ERK signaling is required for VEGF-A/VEGFR2-induced differentiation of porcine adipose-derived mesenchymal stem cells into endothelial cells

Abstract

Background: Cell-based therapy that can rejuvenate the endothelium with stimulated adipose-derived mesenchymal stem cells (AMSCs) is a promising therapeutic strategy for the re-endothelialization of denuded arteries at the stenting site. Previously, we have shown that silencing of MMP-2 and MMP-14 inhibits vascular endothelial growth factor receptor type 2 (VEGFR2) cleavage, and induces differentiation of AMSCs toward the endothelial cell (EC) lineage. In this study, we examined the underlying signaling pathways that regulate differentiation of AMSCs to ECs in vitro through VEGFR2.

Methods: AMSCs were isolated from porcine abdominal adipose tissue. The isolated AMSCs were characterized by positive expression of CD29, CD44, and CD90 and negative expression of CD11b and CD45. The isolated MSCs were transfected with siRNA to silence MMP-2, MMP-14, and angiotensin receptor 2 (ATR2). Cells were suspended either in endothelial basal media (EBM) or endothelial growth media (EGM) with various treatments. Flow cytometry was performed to examine the expression of EC markers, and western blot analysis was performed to examine the expression and activity of various kinases. Scratch assay was performed to examine the cell migration. Data were analyzed by ANOVA using PRISM GraphPad.

Results: After 10 days of stimulation for EC differentiation, the morphology of AMSCs changed to a morphology similar to that of ECs. Silencing MMP-2 and MMP-14 resulted in significant decrease in the number of migrated cells compared with the EGM-only group. ATR2 siRNA transfection did not affect the migration and differentiation of AMSCs to ECs. Stimulation of AMSCs for EC differentiation with or without MMP-2 or MMP-14 siRNA resulted in significant increase in p-ERK, and significant decrease in p-JNK. There was no significant change in p-p38 in all three groups compared with the EBM group. ERK inhibition resulted in significant decrease in the expression of EC markers in the EGM, EGM + MMP-2 siRNA, and EGM + MMP-14 siRNA groups. The VEGFR2 kinase inhibitor induced a dose-dependent inhibition of ERK.

Conclusion: The ERK signaling pathway is critical for VEGF-A/VEGFR2-induced differentiation of AMSCs into ECs. These findings provide new insights into the role of the ERK signaling pathway in AMSC differentiation to ECs for potential clinical use in cardiovascular diseases.

Keywords: Adipose-derived mesenchymal stem cells; Angiotensin type-2 receptor; Endothelial cell differentiation; Extracellular signal-regulated kinase 1/2; Mitogen-activated protein kinase; Stress activated protein kinase-2; Vascular endothelial growth factor receptor type 2; c-Jun NH2-terminal kinase.

Fig. 1

Immunophenotyping of AMSCs and differentiation to ECs. I Immunophenotyping of AMSCs. Flow cytometry data showed no expression for CD45 (A) and CD11b (B), and high expression for the MSC markers CD29 (C), CD44 (D), and CD90 (E). Blue peaks, profile of the isotype control. Flow cytometry was carried out on a FACS Aria Flow Cytometry System. II Endothelial cell differentiation. (A) AMSCs in culture with DMEM. Endothelial cell differentiation after 10 days of stimulation with EGM (B), EGM + MMP-2 siRNA (C), and EGM + MMP-14 siRNA (D). Change in the morphology of cells after 10 days of stimulation for endothelial cell differentiation (Color figure online)

Fig. 2

Migration assay. AMSCs in EBM, EGM, EGM + MMP-2 siRNA, EGM + MMP-14 siRNA, and EGM + ATR2 siRNA showed migration activity after 24 hours of making the scratches (a). MMP-2 and MMP-14 siRNA resulted in significant decrease in the number of migrated cells per field compared with EGM-only cells, whereas ATR2 siRNA silencing had no effect on the migration activity of AMSCs during EC differentiation (b). **p < 0.01. EBM endothelial cell basal medium, EGM endothelial cell growth medium, MMP matrix metalloproteinase, ATR2 angiotensin receptor R2

Fig. 3

ATR2 siRNA transfection and immunophenotyping for EC markers. I Concentration selection for siRNA transfection. Three different concentrations (10, 35, and 50 nM) of ATR2 siRNA were used according to the manufacturer’s protocol. Western blot analysis showed inhibition of ATR2 by 10, 35, and 50 nM of ATR2 siRNA. However, 50 nM of ATR2 siRNA showed the highest inhibition among all three different concentrations (A). ATR2 silencing by siRNA transfection with EGM compared with AMSCs with EGM and EGM + scrambled siRNA (negative control) (B). GAPDH was used as a housekeeping gene. II Flow cytometric analysis of PECAM1 (CD31) in four different groups; control group with EGM (A), AMSCs with EGM and MMP-2 siRNA (B), AMSCs with EGM and MMP-14 siRNA (C), and HUVECs as the positive control (D). Cell transfection with 5 μM of ATR2 siRNA for EGM (E), AMSCs with EGM and MMP-2 siRNA (F), and AMSCs with EGM and MMP-14 siRNA (G). Flow cytometry data were analyzed to show the significant differences between the groups (H). III Flow cytometric analysis of VE-cadherin (CD144) in four different groups: control group AMSCs with EGM (A), AMSCs with EGM and MMP-2 siRNA (B), AMSCs with EGM and MMP-14 siRNA (C), and HUVECs as the positive control (D). Cell transfection with 5 μM of ATR2 siRNA for EGM (E), AMSCs with EGM and MMP-2 siRNA (F), and AMSCs with EGM and MMP-14 siRNA (G). Flow cytometry data were analyzed to show the significant differences between the groups (H). **p < 0.01. EBM endothelial cell basal medium, EGM endothelial cell growth medium, MMP matrix metalloproteinase, ATR2 angiotensin receptor R2, HUVEC human umbilical vein endothelial cell

Fig. 4

Phosphorylation of JNK, p38, and ERK after 10 days of AMSC differentiation to ECs. Western blot detection of p-JNK (a), p-p38 (b), and p-ERK1/2 (c) in AMSC lysates after 10 days of AMSC stimulation for EC differentiation with EBM (control), EGM, EGM + MMP-2 siRNA, and EGM + MMP-14 siRNA. Phospho-proteins were normalized to their total protein expressions. *p < 0.05, **p < 0.01, ***p < 0.001. EBM endothelial cell basal medium, EGM endothelial cell growth medium, MMP matrix metalloproteinase, ERK extracellular signal-regulated kinase, JNK c-Jun NH2-terminal kinase, p38 stress activated protein kinase-2

Fig. 5

Inhibition of ERK phosphorylation and immunophenotyping for EC markers. I Concentration -dependent effect of ERK inhibitor (U0126). Three different concentrations (0.5, 1.0, and 5.0 μM) of U0126 were used. Western blot analysis showed significant inhibition of p-ERK by 1.0 and 5.0 μM of U0126. However, 5.0 μM of U0126 showed the highest inhibition among all three different concentrations. Phospho-ERK was normalized to its total protein expression. II Flow cytometric analysis of PECAM1 (CD31) with ERK inhibitor (U0126). Three different groups treated with 5.0 μM of U0126: AMSCs with EGM (A), AMSCs with EGM and MMP-2 siRNA (B), and AMSCs with EGM and MMP-14 siRNA (C). Flow cytometry data were analyzed to show the significant differences between the groups (D). III Flow cytometric analysis of VE-cadherin (CD144) with ERK inhibitor (U0126). Three different groups were treated with 5.0 μM of U0126: AMSCs with EGM (A), AMSCs with EGM and MMP-2 siRNA (B), and AMSCs with EGM and MMP-14 siRNA (C). Flow cytometry data were analyzed to show the significant differences with or without U0126 (D). *p < 0.05, **p < 0.01, ***p < 0.001. EBM endothelial cell basal medium, EGM endothelial cell growth medium, MMP matrix metalloproteinase, ERK extracellular signal-regulated kinase, HUVEC human umbilical vein endothelial cell

Fig. 6

Dose-dependent inhibition of ERK by VEGFR2 kinase inhibitor. Western blot analysis of p-ERK with three different concentration of VEGFR2 kinase inhibitor (0.1, 2.0, and 5.0 μM), showed significant decrease in ERK phosphorylation with 5.0 μM of VEGFR2 kinase inhibitor. Phospho-ERK was normalized to its total protein expression. *p < 0.05. EBM endothelial cell basal medium, EGM endothelial cell growth medium, ERK extracellular signal-regulated kinase, VEGFR2 vascular endothelial growth factor receptor-2

Fig. 7

Signaling transduction pathway of AMSC differentiation to ECs and negative crosstalk between ERK and JNK. ERK receives signals from VEGFR2, and induces the transcription of EC markers during AMSC stimulation for EC differentiation. ERK activation phosphorylates MKP-7 and blocks the JNK signaling pathway. ERK extracellular signal-regulated kinase, VEGF vascular endothelial growth factor, VEGFR2 vascular endothelial growth factor receptor-2, JNK c-Jun NH2-terminal kinase, MKP-7 MAP kinase phosphatase-7, EC endothelial cell