Research ArticleNanomedicine

MK2 inhibitory peptide delivered in nanopolyplexes prevents vascular graft intimal hyperplasia

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Science Translational Medicine  10 Jun 2015:
Vol. 7, Issue 291, pp. 291ra95
DOI: 10.1126/scitranslmed.aaa4549
  • Fig. 1. MK2i-NP synthesis and characterization.

    (A) MK2i-NP synthesis was achieved through electrostatic complexation of the MK2i peptide and PPAA. (B) Hypothesized MK2i-NP mechanism of endosomal escape. (C) Comparison of different MK2i formulations: endosomolytic MK2i-NPs formulated with PPAA, and NE-MK2i-NPs formulated with PAA. MK2i-NPs and NE-MK2i-NPs were formulated with the shown MK2i peptide sequence (red, modified TAT mimetic CPP sequence; green, MK2 inhibitory sequence). (D) ζ-potential of polyplexes prepared at different CRs ([NH3+]/[COO]). “Alexa” denotes NPs formulated with an Alexa Fluor 488–conjugated MK2i peptide. NE denotes nonendosomolytic MK2i-NPs. Data are means ± SEM (n = 3 independent measurements). (E) Dynamic light scattering (DLS) analysis of MK2i-NP disassembly at pH values low enough to protonate the PPAA polymer.

  • Fig. 2. MK2i-NP formulation increases cellular uptake, extends intracellular retention, and reduces endolysosomal colocalization of MK2i.

    (A) Representative flow cytometry histograms of MK2i internalization when delivered as a free peptide or via NPs. (B) Red blood cell hemolysis assay for pH-dependent membrane-disruptive activity. Data are means ± SEM (n = 4 technical replicates). (C) MK2i Alexa Fluor 488–labeled MK2i colocalization with LysoTracker red in VSMCs over time after 2 hours of treatment determined by calculating Mander’s coefficients. Data are means ± SEM (n ≥ 3 separate images). P values determined by one-way analysis of variance (ANOVA). (D) Representative confocal microscopy images of Alexa Fluor 488–labeled MK2i colocalization with LysoTracker in VSMCs. Scale bars, 20 μm. (E) Quantification of intracellular compartment size of VSMCs treated with each MK2i formulation. Data are means ± SEM (n ≥ 50 separate intracellular compartments per treatment group). P values determined via single-factor ANOVA.

  • Fig. 3. MK2i-NP formulation increases peptide delivery to human vein.

    (A) Immunofluorescence microscopy images and zoomed insets of HSV cross sections treated with Alexa Fluor 568–labeled MK2i, MK2i-NPs, or NE-MK2i-NPs (all red) and stained for the VSMC marker α-SMA and the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI). (B) Immunofluorescence microscopy images and zoomed insets of HSV cross sections treated with Alexa Fluor 568–labeled MK2i, MK2i-NPs, or NE-MK2i-NPs (all red) and stained for the endothelial marker CD31 and DAPI. (C) Pixel intensity distribution derived from the red fluorescent channel of entire (unzoomed) cross-sectional images in (A) and (B). Plots are representative of n = 3 HSV cross sections from a single donor.

  • Fig. 4. Treatment with MK2i-NP reduces neointima formation and alters phosphorylation of molecules downstream of MK2 in HSV ex vivo.

    (A) p38 MAPK–MK2 signaling pathway in smooth muscle. (B to E) Representative Western blots showing the phosphorylation of MK2 substrates hnRNP A0, CREB, and HSP27 with and without treatment. Quantification of Western blots for phospho–hnRNP A0 (C), phospho-CREB (D), and phospho-HSP27 (E) in HSV. Data are means ± SEM (n = 3 separate biological replicates from three separate donors). P values determined by single-factor ANOVA. (F) Neointima were visualized using Verhoeff–van Gieson (VVG) staining of HSV samples that were treated for 2 hours and maintained in organ culture for 14 days. Red bars demarcate intimal thickness. Scale bars, 100 μm. (G) Intimal thickness in HSV samples was quantified after 14 days in organ culture. Data are means ± SEM (n ≥ 3 separate biological replicates from three separate donors). *P ≤ 0.01, **P ≤ 0.001 compared to no treatment (NT) controls; P ≤ 0.05; single-factor ANOVA.

  • Fig. 5. NP formulation enhances MK2i bioactivity and duration of action in vitro.

    (A) TNFα production in human VSMCs stimulated with 10 μM angiotensin II (ANG II) for 4 hours, treated with MK2i formulations for 2 hours, and incubated in fresh medium for 24 hours. (B) IL-6 production in VSMCs stimulated with TNFα (20 ng/ml) for 4 hours, treated with MK2i formulations for 2 hours, and incubated in fresh medium for 24 hours. Data in (A) and (B) are means ± SEM (n = 4 technical replicates); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 compared to NT, P ≤ 0.05, ††P ≤ 0.001; single-factor ANOVA. (C and D) Quantification and representative images of VSMC migration immediately after (C) and 5 days after (D) treatment removal using a Boyden Transwell migration assay. Data are means ± SEM (n = 3 technical replicates from two separate experiments). P values determined by single-factor ANOVA.

  • Fig. 6. Intraoperative treatment with MK2i-NPs reduces neointima formation and promotes a contractile smooth muscle cell phenotype in vivo in transplanted rabbit vein grafts.

    Rabbit jugular vein explants were treated ex vivo for 30 min with MK2i or MK2i-NP (30 μM peptide) before transplant into the carotid artery (n ≥ 7 grafts per treatment group). Histological analyses were done on graft tissues harvested 28 days later. (A) Neointima was visualized using VVG staining of vein grafts. Red bars demarcate intimal thickness on the representative images. Scale bars, 100 μm. (B) Proliferation of intimal cells quantified using PCNA immunohistochemistry. (C) Intimal expression of the contractile marker α-SMA. (D) Intimal expression of the synthetic vascular smooth muscle phenotypic marker vimentin. (E) RAM-11+ macrophages in jugular vein graft sections. Left column scale bar, 100 μm; right column zoomed view scale bar, 50 μm. Data in (B) to (E) are means normalized to total number of cells in intima (that is, number of nuclei) ± SEM. All P values determined by single-factor ANOVA.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/291/291ra95/DC1

    Methods

    Fig. S1. Polymer characterization with GPC and NMR.

    Fig. S2. NP size and morphology.

    Fig. S3. MK2i uptake and intracellular retention in VSMCs.

    Fig. S4. MK2i uptake in ECs and effects on EC and VSMC migration.

    Fig. S5. The effects MK2i-NP and free MK2i on HSV viability.

    Fig. S6. Viability of MK2i-treated VSMCs used for inflammatory cytokine analysis.

    Fig. S7. VSMC proliferation assay as a control for migration experiments.

    Fig. S8. MK2i-NP versus MK2i treatment effects on VSMC and EC MCP-1 production over time.

    Table S1. NP library characterization.

    References (3638)

  • Supplementary Material for:

    MK2 inhibitory peptide delivered in nanopolyplexes prevents vascular graft intimal hyperplasia

    Brian C. Evans, Kyle M. Hocking, Michael J. Osgood, Igor Voskresensky, Julia Dmowska, Kameron V. Kilchrist, Colleen M. Brophy, Craig L. Duvall*

    *Corresponding author. E-mail: craig.duvall{at}vanderbilt.edu

    Published 10 June 2015, Sci. Transl. Med. 7, 291ra95 (2015)
    DOI: 10.1126/scitranslmed.aaa4549

    This PDF file includes:

    • Methods
    • Fig. S1. Polymer characterization with GPC and NMR.
    • Fig. S2. NP size and morphology.
    • Fig. S3. MK2i uptake and intracellular retention in VSMCs.
    • Fig. S4. MK2i uptake in ECs and effects on EC and VSMC migration.
    • Fig. S5. The effects MK2i-NP and free MK2i on HSV viability.
    • Fig. S6. Viability of MK2i-treated VSMCs used for inflammatory cytokine analysis.
    • Fig. S7. VSMC proliferation assay as a control for migration experiments.
    • Fig. S8. MK2i-NP versus MK2i treatment effects on VSMC and EC MCP-1 production over time.
    • Table S1. NP library characterization.
    • References (3638)

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