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Background: Human pluripotent stem cell (hPSC)–derived cardiomyocytes (hPSC-CMs) have tremendous promise for application in cardiac regeneration, but their translational potential is limited by an immature phenotype. We hypothesized that large-scale manufacturing of mature hPSC-CMs could be achieved through culture on polydimethylsiloxane (PDMS)-lined roller bottles and that the transplantation of these cells would mediate better structural and functional outcomes than with conventional immature hPSC-CM populations. Methods: We comprehensively phenotyped hPSC-CMs after in vitro maturation for 20 and 40 days on either PDMS or standard tissue culture plastic substrates. All hPSC-CMs were generated from a transgenic hPSC line that stably expressed a voltage-sensitive fluorescent reporter to facilitate in vitro and in vivo electrophysiological studies, and cardiomyocyte populations were also analyzed in vitro by immunocytochemistry, ultrastructure and fluorescent calcium imaging, and bulk and single-cell transcriptomics. We next compared outcomes after the transplantation of these populations into a guinea pig model of myocardial infarction using end points including histology, optical mapping of graft- and host-derived action potentials, echocardiography, and telemetric electrocardiographic monitoring. Results: We demonstrated the economic generation of >1×10 8 mature hPSC-CMs per PDMS-lined roller bottle. Compared with their counterparts generated on tissue culture plastic substrates, PDMS-matured hPSC-CMs exhibited increased cardiac gene expression and more mature structural and functional properties in vitro. More important, intracardiac grafts formed with PDMS-matured myocytes showed greatly enhanced structure and alignment, better host-graft electromechanical integration, less proarrhythmic behavior, and greater beneficial effects on contractile function. Conclusions: We describe practical methods for the scaled generation of mature hPSC-CMs and provide the first evidence that the transplantation of more mature cardiomyocytes yields better outcomes in vivo.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) have tremendous promise for application in cardiac repair, but their immature phenotype greatly limits their translational potential. The present study was designed to two hypotheses: 1) that previously reported methods to promote the maturation of hESC-CMs by culture on soft polydimethylsiloxane (PDMS) substrates can be upscaled to the quantities required for transplantation studies; and 2) that PDMS-matured hESC-CMs will stably engraft in injured hearts and form graft myocardium with enhanced structural and functional properties. First, we cultured hESC-CMs on either PDMS or tissue culture plastic (TCP) for 20 and 40 days, then phenotyped the resultant populations. All hESC-CMs were engineered to express the fluorescent voltage-sensitive protein ASAP1 to facilitate in vitro and in vivo electrophysiological studies. Relative to their counterparts on TCP, hESC-CMs on PDMS at both time-points exhibited increased cardiac gene expression as well as a more mature structural and electrophysiological phenotype in vitro. Single-cell transcriptomics confirmed enrichment of cardiac maturation markers including gene pathways involved in cardiac contraction, extracellular matrix organization, sarcomerogenesis, and adult heart development in PDMS versus TCP cultures. Next, we transplanted day 20 or 40 TCP vs PDMS ASAP1+ hESC-CMs into injured guinea pig hearts. Recipient hearts were later analyzed by ex vivo optical voltage mapping studies and histology. While CMs from both substrates showed similar capacity for engraftment, grafts formed with PDMS-matured myocytes had more mature structural properties including enhanced alignment, sarcomere lengths and maturation marker expression. Most importantly, graft formed with PDMS-matured myocytes showed improved electrophysiological properties including better host-graft electromechanical integration and more rapid and uniform propagation. We conclude that large quantities of matured hESC-CMs can indeed be economically produced by these methods. Moreover, PDMS-matured myocytes form large intramyocardial grafts with enhanced cardiac structure and electrical function, thereby establishing that maturation prior to transplantation meaningfully improves outcomes in vivo.

Objective To create an experimental chronic total occlusion (CTO) model with calcification by dietary modification (cholesterol, calcium carbonate, vitamin D) and local injection of pro-calcification factors (dipotassium phosphate, calcium chloride, and bone morphogenetic protein-2 [BMP-2]). Background Percutaneous revascularization of CTOs frequently fails in heavily calcified occlusions. Development of novel approaches requires a reproducible preclinical model of calcified CTO. Methods CTOs were created in 18 femoral arteries of 9 New Zealand White rabbits using the thrombin injection model. Dietary interventions included a high cholesterol diet (0.5% or 0.25%), calcium carbonate (150 mg × 3-5 days/week), and vitamin D (50,000 U × 3-5 days/week). In selected animals, BMP-2 (1-4 μg), dipotassium phosphate, and calcium chloride were injected locally at the time of CTO creation. Animals were sacrificed at 2 weeks (n = 4 arteries), 6 weeks (n = 4 arteries), and 10-12 weeks (n = 14 arteries). Results CTOs showed evidence of chronic lipid feeding (foam cells) and chronic inflammation (intimal/medial fibrosis and microvessels, inflammatory cells, internal elastic lamina disruption). In calcium/vitamin D supplemented rabbits, mineralization (calcification and/or ossification) was evident as early as 2 weeks post CTO creation, and in 78% of the overall arteries. Mineralization changes were not present in the absence of calcium/vitamin D dietary supplements. Mineralization occurred in 85% of BMP-treated arteries and 60% of arteries without BMP. Conclusions Complex mineralization occurs in preclinical CTO models with dietary supplementation of cholesterol with vitamin D and calcium.

Aims : Bipolar electrogram (BiEGM)-based substrate maps are heavily influenced by direction of a wavefront to the mapping bipole. In this study, we evaluate high-resolution, orientation-independent peak-to-peak voltage (Vpp) maps obtained with an equi-spaced electrode array and omnipolar EGMs (OTEGMs), measure its beat-to-beat consistency, and assess its ability to delineate diseased areas within the myocardium compared against traditional BiEGMs on two orientations: along (AL) and across (AC) array splines. Methods and results: The endocardium of the left ventricle of 10 pigs (three healthy and seven infarcted) were each mapped using an Advisor™ HD grid with a research EnSite Precision™ system. Cardiac magnetic resonance images with late gadolinium enhancement were registered with electroanatomical maps and were used for gross scar delineation. Over healthy areas, OTEGM Vpp values are larger than AL bipoles by 27% and AC bipoles by 26%, and over infarcted areas OTEGM Vpp values are 23% larger than AL bipoles and 27% larger than AC bipoles (P

Perlecan is a heparan sulfate proteoglycan (HSPG) constituent of the extracellular matrix with roles in cell growth, differentiation, and angiogenesis. The role of the HS side chains in regulating in vivo angiogenesis after hind-limb ischemia is unknown.Heparan sulfate (HS)-deficient perlecan (Hspg2(Δ3/Δ3)) mice (n = 35), containing normal perlecan core protein but deficient in HS side chains, and wild-type (n = 33) littermates underwent surgical induction of hind-limb ischemia. Laser Doppler perfusion imaging (LDPI) and contrast-enhanced ultrasonography (CEU) provided serial assessment of hind-limb perfusion. Harvested muscles underwent immunostaining for endothelial cell density (CD31), real-time reverse transcription polymerase chain reaction RT-PCR for vascular endothelial growth factor (VEGF) mRNA expression and western blot analysis for VEGF and fibroblast growth factor (FGF)2 protein expression at days 2 and 28.Serial LDPI showed significantly greater perfusion recovery in ischemic limbs of wild-type compared with Hspg2(Δ3/Δ3) mice. CEU showed that normalized microvascular perfusion was increased in wild-type compared with Hspg2(Δ3/Δ3) mice at day 28 (0.67 ± 0.12 vs 0.26 ± 0.08; P = 0.001). CD31-positive cell counts were significantly higher in wild-type compared with Hspg2(Δ3/Δ3) mice on day 28 (122 ± 30 cells vs 84 ± 34 cells per high-power field [HPF]; P0.05). Endogenous VEGF mRNA expression (P0.05) and VEGF protein expression (P0.002) were significantly decreased in the ischemic limbs of Hspg2(Δ3/Δ3) mice compared with wild-type mice at day 2 and day 28, respectively. FGF2 protein expression showed no significant differences.These results suggest that the HS side chains in perlecan are important mediators of the angiogenic response to ischemia through a mechanism that involves upregulation of VEGF expression.

Perlecan is a proteoglycan composed of a 470-kDa core protein linked to three heparan sulfate (HS) glycosaminoglycan chains. The intact proteoglycan inhibits the smooth muscle cell (SMC) response to vascular injury. Hspg2Δ3/Δ3 (MΔ3/Δ3) mice produce a mutant perlecan lacking the HS side chains. The objective of this study was to determine differences between these two types of perlecan in modifying SMC activities to the arterial injury response, in order to define the specific role of the HS side chains. In vitro proliferative and migratory activities were compared in SMC isolated from MΔ3/Δ3 and wild-type mice. Proliferation of MΔ3/Δ3 SMC was 1.5× greater than in wild type ( P < 0.001), increased by addition of growth factors, and showed a 42% greater migratory response than wild-type cells to PDGF-BB ( P < 0.001). In MΔ3/Δ3 SMC adhesion to fibronectin, and collagen types I and IV was significantly greater than wild type. Addition of DRL-12582, an inducer of perlecan expression, decreased proliferation and migratory response to PDGF-BB stimulation in wild-type SMC compared with MΔ3/Δ3. In an in vivo carotid artery wire injury model, the medial thickness, medial area/lumen ratio, and macrophage infiltration were significantly increased in the MΔ3/Δ3 mice, indicating a prominent role of the HS side chain in limiting vascular injury response. Mutant perlecan that lacks HS side chains had a marked reduction in the inhibition of in vitro SMC function and the in vivo arterial response to injury, indicating the critical role of HS side chains in perlecan function in the vessel wall.

AIMS Patients with symptomatic chronic total occlusions (CTO) remain a therapeutic challenge. Enhancement of intraluminal neovascularisation by pro-angiogenic therapies has been proposed as a new strategy to improve percutaneous revascularisation. The aim of this study was to investigate the effects of intraluminal injection of bone marrow-derived cells (BMC) into experimental CTO. METHODS AND RESULTS CTO were created in the femoral arteries of 43 New Zealand White rabbits using the thrombin injection model. At 12 weeks following CTO creation, 33 rabbits were injected with either cultured BMC (n=19) or control DMEM alone (n=14) directly into the CTO. Ten rabbits were used for cell tracking (seven BMC and three control). BMC labelled with fluorescent Qdot® nanocrystals were identified in the CTO up to one week after injection. Animals were sacrificed at three to five weeks post-treatment and arterial samples were excised for micro-CT imaging and histologic morphometric analysis. There was a significant but modest increase in neovascularisation in BMC-treated arteries compared to controls (7.47±4.75% vs. 4.35±2.97%, p

Aims To create a large animal coronary chronic total occlusion (CTO) model. Presence of microvessels within the CTO lumen facilitates guidewire crossing. The patterns and time profiles of matrix changes and microvessel formation during coronary CTO maturation are unknown. Methods and results CTO were created in 15 swine by percutaneous deployment of a collagen plug. Matrix changes were assessed by histology. Intraluminal neovascularisation was assessed by histology and several imaging modalities, including conventional and 3D spin angiography, micro-computed tomography (micro-CT) imaging, and contrast-enhanced magnetic resonance imaging (MRI), at six and 12 weeks following CTO creation. Matrix changes included an intense inflammatory reaction at six weeks which had partially abated by 12 weeks. A proteoglycan-rich matrix at six weeks was partially replaced with collagen by 12 weeks. Similar changes were noted in the proximal cap which was acellular. Three patterns of microvessel formation were identified and defined based on the presence and extent of a "lead" neovessel. No major differences in pattern or extent of neovascularisation were noted between six and 12 weeks. Conclusions Heterogeneity in neovascularisation patterns occurs during coronary CTO development in a porcine model. Non-invasive imaging to determine the predominant type of neovascularisation prior to and during CTO revascularisation may improve guidewire crossing success rates. This model may be useful for further exploration of CTO pathophysiology, and may aid in further refinements of in vivo imaging of CTO and development of novel therapeutic approaches to revascularisation of CTO, such as manipulations of the proximal cap, matrix composition, neovessel induction, and device testing.

ObjectiveRecombinant factor VIIa can decrease postoperative bleeding after cardiac surgery. However, the potential for recombinant factor VIIa to cause early vascular graft occlusion at the site of new vascular anastomoses has not been fully explored. We hypothesized that recombinant factor VIIa would cause a dose-dependent reduction in vascular graft patency in rabbits.MethodsReversed end-to-end interpositional vein grafts were sutured into the carotid artery of heparinized rabbits, and then recombinant factor VIIa (300 μg/kg, 90 μg/kg, or 20 μg/kg intravenously) or placebo was administered (n = 16/group). Graft patency was assessed at 24 hours using a vascular ultrasound probe. Factor VII activity levels were measured using a prothrombin time-based assay. In different rabbits, the patency of venous end-to-side anastomoses and simple carotid arterial repairs was assessed (recombinant factor VIIa, 300 μg/kg vs placebo, n = 8/group). Data were analyzed using Fisher’s exact test, t tests, or analysis of variance.ResultsPhysiologic variables (activated clotting time, hemoglobin, pH, Pao2) and vessel diameter were not different between groups. Vein graft patency was reduced (93.8%, 81.2%, 13.8%, and 6.3%) as factor VII activity levels increased (1.8 ± 0.4, 4.4 ± 2.1, 11.8 ± 4.7, and 23.6 ± 16.9 U/mL, respectively) with increasing doses of recombinant factor VIIa administered (0, 20, 90, and 300 μg/kg, respectively, P

We evaluated the role of p15(Ink4), a member of the INK4 family of CDK inhibitors on vascular smooth muscle cells (VSMCs) proliferation, cell cycle progression and intimal hyperplasia after stenting. Aortic VSMCs transduced with either adenovirus encoding for p15(Ink4) or β-galactosidase were assessed for DNA synthesis, cell cycle progression, and pRb phosphorylation. Rabbit carotid arteries were stented and treated with peri-adventitial delivery of saline or adenovirus encoding for p15(Ink4) or β-galactosidase. p15(Ink4) transgene and protein expression were evaluated at 24 h and 72 h, respectively. In-stent cell proliferation was evaluated by BrdU at day 7. Histomorphometric analysis of in-stent intimal hyperplasia was performed at 10 weeks. Human p15(Ink4) DNA was detected in transduced VSMCs at 24h. p15(Ink4) over-expression reduced VSMCs DNA synthesis by 60%. Cell cycle progression was inhibited, with a 30% increase in G1 population accompanied by inhibition of pRb phosphorylation. Human p15(Ink4) transgene was identified in transduced stented arteries but not in control arteries. p15(Ink4) immunostaining was increased and cell proliferation significantly reduced by 50% in p15(Ink4) transduced arteries. Intimal cross-sectional area (CSA) of p15(Ink4)-treated group was significantly lower than the β-gal treated and non-transduced groups (p=0.008). There were no differences in the intimal or medial inflammatory response between groups. p15(Ink4) over-expression blocks cell cycle progression leading to inhibition of VSMCs proliferation. Peri-adventitial delivery of p15(Ink4) significantly inhibits in-stent intimal hyperplasia.

Objectives The purpose of this study was to characterize the 3-dimensional structure of intravascular and extravascular microvessels during chronic total occlusion (CTO) maturation in a rabbit model. Background Intravascular microchannels are an important component of a CTO and may predict guidewire crossability. However, temporal changes in the structure and geographic localization of these microvessels are poorly understood. Methods A total of 39 occlusions were created in a rabbit femoral artery thrombin model. Animals were sacrificed at 2, 6, 12, and 24 weeks (n ≥8 occlusions per time point). The arteries were filled with a low viscosity radio-opaque polymer compound (Microfil) at 150 mm Hg pressure. Samples were scanned in a micro-computed tomography system to obtain high-resolution volumetric images. Analysis was performed in an image processing package that allowed for labeling of multiple materials. Results Two distinct types of microvessels were observed: circumferentially oriented “extravascular” and longitudinally oriented “intravascular” microvessels. Extravascular microvessels were evident along the entire CTO length and maximal at the 2-week time point. There was a gradual and progressive reduction in extravascular microvessels over time, with very minimal microvessels evident beyond 12 weeks. In contrast, intravascular microvessel formation was delayed, with peak vascular volume at 6 weeks, followed by modest reductions at later time points. Intravascular microvessel formation was more prominent in the body compared with that in the proximal and distal ends of the CTO. Sharply angulated connections between the intravascular and extravascular microvessels were present at all time points, but most prominent at 6 weeks. At later time points, the individual intravascular microvessels became finer and more tortuous, although the continuity of these microvessels remained constant beyond 2 weeks. Conclusions Differences are present in the temporal and geographic patterns of intravascular and extravascular microvessel formation during CTO maturation.

AIMS Perlecan is the major heparan sulfate proteoglycan in the arterial wall. Previous studies have suggested that perlecan is a potent inhibitor of smooth muscle cell (SMC) activity. Therefore, perlecan overexpression may serve as a therapeutic modality to prevent in-stent restenosis (ISR). We have investigated a novel compound (RUS3108), identified in a SMC based screen to induce perlecan synthesis in SMC. The aims of this study were to assess the in vitro effects of RUS3108 and the effects of RUS3108-eluting stents in preventing ISR. METHODS AND RESULTS Rabbit aortic SMC and bovine aortic endothelial cells (EC) were used in this study. Immunohistochemistry showed that RUS3108-treated SMC over-expressed perlecan indicating the drug effects. Furthermore, RUS3108 induced a SMC differentiated phenotype by SMembryonic staining. RUS3108 (1 microM) inhibited 3H-thymidine incorporation by >50%, which was completely reversed by a perlecan antibody. RUS3108 also inhibited SMC migration (Boyden chamber) and MMP-9 activity. In contrast, RUS3108 (100nM) modestly stimulated EC 3H-thymidine incorporation by 22% (p

Background Distal coronary embolization (DCE) of thrombotic material occurs frequently during percutaneous interventions for acute myocardial infarction and can alter coronary flow grades. The significance of DCE on infarct size and myocardial function remains unsettled. The aims of this study were to evaluate the effects of DCE sufficient to cause no-reflow on infarct size, cardiac function and ventricular remodeling in a porcine acute myocardial infarction model. Methods and results Female Yorkshire pigs underwent 60 min balloon occlusion of the left anterior descending coronary artery followed by reperfusion and injection of either microthrombi (prepared from autologous porcine blood) sufficient to cause no-reflow (DCE), or saline (control). Animals were sacrificed at 3 h (n = 5), 3 days (n = 20) or 6 weeks (n = 20) post-AMI. Cardiovascular magnetic resonance (CMR), serum troponin-I, and cardiac gelatinase (MMP) and survival kinase (Akt) activities were assessed. At 3d, DCE increased infarct size (CMR: 18.8 % vs. 14.5 %, p = 0.04; serum troponin-I: 13.3 vs. 6.9 ng/uL, p

Purpose Chronic total occlusions (CTO) remain a major limitation of percutaneous interventions. Procedural failure is usually due to the inability to cross the lesion with a guide wire. We have previously shown that local administration of a laboratory-grade collagenase followed by a 72-h waiting period may facilitate guide-wire crossing. The aim of the present study was to evaluate the efficacy and toxicity of a human-grade purified collagenase, suitable for clinical use, in facilitating guide-wire crossing in a rabbit model of femoral artery CTO. Methods and results A chronic total arterial occlusion was constructed in femoral arteries of New Zealand white rabbits. The local administration of purified collagenase solution (150 μg) via an over-the-wire balloon system was performed in 10 CTO. Guide-wire crossing was attempted after 24 h and was successful in all cases. Different doses (50–500 μg) were administered to an additional 17 rabbits to assess collagenase effects. Local subcutaneous bruising was observed at higher doses. Histological evaluation showed no damage to the arterial wall structure. Arterial extracts from collagenase-treated arteries showed increased MMP-2 and MMP-9 activities and higher levels of local MMP-1 and degraded collagen. Conclusions Local administration of a human-grade purified collagenase degrades collagen in CTO and is highly effective for the facilitation of guide-wire crossing in CTO.

Background— Chronic total occlusions (CTOs) are associated with significant angina, impaired left ventricular function, and worse long-term outcomes. Percutaneous coronary interventions in CTO are unsuccessful in up to 50% of cases, primarily because of inability to cross the lesion with a guide wire. Collagen is the predominant component of the atherosclerotic plaque. The objective of this study was to determine the efficacy and toxicity of local delivery of a collagen-degrading enzyme to facilitate guide wire crossing in CTO. Methods and Results— Type IA collagenase (100 or 450 μg) or placebo was locally administered to 45 CTOs in a rabbit femoral artery model. Mean occlusion duration was 16±5 weeks. Attempts to cross the CTO (mean length, 28±9 mm) with conventional guide wires were assessed at 72 hours after treatment. An additional 3 arteries per group were assessed for collagenase effects at 24 hours after treatment. Successful guide wire crossings were significantly higher in collagenase-treated arteries (13 of 21, 62%) than in placebo-treated arteries (7 of 24, 29%) ( P =0.028). No adverse effects on arterial structure were observed in collagenase-treated arteries. At 24 hours, collagenase-treated arteries demonstrated increased collagenase protein, gelatinase activity, and collagen fragments. Conclusions— Local delivery of collagenase can safely facilitate guide wire crossing of CTO. This novel approach could lead to higher percutaneous coronary intervention success rates in CTO.

Objective: Chronic Total Occlusions (CTO) remains a challenge for Percutaneous Coronary Intervention (PCI). Initial studies of bacterial collagenase injections into experimental arterial CTOs and human coronary CTOs have indicated very promising effects to facilitate guide wire crossing in previously failed cases. The aim of the current study was to assess the vascular and cardiac effects of intracoronary collagenase injections in a porcine model. Methods: Intracoronary injections of collagenase were done in the Left Anterior Descending (LAD) artery of 21 Yorkshire pigs through an over-the-wire balloon catheter. Two groups were studied: 24 hours after injection (n=14) and 30 days after injection (n=6). During balloon inflation, 8 ml of a phosphate buffered solution (PBS) containing either collagenase (range 50-3200 μg) or PBS alone (control) was injected. After sacrifice, hearts were photographed immediately and then fixed in 10% buffered formalin. Myocardial cross-sections were stained with H&E and Movat. Results: One pig died acutely due to arterial dissection during balloon inflation. In collagenase-treated pigs, localized epicardial and myocardial haemorrhages were evident at 24 hr in the LAD distribution at doses more than 800 μg. The extent of haemorrhages was related to the dose and was generally mild at doses less than1600 μg and moderate at doses more than 1600 μg. At 30 d, no evidence of haemorrhages was present. Mild myocardial fibrosis, likely due to ischemia, was present in both placebo and treatment groups. Balloon damaged LAD arteries showed mild intimal hyperplasia but no specific effects related to collagenase. Conclusions: A single intracoronary injection of a bacterial collagenase formulation into normal coronary arteries caused dose-related, localized epicardardial and myocardial haemorrhages, which were well tolerated without later sequelae.

Aims Percutaneous revascularisation of chronic total occlusions (CTO) is limited by failure of guidewire crossing. Neovascularisation within the proximal CTO segment may be important for guidewire crossing and dramatically declines in CTO beyond six weeks of age. The aims of the current study were to determine whether local delivery of a pro-angiogenic growth factor increases neovascularisation in mature CTO and facilitates guidewire crossings. Methods and results CTO (n=51) were created in the femoral arteries of 44 New Zealand white rabbits using the thrombin injection model. At 12 weeks, CTO were treated with poly-lactic-glycolic-acid (PLGA) microspheres containing either bovine serum albumin (BSA) (n=15) or recombinant mouse VEGF164 (n=14), or received no intervention (controls, n=12). Contrast-enhanced magnetic resonance angiography (CEMRA) was performed prior to treatment and at three weeks post treatment. Animals were sacrificed at three weeks post treatment and arterial samples were excised for micro-computed tomography imaging (µCT) and histologic morphometric analysis. Guidewire crossing was assessed at three weeks post treatment in an additional 10 VEGF164-treated CTO. In comparison to BSA-treated and control non-intervened CTO, VEGF164-treated CTO showed a significant increase in relative blood volume index in the proximal segment of the CTO lesion as determined by CEMRA and by µCT. Histologic measurements of microvessel area were also higher in VEGF164-treated CTO. Guidewire crossing across the proximal fibrous cap was successful in eight out of 10 VEGF164-treated CTO. Conclusions Angiogenic therapy appears to be a promising strategy to improve neovascularisation and guidewire crossing rates in CTO.

Vein grafts (VG) account for more than 70% of conduits used in aortocoronary bypass surgery, but have high failure rates by 10 years. We have previously shown in an experimental model that VG undergo marked expansion by 4 weeks, which was followed by increased total LDL retention at 12 weeks. The objective of this study was to determine whether statin therapy would prevent these expansive remodelling changes. Methods and Results Reversed jugular vein-to-common carotid artery interposition graft were constructed in 24 cholesterol-fed (0.5%) rabbits. Rabbits were randomized to either control or atorvastatin (5 mg/kg/day) groups, starting two weeks prior to vein graft implantation and continuing until sacrifice at 1 or 12 weeks post-surgery. Ultrasound measurements of arterial luminal cross-sectional area (LCSA) were done at day 3 and at 4, 8 and 12 weeks post-surgery. Histomorphometric measurements were performed following sacrifice at 12 weeks. Atorvastatin treatment significantly decreased total plasma cholesterol levels at 12 weeks (6.70±4.19mmol/L vs placebo 38.65±10.55mmol/L, p2 vs placebo 77.6±10.68mm 2 , p2 vs placebo 9.57±2.43mm 2 , p Conclusion: Atorvastatin prevents expansive remodelling in arterialized VG, likely through inhibition of macrophage infiltration and reduction of tissue proteolytic activity. This may be an important mechanism preventing VG atherosclerosis and late VG failure.

Venous grafts (VG) have high failure rates by 10 years in aortocoronary bypass surgery. We have previously shown that expansive remodeling followed by increased LDL retention are early atherosclerotic changes in experimental VG placed in the arterial circulation. The objective of this study was to determine whether statin therapy prevents these expansive remodeling changes.Reversed jugular vein-to-common carotid artery interposition graft was constructed in 27 cholesterol-fed (0.5%) rabbits. Rabbits were randomized either to control or atorvastatin (5 mg/kg/day) groups, starting two weeks prior to vein graft implantation and continuing until sacrifice at 1 or 12 weeks post-surgery. Ultrasound measurements of arterial luminal cross-sectional area (CSA) were done at day 3 and at 4, 8 and 12 weeks post-surgery. Histomorphometric measurements were performed following sacrifice at 12 weeks. Atorvastatin treatment significantly decreased total plasma cholesterol levels at 4, 8 and 12 weeks (12 weeks: 6.7 ± 4.2 mmol/L versus control 38.7 ± 10.6 mmol/L, p0.0002). Atorvastatin significantly reduced expansive remodeling at 4, 8 and 12 weeks (lumen CSA: 44.6 ± 6.6 mm(2) versus control 77.6 ± 10.7 mm(2), p0.0001). Intimal CSA by histomorphometry was also significantly reduced by atorvastatin at 12 weeks (5.59 ± 2.19 mm(2) versus control 9.57 ± 2.43 mm(2), p0.01). VG macrophage infiltration, MMP-2 activity and metalloelastase activity were reduced in the atorvastatin treated group.Atorvastatin inhibits both expansive remodeling and intimal hyperplasia in arterialized VG, likely through inhibition of macrophage infiltration and reduction of tissue proteolytic activity. The mechanism proposed above may be important for preventing VG atherosclerosis and late VG failure.

Methods In nine pigs a CTO was created by percutaneously inserting a fibrin plug (AngiosealΤΜ) into the mid-todistal left anterior descending artery (LAD). Animals were studied six (n=3) or twelve weeks (n=6) later prior to sacrifice. An x-ray angiogram confirmed the LAD CTO at those time points. Cardiac MR (CMR) studies were then conducted on a 1.5T (n=6) or on a 3.0T MRI system (n=3), which included SSFP short axis sections for wall motion and post-gadolinium LGE-MRIs for viability. After sacrifice, both right and left coronary systems were injected with Microfil. X-ray or MSCT angiography of the fixed heart was obtained. A longitudinal cardiac section including the CTO lesion, proximal/distal LAD and the borders of infarction was removed and prepared in gel, then imaged in a microCT system at 27 micron resolution. LV functional parameters including wall thickness at end-systole (WTES) and end-diastole (WTED) were measured in border zone, infarct and remote region. Systolic wall thickening (SWT) was calculated using as (WTES-WTED) x100/ WTED. CMR and micro-CT data were processed using commercial software. The extent of collaterals on micro-CT images was rated qualitatively using a score from 0 to 3, indicating that no, minimal, moderate, or extensive collaterals were observed. A Student’s t-test was used for the statistical significance of differences between measurements at 6 and 12 weeks.

In acute myocardial infarction (AMI), the aim of any therapeutic intervention is to reduce the infarct size and attenuate adverse remodeling. The type and extent of infarction encountered clinically [transmural, hemorrhagic, heterogeneous, with microvascular obstruction (MVO)], is primarily determined by the severity of the initial ischemic insult. Understanding the in-vivo pathophysiological mechanisms after AMI as a function of severity will be key in predicting functional recovery, prognosis and assessing the efficacy of novel therapies.

Events contributing to restenosis after coronary interventions include platelet aggregation, inflammatory cell infiltration, growth factor release, and accumulation of smooth muscle cells (SMCs) and extracellular matrix (ECM). The ECM is composed of various collagen subtypes and proteoglycans and over time constitutes the major component of the mature restenotic plaque. The pathophysiology of collagen accumulation in the ECM during arterial restenosis is reviewed. Factors regulating collagen synthesis and degradation, including various cytokines and growth factors involved in the process, may be targets for therapies aimed at prevention of in-stent restenosis.

INTRODUCTION: Recombinant factor VIIa (rVIIa) has been used to decrease postoperative bleeding in cardiac surgical patients. However, there is potential for rVIIa to interact with tissue factor expression at the site of new anastomoses resulting in early graft occlusion. This safety study tested the hypothesis that the administration of rVIIa dose-dependently reduces graft patency at the site of new vascular anastomoses comparable in size to human coronaries, in a rabbit model. METHODS: After ACC approval, a reversed venous graft was fashioned in the right carotid artery using autologous right jugular vein with 9 – 0 sutures in 64 anesthetized and heparinized rabbits. Animals were then given either one of three doses of rVIIa (300 ug/kg, 90 ug/kg or 20 ug/kg IV) or placebo (n=16/group). The primary outcome was graft patency, measured by a blinded observer 24 hours postoperatively using vascular ultrasound (HDI 5000cv, 15 MHz probe) and/or direct inspection. Factor VII activity levels were measured from citrated plasma samples with a 1-stage prothrombin time-based assay using rabbit thromboplastin. Data was analyzed using chi-square, fishers exact test, or ANOVA where appropriate, with p RESULTS: Physiologic variables (ACT, hemoglobin, pH, pCO2) and vessel diameter were similar between groups. Graft patency and flow were significantly reduced, and plasma factor VII levels significantly increased in the rVIIa treated rabbits in a dose-dependent fashion. DISCUSSION: The study suggests that high doses of rVIIa (300 and 90 ug/kg) are associated with an increased incidence of occlusion of new vascular grafts. A clear dose-response effect was observed, suggesting that higher doses may be associated with increased thrombotic outcomes.

Arterial chronic total occlusions (CTO) are a common and clinically relevant problem in patients with coronary artery disease. Percutaneous coronary intervention (PCI) success rates in a wide range of CTO are low, primarily due to inability of guidewire crossing. The pathophysiology of CTO is poorly understood and limits our ability to introduce innovative therapies. Recent studies from our laboratory have suggested that microvessel formation within arterial CTO is a complex process with temporal and regional differences. Moreover, there is evidence from pilot studies that the presence of either microvessels or the particular extracellular matrix environment in the adjacent perivascular tissue can facilitate guidewire crossing and successful PCI. Currently, studies are underway in our experimental CTO model to delineate the pathophysiology of microvessel formation in CTO and its potential role in PCI.

Although the application of cold energy, cryotherapy, has been shown to cause selective damage to cellular components with preservation of matrix structure resulting in less fibrosis in a variety of tissues, the effects of intravascular cryotherapy on vessel wall repair after balloon angioplasty are unknown. We sought to characterize the effects of cryotherapy application on vessel wall repair after balloon angioplasty and study the relationship between collagen accumulation in the vessel wall and late lumen loss as assessed by serial intravascular ultrasound.The immediate, early (72 h) and late (10 weeks) effects of three intravascular cryotherapy application time periods (60, 120 and 240 s) after iliac artery balloon angioplasty ('cryotherapy') were compared with balloon angioplasty alone ('control') in 59 rabbits. Arterial lumen area was measured by intravascular ultrasound immediately after the procedure, at 72 h and at 10 weeks. Collagen content was calculated separately for intima and media/adventitia layers and correlated with late lumen loss.Cryotherapy produced average vessel wall temperature of -26 degrees C (range, -20 to -45 degrees C) and resulted in significantly larger lumen cross-sectional area (CSA) immediately after application (5.74+/-1.18 vs. 4.14+/-0.75 mm(2), P=0.008) but was not different than control arteries at 10 weeks. At 72 h, there was extensive cell loss in the medial and adventitial layers accompanied by increased macrophage infiltration in cryotherapy treated arteries compared to control. At 10 weeks, intimal hyperplasia was increased 2-fold in cryotherapy treated arteries. Collagen content was increased 2-fold in the medial/adventitial layers, and nearly 3-fold in the intima of cryotherapy treated arteries. Collagen content in arterial intima (P=0.01) as well as media/adventitia (P=0.005) positively correlated with late lumen loss. Foci of chondro- and osseous metaplasia and calcification were evident at the medial-adventitial junction in cryotherapy treated arteries at 10 weeks.Intravascular cryotherapy induced early arterial wall cell loss and late intimal hyperplasia, vascular fibrosis and chondro- and osseous metaplastic changes with no late beneficial effects on lumen area compared to balloon angioplasty alone. Collagen accumulation in all three layers of the vessel wall contributes to the development of late inward remodeling after balloon angioplasty.

s S59 implantation attenuated the decline of fractional shortening after MI and the scar was significantly thicker in both BMMC groups compared to the media injected controls (p

ObjectivesThe aim of this study was to delineate the temporal profile of adventitial microvessel (Ad-MV) formation after stenting, its relationship to arterial wall hypoxia, and the effects of a tyrosine kinase inhibitor (TKI), SU11218, on Ad-MV and in-stent intimal hyperplasia (IH).BackgroundAdventitial microvessels have been reported after arterial injury; however, the underlying stimulus for this response and its relationship to IH is unknown.MethodsCoronary stenting was performed in 40 pigs randomized to SU11218 (n = 20) or placebo (n = 20). Vessel wall hypoxia was assessed by pimonidazole adducts and hypoxia-inducible factor (HIF)-1 alpha expression. Adventitial microvessels were quantified by three-dimensional microscopic computed tomography (3D micro CT). Intimal hyperplasia was measured by intravascular ultrasound (IVUS), 3D micro CT, and morphometry. The effects of SU11218 were assessed in vitro on smooth muscle cell (SMC) and endothelial cell (EC) functions and in vivo on Ad-MV and IH.ResultsHypoxia was evident in the vessel wall at 48 h and persisted for four weeks. Adventitial microvessels increased significantly at one week (24 ± 7 microvessels/segment) and four weeks (23 ± 7 microvessels/segment) compared with uninjured arteries (16 ± 2 microvessels/segment; p < 0.001) and correlated with IH (r = 0.77, p < 0.001). The TKI SU11218 inhibited platelet-derived growth factor receptor–beta phosphorylation, EC and SMC DNA synthesis, and migration in a dose-dependent manner in vitro and significantly inhibited Ad-MV (16 ± 5 vs. 23 ± 7 microvessels/segment in placebo, p < 0.001) and produced approximately 80% reduction in IH (0.52 ± 0.51 mm2vs. 2.47 ± 1.66 mm2in placebo, p < 0.001) at four weeks in vivo.ConclusionsArterial stenting causes arterial wall hypoxia followed by Ad-MV formation. The TKI SU11218 inhibits both Ad-MV formation and IH and represents a promising therapeutic agent to prevent in-stent restenosis.