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. 2021 Aug 24:12:712400.
doi: 10.3389/fphys.2021.712400. eCollection 2021.

GRIA2/ENPP3 Regulates the Proliferation and Migration of Vascular Smooth Muscle Cells in the Restenosis Process Post-PTA in Lower Extremity Arteries

Mi Zhou  1 , Lixing Qi  1 , Yongquan Gu  1
Affiliations

GRIA2/ENPP3 Regulates the Proliferation and Migration of Vascular Smooth Muscle Cells in the Restenosis Process Post-PTA in Lower Extremity Arteries

Mi Zhou et al. Front Physiol. .

Abstract

Restenosis is the main restriction on the long-term efficacy of percutaneous transluminal angioplasty (PTA) therapy for peripheral artery disease (PAD). Interventions to prevent restenosis are poor, and the exact mechanism is unclear. Here, we aimed to elucidate the role of GRIA2 in the restenosis process post-PTA in lower extremity arteries. We searched the differentially expressed genes (DEGs) between atherosclerotic and restenotic artery plaques in the Gene Expression Omnibus (GEO), and five DEGs were identified. Combined with Gene Ontology (GO) enrichment analysis, GRIA2 was significantly correlated with the restenosis process. Tissue samples were used to examine GRIA2 expression by immunofluorescence staining of atherosclerotic and restenotic artery plaques. The regulation of GRIA2 in vascular smooth muscle cells (VSMCs) was confirmed by lentiviral transfection. Overexpression of GRIA2 promoted the proliferation and migration of VSMCs. Using Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction (PPI) network, a strong connection between ENPP3 and GRIA2 was discovered. In vitro results showed that the high expression of GRIA2 in VSMCs enhanced the expression of ENPP3, while downregulation of GRIA2 downregulated ENPP3. GRIA2 is highly differentially expressed in restenotic arterial plaques, promoting the proliferation and migration of VSMCs through upregulation of ENPP3. These discoveries will help us to obtain a better understanding of restenosis in lower extremity arteries.

Keywords: GRIA2; percutaneous transluminal angioplasty; peripheral artery disease; restenosis; vascular smooth muscle cells.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Integrated analysis of GEO datasets identified DEGs. (A) Venn diagrams of common DGEs between two combined datasets (GSE23314 and GSE53274). Each circle represents a dataset, and the overlap between the circles is the overlap between the datasets. (B) PPI network integration of the common DGEs. Each node is a gene, and the connections between nodes represent the interaction of these biological molecules. ENPP3 (black arrow).
Figure 2
Figure 2
GRIA2 is highly expressed in VSMCs in restenosis. (A) Double immunofluorescence staining of GRIA2 (red) and VSMCs (green, stained with α-SMA) in restenotic plaque tissues, atherosclerotic plaque tissues and healthy control artery tissues is shown (n=3). Intensity of fluorescence is the statistical calculations in the intima. Images are representative, and (B) the bar graph shows the average data of three independent experiments (n=3). Staining was performed at 100×magnification; the right part of the picture was magnified to 200× (scale bar, 50μm). AS: atherosclerotic; RS: restenotic; CON: control; VSMCs: vascular smooth muscle cells. *Statistically significant difference (p < 0.05); **Statistically significant difference (p < 0.01); ***Statistically significant difference (p < 0.001).
Figure 3
Figure 3
The regulation of GRIA2 in VSMCs. (A) GRIA2 protein expression was detected by western blot after cDNA and shRNA regulation of GRIA2. Blots are representative, and the bar graph shows the average data (n=3). (B) GRIA2 mRNA expression was detected by real-time PCR after transfection with lentivirus carrying GRIA2 shRNA, cDNA or empty vector (n=3). (C) EdU assay showed that increased EdU-positive VSMCs overexpressing GRIA2 were observed (scale bar, 50μm). Images are representative, and the bar graph shows the average data of independent experiments (n=3). (D) Rate of EdU-positive VSMCs (n=3). (E) Transwell migration assay of VSMCs after upregulating GRIA2 expression (scale bar, 100μm). Images are representative, and the bar graph shows the average data of three independent experiments (n=3). (F) Quantitative data on the migration of VSMCs (n=3). **Statistically significant difference (p<0.01) and ***statistically significant difference (p<0.001)
Figure 4
Figure 4
GRIA2 interacted with ENPP3 in restenosis. (A) Immunohistochemical analysis and quantitative mRNA analysis showed the expression of ENPP3 in restenotic plaque tissues, atherosclerotic plaque tissues and healthy control artery tissues (n=3). Optical density (OD) values are the statistical calculations in the intima. Images are representative, and the bar graph shows the average data of three independent experiments (n=3). Staining was performed at 200×magnification (scale bar, 50μm). (B) The expression of ENPP3 in VSMCs after upregulating GRIA2 expression. Blots are representative, and the bar graph shows the average data (n=3). *Statistically significant difference (p<0.05), **statistically significant difference (p<0.01), and ***statistically significant difference (p<0.001). AS: atherosclerotic; RS: restenotic; CON: control; VSMCs: vascular smooth muscle cells.
Figure 5
Figure 5
GRIA2 interacted with ENPP3 and regulated the proliferation and migration of VSMCs. (A,B) siENPP3 and ENPP3 plasmids and siNC and shRNA NC plasmids were transfected into GRIA2 lentivirus-transfected VSMCs. The EdU assay illustrated the proliferation of VSMCs (scale bar, 50μm; n=3). (C,D) Transwell assays illustrated the migration of VSMCs (scale bar, 100μm; n=3). Images are representative, and the bar graph shows the average data of four independent experiments (n=3). Images of EdU and Transwell migration assays were taken at 200× magnification. Data are presented as the mean±SEM. **Statistically significant difference (p<0.01) and ***statistically significant difference (p<0.001). VSMCs: vascular smooth muscle cells.
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