Assessment of a pro-healing stent in an animal model of early neoatherosclerosis.

BACKGROUND: Neoatherosclerosis represents an accelerated manifestation of atherosclerosis in nascent neointima after stenting, associated with adverse events. We investigated whether improved reendothelialization using RGD-coated stents results in diminished vascular permeability and reduced foam cell formation compared to standard DES in atherosclerotic rabbits. METHODS AND RESULTS: Neointimal foam cell formation was induced in rabbits (n = 7). Enhanced endothelial integrity in RGD-coated stents resulted in decreased vascular permeability relative to DES, which was further confirmed by SEM and TEM. Cell culture experiments examined the effect of everolimus on endothelial integrity. Increasing concentrations of everolimus resulted in a dose-dependent decrease of endothelial cell junctions and foam cell transformation of monocytes, confirming the relevance of endothelial integrity in preventing permeability of LDL. CONCLUSION: Incomplete endothelial integrity was confirmed as a key factor of neointimal foam cell formation following stent implantation. Pro-healing stent coatings may facilitate reendothelialization and reduce the risk of neoatherosclerosis.

Ex Vivo Assessment of Coronary Atherosclerotic Plaque by Grating-Based Phase-Contrast Computed Tomography: Correlation With Optical Coherence Tomography.

OBJECTIVES: The aim of this study was to determine the diagnostic accuracy of grating-based phase-contrast computed tomography (gb-PCCT) to classify and quantify coronary vessel characteristics in comparison with optical coherence tomography (OCT) and histopathology in an ex vivo setting. MATERIALS AND METHODS: After excision from 5 heart specimens, 15 human coronary arteries underwent gb-PCCT examination using an experimental imaging setup consisting of a rotating molybdenum anode x-ray tube, a Talbot-Lau grating interferometer, and a single photon counting detector. Subsequently, all vessels were imaged by OCT and histopathologically processed. Optical coherence tomography, gb-PCCT, and histopathology images were manually matched using anatomical landmarks. Optical coherence tomography and gb-PCCT were reviewed by 2 independent observers blinded to histopathology. Vessel, lumen, and plaque area were measured, and plaque characteristics (lipid rich, calcified, and fibrous) were determined for each section. Measures of diagnostic accuracy were derived, applying histopathology as the standard of reference. RESULTS: Of a total of 286 assessed cross sections, 241 corresponding sections were included in the statistical analysis. Quantitative measures derived from gb-PCCT were significantly higher than from OCT (P < 0.001) and were strongly correlated with histopathology (Pearson r ≥0.85 for gb-PCCT and ≥0.61 for OCT, respectively). Results of Bland-Altman analysis demonstrated smaller mean differences between OCT and histopathology than for gb-PCCT and histopathology. Limits of agreement were narrower for gb-PCCT with regard to lumen area, for OCT with regard to plaque area, and were comparable with regard to vessel area. Based on histopathology, 228/241 (94.6%) sections were classified as fibrous, calcified, or lipid rich. The diagnostic accuracy of gb-PCCT was excellent for the detection of all plaque components (sensitivity, ≥0.95; specificity, ≥0.94), whereas the results for OCT showed sensitivities of ≥0.73 and specificities of ≥0.66. CONCLUSIONS: In this ex vivo setting, gb-PCCT provides excellent results in the assessment of coronary atherosclerotic plaque characteristics and vessel dimensions in comparison to OCT and histopathology. Thus, the technique may serve as adjunct nondestructive modality for advanced plaque characterization in an experimental setting.

The integrin ligand c(RGDf(NMe)Nal) reduces neointimal hyperplasia in a polymer-free drug-eluting stent system.

The use of highly active and selective integrin ligands in combination with stent implantation is emerging as a promising alternative to the release of classical immunosuppressive drugs by current drug-eluting stents (DES), which has been associated with delayed vascular healing and late stent thrombosis. Herein we present the development and biological evaluation of the integrin ligand c(RGDf(NMe)Nal) as a potent anti-proliferative molecule that targets coronary artery smooth muscle cells (CASMCs). This peptide showed an antagonistic activity for αvß3 and αvß5 in the low-nanomolar range, and selectivity against the platelet receptor αIIbß3. In vitro, it efficiently inhibited the proliferation of CASMCs, displaying higher potency than the anti-tumor drug candidate cilengitide. This peptide was then loaded into a polymer-free bare metal stent (BMS), and its release studied at different time points. Up to seven days of elution, the peptide-coated stents retained high anti-proliferative activity toward CASMCs. Finally, the peptide was examined in vivo in a polymer-free DES system in a rabbit iliac artery model. After 28 days of implantation, histopathological analysis revealed that the peptide clearly decreased neointimal growth and improved vessel healing and re-endothelialization compared with the FDA-approved Cypher DES. Our study shows that this type of lipophilic integrin ligand, when eluted from a polymer-free stent system, has the potential to successfully decrease in-stent restenosis in the absence of delayed vascular healing.

Automatic characterization of neointimal tissue by intravascular optical coherence tomography.

Intravascular optical coherence tomography (IVOCT) is rapidly becoming the method of choice for assessing vessel healing after stent implantation due to its unique axial resolution <20 µm. The amount of neointimal coverage is an important parameter. In addition, the characterization of neointimal tissue maturity is also of importance for an accurate analysis, especially in the case of drug-eluting and bioresorbable stent devices. Previous studies indicated that well-organized mature neointimal tissue appears as a high-intensity, smooth, and homogeneous region in IVOCT images, while lower-intensity signal areas might correspond to immature tissue mainly composed of acellular material. A new method for automatic neointimal tissue characterization, based on statistical texture analysis and a supervised classification technique, is presented. Algorithm training and validation were obtained through the use of 53 IVOCT images supported by histology data from atherosclerotic New Zealand White rabbits. A pixel-wise classification accuracy of 87% and a two-dimensional region-based analysis accuracy of 92% (with sensitivity and specificity of 91% and 93%, respectively) were found, suggesting that a reliable automatic characterization of neointimal tissue was achieved. This may potentially expand the clinical value of IVOCT in assessing the completeness of stent healing and speed up the current analysis methodologies (which are, due to their time- and energy-consuming character, not suitable for application in large clinical trials and clinical practice), potentially allowing for a wider use of IVOCT technology.

Pathophysiology of renal denervation procedures: from renal nerve anatomy to procedural parameters.

Endovascular renal denervation techniques have been clinically adopted for the treatment of resistant arterial hypertension with great success. Despite the favourable early results achieved with this technology, a clear understanding of the pathophysiology underlying this novel treatment is lacking. In addition, non-responsiveness to renal denervation remains a nidus for treatment failure in distinct patients. In search of meaningful surrogate parameters relating to treatment responsiveness, the current article reviews the existing knowledge on renal nerve anatomy, changes occurring after denervation and procedural parameters collected during denervation. From preclinical experience, the most reliable morphological parameter reflecting successful renal denervation is the presence of axonal degeneration. Most procedural and clinical parameters need extended investigation before adopting them as potential surrogate parameters for successful renal denervation. As a consequence, there is an imperative need for dedicated research revealing the pathophysiology of renal denervation procedures. In this regard, close co-operation of engineers, researchers and clinicians is warranted to turn renal denervation into a milestone treatment of arterial hypertension.

Tissue characterization after drug-eluting stent implantation using optical coherence tomography.

OBJECTIVE: To validate optical coherence tomography (OCT) imaging for assessment of vascular healing in a preclinical animal model and human autopsy cases and to translate the findings to the assessment of vascular healing after drug-eluting stent implantation in clinical practice. APPROACH AND RESULTS: Drug-eluting and bare metal stents were imaged 28 and 42 days after implantation in atherosclerotic rabbits using OCT and simultaneously evaluated by histology. After coregistration with histology, gray-scale signal intensity (GSI) was measured for identified mature or immature neointimal tissue. Autopsy specimens were imaged with OCT and GSI values correlated with histology. Finally, prospective OCT imaging and GSI measurements were acquired in 10 patients undergoing follow-up 6 months after stenting with drug-eluting stents. Histopathologic and OCT morphometric analysis of implanted stents showed excellent correlation. Neointimal growth and vessel healing at 28 days in the animal model best correlated with human stented arteries at 6 months. In animal and human autopsy specimens, mature neointimal tissue consistently showed higher GSI values. Receiver operating characteristic curve analysis displayed high sensitivity and specificity for detection of mature neointima in animal (96% and 79%, respectively) and human autopsy (89% and 71%, respectively) data. In patients undergoing OCT follow-up 6 months after drug-eluting stent implantation, prospective GSI analysis revealed that a minimum of 27.7% of areas above stent struts represented mature neointima. CONCLUSIONS: Novel GSI analysis of OCT imaging data allows distinction between mature and immature neointimal tissue in animal models, autopsy specimens, and patients undergoing invasive surveillance in simple atherosclerotic lesions.

Preclinical evaluation of a novel drug-eluting balloon in an animal model of in-stent stenosis.

Despite advances in contemporary stent technology, in-stent restenosis (ISR) remains the major limitation following revascularization procedures. We developed a porcine model of ISR to specifically investigate the preclinical outcomes of a novel drug-eluting balloon (DEB) in this particular setting. Fifteen pigs received bare metal stents in each of the major coronary arteries for 28 days to induce neointimal growth. Following repeat angiography, animals were allocated to fourdifferent treatment groups. The control group consisted of a bare angioplasty catheter, while the Pantera Lux™ (3.0 µg/mm(2) paclitaxel) (30 s inflation) was compared to two consecutive deployments of the Pantera Lux™ (60 s inflation each) and the commercial SeQuent(®) Please balloon (60 s inflation). Twenty-eight days following balloon deployment, the animals underwent repeat angiography and were subsequently sacrificed for histopathologic assessment. There was a trend in reduction of percent diameter stenosis in the DEB group versus control (13.9% vs. 20.4%), while longer inflation duration or consecutive DEB deployment had no additional growth-limiting effect. Neointimal thickness was reduced from 0.38 ± 0.13 to 0.30 ± 0.09 mm in the control versus DEB group. All DEB groups showed delayed vascular healing characterized by dose-dependent increases in fibrin deposition and neointimal cell vacuity. Investigation of DEB in a porcine model of ISR is feasible and more accurately represents human disease conditions. The magnitude of neointima suppression is lower than that observed in non-diseased animal models and is accompanied by delayed vascular healing.

Vascular healing in drug-eluting stents: differential drug-associated response of limus-eluting stents in a preclinical model of stent implantation.

AIMS: We aimed to assess the influence of different sirolimus analogues released from a uniform stent platform on re-endothelialisation and vascular healing responses. METHODS AND RESULTS: Bare metal stents (BMS) were coated with a fluoropolymer containing everolimus (EES), sirolimus (SES) or zotarolimus (ZES) to generate drug-eluting stents (DES) with identical stent backbones, drug loads and release kinetics. DES constructs and control BMS were implanted into the iliac arteries of rabbits and were analysed at 14 days by scanning electron microscopy (SEM) and confocal microscopy for en face evaluation of endothelialisation (n=6 for each stent), or at 28 days to determine histomorphometric characteristics (n=11 for each stent). SEM analysis revealed low degrees of strut re-endothelialisation within the DES without differences among groups, while the BMS control showed almost complete endothelialisation. Percent stenosis was significantly reduced in all DES compared to BMS. Strut-based fibrin analysis revealed significantly greater deposition in the DES compared to BMS, with EES showing maximum fibrin deposition among the DES groups. CONCLUSIONS: Sirolimus and its derivatives have similar effects on endothelial regrowth and neointimal thickening. The observation of greatest fibrin deposition in the experimental EES group indicates that everolimus may affect vascular healing differently.

Morphological assessment of renal arteries after radiofrequency catheter-based sympathetic denervation in a porcine model.

OBJECTIVES: Catheter-based renal artery denervation has been successfully introduced as alternative treatment for patients suffering from drug-resistant essential hypertension. However, the local morphological changes within the vessel wall accompanying this technique remain elusive and we sought to characterize these by utilizing the simplicity radiofrequency catheter approach. METHODS: Following treatment of seven pigs, renal arteries were assigned to either the acute (n  =  6), subacute (10-day follow-up, n  =  6) or control (untreated, n  =  2) group. At follow-up blood analysis, final angiography and optical coherence tomography (OCT)-imaging of the treated arteries were performed and renal arteries and kidneys were processed for histopathology and immunohistochemistry. RESULTS: Radiofrequency-derived energy application to the vessel wall induced transmural tissue coagulation and loss of endothelium resulting in local thrombus formation also detectable by OCT. At 10 days, the luminal surface was almost completely re-endothelialized. Mural wall damage was replaced by fibrotic tissue and the adventitial layer showed strong inflammatory infiltration including vasculogenesis. Remnant autonomic nerve fascicles within the lesion segments of the subacute group displayed enhanced vacuolic degeneration and an impaired neurofilament protein immunostaining pattern. Examination of the kidneys revealed no abnormalities and blood parameters remained within the physiological range. CONCLUSION: Catheter-based application of radiofrequency energy resulted in circumscribed transmural injury within the arterial wall affecting autonomic nerve fascicles delayed to treatment. Acute loss of endothelialization resulted in thrombus formation leaving kidney perfusion apparently unimpaired.

Histopathological comparison of biodegradable polymer and permanent polymer based sirolimus eluting stents in a porcine model of coronary stent implantation.

Biodegradable stent coatings were recently introduced as a potential solution to overcome sustained inflammatory responses observed with permanent polymer-based drug-eluting stents. In a preliminary study, selected biodegradable or permanent polymer-based sirolimus-eluting stent (SES) formulations were screened for effectiveness in comparison to bare metal stents (BMS) at 28 days. Subsequently, the most favourable SES formulation was compared to commercially available SES (Cypher™) at 28, 90 and 180 days to investigate the histopathologic response as well as tissue, blood and organ pharmacokinetics. Overlapping SES implantation was conducted to evaluate vascular healing at 28 days in this particular setting. SES with biodegradable poly (L-lactide) polymer (PLLA) or poly(lactide-co-glycolide) showed the most favourable outcome with regards to reductions in neointimal area in comparison to BMS at 28 days. The PLLA SES showed a similar reduction in neointimal area compared to Cypher™ at 28 days, with significant greater reductions at 90 and 180 days (1.7 ± 0.7 mm² vs. 3.1 ± 1.5 mm², p=0.03 and 1.8 ± 1.2 mm² vs. 3.0 ± 1.5 mm², p=0.01, respectively). Sirolimus vascular tissue concentrations were detectable up to 90 days following implantation. Overlapping stented segments showed favourable histopathologic results with respect to fibrin deposition and endothelialisation at 28 days. In conclusion, the use of PLLA as drug-eluting matrix resulted in mild inflammatory responses in the presence of effective sirolimus tissue concentrations. The greater efficacy observed at long-term follow-up in PLLA SES compared to Cypher™ may be a multifactorial result of stent design, polymer biocompatibility and improved release kinetics.

Vascular effects of paclitaxel following drug-eluting balloon angioplasty in a porcine coronary model: the importance of excipients.

AIMS: The vascular effects of drug- eluting balloon (DEB) deployment in the absence of coronary stents have not been characterised. This study evaluated potential vascular effects of paclitaxel-coated angioplasty balloons using different excipients in the absence of additional stents. METHODS AND RESULTS: A total 45 porcine arteries were treated with paclitaxel-coated DEBs using four different excipients (all 3.0 µg/mm2): A) iopromide (n=9), B) ATEC excipient (n=8), C) BTHC excipient (n=10), D) lecithine excipient (n=10). Uncoated bare angioplasty balloons served as controls (n=8). Histology, histomorphometry, and quantitative angiography analysis were performed 28 days following intervention. Tissue concentrations of paclitaxel were measured in selected animals using BTHC excipient (n=39 arteries) and reached maximum concentrations of 165 ng/mg 30 min after delivery in coronary target tissue. There were no differences in efficacy endpoints using histomorphology or quantitative angiography between groups. In contrast, however, treatment with DEBs using BTHC excipient or iopromide was associated with increased fibrin deposition and inflammation indicating delayed vascular healing. DEBs using lecithin excipient or uncoated angioplasty balloons did not induce any comparable vascular effects. CONCLUSIONS: Effective excipients are necessary to accomplish successful balloon facilitated paclitaxel delivery, which is associated with delayed vascular healing as a sign of successful drug transfer. The potential of DEBs to diminish restenosis following angioplasty may be insufficient in the absence of additional stents.

Histopathologic evaluation of nitinol self-expanding stents in an animal model of advanced atherosclerotic lesions.

AIMS: The histologic response to self-expanding stent implantation into advanced atherosclerotic lesions has not been systematically investigated. We tested the hypothesis of whether gradual expansion of advanced atherosclerotic plaques by self-expanding stents would be an appropriate method to seal atherosclerotic lesions without causing plaque disruption as is usually observed with balloon-expandable stents. METHODS AND RESULTS: Twelve New Zealand white rabbits were fed an atherogenic diet (1% cholesterol) followed by arterial denudation and injection of washed autologous erythrocytes. Nitinol self-expanding stents of two different stent designs and strengths (n=12 for SX and n=12 for Micro-SX) were implanted into the previously formed lesions within the abdominal aorta six weeks following injection of erythrocytes. Four weeks following stent implantation, animals were sacrificed, specimens harvested and processed for histology. Histomorphometry was performed on stented and adjacent non-stented regions. Atherosclerotic lesions were composed of foam cells, cholesterol clefts and necrotic plaque. While SX stents showed an unfavourable outcome with respect to vessel remodelling and the percentage of uncovered stent struts, Micro-SX stents had fewer uncovered stent struts, less positive remodelling and less plaque injury. CONCLUSIONS: Nitinol Micro-SX self-expanding stents might be a valuable approach to seal high risk atherosclerotic lesions.

The pre-clinical assessment of rapamycin-eluting, durable polymer-free stent coating concepts.

All four currently FDA-approved drug-eluting stents (DESs) contain a durable polymeric coating which can negatively impact vascular healing processes and eventually lead to adverse cardiac events. Aim of this study was the pre-clinical assessment of two novel rapamycin-eluting stent (RES) coating technologies that abstain from use of a durable polymer. Two distinctive RES coating technologies were evaluated in vitro and in the porcine coronary artery stent model. The R-poly(S) stent platform elutes rapamycin from a biodegradable polymer that is top coated with the resin shellac to minimize the amount of polymer. The R-pro(S) stent platform allows dual drug release of rapamycin and probucol, blended by shellac. HPLC-based determination of pharmacokinetics indicated drug release for more than 28 days. At 30 days, neointimal formation was found to be significantly decreased for both DESs compared to bare-metal stents. Assessment of vascular healing revealed absence of increased inflammation in both DESs, which is commonly observed in DES with non-erodible polymeric coating. In conclusion, the pre-clinical assessment of RESs with resin-based or dual drug coating indicated an adequate efficacy profile as well as a beneficial effect for vascular healing processes. These results encourage the transfer of these technologies to clinical evaluation.

Site-specific targeting of nanoparticle prednisolone reduces in-stent restenosis in a rabbit model of established atheroma.

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 (22.5+/-4.4 versus 31.0+/-8.4 and 29.5+/-8.1%, P<0.03). CONCLUSIONS: 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.