MIA SH3 Domain ER Export Factor 3 Deficiency Prevents Neointimal Formation by Restoring BAT-Like PVAT and Decreasing VSMC Proliferation and Migration

Figures

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  Figure 1. MIA3 expression is increased in proliferative vascular smooth muscle cells (VSMCs). (A) mRNA levels of MIA3, α-SMA (smooth muscle α-actin), and Cyclin D1 by quantitative real-time reverse transcription polymerase chain reaction in VSMCs after treatment with medium containing 10% fetal bovine serum for 24 h and the control FBS-starve VSMCs. Data are represented as the mean ± SEM, n = 3. *p < 0.05, **p < 0.01, t-test. (B) At 48 h after FBS treatment, VSMC extracts were collected for determining the protein levels of MIA3, α-SMA (smooth muscle α-actin) and Cyclin D1 by Western blotting. GAPDH served as loading control expression. (C) Quantification of protein expression normalized to the levels of GAPDH. Data are represented as the mean ± SEM, n = 3. *p < 0.05, t-test. (D) Immunofluorescent staining of MIA3 in VSMCs after treatment with medium containing 10% fetal bovine serum for 24 h and the control FBS-starve VSMCs. DAPI represents indicates 4’,6-diamidino-2-phenylindole throughout the article; scale bar: 50 μm. (E) mRNA levels of MIA3 by quantitative real-time reserve transcription polymerase chain reaction in femoral artery after wire injury for 28 days. Data are represented as…

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  Figure 2. Knockdown of MIA3 inhibits vascular smooth muscle cell (VSMC) proliferation. (A) MIA3 mRNA levels in human aortic smooth muscle cells (HASMCs) with small interfering RNA (siRNA) MIA3 and scramble siRNA. Data are represented as the mean ± SEM, n = 3. **p < 0.01, t-test. (B) MIA3/TANGO1 protein levels in human aortic smooth muscle cells (HASMCs) with small interfering RNA (siRNA) MIA3 and scramble siRNA with quantitative data at right. GAPDH served as loading control expression. Data are represented as the mean ± SEM, n = 3. *p < 0.05, t-test. (C, D) VSMC proliferation was determined by cell counts (C) and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay (D) in VSMCs with or without transfection of MIA3-specific siRNAs. All data were indicated as the means ± SEM, n = 3. **p < 0.01, t-test. (E) VSMC proliferation was determined by 5-ethynyl-2’deoxyuridine (EdU) incorporation in VSMCs with or without transfection of MIA3-specific siRNAs. Percentage of EdU staining-positive cells was quantified on the right. Scale bar: 50 μm. Data are represented as the mean ± SEM, n = 3. **p < 0.01, t-test. (F) VSMC proliferation was determined by PI-cell cycle analysis in…

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  Figure 3. Knockdown of MIA3 inhibits vascular smooth muscle cell (VSMC) migration. (A) The wound-induced cell migration assay was performed in VSMCs with or without transfection of MIA3-specific small interfering RNA. The relative blank wound areas in the left were quantified on the right. Scale bar: 100 μm. Data are represented as the mean ± SEM, n = 3. n.s. indicates nonsignificant. **p < 0.01, t-test. (B) The transwell assay in VSMCs with MIA3 siRNA or scramble siRNA. The transferred and stained cells were counted on the right. Scale bar: 50 μm. Data are represented as the mean ± SEM, n = 3. t-test.

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  Figure 4. MIA3 overexpression promotes vascular smooth muscle cell (VSMC) proliferation and migration. (A) MIA3 mRNA levels in human aortic smooth muscle cells (HASMCs) transfected with MIA3 overexpressing vector (OE) and control empty vector. Data are represented as the mean ± SEM, n = 3. **p < 0.01, t-test. (B) MIA3 protein levels in human aortic smooth muscle cells (HASMCs) transfected with MIA3 overexpressing vector and control empty vector with quantitative data on the right. GAPDH served as loading control expression. Data are represented as the mean ± SEM, n = 3. *p < 0.05, t-test. (C, D) VSMC proliferation was determined by cell counts (C) and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay (D) in VSMCs with or without transfection of MIA3 overexpressing vector. All data were indicated as the means ± SEM, n = 3. **p < 0.01, t-test. (E) VSMC proliferation was determined by 5-ethynyl-2’-deoxyuridine (EdU) incorporation in VSMCs with or without transfection of MIA3 overexpressing vector. Percentage of EdU staining–positive cells was quantified on the right. Scale bar: 50 μm. Data are represented as the mean ± SEM, n = 3. **p < 0.01, t-test. (F) The…

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  Figure 5. Knockdown MIA3 ameliorates femoral artery wire injury-induced neointimal hyperplasia in mice. (A) Quantitative real-time reserve transcription polymerase chain reaction was performed to confirm the decreased expression of MIA3 in the injured femoral arteries. Data are represented as the mean ± SEM, n = 5. **p < 0.01. t-test. (B) Representative hematoxylin and eosin (H&E) staining of mouse femoral arteries after wire injury for 28 days from mice infected with MIA3 shRNA lentivirus and scramble shRNA lentivirus. Scale bar: 100 μm. (C) The neointima area, intima-to-media ratio, % stenosis, and media area of wire-injured femoral arteries from mice infected with MIA3 shRNA lentivirus and scramble shRNA lentivirus. *p < 0.05, **p < 0.01, n.s. indicates nonsignificant. n = 5, t-test. (D) Representative Masson trichrome staining of mouse femoral arteries after wire injury for 28 days from mice infected with MIA3 shRNA lentivirus and scramble shRNA lentivirus, with quantification data on the right. Scale bar: 100 μm. Data are represented as the mean ± SEM, n = 5. **p < 0.01. t-test. (E) Knockdown MIA3 with shRNA lentivirus exhibit a significant reduction of Ki67-positive cells in the…

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  Figure 6. Knockdown MIA3 increases the expression of uncoupling protein-1 in the perivascular adipocytes. (A) Knockdown MIA3 with shRNA lentivirus exhibited significant increase of PLIN and UCP-1 double-positive cells in the neointima at 28 days after femoral artery wire injury, with representative images (left) and quantification data (right; n = 5 for each group). Data are represented as the mean ± SEM. *p < 0.05. t-test. (B) Knockdown MIA3 with siRNA in VSMCs significantly decreased expression of IL-1β, IL18, CCL7, and CxCL8 by quantitative real-time reserve transcription polymerase chain reaction. Data are represented as the mean ± SEM, n = 3. *p < 0.05, **p < 0.01, t-test.