Int J Stem Cells.  2022 Nov;15(4):372-383. 10.15283/ijsc22024.

LIPUS Promotes Endothelial Differentiation and Angiogenesis of Periodontal Ligament Stem Cells by Activating Piezo1

• Hu R
• Yang Zy
• Li Yh
• Zhou Z

• KMID: 2536207
• DOI: http://doi.org/10.15283/ijsc22024

Abstract

Background and Objectives
Low-intensity pulsed ultrasound (LIPUS) promotes differentiation and regulates biological functions of various stem cells, but its effect on the endothelial differentiation of periodontal ligament stem cells (PDLSCs) is unclear. This study investigated the effect of LIPUS on endothelial differentiation and angiogenesis in PDLSCs and the role of the mechanically sensitive ion channel Piezo1 in this process.
Methods and Results
PDLSCs obtained from healthy people were used for endothelial induction, and 10 μg/ml lipopolysaccharide (LPS) was used to simulate the inflammatory state. The induced cells were treated with LIPUS (50 mW/cm² , 1.5 MHz) to study its effect on the endothelial differentiation of PDLSCs and the tube formation of differentiated cells. PCR, flow cytometry, immunofluorescence, and Matrigel tube formation assays were used to detect the differentiation and tube formation of PDLSCs. GsMTx4 was used to inhibit the expression of Piezo1, and the role of the Piezo1 pathway in the endothelial differentiation and microvascular formation of PDLSCs after LIPUS treatment was studied. The data showed that LIPUS increased endothelial differentiation and angiogenesis in PDLSCs under inflammatory or noninflammatory conditions. The use of an inhibitor weakened the effect of LIPUS.
Conclusions
This study demonstrated that LIPUS can activate the expression of Piezo1 and promote the endothelial differentiation and microvascular formation of PDLSCs.

Keyword

PDLSC; LIPUS; Piezo1; Endothelial differentiation; Vessel

Figure

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  Fig. 1 Cell culture and identification. (A) Spindle-like PDLSCs crawling out from the tissue block, magnification: 4×. (B) Identification of PDLSC multidirectional differentiation: I: PDLSCs with no treatment, magnification: 4×; II: PDLSCs cultured in adipogenic induction medium for 14 days and tested by oil red O staining, magnification: 40×; III: PDLSCs cultured in osteogenic induction medium for 21 days and tested by Alizarin red S staining, magnification: 4×. (C) Identification of PDLSC surface markers: CD73, CD90, CD105, CD31, CD45.

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  Fig. 2 LIPUS promotes osteogenic differentiation of PDLSCs. (A) The ALP miRNA expression of PDLSCs with osteogenic induction for 7 days that were treated with LIPUS at different intensities, **p<0.005. (B) The CD31 miRNA expression of PDLSCs with osteogenic induction that were treated with LIPUS at different intensities for 7 days, **p<0.005. (C) Alizarin red staining results of PDLSCs with osteogenic induction and treatment with LIPUS for 14 days: I. control; II: treated with LIPUS, magnification: normal or 4×.

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  Fig. 3 LIPUS promotes endothelial differentiation and angiogenesis of PDLSCs. (A) Cell morphology comparison following endothelial induction for 14 days: I: PDLSCs with no treatment; II: HUVECs; III: PDLSCs with endothelial induction for 14 days; magnification: 4×. (B) CD31 miRNA expression of PDLSCs with endothelial induction and treatment with LIPUS at different intensities for 14 days, ***p< 0.0005. (C) Flow cytometric results: CD31 expression of PDLSCs with endothelial induction and treatment with LIPUS (50 mW/cm2) for 14 days: I: control; II: treated by LIPUS. (D) Tube formation results: PDLSCs with endothelial induction and treatment with LIPUS (50 mW/cm2) for 14 days: I: control; II: treated by LIPUS, magnification: 4×, a∼c: Statistical analysis results: V: endothelial induction in noninflammatory conditions, ***p<0.0005.

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  Fig. 4 LIPUS promotes endothelial differentiation and angiogenesis of PDLSCs under inflammatory conditions. (A) IL-6 mRNA expression of PDLSCs induced by LPS for 24 h. (B) LPS at different concentration was used to continuously induce cells for 7 days, ***p<0.0005, and CCK-8 assays were used to test cell proliferation, shown as the OD value, **p<0.005. (C) Flow cytometric results: CD31 expression of PDLSCs with endothelial induction and treatment with LIPUS (50 mW/cm2) for 14 days in inflammatory conditions: I: control; II: treated with LIPUS. (D) Tube formation assay results: PDLSCs with endothelial induction and treatment with LIPUS (50 mW/cm2) for 14 days in inflammatory conditions: I: control; II: treated with LIPUS, magnification: 4×; a∼c: Statistical analysis results: LV, endothelial induction in inflammatory conditions, *p<0.05, **p<0.005.

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  Fig. 5 LIPUS promotes the expression of Piezo1, and GsMTx4 inhibits this effect. (A) Piezo1 miRNA expression of PDLSCs with endothelial induction and treatment with LIPUS (50 mW/cm2) for 14 days, **p<0.005. (B) Piezo1 miRNA expression of PDLSCs cocultured with different concentrations of GsMTx4 and after LIPUS (50 mW/cm2) treatment for 24 h, **p<0.005. (C) Western blot results and statistical results: Piezo1 expression of PDLSCs with endothelial induction and treatment with GsMTx4 and LIPUS (50 mW/cm2) for 14 days: V: endo-thelial induction in noninflammatory condition, VG: endothelial induction and cocultured with GsMTx4 in noninflammatory condition, *p< 0.05, ***p<0.0005. (D) Immunofluorescence staining results: Piezo1 expression of PDLSCs with endothelial induction and treatment with GsMTx4 and LIPUS (50 mW/cm2) in inflammatory or noninflammatory conditions for 14 days, magnification: 4×, a, b: Statistical analysis results: V: endothelial induction in noninflammatory conditions, VG: endothelial induction and coculture with GsMTx4 in noninflammatory conditions, LV: endothelial induction in inflammatory conditions, LVG: endothelial induction and coculture with GsMTx4 in inflammatory conditions, *p<0.05, ***p<0.0005.

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  Fig. 6 Piezo1 regulates the endothelial differentiation and angiogenesis of PDLSCs treated with LIPUS. (A) CD31 miRNA expression of PDLSCs treated with GsMTx4 with endothelial induction and LIPUS treatment (50 mW/cm2) under inflammatory or noninflammatory conditions for 14 days, **p<0.005, ***p<0.0005. a: noninflammatory conditions; b: inflammatory conditions. (B) Flow cytometric results of PDLSCs: CD31 expression with treatment with GsMTx4 under endothelial induction and LIPUS treatment (50 mW/cm2) under inflammatory or noninflam-matory conditions for 14 days: I: V: endothelial induction under noninflammatory conditions, II: VG: endothelial induction and coculture with GsMTx4 under noninflammatory conditions, III: LV: endothelial induction under inflammatory conditions, IV: LVG: endothelial induction and coculture with GsMTx4 under inflammatory conditions. (C) The tube formation results of PDLSCs containing GsMTx4 under endothelial induction and treatment with LIPUS (50 mW/cm2) under inflammatory or noninflammatory conditions: The grouping is the same as in Fig. 6B, magnification: 4×; a∼c: Statistical analysis, **p<0.005, ***p<0.0005. (D) Immunofluorescence staining results of PDLSCs: CD31 expression of PDLSCs with GsMTx4 under endothelial induction and treatment with LIPUS (50 mW/cm2) in inflammatory or noninflammatory conditions for 14 days. The grouping is the same as in Fig. 5D, magnification: 4×. a, b: Statistical results, **p<0.005, ***p<0.0005.

Reference

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