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Polymeric-based perivascular delivery of a nitric oxide donor inhibits intimal thickening after balloon denudation arterial injury: role of nuclear factor-kappaB¹

Sanjay Kaul, MD^* , Bojan Cercek, MD, FACC^* , Jan Rengstrom, MD^*, Xiao-Ping Xu, MD^* , Mia D. Molloy, BS^* , Paul Dimayuga, BS^* , Akik K. Parikh, MD^* , Michael C. Fishbein, MD , Jan Nilsson, MD^*, Tripathi B. Rajavashisth, PhD^* and Prediman K. Shah, MD, FACC^*

^* Vascular Physiology and Thrombosis Laboratory of the Atherosclerosis Research Center, Cedars-Sinai Medical Center, Los Angeles, California, USA
Burns and Allen Research Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA
Division of Cardiology, Cedars-Sinai Medical Center, Los Angeles, California, USA
^* Karolinska Institute, Stockholm, Sweden

View larger version (25K): [in a new window]   Figure 1 Figure illustrating the experimental design. The study consisted of four separate protocols. In study protocol 1, the effect of NO donor on neointimal thickening was evaluated by histomorphometry at day 14 after balloon injury (n = 28). In study protocol 2, PCNA immunohistochemistry was used to assess DNA synthesis and VSMC proliferation at day 3 after balloon injury (n = 18). Vascular cGMP levels were also measured by radioimmunoassay in protocol 2 (n = 11). In study protocol 3, activation of NF-kappaB, was examined by EMSA and supershift assays at 6 hours after balloon injury (n = 27). Platelet aggregation and bleeding time were also evaluated in some rats in protocols 2 (n = 6) and 3 (n = 5). In study protocol 4, the in vitro release profile of NO form the polymeric delivery system was examined in isolated arteries (n = 3). cGMP = cyclic guanosine monophosphate; EMSA = electrophoretic mobility shift assay; NF-kappaB = nuclear factor kappa B; PCNA = proliferating cell nuclear antigen; SPER/NO = spermine/NO (NO donor); SPER = spermine (vehicle for the NO donor); VSMC = vascular smooth muscle cells.

View larger version (94K): [in a new window]   Figure 2 Photomicrographs demonstrating the effects on balloon injury-induced neointimal thickening. EEL = external elastic lamina; IEL = internal elastic lamina; NO = nitric oxide; spermine/NO = NO donor; spermine = vehicle for the NO donor. (Hematoxylin & eosin; x 100, reduced by 70%.)

View larger version (38K): [in a new window]   Figure 3 (A and B) Bar graphs demonstrating the effects on histomorphometric parameters. In the first group of rats, the injured arteries on one side were treated with the NO donor-polymer mixture (injury + polymer + spermine/NO) and a mixture of spermine, the carrier vehicle for the NO donor, and the polymer was applied to the contralateral injured artery (injury + polymer + spermine). In the second group, the artery on one side was injured without any treatment (injury, no treatment) while the other artery was left uninjured without any treatment (no injury, no treatment). The third group had the polymer alone applied around the injured artery on one side (injury + polymer) while the contralateral uninjured artery was treated with the NO donor/polymer mixture (no injury + polymer + spermine/NO). Data are mean ± SEM: n = number of animals. P values are based on analysis of variance with Tukey’s multiple-comparison test except for A where: * is p < 0.05 vs. injury + polymer + spermine based on planned single-pair comparison. NO = nitric oxide. Open box = no injury, no treatment (n = 8); solid box = injury, no treatment (n = 8); gray box = injury plus polymer (n = 6); slanted line box = injury plus polymer plus spermine (n = 14); dotted box = injury plus polymer plus spermine/NO (n = 14).

View larger version (14K): [in a new window]   Figure 4 Bar graphs demonstrating the effects on cell proliferation as assessed by PCNA immunohistochemistry. Data (mean ± SEM) are expressed as the number of PCNA-positive cells per section averaged over three sections per artery. N = 6 in each group. *p < 0.005 vs. no treatment or polymer + spermine. PCNA = proliferating cell nuclear antigen. Open box = no injury; solid box = injury.

View larger version (38K): [in a new window]   Figure 5 Electrophoretic mobility shift assays demonstrating the effects of NO donor, spermine/NO, on NF-kappaB DNA binding activity (representative of three similar experiments). Supershifted immunoreactive band using antibodies against the p50 and p65 subunits of NF-kappaB are indicated by the arrows. NF-kappaB = nuclear factor kappa B; NO = nitric oxide; Spermine = the carrier vehicle for the NO donor.

View larger version (14K): [in a new window]   Figure 6 Bar graphs demonstrating the effects on vascular cGMP levels in rats with no treatment (n = 3), rats treated with polymer + spermine/NO (n = 5) and polymer + spermine (n = 3). Data are mean ± SEM. *p < 0.01 vs. no treatment or polymer + spermine. cGMP = cyclic guanosine monophosphate. Open box = no injury; solid box = injury.