN) following the manufacturer’s protocol as previously described.

Techniques: Inhibition, Fluorescence, Control