ADCY9 (Adenylate Cyclase Type 9) Inactivation Protects From Atherosclerosis Only in the Absence of CETP (Cholesteryl Ester Transfer Protein).

BACKGROUND: Pharmacogenomic studies have shown that ADCY9 genotype determines the effects of the CETP (cholesteryl ester transfer protein) inhibitor dalcetrapib on cardiovascular events and atherosclerosis imaging. The underlying mechanisms responsible for the interactions between ADCY9 and CETP activity have not yet been determined. METHODS: Adcy9-inactivated ( Adcy9Gt/Gt) and wild-type (WT) mice, that were or not transgenic for the CETP gene (CETPtg Adcy9Gt/Gt and CETPtg Adcy9WT), were submitted to an atherogenic protocol (injection of an AAV8 [adeno-associated virus serotype 8] expressing a PCSK9 [proprotein convertase subtilisin/kexin type 9] gain-of-function variant and 0.75% cholesterol diet for 16 weeks). Atherosclerosis, vasorelaxation, telemetry, and adipose tissue magnetic resonance imaging were evaluated. RESULTS: Adcy9Gt/Gt mice had a 65% reduction in aortic atherosclerosis compared to WT ( P<0.01). CD68 (cluster of differentiation 68)-positive macrophage accumulation and proliferation in plaques were reduced in Adcy9Gt/Gt mice compared to WT animals ( P<0.05 for both). Femoral artery endothelial-dependent vasorelaxation was improved in Adcy9Gt/Gt mice (versus WT, P<0.01). Selective pharmacological blockade showed that the nitric oxide, cyclooxygenase, and endothelial-dependent hyperpolarization pathways were all responsible for the improvement of vasodilatation in Adcy9Gt/Gt ( P<0.01 for all). Aortic endothelium from Adcy9Gt/Gt mice allowed significantly less adhesion of splenocytes compared to WT ( P<0.05). Adcy9Gt/Gt mice gained more weight than WT with the atherogenic diet; this was associated with an increase in whole body adipose tissue volume ( P<0.01 for both). Feed efficiency was increased in Adcy9Gt/Gt compared to WT mice ( P<0.01), which was accompanied by prolonged cardiac RR interval ( P<0.05) and improved nocturnal heart rate variability ( P=0.0572). Adcy9 inactivation-induced effects on atherosclerosis, endothelial function, weight gain, adipose tissue volume, and feed efficiency were lost in CETPtg Adcy9Gt/Gt mice ( P>0.05 versus CETPtg Adcy9WT). CONCLUSIONS: Adcy9 inactivation protects against atherosclerosis, but only in the absence of CETP activity. This atheroprotection may be explained by decreased macrophage accumulation and proliferation in the arterial wall, and improved endothelial function and autonomic tone.

Validating Intravascular Imaging with Serial Optical Coherence Tomography and Confocal Fluorescence Microscopy.

Atherosclerotic cardiovascular diseases are characterized by the formation of a plaque in the arterial wall. Intravascular ultrasound (IVUS) provides high-resolution images allowing delineation of atherosclerotic plaques. When combined with near infrared fluorescence (NIRF), the plaque can also be studied at a molecular level with a large variety of biomarkers. In this work, we present a system enabling automated volumetric histology imaging of excised aortas that can spatially correlate results with combined IVUS/NIRF imaging of lipid-rich atheroma in cholesterol-fed rabbits. Pullbacks in the rabbit aortas were performed with a dual modality IVUS/NIRF catheter developed by our group. Ex vivo three-dimensional (3D) histology was performed combining optical coherence tomography (OCT) and confocal fluorescence microscopy, providing high-resolution anatomical and molecular information, respectively, to validate in vivo findings. The microscope was combined with a serial slicer allowing for the imaging of the whole vessel automatically. Colocalization of in vivo and ex vivo results is demonstrated. Slices can then be recovered to be tested in conventional histology.

Cardiovascular effects of oxytocin infusion in a porcine model of myocardial infarct.

The effects of oxytocin (OT) on cardiovascular endpoints were assessed in a myocardial infarct (MI) model. OT (10 ng.kg(-1).hour(-1)) or saline infusion was initiated at reperfusion (D0) or 8 days (D8) after MI. Our hypothesis was that OT administration to individuals with a low pretreatment OT levels (PTOT) may be beneficial, whereas individuals with an elevated PTOT would be prone to adverse effects. Starting OT on D0 reduced left ventricular fraction shortening evaluated 8 days post MI and had no effect on infarct size. OT initiated on D8 in animals with high PTOT decreased ejection fraction (EF) and increased left ventricular end-systolic diameter at 28 days post MI but had no significant effects on EF and left ventricular end-systolic diameter in low PTOT animals. OT infusion reduced OT receptor protein expression in high PTOT animals but not in low PTOT animals. Among placebo-treated individuals, low PTOT presented a trend toward reduced EF and larger infarct size compared with high PTOT. MI areas of fibrosis presented lower Annexin V expression compared with MI with cardiomyocyte predominance. Pretreatment endogenous OT levels and timing of OT administration post MI seem to impact outcome in this porcine model, and further investigations are warranted to define potential role of OT in cardiac regenerative therapy.

In Vivo Near-Infrared Fluorescence Imaging of Atherosclerosis Using Local Delivery of Novel Targeted Molecular Probes.

This study aimed to evaluate the feasibility and accuracy of a technique for atherosclerosis imaging using local delivery of relatively small quantities (0.04-0.4 mg/kg) of labeled-specific imaging tracers targeting ICAM-1 and unpolymerized type I collagen or negative controls in 13 rabbits with atheroma induced by balloon injury in the abdominal aorta and a 12-week high-cholesterol diet. Immediately after local infusion, in vivo intravascular ultrasonography (IVUS)-NIRF imaging was performed at different time-points over a 40-minute period. The in vivo peak NIRF signal was significantly higher in the molecular tracer-injected rabbits than in the control-injected animals (P < 0.05). Ex vivo peak NIRF signal was significantly higher in the ICAM-1 probe-injected rabbits than in controls (P = 0.04), but not in the collagen probe-injected group (P = 0.29). NIRF signal discrimination following dual-probe delivery was also shown to be feasible in a single animal and thus offers the possibility of combining several distinct biological imaging agents in future studies. This innovative imaging strategy using in vivo local delivery of low concentrations of labeled molecular tracers followed by IVUS-NIRF catheter-based imaging holds potential for detection of vulnerable human coronary artery plaques.

Local delivery of 17-beta-estradiol modulates collagen content in coronary porcine arteries after PTCA and stent implantation.

BACKGROUND: Percutaneous transluminal coronary angioplasty (PTCA) and stent implantation are associated with intimal hyperplasia and extracellular matrix (ECM) accumulation, resulting in restenosis. We showed that local delivery of 17-beta-estradiol (17betaE) reduced restenosis following PTCA and stent implantation by 47 and 23%, respectively. Because estrogens decreased type I and type III collagen synthesis in vitro, we hypothesized that local delivery of 17betaE may influence intimal hyperplasia formation by modulating ECM expression. METHODS: Porcine coronary arteries underwent PTCA or stenting and were randomly assigned to 17betaE or placebo. After 28 days, animals were sacrificed for histology and collagen type I and III content analysis. RESULTS: Both collagen subtypes increased in the media by 1.7 to 2.6-fold after PTCA and by 15.7 to 16.1-fold after stenting, as compared to PTCA segments. In the neointima, the ratio of collagen type III to type I was 2.7 in stented arteries and only 0.3 in PTCA arteries. In the neointima of 17betaE-treated animals, collagen type I (but not type III) content upregulation was limited by 53% after PTCA and by 74% after stenting. CONCLUSION: Local delivery of 17betaE reduces restenosis, in part by decreasing the density of collagen type I in the neointima in PTCA and stented arteries.

Stent implantation in coronary porcine arteries is associated with early activation of TNFalpha and TNFalpha receptor II expression.

Inflammation present in restenosis after angioplasty is associated with production of cytokines such as tumor necrosis factor (TNFalpha). However, limited data exist on the possible increase in TNFalpha and TNFalpha receptor expression induced during the chronic phase after stenting. To this end, swine underwent balloon denudation (PTCA) and stent implantation in coronary arteries. At day 1, 7 or 28 post-procedure, sections from injured and reference vessel segments were evaluated for extent of pathology and expression of TNFalpha and TNFalpha receptors (RI and RII). Restenosis assessed at days 7 and 28 showed, respectively, two- and six-fold more neointimal (NI) area in stented than in PTCA segments. Unlike reference segments, TNFalpha-positive cells were detected in both the media and the NI of injured segments, with a significant increase over the 28-day time frame. Stenting was associated with an eight-fold enhancement in TNFalpha expression over PTCA. TNFalpha expression and NI area tended to correlate in injured segments. Furthermore, the pattern of expression of TNFalpha-RII, but not TNFalpha-RI, resembled that of TNFalpha itself. These results implicate TNFalpha and TNFalpha-RII as important actors in both the acute and the chronic phases of inflammation following stent implantation.

Local delivery of 17beta-estradiol improves reendothelialization and decreases inflammation after coronary stenting in a porcine model.

In the current study, we investigated the effect of local intravascular delivery of 17beta-estradiol (17beta-E) on subsequent in-stent neointimal hyperplasia. Twenty-seven stents were implanted in coronary arteries of juvenile swine. Coronary arteries were randomized to local treatment with 17beta-E or no drug therapy (control-vehicle treated). Twenty-eight days post-treatment, angiographic images revealed an improved minimal lumen diameter (2.2 +/- 0.2 vs. 1.3 +/- 0.2 mm, P < 0.005) and a reduction of late lumen loss (1.7 +/- 0.2 vs. 2.3 +/- 0.1 mm, P < 0.01) in 17beta-E-treated vessels compared to control-vehicle treated. Histological analyses showed a reduction of stenosis (51.49 +/- 6.75 vs. 70.86 +/- 6.24%, P < 0.05), mean neointimal thickness (0.51 +/- 0.07 vs. 0.83 +/- 0.14 mm, P < 0.05) and inflammation score (1.29 +/- 0.28 vs. 2.85 +/- 0.40, P < 0.05) in 17beta-E-treated arteries compared to control-vehicle treated arteries. Immunohistochemistry analyses revealed a reduction of proliferating smooth muscle cells and increased in-stent reendothelialization in 17beta-E-treated arteries. Finally, we observed a correlation between neointimal hyperplasia and inflammation score, which in turn, was inversely related to reendothelialization. Locally delivered, 17beta-E is inhibiting the inflammatory response and smooth muscle cells proliferation and improving vascular reendothelialization which together are contributing to reduce in-stent restenosis in a porcine coronary injury model. Together, these data demonstrate the potential clinical application of 17beta-estradiol to improve vascular healing and prevent in-stent restenosis.

Percutaneous endoluminal arterial cryoenergy improves vascular remodelling after angioplasty.

This study aimed to investigate the effect of percutaneous endoluminal arterial cryoenergy immediately after balloon angioplasty on vascular remodeling. Restenosis, the main complication after coronary artery angioplasty, is a complex phenomenon in which vascular remodeling plays a prominent role. Observations of reduced scarring in freeze-induced wounds suggest potential value for cryoenergy in the prevention of restenosis. Juvenile swine underwent a first oversized balloon injury in both carotid arteries (CA) (3 injury sites/artery) and a second injury at day 14. At that time, one CA was randomly assigned to endoluminal cryoenergy of the sequential segments (proximal, medial, distal) at -15 degrees C, -30 degrees C, and -50 degrees C for 120 sec, and the other CA was used as control. Animals were sacrificed 28 days after the second procedure. Compared with intact reference segments, angioplasty reduced both the luminal (LA) and the external elastic lamina (EEL) areas from 6.66+/-0.59 to 3.13+/-0.54 mm(2) (p<0.05) and from 8.81+/-0.81 to 6.48+/-0.52 mm(2) (p<0.05), respectively. Cryoenergy, at the temperature with maximal benefits (-50 degrees C), caused a temperature-related protection, as the LA was maintained (6.79+/-0.89 versus 7.18+/-0.78 mm(2) for reference) and the EEL area increased from 9.12+/-0.78 to 12.98+/-1.07 mm(2), p<0.05. Moreover, cryoenergy increased the density of collagen III (p<0.05) which correlated with the maintenance of the LA (r=0.8045, p<0.009). Cryoenergy prevents late luminal loss after double-injury angioplasty by improving vascular remodeling, and is an interesting new therapeutic approach for the prevention of restenosis after angioplasty. The increased synthesis of collagen III appears to be involved in this phenomenon and could be a potential method of stabilizing the vulnerable plaque.

Prevention of in-stent restenosis via reduction of thrombo-inflammatory reactions with recombinant P-selectin glycoprotein ligand-1.

The binding of leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) to platelet P-selectin is central to post-angioplasty restenosis. Although intracoronary stents limit the mechanical component of restenosis, they cause marked thrombo-inflammation and neointimal proliferation leading to greater late luminal loss. We sought to demonstrate that P-selectin antagonism, using recombinant PSGL-1 (rPSGL-Ig), is effective in reducing platelet-leukocyte reactions and in-stent restenosis in double-injured porcine coronary arteries. Two weeks after initial injury by angioplasty to the coronary arteries, stents were implanted at the injury-induced lesion site, 15 min after an i.v. bolus administration of a vehicle or rPSGL-Ig (1 mg/kg). Four weeks later, adhesion of (51)Cr-platelets and (111)In-neutrophils and histomorphometric analyses were performed. In-stent residual lumen was almost 3 fold larger in rPSGL-Ig-treated arteries (3.1 +/- 0.4 mm(2)) as compared to control (1.1 +/- 0.2 mm(2)), which correspond to 64% vascular stenosis in control with no change in rPSGL-Ig animals. For a similar injury score, in-stent neointima was significantly reduced by 30 to 40% in the rPSGL-Ig group and quantitative coronary angiography showed a significant 35% reduction in late lumen loss. These effects of rPSGL-Ig were associated with a respective 70% and 53% reduction in platelet and neutrophil adhesion. In conclusion, pretreatment with rPSGL-Ig reduces thrombo-inflammatory responses, neointimal proliferation, and in-stent restenosis. P-selectin antagonism offers a promising therapy to improve clinical outcomes of coronary stenting.

Endovascular cryotherapy accentuates the accumulation of the fibrillar collagen types I and III after percutaneous transluminal angioplasty in pigs.

PURPOSE: To investigate the effect of endovascular cryotherapy (Cryo) on the density of collagen types I (CI) and III (CIII), which are involved in the dynamic modulation of extracellular matrix (ECM) after percutaneous transluminal angioplasty (PTA). METHODS: Twenty-one juvenile farm swine and 10 miniswine underwent PTA of the femoral arteries with and without Cryo (-50 degrees C for 2 minutes). Quantitative angiography, histomorphometry, and quantification of CI and CIII were performed at 1 week (n=7), 1 month (n=7), 3 months (n=7), and 6 months (n=10). RESULTS: PTA decreased the minimal luminal diameter (MLD) (range 3.48+/-0.18 to 4.2+/-0.39 mm) compared to baseline values (range 3.67+/-0.15 to 4.59+/-0.23 mm), but the application of Cryo maintained the MLDs at preprocedural levels (range 3.88+/-0.31 to 4.58+/-0.21 mm). At the time of sacrifice, the MLDs were similar in PTA and Cryo-treated arteries, but the external elastic lamina was significantly greater after Cryo application (range 10.17+/-0.54 to 14.34+/-0.76 mm2) than after PTA (range 8.69+/-0.70 to 11.77+/-0.73 mm2, p<0.05). Cryo did not alter the luminal area or prevent neointimal growth. A time-dependent increase of both CI and CIII was observed as early as 1 week after PTA, peaking at 3 months, and declining thereafter. Cryo accentuated this increase at all time points. CONCLUSION: The application of Cryo accentuates the accumulation of CI and CIII in PTA-treated femoral arteries. This effect may be of clinical relevance in the stabilization of peripheral atherosclerotic plaque.

Optimization of regional intraarterial naked DNA-mediated transgene delivery to skeletal muscles in a large animal model.

Effective gene therapy for muscular dystrophy will likely require intravascular administration. Although plasmid DNA (pDNA) contained within a large volume and rapidly infused into a major artery can achieve gene transfer within downstream muscles, this is associated with substantial muscle edema. Here we hypothesized that excessive edema-related increases in intramuscular pressure (IM pressure) developed during intraarterial pDNA injections could hinder successful gene delivery. Accordingly, we monitored IM pressure during injection of pDNA carrying a LacZ transgene into the femoral artery of rats and pigs. Large variations in IM pressure were found between different muscles. There was a significant inverse relationship between IM pressure and the subsequent level of gene transfer to muscle. Modification of the injection protocol to reduce IM pressure led to greatly increased pDNA-mediated gene expression and reduced muscle damage in pigs. Under the most optimized conditions, average transfection within eight different muscles of the pig hind limb amounted to 22% of all fibers, attaining a maximum of 60% in the gastrocnemius muscle. We conclude that IM pressure monitoring is a simple and useful procedure, which can be applied in both small and large animals to help optimize pDNA-mediated gene transfer to skeletal muscles by the intraarterial route.

Chronic platelet and neutrophil adhesion: a causal role for neointimal hyperplasia in in-stent restenosis.

PURPOSE: To investigate the relationship between platelets and neutrophils and the progression of neointimal hyperplasia after angioplasty versus stenting of porcine coronary arteries. METHODS: Balloon angioplasty or implantation of a balloon-expandable stent was randomly performed in the left and right coronary arteries of 16 Yorkshire swine; the animals were euthanized 1 hour (n=6), 24 hours (n=4), or 1 month (n=6) after the interventions. The adhesion of chromium 51-labeled platelets and indium 111-labeled neutrophils was quantified (per cm2), and histological and morphometric analyses were performed. RESULTS: The acute adhesion of platelets and neutrophils observed on the injured segments 1 hour after the interventions was similar between the treated groups. However, at 24 hours, stenting was associated with 2-fold more platelets and 3-fold more neutrophils (p<0.05) than was dilation, and stented arteries remained more thrombogenic at 1 month (p<0.05). Neointimal formation was more intense after stent implantation (3.80+/-0.77 mm, p<0.05) than after dilation (0.81+/-0.21 mm), and it correlated positively with the adhesion of platelets (r=0.81, p<0.002) and neutrophils (r=0.69, p<0.01). CONCLUSIONS: These results indicate that stent implantation is associated with a more intense acute and chronic, low-grade inflammatory response than is dilation. It appears that the chronic inflammatory response is, at least in part, platelet- and neutrophil-driven and contributes to the progression of neointimal proliferation after stenting.