Int J Stem Cells.  2018 Nov;11(2):168-176. 10.15283/ijsc18079.

rBMSCs/ITGA5B1 Promotes Human Vascular Smooth Muscle Cell Differentiation via Enhancing Nitric Oxide Production

• Zhang Y
• Ding J
• Xu C
• Yang H
• Xia P
• Ma S
• Chen H

• KMID: 2429549
• DOI: http://doi.org/10.15283/ijsc18079

Abstract

BACKGROUND AND OBJECTIVES
Previous studies have shown that integrins alpha5beta1 (ITGA5B1) gene-modified rat bone marrow mesenchymal stem cells (rBMSCs) could prevent cell anoikis and increase the nitric oxide (NO) production. Here we examined the capability of rBMSCs/ITGA5B1 on the phenotype modulation of Human Pulmonary Artery Smooth Muscle Cell (HPASMC) in vitro.
METHODS AND RESULTS
The synthetic (dedifferentiated) phenotype of HPASMC was induced by monocrotaline (MCT, 1μM) for 24 h and then co-cultured with rBMSCs/ITGA5B1 in a transwell culture system. The activation of NO/cGMP (nitric oxide/Guanosine-3"², 5"²-cyclic monophosphate) signaling was investigated in HPASMC. The changes of pro-inflammatory factors, oxidative stress, vasodilator, vasoconstrictor, contractile and synthetic genes, and the morphological changes of HPASMC were investigated. The results of this study showed that the NO/cGMP signal, endothelial nitric oxide synthase (eNOS) expression, the expression of the vasoprotective genes heme oxygenase-1 (HMOX1) and prostaglandin-endoperoxide synthase 2 (PTGS2) were increased, but the expression of transforming growth factor-β1 (TGF-β1), CCAAT/enhancer-binding proteins delta (Cebpd), Krüppel-like factor 4 (KLF4), and activating transcription factor 4 (ATF4) were reduced in MCT treated HPASMC co-cultured with rBMSCs/ITGA5B1. The synthetic smooth muscle cells (SMCs) phenotype markers thrombospondin-1, epiregulin and the vasoconstrictor endothelin (ET)-1, thromboxane A2 receptor (TbxA2R) were down-regulated, whereas the contractile SMCs phenotype marker transgelin expression was up-regulated by rBMSCs/ITGA5B1. Furthermore, rBMSCs/ITGA5B1 promoted the morphological restoration from synthetic (dedifferentiation) to contractile (differentiation) phenotype in MCT treated HPASMC.
CONCLUSIONS
rBMSCs/ITGA5B1 could inhibit inflammation and oxidative stress related genes to promote the HPASMC cell differentiation by activation NO/cGMP signal.

Keyword

Bone marrow derived mesenchymal stem cells; Integrin; Nitric oxide; Phenotypic transition; Human vascular smooth muscle cell (HPASMC)

MeSH Terms

Activating Transcription Factor 4
Animals
Anoikis
Bone Marrow
Cell Differentiation*
Endothelins
Epiregulin
Genes, Synthetic
Heme Oxygenase-1
Humans*
In Vitro Techniques
Inflammation
Integrins
Mesenchymal Stromal Cells
Monocrotaline
Muscle, Smooth, Vascular*
Myocytes, Smooth Muscle
Nitric Oxide Synthase Type III
Nitric Oxide*
Oxidative Stress
Phenotype
Prostaglandin-Endoperoxide Synthases
Pulmonary Artery
Rats
Receptors, Thromboxane A2, Prostaglandin H2
Activating Transcription Factor 4
Endothelins
Epiregulin
Heme Oxygenase-1
Integrins
Monocrotaline
Nitric Oxide
Nitric Oxide Synthase Type III
Prostaglandin-Endoperoxide Synthases
Receptors, Thromboxane A2, Prostaglandin H2

Figure

• 

  Fig. 1 The effect of rBMSCs/ITGA5B1 on NO/cGMP signal, eNOS expression and ROS production. (A) NO production was evaluated by measuring nitrite (the stable end product of NO) concentration by a nitric oxide colorimetric assay. (B) The mRNA expression of cGMP. (C) ROS production. ROS level was detected by the fluorescent probe DCFH-DA. The FITC-A Mean represents for the average fluorescence intensity. (D) The expression of eNOS protein was investigated by western-blot. 1: Control groups, 2: Model groups, 3: Vector groups, 4: ITGA5B1 groups. *p<0.05, **p<0.01.

• 

  Fig. 2 The regulation effects of rBMSCs/ITGA5B1 on the expression of stress response genes, pro-inflammatory factors and vasoconstrictors. (A, B) The mRNA expression of ATF4, HMOX1, PTGS2 and TbxA2R. (C, D) The protein expression of pro-inflammatory factors and vasoconstrictor. 1: Control groups, 2: Vector groups, 3: Model groups, 4: ITGA5B1 groups.

• 

  Fig. 3 Effect of rBMSCs/ITGA5B1 on morphological changes of MCT induced HPASMC. Compared to control groups, the cells showed fibroblast like and hypertrophy in model groups, whereas the cell shape in ITGA5B1 groups showed spindle-shaped with small size and similar in morphology to control groups. Bar=100 μm (10×).

• 

  Fig. 4 The mRNA expression of the thrombospondin, transgelin and epiregulin. The synthetic SMCs phenotype markers thrombospondin and epiregulin was reduced while the contractile SMCs phenotype marker transgelin expression was up-regulated by rBMSCs/ITGA5B1.

Reference

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