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Muneera R. Kapadia, MD, Lesley W. Chow, BS, Nick D

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1 Nitric oxide and nanotechnology: A novel approach to inhibit neointimal hyperplasia 
Muneera R. Kapadia, MD, Lesley W. Chow, BS, Nick D. Tsihlis, PhD, Sadaf S. Ahanchi, MD, Jason W. Eng, Jozef Murar, BA, Janet Martinez, AAS, Daniel A. Popowich, MD, Qun Jiang, MD, Joseph A. Hrabie, PhD, Joseph E. Saavedra, PhD, Larry K. Keefer, PhD, James F. Hulvat, PhD, Samuel I. Stupp, PhD, Melina R. Kibbe, MD  Journal of Vascular Surgery  Volume 47, Issue 1, Pages (January 2008) DOI: /j.jvs Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

2 Fig 1 Our approach to inhibiting neointimal hyperplasia uses two components: (A) diazeniumdiolate nitric oxide donors, PROLI/NO, PAPA/NO, DPTA/NO, and DETA/NO (half-lives listed are for physiologic conditions, 37°C and pH 7.4) and (B) heparin-binding peptide amphiphile. C, The peptide amphiphiles spontaneously form nanofibers in aqueous solutions when exposed to heparin, as depicted in this drawing. PROLI/NO, Disodium 1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate; PAPA/NO, 1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate; DPTA/NO, 1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)]diazen-1-ium-1,2-diolate; DETA/NO, 1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

3 Fig 2 A, Nitrite release from nanofiber, PROLI/NO nanofiber, and DPTA/NO nanofiber gels in vitro, determined using the Griess reaction (n = 2 per group). Data are representative of three separate experiments. Scanning electron microscopy images (×4000 original magnification) of the (B) nanofiber gel, (C) PROLI/NO nanofiber gel, and (D) DPTA/NO nanofiber gel. NO, Nitric oxide. See Fig 1 for the expansion of the abbreviations PROLI/NO and DPTA/NO. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

4 Fig 3 A, B, Effect of PROLI/NO and DPTA/NO on vascular smooth muscle cell proliferation and (C, D) cell death in vitro. Proliferation and cell death were quantified using tritiated thymidine and the Guava Personal Cell Analysis (PCA) system (Hayward, Calif), respectively, (n = 3 per group). Data are representative of 3 separate experiments. See Fig 1 for expansion of the abbreviations PROLI/NO and DPTA/NO. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

5 Fig 4 Rat carotid artery sections from uninjured, injury alone, nanofiber gel, PROLI/NO nanofiber gel, and DPTA/NO nanofiber gel animals euthanized at 14 days (n = 6 per group). A, Representative sections (×100 original magnification) from each group using routine staining with hematoxylin and eosin (H&E) and Verhoff-van Gieson (VvG). B, Graphic representation of intima/media (I/M) area ratio presented with the standard error of the mean. Morphometric analysis conducted on 6 sections per rat. Units are arbitrary. IA, Injury alone, NG, nanofiber gel, PNG, PROLI/NO nanofiber gel; DNG, DPTA/NO nanofiber gel. NO, Nitric oxide. See Fig 1 for the complete expansion of the abbreviations PROLI/NO and DPTA/NO. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

6 Fig 5 Immunofluorescence staining for markers of inflammation and proliferation. A, Representative sections (×400 original magnification) from uninjured, injury alone, nanofiber gel, PROLI/NO nanofiber gel, and DPTA/NO nanofiber gel treatment groups (n = 2 per group, 3 sections per animal). Positive staining is indicated by red and elastic lamina is green. Graphic representation of scoring by 8 blinded observers (scale, 0-3) for (B) monocytes (anti-ED1), (C) leukocytes (anti-CD45), and (D) proliferation (anti-Ki67). Each data point represents scoring for a particular animal. U, Uninjured; IA, injury alone; NG, nanofiber gel; NO, nitric oxide; PNG, PROLI/NO nanofiber gel; and DNG, DPTA/NO nanofiber gel. See Fig 1 for the complete expansion of the abbreviations PROLI/NO and DPTA/NO. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

7 Fig 6 Re-endothelialization of injured rat carotid arteries. Groups include injury alone and injury with nanofiber, PROLI/NO nanofiber, or DPTA/NO nanofiber gels (n = 3 per group). Arteries were procured at 7 days postinjury. Arterial areas lacking endothelium stain blue (arrow). See Fig 1 for the complete expansion of the abbreviations PROLI/NO and DPTA/NO. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

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