Intraluminal delivery of thrombospondin-2 small interfering RNA inhibits the vascular response to injury in a rat carotid balloon angioplasty model

Abstract

In an effort to inhibit the response to vascular injury that leads to intimal hyperplasia, this study investigated the in vivo efficacy of intraluminal delivery of thrombospondin-2 (TSP-2) small interfering RNA (siRNA). Common carotid artery (CCA) balloon angioplasty injury was performed in rats. Immediately after denudation, CCA was transfected intraluminally (15 min) with one of the following: polyethylenimine (PEI)+TSP-2 siRNA, saline, PEI only, or PEI+control siRNA. CCA was analyzed at 24 h or 21 d by using quantitative real-time PCR and immunohistochemistry. TSP-2 gene and protein expression were significantly up-regulated after endothelial denudation at 24 h and 21 d compared with contralateral untreated, nondenuded CCA. Treatment with PEI+TSP-2 siRNA significantly suppressed TSP-2 gene expression (3.1-fold) at 24 h and TSP-2 protein expression, cell proliferation, and collagen deposition up to 21 d. These changes could be attributed to changes in TGF-β and matrix metalloproteinase-9, the downstream effectors of TSP-2. TSP-2 knockdown induced anti-inflammatory M2 macrophage polarization at 21 d; however, it did not significantly affect intima/media ratios. In summary, these data demonstrate effective siRNA transfection of the injured arterial wall and provide a clinically effective and translationally applicable therapeutic strategy that involves nonviral siRNA delivery to ameliorate the response to vascular injury.-Bodewes, T. C. F., Johnson, J. M., Auster, M., Huynh, C., Muralidharan, S., Contreras, M., LoGerfo, F. W., Pradhan-Nabzdyk, L. Intraluminal delivery of thrombospondin-2 small interfering RNA inhibits the vascular response to injury in a rat carotid balloon angioplasty model.

Keywords: arterial injury; extracellular matrix protein; intimal hyperplasia; siRNA; vascular remodeling.

Figure 1.

Relative gene expression of TSP-2. Fold change in TSP-2 mRNA expression in all groups relative to nondenuded contralateral CCA at 24 h (A) and 21 d (B). Data are presented as means ± sem; n = 5. *P < 0.05.

Figure 2.

TSP-2 protein expression. A, B) Quantification of TSP-2 protein expression in all groups at 21 d on the basis of density (A) and dissemination scores (B). Data are expressed as arbitrary scores (1–5) and presented as means ± sem; n = 4. C) Representative images at 21 d of all denuded CCA stained for TSP-2 protein (red), nuclei (blue), and elastic lamina (green autofluorescence). Original magnification, ×400. Scale bars, 50 μm. *P < 0.05.

Figure 3.

Ki-67 immunostaining for cell proliferation. A) Quantitative analysis of cell proliferation in all groups at 21 d expressed as the number of cells per square millimeter. Data are presented as means ± sem; n = 4. B) Representative images at 21 d of denuded CCA in the saline and PEI+TSP-2 siRNA group. Proliferating cells are indicated with black arrows. Original magnification, ×400. Scale bars, 50 μm. *P < 0.05.

Figure 4.

Collagen deposition. A) Quantitative analysis of collagen deposition in all groups at 21 d expressed as a percentage of total area of the cross-section. Data are presented as means ± sem; n = 4. B) Representative images at 21 d of Masson’s Trichrome–stained denuded CCA in the saline and PEI+TSP-2 siRNA groups. Collagen (blue); cytoplasm, keratin, and muscle (red); and nuclei (black). Original magnification, ×100. Scale bars, 100 μm. *P < 0.05.

Figure 5.

Total macrophage infiltration. Quantification of total macrophage infiltration in all groups expressed as CD68⁺ cells at 24 h (A) and 21 d (B). Data are presented as means ± sem; n = 4–6. *P < 0.05.

Figure 6.

Macrophage polarization. A, B) Quantification of macrophage polarization in all denuded CCA expressed as M1/M2 ratio at 24 h (A) and 21 d (B). Data are presented as means ± sem; n = 4. C, D) Representative images at 21 d of denuded CCA in all groups depicting M1 and M2 macrophage polarization. Proinflammatory M1 macrophages were costained with DAPI (blue), CD68 (green), and iNOS (red) (C), whereas anti-inflammatory protissue repair M2 macrophages were costained with DAPI (blue), CD68 (green), and CD206 (red) (D). White arrows indicate the respective macrophages. Original magnification, ×400. Scale bars, 50 μm. *P < 0.05.

Figure 7.

Downstream targets. A) Quantification of TGF-β protein expression in all groups at 21 d on the basis of arbitrary density scores (1–5). Data are presented as means ± sem; n = 4. B) Representative images at 21 d of denuded CCA in the saline and PEI+TSP-2 siRNA groups stained for TGF-β (red), nuclei (blue), and elastic lamina (green autofluorescence). Original magnification, ×400. Scale bars, 50 μm. C) Quantification of MMP-9 expression in all groups 21 d expressed as a percentage of the intima. Data are presented as means ± sem; n = 4. D) Representative images at 21 d of denuded CCA in the saline and PEI+TSP-2 siRNA groups stained for MMP-9 (red), nuclei (blue), and elastic lamina (green autofluorescence). Original magnification, ×100. Scale bars, 200 μm. *P < 0.05.

Figure 8.

Morphometric analysis. A) Intima/media area ratios in all groups at 21 d. Data are presented as means ± sem; n = 7. B) Representative cross-sections at 21 d of denuded CCA in the saline and PEI+TSP-2 siRNA groups. Sections were stained with Verhoef-van Giesson. Intima was defined by the area between lumen and inner elastic lamina, whereas the area between the inner and outer elastic lamina represents the media. Original magnification, ×40. Scale bars, 200 μm. *P < 0.05.