Plasma Small Extracellular Vesicle-Carried miRNA-501-5p Promotes Vascular Smooth Muscle Cell Phenotypic Modulation-Mediated In-Stent Restenosis

Abstract

Vascular smooth muscle cell (VSMC) phenotypic modulation plays an important role in the occurrence and development of in-stent restenosis (ISR), the underlying mechanism of which remains a key issue needing to be urgently addressed. This study is designed to investigate the role of plasma small extracellular vesicles (sEV) in VSMC phenotypic modulation. sEV were isolated from the plasma of patients with ISR (ISR-sEV) or not (Ctl-sEV) 1 year after coronary stent implantation using differential ultracentrifugation. Plasma sEV in ISR patients are elevated markedly and decrease the expression of VSMC contractile markers α-SMA and calponin and increase VSMC proliferation. miRNA sequencing and qRT-PCR validation identified that miRNA-501-5p was the highest expressed miRNA in the plasma ISR-sEV compared with Ctl-sEV. Then, we found that sEV-carried miRNA-501-5p level was significantly higher in ISR patients, and the level of plasma sEV-carried miRNA-501-5p linearly correlated with the degree of restenosis (R ² = 0.62). Moreover, miRNA-501-5p inhibition significantly increased the expression of VSMC contractile markers α-SMA and calponin and suppressed VSMC proliferation and migration; in vivo inhibition of miRNA-501-5p could also blunt carotid artery balloon injury induced VSMC phenotypic modulation in rats. Mechanically, miRNA-501-5p promoted plasma sEV-induced VSMC proliferation by targeting Smad3. Notably, endothelial cells might be the major origins of miRNA-501-5p. Collectively, these findings showed that plasma sEV-carried miRNA-501-5p promotes VSMC phenotypic modulation-mediated ISR through targeting Smad3.

Figure 1

Characterization and function of plasma sEV from patients and rats. The isolated small extracellular vesicles (sEV) from patients with in-stent restenosis (ISR) or not (control) were used for the characterization. (a) Representative transmission electron microscopic (TEM) images of sEV. (b) Representative results of nanoparticle tracking analysis (NTA) demonstrating the particle distribution of sEV (n = 3). (c) Representative western blots identifying the biomarkers of sEV including CD63 and TSG101 in the same amount of plasma sEV. (d) Representative hematoxylin-eosin stained images of carotid arteries from rat carotid artery balloon injury (CABI) model or sham group (n = 6). (e) Representative TEM images of sEV from rats. (f) NTA showing the concentration of sEV (n = 3). Values are mean ± SEM. ^∗∗P < 0.01 and ^∗∗∗P < 0.001. Ctl-sEV: sEV purified from the plasma of patients without ISR after stent implantation; ISR-sEV: sEV purified from the plasma of patients with ISR after stent implantation; Sham-sEV: sEV purified from the plasma of rats with sham procedure; CABI-sEV: sEV purified from the plasma of rats with carrotid artery stenosis after balloon injury.

Figure 2

The effect of plasma sEV from patients on vascular smooth muscle cells proliferation and migration. (a) Primary human aortic smooth muscle cells (HASMC) were cultured in the presence of PKH26-labeled sEV. Representative confocal images showing the red sEV and the blue nuclei (DAPI) of HASMC. (b) Representative blots and quantified data showing the expression of vascular smooth muscle cell- (VSMC-) specific contractile marker (α-SMA and calponin) in HASMC treated with plasma sEV. (c) Representative confocal images showing immunofluorescent staining for VSMC-specific contractile marker (α-SMA, green) to determine VSMC identity. Nuclei were stained with DAPI (blue). The VSMC proliferation was evaluated with Edu-positive cells (d) and MTT assay (e) in HASMC treated with plasma sEV. The VSMC migration was measured using Transwell (f) and wound healing assay (g) in HASMC treated with plasma sEV. (h) The reduced mRNA expression of VSMC-specific contractile markers (α-SMA and calponin) in CABI group (n = 6). Data are normalized to U6. (i) The further reduced mRNA expression of VSMC-specific contractile marker (α-SMA and calponin) in CABI rats treated with sEV (n = 6). Values are mean ± SEM. ^∗∗P < 0.01 and ^∗∗∗P < 0.001.

Figure 3

miRNA microarray assay and the relationship of miRNA-501-5p and in-stent restenosis. (a) miRNA microarray assays were performed in plasma sEV purified from patients with in-stent restenosis (ISR) or not (n = 3). (b) qRT-PCR analysis of the 13 upregulated miRNAs in ISR-sEV and Ctl-sEV (n = 3). Data are normalized to cel-miRNA-39. (c) qRT-PCR analysis of plasma sEV-carried miRNA-501-5p level in patients with ISR or not (n = 20). Data are normalized to cel-miRNA-39. (d) The relationship between plasma sEV-carried miRNA-501-5p level and late lumen loss (reflecting the degree of restenosis) (n = 20). Data are normalized to cel-miRNA-39. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 4

The effect of miRNA-501-5p on smooth muscle cell proliferation and migration. (a) Representative blots and quantified data showing the expression of vascular smooth muscle cell- (VSMC-) specific contractile marker (α-SMA and calponin) in primary human aortic smooth muscle cells (HASMC) transfected with miRNA-501-5p mimic. (b) Representative confocal images showing immunofluorescent staining for VSMC-specific contractile marker (α-SMA, green) to determine VSMC identity. Nuclei were stained with DAPI (blue). The VSMC proliferation was evaluated with Edu-positive cells (c) and MTT assay (d) in HASMC transfected with miRNA-501-5p mimic. The VSMC migration was measured using Transwell (e) and wound healing assay (f) in HASMC transfected with miRNA-501-5p mimic. Effects of miRNA-501-5p inhibitor on MTT assay (g), immunofluorescent staining of α-SMA (h), Edu assay (i), and transwell assay (j), in HASMC treated with plasma sEV, then transfected with miRNA-501-5p inhibitor or not. n = 8 per group in (d) and (g); n = 3 per group in other groups. NC: negative control. Values are mean ± SEM. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 5

Plasma sEV-carried miRNA-501-5p promotes rat carotid artery stenosis. (a) qRT-PCR analysis of pre-miRNA-501-5p and miRNA-501-5p in carotid artery from rats with carotid artery balloon injury (CABI) or sham procedure. Data are normalized to U6. (b) qRT-PCR analysis of pre-miRNA-501-5p and miRNA-501-5p in carotid artery from CABI rats treated with plasma sEV. Data are normalized to U6. (c) Representative hematoxylin-eosin stained images of carotid arteries from CABI rats treated with plasma sEV or antagomir-501-5p. (d) Representative Masson stained images of carotid arteries from CABI rats treated with plasma sEV or antagomir-501-5p. (e and f) Quantitative analysis for media thickness and the ratio of media thickness and lumen diameter. n = 6 for each group. Values are mean ± SEM. ^∗∗P < 0.01 and ^∗∗∗P < 0.001.

Figure 6

Smad3 is target gene of plasma sEV-carried miRNA-501-5p in smooth muscle cells. (a) Screening scheme for putative target gene of miRNA-501-5p. (b) Schematic of miRNA-501-5p putative target sites in the 3′URT of Smad3 and the sequences of mutant UTRs. (c) Dual luciferase reporter assay of 293T cells cotransfected with miRNA-501-5p mimic or negative control (NC) (n = 3). (d) Representative blots and quantified data showing protein expressions of Smad2, Smad3, and Smad4 in human aortic smooth muscle cells (HASMC) transfected with miRNA-501-5p mimic or NC (n = 3). (e) qRT-PCR analysis of Smad2, Smad3, and Smad4 in carotid artery from rats with carotid artery balloon injury (CABI) undergoing sEV injection (n = 6). Data are normalized to U6. (f) qRT-PCR analysis of Smad2, Smad3, and Smad4 in carotid artery from rats with carotid artery balloon injury (CABI) undergoing antagomir-501-5p injection (n = 6). Data are normalized to U6. Values are mean ± SEM. ^∗∗P < 0.01 and ^∗∗∗P < 0.001.

Figure 7

The potential origin of miRNA-501-5p and the proposed schematic. qRT-PCR analysis of miRNA-501-5p (a) and pre-miRNA-501-5p (b) in tissues from rats with carotid artery balloon injury (CABI) or not. Data are normalized to U6 (n = 6). (c) qRT-PCR analysis showing that the expressions of miRNA-501-5p and pre-miRNA-501-5p are significantly higher in rat aorta with endothelium (endo+) than that without endothelium (endo-). Data are normalized to U6 (n = 6). (d) Drug-eluting stent implantation increases the production of miRNA-501-5p in multiple tissues, especially in endothelial cells. miRNA-501-5p, cargoed in sEV, is delivered to the vascular smooth muscle cells (VSMC) by plasma sEV and promotes VSMC proliferation and migration by targeting Smad3. Values are mean ± SEM. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.