Nitric oxide and nanotechnology: a novel approach to inhibit neointimal hyperplasia.
OBJECTIVE
Nitric oxide (NO) has been shown to inhibit neointimal hyperplasia after arterial interventions in several animal models. To date, however, NO-based therapies have not been used in the clinical arena. Our objective was to combine nanofiber delivery vehicles with NO chemistry to create a novel, more potent NO-releasing therapy that can be used clinically. Thus, the aim of this study was to evaluate the perivascular application of spontaneously self-assembling NO-releasing nanofiber gels. Our hypothesis was that this application would prevent neointimal hyperplasia.


METHODS
Gels consisted of a peptide amphiphile, heparin, and a diazeniumdiolate NO donor (1-[N-(3-Aminopropyl)-N-(3-ammoniopropyl)]diazen-1-ium-1,2-diolate [DPTA/NO] or disodium 1-[(2-Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate [PROLI/NO]). Nitric oxide release from the gels was evaluated by the Griess reaction, and scanning electron microscopy confirmed nanofiber formation. Vascular smooth muscle cell (VSMC) proliferation and cell death were assessed in vitro by (3)H-thymidine incorporation and Personal Cell Analysis (PCA) system (Guava Technologies, Hayward, Calif). For the in vivo work, gels were modified by reducing the free-water content. Neointimal hyperplasia after periadventitial gel application was evaluated using the rat carotid artery injury model at 14 days (n = 6 per group). Inflammation and proliferation were examined in vivo with immunofluorescent staining against CD45, ED1, and Ki67 at 3 days (n = 2 per group), and graded by blinded observers. Endothelialization was assessed by Evans blue injection at 7 days (n = 3 per group).


RESULTS
Both DPTA/NO and PROLI/NO, combined with the peptide amphiphile and heparin, formed nanofiber gels and released NO for 4 days. In vitro, DPTA/NO inhibited VSMC proliferation and induced cell death to a greater extent than PROLI/NO. However, the DPTA/NO nanofiber gel only reduced neointimal hyperplasia by 45% (intima/media [I/M] area ratio, 0.45 +/- 0.07), whereas the PROLI/NO nanofiber gel reduced neointimal hyperplasia by 77% (I/M area ratio, 0.19 +/- 0.03, P < .05) vs control (injury alone I/M area ratio, 0.83 +/- 0.07; P < .05). Both DPTA/NO and PROLI/NO nanofiber gels significantly inhibited proliferation in vivo (1.06 +/- 0.30 and 0.19 +/- 0.11 vs injury alone, 2.02 +/- 0.20, P < .05), yet had minimal effect on apoptosis. Only the PROLI/NO nanofiber gel inhibited inflammation (monocytes and leukocytes). Both NO-releasing nanofiber gels stimulated re-endothelialization.


CONCLUSIONS
Perivascular application of NO-releasing self-assembling nanofiber gels is an effective and simple therapy to prevent neointimal hyperplasia after arterial injury. Our study demonstrates that the PROLI/NO nanofiber gel most effectively prevented neointimal hyperplasia and resulted in less inflammation than the DPTA/NO nanofiber gel. This therapy has great clinical potential to prevent neointimal hyperplasia after open vascular interventions in patients.
Kapadia
Muneera R
MR
Division of Vascular Surgery, Northwestern University, 201 E. Huron Street, Chicago, IL 60611, USA.
Chow
Lesley W
LW
Tsihlis
Nick D
ND
Ahanchi
Sadaf S
SS
Eng
Jason W
JW
Murar
Jozef
J
Martinez
Janet
J
Popowich
Daniel A
DA
Jiang
Qun
Q
Hrabie
Joseph A
JA
Saavedra
Joseph E
JE
Keefer
Larry K
LK
Hulvat
James F
JF
Stupp
Samuel I
SI
Kibbe
Melina R
MR
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002339:Carotid Arteries / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D020212:Carotid Artery Injuries / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D016923:Cell Death / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D003692:Delayed-Action Preparations; D004195:Disease Models, Animal; D004337:Drug Carriers; D004339:Drug Compounding; D004730:Endothelium, Vascular / Q000187:drug effects; D005782:Gels; D006965:Hyperplasia; D008297:Male; D008958:Models, Molecular; D015394:Molecular Structure; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D036103:Nanotechnology / Q000379:methods*; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D017539:Tunica Intima / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology
D003692:Delayed-Action Preparations; D004337:Drug Carriers; D005782:Gels; D020030:Nitric Oxide Donors; C102043:proline-nitric oxide; D009569:Nitric Oxide; D011392:Proline