Reduction of intimal hyperplasia in injured rat arteries promoted by catheter balloons coated with polyelectrolyte multilayers that contain plasmid DNA encoding PKCδ

Abstract

New therapeutic approaches that eliminate or reduce the occurrence of intimal hyperplasia following balloon angioplasty could improve the efficacy of vascular interventions and improve the quality of life of patients suffering from vascular diseases. Here, we report that treatment of arteries using catheter balloons coated with thin polyelectrolyte-based films ('polyelectrolyte multilayers', PEMs) can substantially reduce intimal hyperplasia in an in vivo rat model of vascular injury. We used a layer-by-layer (LbL) process to coat the surfaces of inflatable catheter balloons with PEMs composed of nanolayers of a cationic poly(β-amino ester) (polymer 1) and plasmid DNA (pPKCδ) encoding the δ isoform of protein kinase C (PKCδ), a regulator of apoptosis and other cell processes that has been demonstrated to reduce intimal hyperplasia in injured arterial tissue when administered via perfusion using viral vectors. Insertion of balloons coated with polymer 1/pPKCδ multilayers into injured arteries for 20 min resulted in local transfer of DNA and elevated levels of PKCδ expression in the media of treated tissue three days after delivery. IFC and IHC analysis revealed these levels of expression to promote downstream cellular processes associated with up-regulation of apoptosis. Analysis of arterial tissue 14 days after treatment revealed polymer 1/pPKCδ-coated balloons to reduce the occurrence of intimal hyperplasia by ~60% compared to balloons coated with films containing empty plasmid vectors. Our results demonstrate the potential therapeutic value of this nanotechnology-based approach to local gene delivery in the clinically important context of balloon-mediated vascular interventions. These PEM-based methods could also prove useful for other in vivo applications that require short-term, surface-mediated transfer of plasmid DNA.

Figure 1

Representative low magnification fluorescence microscopy images (C–F, I–J) and high magnification LSCM images (A–B,G–H) of rat carotid arteries treated with balloons coated with either (A) a polymer 1/DNA_TMR film 16 bilayers thick, or (C–D, G–J) a polymer 1_OG/DNA_TMR film 16 bilayers thick. TMR fluorescence is false-colored red and Oregon Green (OG) fluorescence is false-colored green; additional green fluorescence arises from natural tissue autofluorescence (e.g., as shown in panel B). Tissue sections shown in A–B were harvested for analysis immediately following treatment with film-coated balloons (i.e., prior to restoration of blood flow; see text). Tissue sections shown in C–H and I–J were harvested 10 hours and 23 hours, respectively, following treatment with film coated balloons. Panels B and E–F show sections of uninjured and untreated artery sections used as negative controls. For all images, the designation ‘L’ indicates the location of the artery lumen. Scale bars for LSCM images (A–B, G–H) are 40 µm. Scale bars for fluorescence microscopy images (C–F, I–J) are 200 µm.

Figure 2

Representative fluorescence microscopy images of rat carotid arteries stained for PKCδ using immunofluorescence staining. The image in (A) shows a montage of low magnification images showing the locations and relative levels of PKCδ expression in a cross-section of an injured artery treated with a balloon coated with a polymer 1/PKCδ film 32 bilayers thick (see text). (B) A higher magnification image of a section of a PKCδ-treated artery. (C) A high magnification image of a control section of tissue from an injured artery that was treated with a polymer 1/pcDNA3 film 32 bilayers thick. This image shows native levels of PKCδ expression in vascular tissue induced by the original balloon injury and subsequent treatment with a film-coated balloon containing a control plasmid. In panels B and C, the designation ‘L’ indicates the location of the artery lumen. Scale bars = 100 µm.

Figure 3

Representative high magnification LSCM images showing TUNEL staining (false-colored red, A-B, D-E) or IHC staining for activated (cleaved) caspase-3 (stained brown, C, F) for sections of injured arterial tissue treated with a balloon coated with either a polymer 1/PKCδ film (A–C) or a polymer 1/pcDNA3 film (D–F) 32 bilayers thick. Images in panels A and D show IFC staining of PKCδ expression (false-colored green). In images A, B, D, and E, cell nuclei are stained and false-colored blue, and the locations of the internal and external elastic lamina defining the boundaries of the media are indicated by white dashed lines. In images C and F, cell nuclei are stained blue with hematoxylin; the locations of representative cells positive for cleaved caspase-3 (brown) are indicated by black arrows. Panels B and E show enlarged images of the regions enclosed by the yellow dashed-line boxes shown in A and C (without the green channel showing PKCδ expression, for clarity). In panels A, C, E, and F, the designation ‘L’ indicates the location of the artery lumen. Scale bars = 20 µm.

Figure 4

Representative high magnification LSCM images showing IFC staining for MCP-1 expression (A and C, false-colored green) and Ki67 expression (B and D, false-colored red) for sections of injured arterial tissue treated with a balloon coated with either a polymer 1/PKCδ film (A, B) or a polymer 1/pcDNA3 film (C, D) 32 bilayers thick. In all images, cell nuclei are stained and false-colored blue, and the locations of the internal and external elastic lamina defining the boundaries of the media are indicated by white dashed lines. Additional IFC staining of tissue sections immediately adjacent to those shown in images A and B showed high levels of PKCδ expression; tissue sections immediately adjacent to those shown in images C and D showed relatively low levels of PKCδ expression (data not shown). In each image, the designation ‘L’ indicates the location of the artery lumen. Scale bars = 20 µm.

Figure 5

(A) Schematic showing the area of the common carotid artery that was injured and/or balloon-treated. Red shading indicates areas of the artery where initial injury was induced by three passages of an inflated angioplasty balloon (see text for details). Regions I, II, and III denote three different regions of arterial tissue used to characterize intimal hyperplasia 14 days after treatment with balloons coated with polymer 1/DNA films 32-bilayers thick. Panels B–D show representative images of H&E-stained tissue sections from Regions I-III for a rat treated with a balloon coated with a film fabricated using pcDNA3 (as a control). Panels E–G show representative images of tissue sections from Regions I-III for a rat treated with a balloon coated with a film fabricated using pPKCδ. The locations of the internal and external elastic lamina are indicated by arrows and arrowheads, respectively.

Figure 6

Plots showing average I/M ratio (A), lumen area (B), and external elastic lamina (EEL) length (C) measured for Regions I, II, and III (as defined above in Figure 5) of injured arteries treated with balloons coated with polymer 1/DNA films (32 bilayers thick) fabricated using either pcDNA3 (control; white bars) or pPKCδ (gray bars). Average values and error bars (shown as standard deviations) were calculated using measurements for three arbitrarily chosen sections from each Region (I, II, and III) depicted in Figure 5A. Two rats treated with polymer 1/pcDNA3 films (n = 6 for statistical analysis for each region) and three rats treated with polymer 1/PKCδ films (n = 9 for statistical analysis for each region) were used to calculate averages, standard deviations, and statistical significance of the data. Statistical analysis was conducted using ANOVA Single Factor analysis of statistical variance with the following reported p values: * p < 0.001; ** p < 0.01; and *** p < 0.10. See Materials and Methods for additional details on calculation of values and analysis of statistical variance.