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Functional regulation of YAP mechanosensitive transcriptional coactivator by Focused Low-Intensity Pulsed Ultrasound (FLIPUS) enhances proliferation of murine mesenchymal precursors.

ABSTRACT: Yes-associated protein (YAP) acts as a mechanotransducer in determining the cell fate of murine C2C12 mesenchymal precursors as investigated after stimulation with ultrasound. We applied Focused Low-Intensity Pulsed Ultrasound (FLIPUS) at a sound frequency of 3.6 MHz, 100 Hz pulse repetition frequency (PRF), 27.8% duty cycle (DC), and 44.5 mW/cm2 acoustic intensity ISATA for 5 minutes and evaluated early cellular responses. FLIPUS decreased the level of phosphorylated YAP on Serine 127, leading to higher levels of active YAP in the nucleus. This in turn enhanced the expression of YAP-target genes associated with actin nucleation and stabilization, cytokinesis, and cell cycle progression. FLIPUS enhanced proliferation of C2C12 cells, whereas silencing of YAP expression abolished the beneficial effects of ultrasound. The expression of the transcription factor MyoD, defining cellular myogenic differentiation, was inhibited by mechanical stimulation. This study shows that ultrasound exposure regulates YAP functioning, which in turn improves the cell proliferative potential, critical for tissue regeneration process.

SUBMITTER: Puts R

PROVIDER: S-EPMC6203358 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Functional regulation of YAP mechanosensitive transcriptional coactivator by Focused Low-Intensity Pulsed Ultrasound (FLIPUS) enhances proliferation of murine mesenchymal precursors.

Puts Regina R Rikeit Paul P Ruschke Karen K Knaus Petra P Schreivogel Sophie S Raum Kay K

PloS one 20181026 10

Yes-associated protein (YAP) acts as a mechanotransducer in determining the cell fate of murine C2C12 mesenchymal precursors as investigated after stimulation with ultrasound. We applied Focused Low-Intensity Pulsed Ultrasound (FLIPUS) at a sound frequency of 3.6 MHz, 100 Hz pulse repetition frequency (PRF), 27.8% duty cycle (DC), and 44.5 mW/cm2 acoustic intensity ISATA for 5 minutes and evaluated early cellular responses. FLIPUS decreased the level of phosphorylated YAP on Serine 127, leading ...[more]

PMID: 30365513

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Project description:Ultrasound is considered a safe and non-invasive tool in regenerative medicine and has been used in the clinic for more than twenty years for applications in bone healing after the approval of the Exogen device, also known as low-intensity pulsed ultrasound (LIPUS). Beyond its effects on bone health, LIPUS has also been investigated for wound healing of soft tissues, with positive results for various cell processes including cell proliferation, migration and angiogenesis. As LIPUS has the potential to treat chronic skin wounds, we sought to evaluate the effects produced by a conventional therapeutic ultrasound device at low intensities (also considered LIPUS) on the migration capacity of mouse and human skin mesenchymal precursors (s-MPs). Cells were stimulated for 3 days (20 minutes per day) using a traditional ultrasound device with the following parameters: 100 mW/cm2 with 20% duty cycle and frequency of 3 MHz. At the parameters used, ultrasound failed to affect s-MP proliferation, with no evident changes in morphology or cell groupings, and no changes at the cytoskeletal level. Further, the migration and invasion ability of s-MPs were unaffected by the ultrasound protocol, and no major changes were detected in the gene/protein expression of ROCK1, integrin β1, laminin β1, type I collagen and transforming growth factor β1. Finally, RNA-seq analysis revealed that only 10 genes were differentially expressed after ultrasound stimulation. Among them, 5 encode for small nuclear RNAs and 2 encode for proteins belonging to the nuclear pore complex. Considering the results overall, while the viability of s-MPs was not affected by ultrasound stimulation and no changes were detected in proliferation/migration, RNA-seq analysis would suggest that s-MPs do respond to ultrasound. The use of 100 mW/cm2 intensity or conventional therapeutic ultrasound devices might not be optimal for the stimulation the properties of cell populations. Future studies should investigate the potential application of ultrasound using variations of the tested parameters.

Project description:BackgroundSteroids are a leading cause of femoral head osteonecrosis. Currently there are no medications available to prevent and/or treat steroid-associated osteonecrosis. Low-intensity pulsed ultrasound (LIPUS) was approved by the FDA for treating delayed union of bone fractures. Some studies have reported that LIPUS can enhance bone formation and local blood flow in an animal model of fracture healing. However, whether the effect of osteogenesis and neovascularization by LIPUS can enhance the repair progress in steroid-associated osteonecrosis is unknown.Questions/purposesWe hypothesized that LIPUS may facilitate osteogenesis and neovascularization in the reparative processes of steroid-associated osteonecrosis. Using a rabbit animal model, we asked whether LIPUS affects (1) bone strength and trabecular architecture; (2) blood vessel number and diameter; and (3) BMP-2 and VEGF expression.MethodsBilateral femoral head necrosis was induced by lipopolysaccharide and methylprednisolone in 24 rabbits. The left femoral heads of rabbits received LIPUS therapy (200 mW/cm(2)) for 20 minutes daily and were classified as the LIPUS group. The right femoral heads of the same rabbits did not receive therapy and were classified as the control group. All rabbits were euthanized 12 weeks after LIPUS therapy. Micro-CT, biomechanical testing, histologic evaluation, immunohistochemistry, quantitative real-time PCR, and Western blot were used for examination of the effects of LIPUS.ResultsTwelve weeks after LIPUS treatment, the loading strength in the control group was 355 ± 38 N (95% CI, 315-394 N), which was lower (p = 0.028) than that in the LIPUS group (441 ± 78 N; 95% CI, 359-524 N). The bone tissue volume density (bone volume/total volume) in the LIPUS group (49.29% ± 12.37%; 95 % CI, 36.31%-62.27%) was higher (p = 0.022) than that in the control group (37.93% ± 8.37%; 95 % CI, 29.15%-46.72%). The percentage of empty osteocyte lacunae in the LIPUS group (17% ± 4%; 95% CI, 15%-20%) was lower (p = 0.002) than that in the control group (26% ± 9%; 95% CI, 21%-32%). The mineral apposition rate (μm/day) in the LIPUS group (2.3 ± 0.8 μm/day; 95% CI, 1.8 2.8 μm/day) was higher (p = 0.001) than that in the control group (1.6 ± 0.3 μm/day; 95% CL, 1.4-1.8 μm/day). The number of blood vessels in the LIPUS group (7.8 ± 3.6/mm(2); 95% CI, 5.5-10.1 mm(2)) was greater (p = 0.025) than the number in the control group (5.7 ± 2.6/mm(2); 95% CI, 4.0-7.3 mm(2)). Messenger RNA (mRNA) and protein expression of BMP-2 in the LIPUS group (75 ± 7, 95% CI, 70-79; and 30 ± 3, 95% CI, 28-31) were higher (both p < 0.001) than those in the control groups (46 ± 5, 95% CI, 43-49; and 15 ± 2, 95% CI, 14-16). However, there were no differences (p = 0.114 and 0.124) in mRNA and protein expression of vascular endothelial growth factor between the control (26 ± 3, 95% CI, 24-28; and 22 ± 6, 95% CI, 18-26) and LIPUS groups (28 ± 2, 95% CI, 26-29; and 23 ± 6, 95% CI, 19-27).ConclusionsThe results of this study indicate that LIPUS promotes osteogenesis and neovascularization, thus promoting bone repair in this steroid-associated osteonecrosis model.Clinical relevanceLIPUS may be a promising modality for the treatment of early-stage steroid-associated osteonecrosis. Further research, including clinical trials to determine whether LIPUS has a therapeutic effect on patients with early-onset steroid-associated osteonecrosis may be warranted.

Dataset Information

Functional regulation of YAP mechanosensitive transcriptional coactivator by Focused Low-Intensity Pulsed Ultrasound (FLIPUS) enhances proliferation of murine mesenchymal precursors.

Publications

Functional regulation of YAP mechanosensitive transcriptional coactivator by Focused Low-Intensity Pulsed Ultrasound (FLIPUS) enhances proliferation of murine mesenchymal precursors.

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