Fig 1.
Expression of Epsin in Carotid Arteries of Mice in Each Group after 7 Days.
(A) Immunohistochemical staining with specific anti‐CD31 Epsin1 and Epsin2 antibodies in mice carotid arteries. The relative quantification of Epsin protein expression. (B) and (C) Image J software was applied to evaluate protein expression according to the grayscale values. (D) H&E‐stained carotid arteries treated with sh(Epsin1 + Epsin2) or shNC at 7 days after injury. (E) and (F) Quantification of neointimal area and neointima/media ratio of carotid arteries treated with sh(Epsin1 + Epsin2) or shNC. t-test, n = 6 in each group. * : P < 0.05 versus the Sham group.
Fig 2.
The effect of knocking down Epsin on EC proliferation and migration.
(A) and (B) Ki67 incorporation assays of EC. Representative immunofluorescence of Ki67 (green) and DAPI (blue). percentages of Ki67 incorporated EC. (C) and (D) Scratch‐wound assays showed the effect of VEGF on the migration of EC infected with siEpsin1 + siEpsin2. (E) and (F): expression levels of Epsin1, Epsin2, VEGFR2 and Erk were determined by Western blotting using the appropriate antibodies. GAPDH served as a loading control. GAPDH served as a loading control. t-test, n = 3, in each group. * : P ＜ 0.05 vs. sinormal control (NC); #: P ＜ 0.05 vs. siEpsin1 + siEpsin2; &: P ＜ 0.05 vs. siNC+VEGF.
Fig 3.
In vitro, Epsin regulates EC mitochondrial dynamics, membrane potential, and function.
(A) MitoTracker Green staining showed the mitochondrial morphology of EC after 24 hours of treatment. (B) and (C) mitochondrial membrane potentials of EC were evaluated using JC-1 staining after treating for 24 h, J-aggregates (red) and monomers (green). (D) and (E) expression levels of Epsin1, Epsin2, Drp1 and Opa1 were determined by Western blotting using the appropriate antibodies. GAPDH served as a loading control. t-test, n = 3, in each group. * : P ＜ 0.05 vs. sinormal control (NC); #: P ＜ 0.05 vs. siEpsin1 + siEpsin2; &: P ＜ 0.05 vs. siNC+VEGF.
Fig 4.
The impact of Epsin knockdown in ECs on the proliferation and migration of co-cultured SMCs.
(A) and (B) Ki67 incorporation assay of SMCs, treated as in A for 48 hours. representative immunofluorescence of Ki67 (green) and DAPI (blue). percentages of Ki67 incorporated EC. (C) and (D) migration ability assessed by wound healing. t-test, n = 3, in each group. * : P ＜ 0.05 vs. sinormal control (NC); #: P＜ 0.05 vs. siEpsin1 + siEpsin2; &: P ＜ 0.05 vs. siNC+VEGF.
Fig 5.
Transfection efficiency, surface characteristics.
(A) A scanning electron microscope results showed surface of bare-metal stents and a collagen layer of shEpsin1 + siEpsin2 stents. (B) photographed from a confocal laser scanning microscope, cells on the stent were transfected. DAPI stained nuclei of the ECs on stent were blue and shRNA-transfected cells were green. Scale bars are 500µm SEM and water contact angle analysis. (C) and (D) Representative images of water contact angle testing on different surfaces. (E) Activated partial thromboplastin time (APTT) and prothrombin time (PT) measurements.
Fig 6.
Carotid artery implantation results of Epsin-eluting stent.
(A) Representative Digital substraction angiography images after stents implantation in swine carotid arteries show no thrombus and stent displacement at perioperative period. AP: anteroposterior position, LP: lateral position. n = 3.(B),(C) and (D) H&E staining (×200) showed IH in the shnormal contrast (NC) stent group and the sh(Epsin1 + Epsin2) void vector group * : P ＜ 0.05 vs. shNC group; n = 3.