Cortical Bone Derived Stem Cells Modulate Cardiac Fibroblast Response via miR-18a in the Heart After Injury

Figures

• 

  FIGURE 1. CBSCs treatment restrict fibrotic scar after MI. (A) Outline of experimental design using an adult mouse heart given a myocardial infarction (MI) and treated with CBSC or Saline injection with measurements taken 3, 7, and 14 days after injection. (B) Fibrosis was decreased in hearts injected with CBSCs compared to PBS using Picrosirius Red stain for fibrosis and quantified based on the amount of fibrosis relative to the total area 14 days after injection, n = 5, *P < 0.05. (C) A decrease in percent of infarct size in heart injected with CBSCs compared to PBS hearts using Masson’s trichrome staining and quantified based on the percentage of infarct size 14 days after injection, n = 5, **P < 0.01. (D) A fibrotic screen of a MI heart treated with PBS or CBSCs showing lower myofibroblast marker expression in CBSC treated hearts compared to PBS treatment. Black bars indicating the control PBS or Saline treatment with adult cardiac fibroblasts, gray bars indicating CBSC treatment with adult cardiac fibroblasts.

• 

  FIGURE 2. CBSC secretome reduces myofibroblast markers in adult cardiac fibroblasts. (A) Outline of experimental design showing adult cardiac fibroblasts isolation (B) Representative image of untreated fibroblasts that have a (spindle-like shaped), fibroblast treated with TGFβ (flattened and broad) indicating myofibroblast transition. The fibroblast reverts their morphology back to untreated fibroblasts after CBSC treatment scale bar represents 20 μM. (C) Decreased myofibroblast marker expression is observed in fibroblast treated with CBSCs and TGFβ measured by quantitative reverse transcription polymerase chain reaction analysis, n = 3, *P < 0.05. (D) Decreased myofibroblast and TGFβ pathway maker expression are observed in fibroblasts treated with CBSCs and CBSCs and TGFβ using Western blot analysis, normalized to GAPDH, *P < 0.05.

• 

  FIGURE 3. The miR-18a-5p prevents adult cardiac fibroblast differentiation and TGF-β stimulation. (A) The increased expression of miR-18-5p in adult hearts after treatment with CBSCs compared to PBS treatment using quantitative reverse transcription polymerase chain reaction analysis, n = 4, *P < 0.05. (B) The increased expression of miR-18-5p in adult cardiac fibroblast treated with CBSC and TGFβ compared to TGFβ alone using quantitative reverse transcription polymerase chain reaction analysis, n = 3, *P < 0.05. (C) Outline of experimental design using adult cardiac fibroblasts treated with the miR-18-5p mimic or inhibitor for 24 h. (D) Decrease in myofibroblast marker expression in fibroblast treated with the mimic of miR-18-5p with TGFβ compared to TGFβ alone and/or the inhibitor of miR-18-5p and TGFβ using quantitative reverse transcription polymerase chain reaction analysis, n = 3, *P < 0.05 All qRT-PCR data is represented as RFC (relative fold change).

• 

  FIGURE 4. The miR-18a-5p prevents fibrosis in the adult heart. (A) A target scan search of the miR-18-5p sequence indicating conserved sequence across species and a miR-18-5p target predication position for pairing of the target region of the myofibroblast marker, CTGF. (B) Decrease in relative luciferase activity of CTGF with the treatment of the mimic of miR-18-5p using a Luc-Pair Duo-Luciferase assay, n = 6. (C) A decrease in myofibroblast marker expression of Acta2 and CTGF respectively with the treatment of the mimic of miR-18-5p with TGFβ using western blot analysis, normalized to GAPDH, n = 6, *P < 0.05. (D) The mechanism of action of CBSCs and the miR-18-5p inhibiting CTGF, preventing the myofibroblast transition and fibrosis.