Background-To enable intravascular detection of inflammation in atherosclerosis, we developed a near-infrared fluorescence (NIRF) catheter-based strategy to sense cysteine protease activity during vascular catheterization. Methods and Results-The NIRF catheter design was based on a clinical coronary artery guidewire. In phantom studies of NIRF plaques, blood produced only a mild (Ͻ30%) attenuation of the fluorescence signal compared with saline, affirming the favorable optical properties of the NIR window. Catheter evaluation in vivo used atherosclerotic rabbits (nϭ11). Rabbits received an injection of a cysteine protease-activatable NIRF imaging agent (Prosense750; excitation/emission, 750/770 nm) or saline. Catheter pullbacks through the blood-filled iliac artery detected NIRF signals 24 hours after injection of the probe. In the protease agent group, the in vivo peak plaque target-to-background ratio was 558% greater than controls (6.8Ϯ1.9 versus 1.3Ϯ0.3, meanϮSEM; PϽ0.05). Ex vivo fluorescence reflectance imaging corroborated these results (target-tobackground ratio, 10.3Ϯ1.8 for agent versus 1.8Ϯ0.3 for saline group; PϽ0.01). In the protease group only, saline flush-modulated NIRF signal profiles further distinguished atheromata from normal segments in vivo (PϽ0.01). Good correlation between the in vivo and ex vivo plaque target-to-background ratio was present (rϭ0.82, PϽ0.01). Histopathological analyses demonstrated strong NIRF signal in plaques only from the protease agent group. NIRF signals colocalized with immunoreactive macrophages and the cysteine protease cathepsin B. Conclusions-An intravascular fluorescence catheter can detect cysteine protease activity in vessels the size of human coronary arteries in real time with an activatable NIRF agent. This strategy could aid in the detection of inflammation and high-risk plaques in small arteries.

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Background— Rapamycin (sirolimus)-eluting stents are associated with reduced restenosis rates in animal studies and initial human trials. The present study evaluated whether orally administered everolimus (a macrolide of the same family as sirolimus) inhibits in-stent neointimal growth in rabbit iliac arteries. Methods and Results— New Zealand white rabbits were randomized to everolimus 1.5 mg/kg per day starting 3 days before stenting and reduced to 1 mg/kg per day from days 14 to 28 (group 1), everolimus 1.5 mg/kg given 1 day before stenting followed by 0.75 mg/kg per day for 28 days (group 2), or matching placebo for each group. Drugs were administered by oral gavage. Stents were deployed in both iliac arteries, and arteries were harvested 28 days after stenting. Group 1 everolimus-treated rabbits experienced weight loss and anorexia, which resolved after the everolimus dose was lowered on day 14. Group 2 animals were healthy for the duration of everolimus dosing. Both everolimus treatment groups significantly reduced in-stent neointimal growth (46% reduction and 42% reduction in intimal thickness in groups 1 and 2, respectively). In group 2 everolimus-treated animals, the neointima was healed or healing, characterized by stent struts covered by a thin neointima, overlying endothelial cells, and only small foci of fibrin. Scanning electron microscopy showed >80% stent surface endothelialization in group 2 everolimus-treated rabbits. Conclusions— Oral everolimus suppresses in-stent neointimal growth in the rabbit iliac artery. At a dose of 1.5 mg/kg given 1 day before stenting followed by 0.75 mg/kg per day for 28 days, everolimus was well tolerated and was associated with significant neointimal healing.

Background-Paclitaxel (PXL)-eluting stents in animals cause incomplete healing and, in some instances, a lack of sustained suppression of neointimal growth. The present study tested the efficacy of a novel systemic delivery nanoparticle PXL for reducing in-stent restenosis. Methods and Results-A saline-reconstituted formulation of PXL stabilized by albumin nanoparticles (nPXL) was tested in 38 New Zealand White rabbits receiving bilateral iliac artery stents. Doses of nPXL (1.0 to 5.0 mg/kg) were administered as a 10-minute intra-arterial infusion; control animals received vehicle (0.9% normal saline). In a follow-up chronic experiment, nPXL 5.0 mg/kg was given at stenting with or without an intravenous 3.5-mg/kg repeat nPXL dose at 28 days; these studies were terminated at 3 months. At 28 days, mean neointimal thickness was reduced (PՅ0.02) by doses of nPXL Ն2.5 mg/kg with evidence of delayed healing. The efficacy of a single dose of nPXL 5.0 mg/kg, however, was lost by 90 days. In contrast, a second repeat dose of nPXL 3.5 mg/kg given 28 days after stenting resulted in sustained suppression of neointimal thickness at 90 days (PՅ0.009 versus single dose nPXL 5.0 mg/kg and controls) with nearly complete neointimal healing. Conclusions-Although systemic nPXL reduces neointimal growth at 28 days, a single repeat dose was required for sustained neointimal suppression. Thus, this novel systemic formulation of PXL may allow adjustment of dose at the stent treatment site and prove to be a useful adjunct for the clinical prevention of in-stent restenosis.

Objective-TRM-484 is a novel drug consisting of nanoparticles of prednisolone with high affinity to chondroitin sulfate proteoglycans (CSPGs). This may allow for neointimal suppression via directed targeting to areas of injury at systemic concentrations low enough to avoid adverse side effects known to occur with oral delivery of steroids. Methods and Results-Atherosclerotic New Zealand white Rabbits were implanted with bare metal stents and randomized to receive intravenous TRM-484 at doses of 1 mg/kg or 0.32 mg/kg starting at the day of stenting and continuing 3 times a week for the duration of the study. Control animals received empty liposomes (placebo) or saline infusion. Stented arterial segments were harvested at 42 days and processed for histomorphometry and immunohistochemistry. Tissue and plasma levels were determined along with confocal microscopic analysis to determine distribution of rhodamine-labeled TRM-484 at various time points. TRM-484 was exclusively observed at sites of stent-induced injury, with absence of drug in contralateral nonstented arteries. Tissue concentration of stented arteries exceeded that of contralateral nonstented arteries by 100-fold 24 hours after administration of 1 mg/kg TRM-484 and resulted in significant reduction of percent stenosis compared to saline and placebo treated rabbits (22.5Ϯ4.4 versus 31.0Ϯ8.4 and 29.5Ϯ8.1%, PϽ0.03). Conclusion-TRM-484 at doses of 1 mg/kg resulted in significant suppression of in-stent neointimal growth in atherosclerotic rabbits. Site-specific targeting by this nanoparticle steroid in injured atherosclerotic areas might be a valuable and cost-effective approach for the prevention of in-stent restenosis. (Arterioscler

Polymer-free stents coated with SES or SES+ED result in less robust neointimal suppression but markedly improved arterial healing compared with CDES in the rabbit model.

Background— Late total occlusion after vascular brachytherapy (VBT) continues to be a serious complication. Delayed reendothelialization was suggested as a pivotal cause, but the time course for complete healing is unknown. Methods and Results— Seventy-two rabbit iliac arteries underwent stent implantation and were treated with γ-radiation using 192 Ir. The prescribed doses were 0 Gy (controls, n=24 arteries), 15 Gy (n=24), or 30 Gy (n=24) at 2 mm. Animals were killed at 1 month (n=24), 3 months (n=24), or 6 months (n=24) and were analyzed for histomorphometry or scanning electron microscopy. Intimal area was reduced after VBT at 3 months with 15 and 30 Gy (0.66±0.07 and 0.66±0.04 mm 2 , respectively) compared with controls (1.01±0.11 mm 2 , P <0.05) and at 6 months with 30 Gy (0.75±0.09 versus 1.28±0.26 mm 2 in controls, P <0.01). Intimal area was similar at 6 months between 15 Gy and controls. At 1 month, 92±4% of the control stented segment was covered with endothelial cells, whereas only 37±4% and 37±8% was covered in the 15- and 30-Gy arteries, respectively. Similarly, at 3 and 6 months, there was a difference in the extent of reendothelialized areas (at 3 months, 95±2%, 32±12%, and 29±13%; and at 6 months, 98±2%, 40±8%, and 35±12% in control, 15-Gy, and 30-Gy arteries, respectively). Excess platelets and leukocytes were seen in irradiated arteries without complete coverage of endothelium. Conclusions— Reendothelialization after VBT is not completed at 6 months after VBT. Special care with prolonged antiplatelet therapy should be considered beyond that time point.

Rationale Sirolimus- (SES) and paclitaxel-eluting coronary stents (PES) are used to reduce restenosis, but have different sites of action. The molecular targets of sirolimus (SRL) overlap with those of the PPARγ agonist, rosiglitazone (RSG), but the consequence of this interaction on endothelialization is unknown. Objective Using the New Zealand White rabbit iliac model of stenting, we examined the effects of RSG on SES, PES and bare metal stent (BMS) endothelialization (ENDO). Methods and Results Animals receiving SES, PES, or BMS, and either RSG (3mg/kg/day) or placebo, were sacrificed at 28 days, and arteries evaluated by scanning electron microscopy (SEM). Fourteen-day organ culture (OC) and western blotting (WB) of iliac arteries, and tissue culture experiments were conducted. ENDO was significantly reduced by RSG in SES, but not in PES or BMS. OC revealed reduced VEGF in SES receiving RSG compared to RSG animals receiving BMS or PES. Quantitative PCR in human aortic endothelial cells (HAECs) revealed that SRL (but not paclitaxel) inhibited RSG-induced VEGF transcription. WB demonstrated that inhibition of molecular signaling in SES+RSG treated arteries was similar to findings in HAECs treated with RSG and siRNA to PPARγ, suggesting that SRL inhibits PPARγ. Transfection of HAECs with mTOR shRNA and with Akt2 siRNA significantly inhibited RSG mediated transcriptional upregulation of heme oxygenase-1 (HO-1), a PPARγ target gene. Chromatin immunoprecipitation assay demonstrated SRL interferes with binding of PPARγ to its response elements in HO-1 promoter. Conclusions mTOR/Akt2 is required for optimal PPARγ activation. Patients who receive SES during concomitant RSG treatment may be at risk for delayed stent healing.