A heparin-rosuvastatin-loaded P(LLA-CL) nanofiber-covered stent inhibits inflammatory smooth-muscle cell viability to reduce in-stent stenosis and thrombosis

Abstract

Background: An endovascular covered-stent has unique advantages in treating complex intracranial aneurysms; however, in-stent stenosis and late thrombosis have become the main factors affecting the efficacy of covered-stent treatment. Smooth-muscle-cell phenotypic modulation plays an important role in late in-stent stenosis and thrombosis. Here, we determined the efficacy of using covered stents loaded with drugs to inhibit smooth-muscle-cell phenotypic modulation and potentially lower the incidence of long-term complications.

Methods: Nanofiber-covered stents were prepared using coaxial electrospinning, with the core solution prepared with 15% heparin and 20 µM rosuvastatin solution (400: 100 µL), and the shell solution prepared with 120 mg/mL hexafluoroisopropanol. We established a rabbit carotid-artery aneurysm model, which was treated with covered stents. Angiography and histology were performed to evaluate the therapeutic efficacy and incidence rate of in-stent stenosis and thrombosis. Phenotype, function, and inflammatory factors of smooth-muscle cells were studied to explore the mechanism of rosuvastatin action in smooth-muscle cells.

Result: Heparin-rosuvastatin-loaded nanofiber scaffold mats inhibited the proliferation of synthetic smooth-muscle cells, and the nanofiber-covered stent effectively treated aneurysms in the absence of notable in-stent stenosis. Additionally, in vitro experiments showed that rosuvastatin inhibited the smooth-muscle-cell phenotypic modulation of platelet-derived growth factor-BB induction and decreased synthetic smooth-muscle-cell viability, as well as secretion of inflammatory cytokines.

Conclusion: Rosuvastatin inhibited the abnormal proliferation of synthetic smooth-muscle cells, and heparin-rosuvastatin-loaded covered stents reduced the incidence of stenosis and late thrombosis, thereby improving the healing rates of stents used for aneurysm treatment.

Keywords: Intracranial aneurysm; Late thrombosis; Long-term arterial stenosis; Nanofiber-covered stent; Rosuvastatin.

Fig. 1

Nanofiber characterizations and mechanical properties. A Characteristics of the nanofiber-covered stents. (a) Schematic diagram of stent-graft fabrication, (b) the nanofiber-covered stent, and (c) SEM images of the nanofiber mats. Scale bar, 10 µm. (d,e) TEM image of the core–shell structure. Scale bar, 250 nm and 20 nm. B Diameter distribution of the nanofiber mats. Scale bar, 25 µm

Fig. 2

Viability and morphology of attached SMCs. a SEM images of synthetic SMCs attached to control, Rosu 50, Rosu 75, and Rosu 100 nanofiber mats. Scale bar, 100 µm. Corresponding magnified image. Scale bar, 25 µm. b Phalloidin-labeled SMCs attached to nanofiber mats. Scale bar, 30 µm. c Hoechst-33342-labeled SMCs attached to nanofiber mats. Scale bar, 150 µm. d Bar graph showing the viabilities of SMCs attached to nanofiber mats after 24 h and 48 h

Fig. 3

Cytokine secretion by attached SMCs. Bar graph shows the levels of inflammatory factors secreted by SMCs attached to nanofiber mats according to MILLIPLEX MAP rat cytokine/chemokine factor panel. Data represent the mean ± SD (n = 3/group). *p < 0.05, ****p < 0.0001

Fig. 4

Images and corresponding scatter charts for balloon-expansion experiments on covered stents fabricated with nanofiber scaffolds spun for different times (3, 5, and 10 min)

Fig. 5

Establishment of the rabbit aneurysm model and short- and long-term follow-ups. A Initiation of the rabbit aneurysm model and stent implantation: (a) Schematic diagram of aneurysm initiation, (b) photograph of the induced aneurysm, (c) angiography of the aneurysm after 30 days, and (d) angiography of aneurysm occlusion after stent implantation. B Angiography illustrations of type A, B, and C aneurysms at follow-up. Bar graph shows the number of different types in control and Rosu 100 groups immediately after implantation and at 3- and 12-month follow-ups. C Angiography of the parent artery at (a,b)3- and (c,d)12-month follow-ups in the control and Rosu 100 groups

Fig. 6

SEM, histology, and toxicity of the nanofiber scaffold mats. A SEM and H&E-stained vascular-section images showing endothelialization of the covered stent at 3 months. B SEM and H&E-stained vascular-section images showing endothelialization of the covered stent at 12 months. Scale bars: (a, c) 150 µm and (b, d, e, f) 100 µm. C H&E-stained vascular-section images showing the effects of control, Rosu 50, Rosu 75, and Rosu 100 nanofiber mats implanted under abdominal subcutaneous tissue for 1 and 3 months. Scale bar, 250 µm

Fig. 7

Cell morphology, viability, and function of PDGF-BB- and rosuvastatin-treated SMCs. a Cell morphology of control, PDGF, PDGF + Rosu, and Rosu groups. Scale bar, 20 µm. b Cell proliferation rate of control, PDGF, PDGF + Rosu, and Rosu groups. Scale bar, 100 µm. Bar graph showing the cell proliferation rate (%). Data represent the mean ± SD (n = 3/group). ****p < 0.0001. c Wound-healing assays and corresponding representative images of SMC migration in control, PDGF, PDGF + Rosu, and Rosu groups according to scratch assay. Scale bar, 100 µm. Data represent the mean ± SD (n = 3/group). *p < 0.05, **p < 0.01, ***p < 0.001. d Transwell assays and corresponding representative images of crystal violet-stained invasive cells in the lower chamber. Scale bar, 100 µm. Data represent the mean ± SD (n = 3/group). *p < 0.05, **p < 0.01

Fig. 8

Effects of PDGF-BB- and rosuvastatin-treated SMCs on phenotype markers and inflammatory cytokines. a Fold changes in mRNA levels of α-SMA, SM22-α, OPN, and inflammatory cytokines. Data represent the mean ± SD (n = 3/group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. b Western blot analysis of SM22-α, OPN, and MMP-9 levels. Data represent the mean ± SD (n = 3/group). *p < 0.05, **p < 0.01. c Inflammatory cytokines analyzed using MILLIPLEX MAP rat cytokine/chemokine factor panel. Data represent the mean ± SD (n = 3/group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001