In Vitro Matured Human Pluripotent Stem Cell-Derived Cardiomyocytes Form Grafts With Enhanced Structure and Function in Injured Hearts.

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 >1×108 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.

A calcified chronic total occlusion preclinical model.

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.

Omnipolarity applied to equi-spaced electrode array for ventricular tachycardia substrate mapping.

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 < 0.05). Omnipolar EGM voltage maps were 37% denser than BiEGM maps. In addition, OTEGM Vpp values are more consistent than bipolar Vpps showing less beat-by-beat variation than BiEGM by 39% and 47% over both infarcted and healthy areas, respectively (P < 0.01). Omnipolar EGM better delineate infarcted areas than traditional BiEGMs from both orientations. CONCLUSION: An equi-spaced electrode grid when combined with omnipolar methodology yielded the largest detectable bipolar-like voltage and is void of directional influences, providing reliable voltage assessment within infarcted and non-infarcted regions of the heart.

Distal coronary embolization following acute myocardial infarction increases early infarct size and late left ventricular wall thinning in a porcine model.

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 < 0.05) and MMP-2 activity levels (0.81 vs. 0.49, p = 0.002), with reduced activation of Akt (0.06 versus 0.26, p = 0.02). At 6 weeks, there were no differences in infarct size, ventricular volume or ejection fraction between the two groups, although infarct transmurality (70% vs. 57%, p< 0.04) and ventricular thinning (percent change in mid anteroseptal wall thickness:-25.6% vs. 0.7%, p = 0.03) were significantly increased in the DCE group. CONCLUSIONS: DCE increased early infarct size, but without affecting later infarct size, cardiac function or ventricular volumes. The significance of the later remodelling changes (ventricular thinning and transmurality) following DCE, possibly due to changes in MMP-2 activity and Akt activation, merits further study.

Heparan sulfate side chains have a critical role in the inhibitory effects of perlecan on vascular smooth muscle cell response to arterial injury.

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. Hspg2(Δ3/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.

Blood-pool MRI assessment of myocardial microvascular reactivity.

Purpose: The ability to non-invasively image myocardial microvascular dilation and constriction is essential to assessing intact function and dysfunction. Yet, conventional measurements based on blood oxygenation are not specific to changes in blood volume. The purpose of this study was to extend to the heart a blood-pool MRI approach for assessing vasomodulation in the presence of blood gas changes and investigate if sex-related differences exist. Methods: Animals [five male and five female healthy Sprague Dawley rats (200-500 g)] were intubated, ventilated, and cycled through room air (normoxia) and hypercapnia (10% CO2) in 10-minute cycles after i.v. injection of blood-pool agent Ablavar (0.3 mmol/kg). Pre-contrast T1 maps and T1-weighted 3D CINE were acquired on a 3 Tesla preclinical MRI scanner, followed by repeated 3D CINE every 5 min until the end of the gas regime. Invasive laser Doppler flowmetry of myocardial perfusion was performed to corroborate MRI results. Results: Myocardial microvascular dilation to hypercapnia and constriction to normoxia were readily visualized on T1 maps. Over 10 min of hypercapnia, female myocardial T1 reduced by 20% (vasodilation), while no significant change was observed in the male myocardium. After return to normoxia, myocardial T1 increased (vasoconstriction) in both sexes (18% in females and 16% in males). Laser Doppler perfusion measurements confirmed vasomodulatory responses observed on MRI. Conclusion: Blood-pool MRI is sensitive and specific to vasomodulation in the myocardial microcirculation. Sex-related differences exist in the healthy myocardium in response to mild hypercapnic stimuli.

Methods for Transepicardial Cell Transplantation in a Swine Myocardial Infarction Model.

The transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) has garnered significant attention as a potential means of restoring lost muscle mass and contractile function in injured hearts. Early preclinical work with hPSC-CMs employed rodent models, but the field has recently advanced to transplantation studies in more translationally relevant large animal models including non-human primates and swine. The pig is a particularly attractive model for such studies because the size, structure, and physiology of the porcine heart is very similar to that of humans. The pig model has reasonably high throughput, is readily amenable to clinically available cell delivery methods and imaging modalities and has been used frequently to test the safety and efficacy of new cardiac therapies. Here, we describe methods that were established in our laboratory for the specific purpose of testing hPSC-CM transplantation in a pig model of subacute myocardial infarction, but these same techniques should be broadly applicable to the transepicardial delivery of other biologicals including other candidate cell populations, biomaterials, and/or viral vectors.

Inhibition of intimal hyperplasia after stenting by over-expression of p15: a member of the INK4 family of cyclin-dependent kinase inhibitors.

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 ß-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 ß-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 ß-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.

Myocardial BOLD imaging at 3 T using quantitative T2: application in a myocardial infarct model.

Left ventricular remodeling as a result of acute myocardial infarction (AMI) is associated with significant morbidity, leading to cardiovascular dysfunction, disability, and death. Despite successful revascularization, coronary vasodilatory dysfunction has been shown in infarcted and remote myocardium of patients following AMI. Our study explored the utility of a T(2)-based blood-oxygen-level-dependent approach in probing regional and longitudinal fluctuations in vasodilatory function in a porcine model of AMI at 3 T. Ten pigs underwent MRI in control state and at day 2, weeks 1-6 following 90 min occlusion followed by reperfusion. The remote myocardium exhibited vasodilatory dysfunction at weeks 1 and 2 that gradually recovered, whereas the infarct zone showed no vasodilatory alterations. Our study suggests that microvascular alterations occurring in infarcted and remote myocardium after AMI might serve as an indicator of adverse left ventricular remodeling. The blood-oxygen-level-dependent technique using quantitative T(2) could potentially be a useful noninvasive tool to evaluate novel therapeutic strategies aimed at limiting vasoconstriction and improving coronary flow reserve after AMI.

Quantitative tracking of edema, hemorrhage, and microvascular obstruction in subacute myocardial infarction in a porcine model by MRI.

Pathophysiological responses after acute myocardial infarction include edema, hemorrhage, and microvascular obstruction along with cellular damage. The in vivo evolution of these processes simultaneously throughout infarct healing has not been well characterized. The purpose of our study was to quantitatively monitor the time course of these mechanisms by MRI in a porcine model of myocardial infarction. Ten pigs underwent MRI before coronary occlusion with subgroups studied at day 2 and weeks 1, 2, 4, and 6 post-infarction. Tissue characterization was performed using quantitative T2 and T2* maps to identify edema and hemorrhage, respectively. Contrast-enhanced MRI was used for infarct/ microvascular obstruction delineation. Inflammation was reflected by T2 fluctuations, however at day 2, edema and hemorrhage had counter-acting effects on T2. Hemorrhage (all forms) and mineralization (calcium) could be identified by T2* in the presence of edema. Simultaneous resolution of microvascular obstruction and T2* abnormality suggested that the two phenomenon were closely associated during the healing process. Our study demonstrates that quantitative T2 and T2* mapping techniques allow regional, longitudinal, and cross-subject comparisons and give insights into histological and tissue remodeling processes. Such in vivo characterization will be important in grading severity and evaluating treatment strategies for myocardial infarction, potentially improving clinical outcomes.

Transplanted microvessels improve pluripotent stem cell-derived cardiomyocyte engraftment and cardiac function after infarction in rats.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an unprecedented opportunity to remuscularize infarcted human hearts. However, studies have shown that most hiPSC-CMs do not survive after transplantation into the ischemic myocardial environment, limiting their regenerative potential and clinical application. We established a method to improve hiPSC-CM survival by cotransplanting ready-made microvessels obtained from adipose tissue. Ready-made microvessels promoted a sixfold increase in hiPSC-CM survival and superior functional recovery when compared to hiPSC-CMs transplanted alone or cotransplanted with a suspension of dissociated endothelial cells in infarcted rat hearts. Microvessels showed unprecedented persistence and integration at both early (~80%, week 1) and late (~60%, week 4) time points, resulting in increased vessel density and graft perfusion, and improved hiPSC-CM maturation. These findings provide an approach to cell-based therapies for myocardial infarction, whereby incorporation of ready-made microvessels can improve functional outcomes in cell replacement therapies.

Human Embryonic Stem Cell-Derived Cardiomyocytes Regenerate the Infarcted Pig Heart but Induce Ventricular Tachyarrhythmias.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) show considerable promise for regenerating injured hearts, and we therefore tested their capacity to stably engraft in a translationally relevant preclinical model, the infarcted pig heart. Transplantation of immature hESC-CMs resulted in substantial myocardial implants within the infarct scar that matured over time, formed vascular networks with the host, and evoked minimal cellular rejection. While arrhythmias were rare in infarcted pigs receiving vehicle alone, hESC-CM recipients experienced frequent monomorphic ventricular tachycardia before reverting back to normal sinus rhythm by 4 weeks post transplantation. Electroanatomical mapping and pacing studies implicated focal mechanisms, rather than macro-reentry, for these graft-related tachyarrhythmias as evidenced by an abnormal centrifugal pattern with earliest electrical activation in histologically confirmed graft tissue. These findings demonstrate the suitability of the pig model for the preclinical development of a hESC-based cardiac therapy and provide new insights into the mechanistic basis of electrical instability following hESC-CM transplantation.

Collagenase plaque digestion for facilitating guide wire crossing in chronic total occlusions.

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 microg) 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.

Human-grade purified collagenase for the treatment of experimental arterial chronic total occlusion.

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 microg) 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 microg) 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.

Effects of intravascular cryotherapy on vessel wall repair in a balloon-injured rabbit iliac artery model.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Poly(methylidene malonate 2.1.2) nanoparticles: a biocompatible polymer that enhances peri-adventitial adenoviral gene delivery.

Vascular gene therapy is currently limited by low and transient levels of gene transfection. The objectives of this study were to determine whether peri-adventitial delivery of adenovirus coupled to nanoparticles could improve transfection efficiency and duration. Adenovirus was absorbed to the surface of nanoparticles that were made from poly(methylidene malonate)2.1.2 (PMM2.1.2). These complexes were found to have good adhesive properties to both cultured vascular smooth muscle cells and to the luminal and adventitial layers of excised rabbit carotid arteries. Adenovirus encoding to beta-galactosidase coupled to PMM2.1.2 nanoparticles or adenovirus alone were delivered locally to the adventitia of rabbit carotid arteries. Transfection rate was assessed histologically by the percentage of beta-galactosidase positive cells in the vessel wall at 1 and at 2 weeks. There was significantly higher transfection rate when adenovirus was complexed with nanoparticles as compared to free adenovirus (At 1 week: 10+/-3.9% beta-gal positive cells vs. 2.4+/-0.3% and at 2 weeks: 4.3+/-4.1% vs. 0%, P<0.005 for all). This difference was present in both the medial and adventitial layers. In conclusion, adenoviral mediated gene therapy was significantly enhanced by adsorbing the virus to PMM2.1.2 nanoparticles. This delivery method may be a good therapeutic strategy for the treatment of various vascular diseases.

Optimization of local methylprednisolone delivery to inhibit inflammatory reaction and neointimal hyperplasia of coated coronary stents.

Polymer coating can optimize the surface characteristics of metallic coronary stents and serve as a vehicle for local drug delivery. Major problems, however, include the lack of biocompatibility of the polymers used and the limited amount of drug that can be loaded onto the stent. Stainless-steel stents were spray-coated or spray-coated combined with a barrier coating using a fluorinated polymethacrylate PFM-P75 impregnated with different methylprednisolone concentrations. When spray-coated with highly concentrated methylprednisolone ( 33%) fluorinated polymethacrylate PFM-P75, the surface became progressively more rough. Adding a barrier coating, however, could decrease these surface irregularities of methylprednisolone-loaded PFM-P75 spray-coated stents. In vitro, most of the methylprednisolone was released in the first 48 hours. A barrier coating could dramatically slow down the drug release from 80% to 13% during the first 48 hours. Histomorphometric analysis showed that the inflammatory response and neointimal hyperplasia of methylprednisolone-loaded stents were lower than in control stents. Neointimal hyperplasia of methylprednisolone-loaded PFM-P75 stents spray-coated with a barrier coating was decreased compared to the non-barrier-coated methylprednisolone-loaded stents. In conclusion, spray coating enables the use of high methylprednisolone concentrations. A barrier coating could significantly slow down the methylprednisolone release. Methylprednisolone-loaded PFM-P75-coated stents could significantly inhibit the inflammatory response and neointimal hyperplasia. The response to methylprednisolone was related to the dose used and the release time of the drug.

Unexpected calcification after direct intravascular injection of autologous bone marrow-derived cells in a chronic total occlusion model.

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<0.05). However, unexpected intravascular calcification was only detected within the CTO in BMC cell treated arteries. Western blot for conditioned medium from BMC showed up-regulation of osteogenic proteins (BMP-2 and -7). CONCLUSIONS: Although direct delivery of BMC into CTO increases neovascularisation, undesirable vascular calcification will limit this therapeutic approach.

Perlecan heparan sulfate proteoglycan is a critical determinant of angiogenesis in response to mouse hind-limb ischemia.

BACKGROUND: 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. METHODS: 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. RESULTS: 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]; P < 0.05). Endogenous VEGF mRNA expression (P < 0.05) and VEGF protein expression (P < 0.002) 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. CONCLUSIONS: 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.

Therapeutic angiogenesis with VEGF164 for facilitation of guidewire crossing in experimental arterial chronic total occlusions.

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.