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Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNALINC00607to be highly enriched in human endothelial cells.LINC00607was induced in response to hypoxia, arteriosclerosis regression in non-human primates, post-atherosclerotic cultured endothelial cells from patients and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout ofLINC00607attenuated VEGF-A-induced angiogenic sprouting.LINC00607knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression ofLINC00607in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq afterLINC00607knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically,LINC00607interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout ofBRG1in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion,LINC00607is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1 to ultimately mediate angiogenesis.

BACKGROUND miR-21 is a central regulator of cardiac fibrosis, and its inhibition in small-animal models has been shown to be an effective antifibrotic strategy in various organs, including the heart. Effective delivery of therapeutic antisense micro-ribonucleic acid (antimiR) molecules to the myocardium in larger organisms is challenging, though, and remains to be established for models of chronic heart failure.OBJECTIVES The aims of this study were to test the applicability and therapeutic efficacy of local, catheter-based delivery of antimiR-21 in a pig model of heart failure and determine its effect on the cardiac transcriptomic signature and cellular composition.METHODS Pigs underwent transient percutaneous occlusion of the left coronary artery and were followed up for 33 days. AntimiR-21 (10 mg) was applied by intracoronary infusion at days 5 and 19 after the injury. Cardiac function was assessed in vivo, followed by histological analyses and deep ribonucleic acid sequencing (RNA-seq) of the myocardium and genetic deconvolution analysis.RESULTS AntimiR-21 effectively suppressed the remodeling-associated increase of miR-21. At 33 days after ischemia/reperfusion injury, LNA-21-treated hearts exhibited reduced cardiac fibrosis and hypertrophy and improved cardiac function. Deep RNA-seq revealed a significant derepression of the miR-21 targetome in antimiR-21-treated myocardium and a suppression of the inflammatory response and mitogen-activated protein kinase signaling. A genetic deconvolution approach built on deep RNA-seq and single-cell RNA-seq data identified reductions in macrophage and fibroblast numbers as the key cell types affected by antimiR-21 treatment.CONCLUSIONS This study provides the first evidence for the feasibility and therapeutic efficacy of miR-21 inhibition in a large animal model of heart failure. (C) 2020 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation.

Due to severely limited donor heart availability, durable mechanical circulatory support remains the only treatment option for many patients with end-stage heart failure. However, treatment complexity persists due to its univentricular support modality and continuous contact with blood. We investigated the function and safety of reBEAT (AdjuCor GmbH), a novel, minimal invasive mechanical circulatory support device that completely avoids blood contact and provides pulsatile, biventricular support.For each animal tested, an accurately sized cardiac implant was manufactured from computed tomography scan analyses. The implant consists of a cardiac sleeve with three inflatable cushions, 6 epicardial electrodes and driveline connecting to an electro-pneumatic, extracorporeal portable driver. Continuous epicardial electrocardiogram signal analysis allows for systolic and diastolic synchronization of biventricular mechanical support. In 7 pigs (weight, 50-80 kg), data were analyzed acutely (under beta-blockade, n = 5) and in a 30-day long-term survival model (n = 2). Acquisition of intracardiac pressures and aortic and pulmonary flow data were used to determine left ventricle and right ventricle stroke work and stroke volume, respectively.Each implant was successfully positioned around the ventricles. Automatic algorithm electrocardiogram signal annotations resulted in precise, real-time mechanical support synchronization with each cardiac cycle. Consequently, progressive improvements in cardiac hemodynamic parameters in acute animals were achieved. Long-term survival demonstrated safe device integration, and clear and stable electrocardiogram signal detection over time.The present study demonstrates biventricular cardiac support with reBEAT. Various demonstrated features are essential for realistic translation into the clinical setting, including safe implantation, anatomical fit, safe device-tissue integration, and real-time electrocardiogram synchronized mechanical support, result in effective device function and long-term safety.

Diabetes and the often accompanying cardiovascular diseases including cardiomyopathy represent a complex disease, that is reluctant to reveal the molecular mechanisms and underlying cellular responses. Current research projects on diabetic cardiomyopathy are predominantly based on animal models, in which there are not only obvious advantages, such as genetics that can be traced over generations and the directly measurable influence of dietary types, but also not despisable disadvantages. Thus, many studies are built up on transgenic rodent models, which are partly comparable to symptoms in humans due to their genetic alterations, but on the other hand are also under discussion regarding their clinical relevance in the translation of biomedical therapeutic approaches. Furthermore, a focus on transgenic rodent models ignores spontaneously occurring diabetes in larger mammals (such as dogs or pigs), which represent with their anatomical similarity to humans regarding their cardiovascular situation appealing models for testing translational approaches. With this in mind, we aim to shed light on the currently most popular animal models for diabetic cardiomyopathy and, by weighing the advantages and disadvantages, provide decision support for future animal experimental work in the field, hence advancing the biomedical translation of promising approaches into clinical application.

Background Pathological cardiac hypertrophy is a result of afterload-increasing pathologies including untreated hypertension and aortic stenosis. It features progressive adverse cardiac remodeling, myocardial dysfunction, capillary rarefaction, and interstitial fibrosis often leading to heart failure. Objectives This study aimed to establish a novel porcine model of pressure-overload–induced heart failure and to determine the effect of inhibition of microribonucleic acid 132 (miR-132) on heart failure development in this model. Methods This study developed a novel porcine model of percutaneous aortic constriction by implantation of a percutaneous reduction stent in the thoracic aorta, inducing progressive remodeling at day 56 (d56) after pressure-overload induction. In this study, an antisense oligonucleotide specifically inhibiting miR-132 (antimiR-132), was regionally applied via intracoronary injection at d0 (percutaneous transverse aortic constriction induction) and d28. Results At d56, antimiR-132 treatment diminished cardiomyocyte cross-sectional area (188.9 ± 2.8 vs. 258.4 ± 9.0 μm2 in untreated hypertrophic hearts) and improved global cardiac function (ejection fraction 48.9 ± 1.0% vs. 36.1 ± 1.7% in control hearts). Moreover, at d56 antimiR-132-treated hearts displayed less increase of interstitial fibrosis compared with sham-operated hearts (Δsham 1.8 ± 0.5%) than control hearts (Δsham 10.8 ± 0.6%). Of note, cardiac platelet and endothelial cell adhesion molecule 1+ capillary density was higher in the antimiR-132–treated hearts (647 ± 20 cells/mm2) compared with in the control group (485 ± 23 cells/mm2). Conclusions The inhibition of miR-132 is a valid strategy in prevention of heart failure progression in hypertrophic heart disease and may be developed as a treatment for heart failure of nonischemic origin.

Aims Increased shedding of extracellular vesicles (EVs)—small, lipid bilayer-delimited particles with a role in paracrine signalling—has been associated with human pathologies, e.g. atherosclerosis, but whether this is true for cardiac diseases is unknown. Methods and results Here, we used the surface antigen CD172a as a specific marker of cardiomyocyte (CM)-derived EVs; the CM origin of CD172a+ EVs was supported by their content of cardiac-specific proteins and heart-enriched microRNAs. We found that patients with aortic stenosis, ischaemic heart disease, or cardiomyopathy had higher circulating CD172a+ cardiac EV counts than did healthy subjects. Cellular stress was a major determinant of EV release from CMs, with hypoxia increasing shedding in in vitro and in vivo experiments. At the functional level, EVs isolated from the supernatant of CMs derived from human-induced pluripotent stem cells and cultured in a hypoxic atmosphere elicited a positive inotropic response in unstressed CMs, an effect we found to be dependent on an increase in the number of EVs expressing ceramide on their surface. Of potential clinical relevance, aortic stenosis patients with the highest counts of circulating cardiac CD172a+ EVs had a more favourable prognosis for transcatheter aortic valve replacement than those with lower counts. Conclusion We identified circulating CD172a+ EVs as cardiac derived, showing their release and function and providing evidence for their prognostic potential in aortic stenosis patients.

Background: Ischemic heart diseases are leading causes of death and reduced life quality worldwide. Although revascularization strategies significantly reduce mortality after acute myocardial infarction (MI), a large number of patients with MI develop chronic heart failure over time. We previously reported that a fragment of the extracellular matrix protein agrin promotes cardiac regeneration after MI in adult mice. Methods: To test the therapeutic potential of agrin in a preclinical porcine model, we performed ischemia–reperfusion injuries using balloon occlusion for 60 minutes followed by a 3-, 7-, or 28-day reperfusion period. Results: We demonstrated that local (antegrade) delivery of recombinant human agrin to the infarcted pig heart can target the affected regions in an efficient and clinically relevant manner. A single dose of recombinant human agrin improved heart function, infarct size, fibrosis, and adverse remodeling parameters 28 days after MI. Short-term MI experiments along with complementary murine studies revealed myocardial protection, improved angiogenesis, inflammatory suppression, and cell cycle reentry as agrin’s mechanisms of action. Conclusions: A single dose of agrin is capable of reducing ischemia–reperfusion injury and improving heart function, demonstrating that agrin could serve as a therapy for patients with acute MI and potentially heart failure.

BACKGROUND Heart failure following myocardial infarction (MI) remains one of the major causes of death worldwide, and its treatment is a crucial challenge of cardiovascular medicine. An attractive therapeutic strategy is to stimulate endogenous mechanisms of myocardial regeneration.OBJECTIVES This study evaluates the potential therapeutic treatment with annexin A1 (AnxA1) to induce cardiac repair after MI.METHODS AnxA1 knockout (AnxA1(-/-)) and wild-type mice underwent MI induced by ligation of the left anterior descending coronary artery. Cardiac functionality was assessed by longitudinal echocardiographic measurements. Histological, fluorescence-activated cell sorting, dot blot analysis, and in vitro/ex vivo studies were used to assess the myocardial neovascularization, macrophage content, and activity in response to AnxA1.RESULTS AnxA1(-/-) mice showed a reduced cardiac functionality and an expansion of proinflammatory macrophages in the ischemic area. Cardiac macrophages from AnxA1(-/-) mice exhibited a dramatically reduced ability to release the proangiogenic mediator vascular endothelial growth factor (VEGF)-A. However, AnxA1 treatment enhanced VEGF-A release from cardiac macrophages, and its delivery in vivo markedly improved cardiac performance. The positive effect of AnxA1 treatment on cardiac performance was abolished in wild-type mice transplanted with bone marrow derived from Cx(3)cr1cre(ERT2)Vegf(flox/flox) or in mice depleted of macrophages. Similarly, cardioprotective effects of AnxA1 were obtained in pigs in which full-length AnxA1 was overexpressed by use of a cardiotropic adeno-associated virus.CONCLUSIONS AnxA1 has a direct action on cardiac macrophage polarization toward a pro-angiogenic, reparative phenotype. AnxA1 stimulated cardiac macrophages to release high amounts of VEGF-A, thus inducing neovascularization and cardiac repair. (C) 2019 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation.

Background: Upregulation of microRNA-21 (miR-21) has been associated with myocardial disease and its systemic inhibition was effective in reducing cardiac fibrosis and dysfunction in rodent models. However, the translation to human disease is challenging, as systemic application of miR inhibitors does not achieve adequate delivery in most organs including the heart. Here, we sought to test the applicability and therapeutic efficacy of local, catheter-based delivery of antimiR-21 in a large animal model of heart failure. Methods and results: Pigs underwent percutaneous occlusion of the left coronary artery for 60 minutes and were followed up for 33 days. A synthetic inhibitor against miR-21 (LNA-antimiR-21, 10 mg per application) was applied locally by intracoronary infusion at days 5 and 19 after the injury. AntimiR-21 effectively suppressed the I/R-induced increase of miR-21. At 33 days after I/R, LNA-21-treated hearts exhibited reduced cardiac fibrosis and hypertrophy, and improved cardiac function (ejection fraction: I/R control 22±1% vs. I/R LNA-21 33±2%). RNA sequencing of ischemic myocardium revealed a significant enrichment for miR-21 targets in anti-miR-21 treated hearts. Mechanistically, antimiR-21 interfered with a number of signalling pathways involved in myocardial remodelling including the MAPK-ERK signalling cascade. Conclusion: Taken together, our study provides the first evidence for the feasibility and therapeutic efficacy of miR-21 inhibition in a large animal model of myocardial ischemia. AntimiR-21 prevented the structural and functional consequences of ischemic myocardial damage.

Background: Heme oxygenase-1 (HO-1) is an inducible stress-responsive enzyme converting heme to bilirubin, carbon monoxide, and free iron, which exerts anti-inflammatory and antiapoptotic e...

miRNA are post-transcriptional gene regulators which modulate RNA silencing and gene expression. In chronic myocardial ischemia, miRNA are altered and dysregulated. MiR92a inhibition in a mouse model of LAD occlusion led to improved neovascularisation and reduced infarct sizes. We aimed to investigate this therapeutic effect of miR92a- inhibition in a pig model of chronic myocardial ischemia. Methods: In pigs (n=5/group) reduction stent graft implantation in the circumflex artery led to a gradual occlusion of the vessel within 28 days (d28) after intervention. Using selective pressure-regulated retroinfusion the nucleic acid-modified antisense miR92a (LNA-92a; 5 mg/KG heart weight) was injected 28 days post implantation. The global myocardial function (Ejection Fraction (EF)) and the left end-diastolic pressure (LVEDP) were obtained at day 28 and 56. At day 56 after stent implantation regional myocardial function (subendocardial segmentshortning) as well as post mortem angiographies for collateral growth were analyzed. For the histological quantification the ischemic tissue was stained for PECAM-1 positive cells (capillaries) and for NG2- positive cells (vessel maturation, pericyte coverage). Results: The regionally applied locked nucleic acid-modified anti-sense miR-92a significantly reduced miR-92a expression in the heart and enhanced capillary coverage in the ischemia. Improved collateral growth (3±1 control vs. 6±1 in LNA-92a treated hearts) and distal perfusion of the occluded coronary artery (Rentrop score: 1.3±0.2 control vs. 2.4±0.2 in LNA-92a treated hearts) was seen after miR-92a inhibition. Moreover the increased LVEDP at day 28 (control: 16±1 mmHg vs. LNA-92a 15±1mmHg) was reduced after regional miRNA-92a inhibition (control: 16±1 mmHg vs. LNA-92a 15±1mmHg) and EF clearly increased after LNA-92a application (39±4% vs. 25±2 % in control). Regional myocardial function, obtained in the ischemic area under increased heart rate, was significantly improved after miR-92a inhibition (SES at hear rate 150bpm: 7±3 % of non-ischemic in control vs. 70±11 %% of non-ischemic in LNA-92a treated animals). Conclusion: Even single application of LNA-92a was able to reduce ischemia dependent impaired myocardial function

Purpose of review To discuss the possible therapeutic options for miRNA as targets in coronary heart disease. Noncoding RNAs regulate gene expression at a posttranscriptional state. Modulation of miRNAs might be a new therapeutic option in coronary heart disease. Recent findings Noncoding RNAs (long and short noncoding RNAs) might be used as biomarkers in cardiovascular disease, as they are differentially regulated and released in the pathophysiological situation of coronary heart disease. In acute myocardial infarction already a lot of miRNAs are investigated as biomarkers, still not superior to high-sensitive Troponin T. In rare cardiovascular diseases such as Tako-Tsubo cardiomyopathy or the different stages of heart failure, development of new biomarkers is even more important. In addition, miRNA inhibition via antimiRs is capable of attenuating cardiovascular disease in small and large animal models. Over-expression of 'protective' miRNAs (miR mimics) improved the outcome of cardiovascular disease in vitro and in first small animal models. Summary Noncoding RNAs are promising new biomarkers for cardiovascular disease. Directly targeting miRNA for disease modulation is possible for the specific inhibition, as well as for overexpression of 'protective' miRNAs. However, additional preclinical and clinical testing has to be performed before this therapy will enter clinical routine.

The purpose of the study is feasibility of dynamic CT perfusion imaging to detect and differentiate ischemic and infarcted myocardium in a large porcine model. 12 Country pigs completed either implantation of a 75 % luminal coronary stenosis in the left anterior descending coronary artery simulating ischemia or balloon-occlusion inducing infarction. Dynamic CT-perfusion imaging (100 kV, 300 mAs), fluorescent microspheres, and histopathology were performed in all models. CT based myocardial blood flow (MBFCT), blood volume (MBVCT) and transit constant (Ktrans), as well as microsphere’s based myocardial blood flow (MBFMic) were derived for each myocardial segment. According to histopathology or microsphere measurements, 20 myocardial segments were classified as infarcted and 23 were ischemic (12 and 14 %, respectively). Across all perfusion states, MBFCT strongly predicted MBFMic (β 0.88 ± 0.12, p

Objectives This study sought to compare dynamic versus single-phase high-pitch computed tomography (CT) acquisitions for the assessment of myocardial perfusion in a porcine model with adjustable degrees of coronary stenosis. Background The incremental value of the 2 different approaches to CT-based myocardial perfusion imaging remains unclear. Methods Country pigs received stent implantation in the left anterior descending coronary artery, in which an adjustable narrowing (50% and 75% stenoses) was created using a balloon catheter. All animals underwent CT-based rest and adenosine-stress myocardial perfusion imaging using dynamic and single-phase high-pitch acquisitions at both degrees of stenosis. Fluorescent microspheres served as a reference standard for myocardial blood flow. Segmental CT-based myocardial blood flow (MBFCT) was derived from dynamic acquisitions. Segmental single-phase enhancement (SPE) was recorded from high-pitch, single-phase examinations. MBFCT and SPE were compared between post-stenotic and reference segments, and receiver-operating characteristic curve analysis was performed. Results Among 6 animals (28 ± 2 kg), there were significant differences of MBFCT and SPE between post-stenotic and reference segments for all acquisitions at 75% stenosis. By contrast, although for 50% stenosis at rest, MBFCT was lower in post-stenotic than in reference segments (0.65 ± 0.10 ml/g/min vs. 0.75 ± 0.16 ml/g/min, p Conclusions At higher degrees of coronary stenosis, both MBFCT and SPE permit an accurate prediction of segmental myocardial hypoperfusion. However, accuracy of MBFCT is higher than that of SPE at 50% stenosis and can be increased by adenosine stress at both degrees of stenosis.

Background— MicroRNAs (miRs) are small noncoding RNAs that posttranscriptionally control gene expression. Small-animal studies suggest that miRs might offer novel therapeutic targets in cardiovascular diseases such as cardioprotection of murine hearts after myocardial infarction via miR-92a inhibitors. Because the functional benefits of miR-92a inhibitors in larger preclinical models are not known, we assessed the therapeutic efficacy of miR-92a inhibition in a porcine model of ischemia and reperfusion. Methods and Results— Pigs (n=5 per group) underwent percutaneous ischemia/reperfusion (60 min/72 h or 7 days, respectively). Locked nucleic acid–modified antisense miR-92a (LNA-92a) was applied either regionally (antegrade or retrograde) with a catheter or systemically (intravenously). LNA-92a significantly ( P P P Conclusions— Regional LNA-92a delivery reduces miR-92a levels and infarct size and postischemic loss of function. LNA-92a exerts cell-protective, proangiogenic, and anti-inflammatory effects. miR-92a inhibition might be a novel therapeutic tool to preserve cardiac function after ischemia.

Background Diabetes mellitus causes microcirculatory rarefaction and may impair the responsiveness of ischemic myocardium to proangiogenic factors. Objectives This study sought to determine whether microvascular destabilization affects organ function and therapeutic neovascularization in diabetes mellitus. Methods The authors obtained myocardial samples from patients with end-stage heart failure at time of transplant, with or without diabetes mellitus. Diabetic (db) and wild-type (wt) pigs were used to analyze myocardial vascularization and function. Chronic ischemia was induced percutaneously (day 0) in the circumflex artery. At day 28, recombinant adeno-associated virus (rAAV) (5 × 1012 viral particles encoding vascular endothelial growth factor-A [VEGF-A] or thymosin beta 4 [Tβ4]) was applied regionally. CD31+ capillaries per high power field (c/hpf) and NG2+ pericyte coverage were analyzed. Global myocardial function (ejection fraction [EF] and left ventricular end-diastolic pressure) was assessed at days 28 and 56. Results Diabetic human myocardial explants revealed capillary rarefaction and pericyte loss compared to nondiabetic explants. Hyperglycemia in db pigs, even without ischemia, induced capillary rarefaction in the myocardium (163 ± 14 c/hpf in db vs. 234 ± 8 c/hpf in wt hearts; p

Translational perspective Cardiomyocytes (CMs) generated from human induced pluripotent stem cells are an evolving platform to understand molecular disease mechanism and evaluate cardiovascular drugs. A major limitation of this system is that they represent a heterogeneous mix of ventricular-, atrial-, and nodal-like CMs. By expressing a voltage-sensitive fluorescent protein under the control of lineage-specific promoters, we developed a convenient system allowing high-throughput subtype-specific optical action potential (AP) imaging in these cells. This enables not only quantification of electrical phenotypes in patient-specific CMs but also subtype-specific investigation of drug effects, which may aid both drug development and safety pharmacology in the cardiovascular field., Aims Cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are increasingly used in disease modelling and drug evaluation. However, they are typically a heterogeneous mix of ventricular-, atrial-, and nodal-like cells based on action potentials (APs) and gene expression. This heterogeneity and the paucity of methods for high-throughput functional phenotyping hinder the full exploitation of their potential. We aimed at developing a method for rapid, sequential, and subtype-specific phenotyping of hiPSC-CMs with respect to AP morphology and single-cell arrhythmias. Methods and results We used cardiac lineage-specific promoters to drive the expression of a voltage-sensitive fluorescent protein (VSFP-CR) in hiPSC-CMs, enabling subtype-specific optical AP recordings. In a patient-specific hiPSC model of long-QT syndrome type 1, AP prolongation and frequent early afterdepolarizations were evident in mutant ventricular- and atrial like, but not in nodal-like hiPSC-CMs compared with their isogenic controls, consistent with the selective expression of the disease-causing gene. Furthermore, we demonstrate the feasibility of sequentially probing a cell over several days to investigate genetic rescue of the disease phenotype and to discern CM subtype-specific drug effects. Conclusion By combining a genetically encoded membrane voltage sensor with promoters that drive its expression in the major subtypes of hiPSC-CMs, we developed a convenient system for disease modelling and drug evaluation in the relevant cell type, which has the potential to advance the emerging utility of hiPSCs in cardiovascular medicine.

Coronary heart disease is still the leading cause of death in industrialized nations, occurring either as acute coronary occlusion and myocardial infarction or as chronic ischaemic cardiomyopathy caused by continuous obstruction of one or more coronary arteries. Even after successful reperfusion, an additional loss of otherwise vital cardiomyocytes may occur in the primary ischaemic area, called lethal reperfusion injury. In experimental settings, delivery of therapeutic agents targeting the reperfusion injury reduces the infarct size by 30%. In addition to the choice of therapeutic agent and time point, the mode of application may be crucial for the therapeutic success. Therefore, this review focuses on the current and future administration techniques for early and late post-myocardial infarction therapies.

Objectives We set out to investigate the ability of cardiotropic adeno-associated viral vector (AAV2.9 = recombinant adeno-associated virus [rAAV]) to induce prolonged expression of vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF)-B in a rabbit hindlimb ischemia model and a pig model of hibernating myocardium. Background Gene therapy to induce angiogenesis and arteriogenesis has produced mixed results. However, long-acting viruses, such as rAAV, as well as combined induction of angiogenesis and vessel maturation might extend the therapeutic potential. Methods In rabbits, 0.5 × 1011particles rAAV.VEGF-A with or without 1 × 1012particles rAAV.PDGF-B were retroinfused at day 7 after femoral artery excision. At days 7 and 35, collateral counts and perfusion were determined, each value given as the day 35/day 7 ratio. Capillary-to-muscle fiber ratio was determined at day 35. In pigs, implantation of a reduction stent graft into the circumflex artery led to complete occlusion at day 28. At this time point, retroinfusion of rAAV.VEGF-A (1 × 1013particles), rAAV.VEGF-A/PDGF-B (2 × 1012and 4 × 1012particles, respectively) or mock transfection was performed. Ejection fraction and left ventricular end-diastolic pressure were assessed at days 28 and 56. Results In rabbits, rAAV.VEGF-A strongly induced angiogenesis (capillary-to-muscle fiber ratio; 1.67 ± 0.09 vs. 1.32 ± 0.11 in rAAV.LacZ-treated limbs, p Conclusions Retroinfusion of rAAV.VEGF-A alone induces angiogenesis, but fails to enhance collateralization and perfusion, unless PDGF-B is cotransfected. In addition to neovascularization, rAAV.VEGF-A/PDGF-B improves regional and global myocardial function in hibernating myocardium.

Background— Prolonged myocardial ischemia results in cardiomyocyte loss despite successful revascularization. We have reported that retrograde application of embryonic endothelial progenitor cells (eEPCs) provides rapid paracrine protection against ischemia-reperfusion injury. Here, we investigated the role of thymosin β4 (Tβ4) as a mediator of eEPC-mediated cardioprotection. Methods and Results— In vitro, neonatal rat cardiomyocytes were subjected to hypoxia-reoxygenation in the absence or presence of eEPCs with or without Tβ4 short hairpin RNA (shRNA) transfection. In vivo, pigs (n=9 per group) underwent percutaneous left anterior descending artery occlusion for 60 minutes on day 1. After 55 minutes of ischemia, control eEPCs (5×10 6 cells) or cells transfected with Tβ4 shRNA when indicated or 15 mg Tβ4 alone were retroinfused into the anterior interventricular vein. Segmental endocardial shortening in the infarct zone at 150-bpm atrial pacing, infarct size (triphenyl tetrazolium chloride viability and methylene blue exclusion), and inflammatory cell influx (myeloperoxidase activity) were determined 24 hours later. Survival of neonatal rat cardiomyocytes increased from 32±4% to 90±2% after eEPC application, an effect sensitive to shRNA transfection compared with Tβ4 (45±7%). In vivo, infarct size decreased with eEPC application (38±4% versus 54±4% of area at risk; P Conclusion— Our findings show that short-term cardioprotection derived by regional application of eEPCs can be attributed, at least in part, to Tβ4.

Objectives We evaluated a novel technique for hybrid patch closure of muscular ventricular septal defects (mVSDs) without cardiopulmonary bypass (CPB) in a pig model. Background So far, surgical and interventional therapies for mVSDs have been associated with significant morbidity, especially in newborns and infants. Thus, it is essential to develop new techniques. Hybrid therapy is an innovative approach for mVSDs that combines the advantages of surgical and interventional techniques. Methods Six pigs underwent left anterolateral thoracotomy to expose the left ventricle (LV). The mVSDs were created under echocardiographic guidance with a 7.5-mm sharp punch instrument that was forwarded via an LV incision. A special designed patch system composed of a patch with a Nitinol frame was passed across the carotid artery into the LV and positioned in front of the mVSD. An instrument resembling a stapler was introduced across the LV wall on the beating heart without use of CPB. The patch was fixed with Nitinol anchors on the septum under echocardiographic and fluoroscopic guidance. Finally, the Nitinol frame was detached from the patch. Results The locations of the defects were apical (n = 1), midmuscular (n = 3), and anterior muscular (n = 2). Closure of the mVSD was successful in 5 of 6 animals confirmed by echocardiography, hemodynamic measurements, and explantation of the heart. Animals were hemodynamically stable throughout the experiment. Conclusions Here, we present a novel technique for hybrid closure of mVSDs without use of CPB. Further development of the patch system is necessary to assess applicability in humans, especially for the target group of newborns and infants.

Objectives We investigated whether retroinfusion of liposomal endothelial nitric oxide synthase (eNOS) S1177D complementary deoxyribonucleic acid (cDNA) would affect neovascularization and function of the ischemic myocardium. Background Recently, we demonstrated the feasibility of liposomal eNOS cDNA transfection via retroinfusion in a model of acute myocardial ischemia/reperfusion. In the present study, we used this approach to target a phosphomimetic eNOS construct (eNOS S1177D) into chronic ischemic myocardium in a pig model of hibernation. Methods Pigs (n = 6/group) were subjected to percutaneous implantation of a reduction stent graft into the left anterior descending artery (LAD), inducing total occlusion within 28 days. At day 28, retroinfusion of saline solution containing liposomal green fluorescent protein or eNOS S1177D cDNA (1.5 mg/animal, 2 x 10 min) was performed. Furthermore, L-nitroarginine-methylester (L-NAME) was applied orally from day 28, where indicated. At day 28 and day 49, fluorescent microspheres were injected into the left atrium for perfusion analysis. Regional functional reserve (at atrial pacing 140/min) was assessed at day 49 by subendocardial segment shortening (SES) (sonomicrometry, percent of ramus circumflexus region). Results The eNOS S1177D overexpression increased endothelial cell proliferation as well as capillary and collateral growth at day 49. Concomitantly, eNOS S1177D overexpression enhanced regional myocardial perfusion from 62 +/- 4% (control) to 77 +/- 3% of circumflex coronary artery-perfused myocardium, unless L-NAME was co-applied (69 +/- 5%). Similarly, eNOS S1177D cDNA improved functional reserve of the LAD (33 +/- 5% vs. 7 +/- 3% of circumflex coronary artery-perfused myocardium), except for L-NAME coapplication (13 +/- 6%). Conclusions Retroinfusion of eNOS S1177D cDNA induces neovascularization via endothelial cell proliferation and collateral growth. The resulting gain of perfusion enables an improved functional reserve of the hibernating myocardium.

Repairing defective cardiac cells is important towards improving heart function. Due to the frequency and severity of ischemic heart disease, management of patients featuring this type of cardiac failure receives significant interest. Previously we discovered that Thymosin β4 (TB4), a 43 amino-acid secreted actin sequestering peptide, is beneficial for myocardial cell survival and coronary re-growth after infarction in adult mammals. Considering the regenerative potential of full-length TB4 in the heart, and that minimal structural variations alter TB4's influence on actin assembly and cell movement, we investigated how various TB4 domains affect cardiac cell behavior and post-ischemic mammalian heart function. We synthesized 17 domain combinations of full-length TB4 and analyzed their impact on embryonic cardiac cells in vitro, and after cardiac infarction in vivo. We discovered the domains of TB4 affect cardiac cell behavior distinctly. We revealed TB4 specific C-terminal tetrapeptide, AGES, increases embryonic cardiac cell migration and myocyte beating in culture, and improves adult mammalian heart function following ischemia. Investigating the molecular background and mechanism we discovered systemic injection of AGES enhances early myocyte survival by activating Akt-mediated signaling mechanisms, increases coronary vessel growth and inhibits inflammation in mice and pigs. Biodistribution analyses revealed cardiomyocytes uptake AGES efficiently in vitro and in vivo projecting a potential independent clinical utilization for the tetrapeptide. Our comprehensive domain investigations also suggest, preservation and/or restoration of cardiomyocyte communication is a target of TB4 and AGES, and critical to improve post-ischemic heart function in pigs. In summary, we identified the C-terminal four amino-acid variable end of TB4 as the essential and responsible domain for the molecule's full benefits in the hypoxic heart. Additionally, we introduced AGES as a novel, systemically applicable drug candidate to aid cardiac infarction in adult mammals.

Aims Percutaneous stent-based ventricle-to-coronary vein bypass (venous VPASS™) is a new approach to chronic venous arterialization as a treatment modality in an otherwise no option patient with coronary artery disease. In this study, the efficacy of venous VPASS™ was compared with catheter-based selective pressure-regulated retro-infusion of arterial blood during acute ischaemia. Methods and results In seven pigs, venous VPASS™ was established using a percutaneous ultrasound-guided puncture from the anterior cardiac vein to the left ventricle, with subsequent implantation of an ePTFE-covered stent graft. During left anterior descending artery (LAD) occlusion, coronary venous pressure in the distal anterior cardiac vein increased to 55±4 mmHg under conditions of venous VPASS™ compared with 78±5 mmHg during selective pressure-regulated retro-infusion. Significant preservation of regional myocardial function was observed during venous VPASS™ (67±6% baseline) and during selective retro-infusion (83±4%) compared with control LAD occlusion (0.4±2%). Conclusion Percutaneous implantation of a PTFE covered stent (venous VPASS™) was feasible and associated with significant preservation of regional myocardial function during acute ischaemia in pigs at reasonable levels of mean coronary venous pressure to avoid tissue damage during chronic application.

Objectives We sought to study adenoviral gene delivery using percutaneous selective pressure-regulated retroinfusion and to compare it directly with surgical and percutaneous intramyocardial delivery (PIMD) for the first time. Background Intramyocardial delivery (IMD) has been recommended to be the preferred gene delivery strategy so far. However, surgical and percutaneous intramyocardial injection lead to incomplete retention of the injected viral vectors and to limited spatial myocardial distribution. Percutaneous selective pressure-regulated retroinfusion of the coronary veins was developed recently to provide an effective and more homogenous regional myocardial gene transfer. Methods In 15 pigs, adenoviral vectors (Ad2-CMV beta-galactosidase [β-gal] 5 × 109pfu) were applied via surgical IMD (n = 5), PIMD (n = 5), and selective pressure-regulated retroinfusion (n = 5). Seven days after gene transfer, myocardial β-gal expression was measured by ELISA. Results Selective retroinfusion into the anterior cardiac vein substantially increased reporter gene expression (1,039 ± 79 pg β-gal/mg protein) in the targeted left anterior descending coronary artery territory when compared with surgical (448 ± 127, p Conclusions Percutaneous selective pressure-regulated retroinfusion compares favorably with surgical and percutaneous intramyocardial injection techniques by providing a more homogenous and even more efficient adenoviral gene delivery.

Rationale: Chemokine-controlled arterial leukocyte recruitment is a crucial process in atherosclerosis. Formyl peptide receptor 2 (FPR2) is a chemoattractant receptor that recognizes proinflammatory and proresolving ligands. The contribution of FPR2 and its proresolving ligand annexin A1 to atherosclerotic lesion formation is largely undefined. Objective: Because of the ambivalence of FPR2 ligands, we here investigate the role of FPR2 and its resolving ligand annexin A1 in atherogenesis. Methods and Results: Deletion of FPR2 or its ligand annexin A1 enhances atherosclerotic lesion formation, arterial myeloid cell adhesion, and recruitment. Mechanistically, we identify annexin A1 as an endogenous inhibitor of integrin activation evoked by the chemokines CCL5, CCL2, and CXCL1. Specifically, the annexin A1 fragment Ac2-26 counteracts conformational activation and clustering of integrins on myeloid cells evoked by CCL5, CCL2, and CXCL1 through inhibiting activation of the small GTPase Rap1. In vivo administration of Ac2-26 largely diminishes arterial recruitment of myeloid cells in a FPR2-dependent fashion. This effect is also observed in the presence of selective antagonists to CCR5, CCR2, or CXCR2, whereas Ac2-26 was without effect when all 3 chemokine receptors were antagonized simultaneously. Finally, repeated treatment with Ac2-26 reduces atherosclerotic lesion sizes and lesional macrophage accumulation. Conclusions: Instructing the annexin A1-FPR2 axis harbors a novel approach to target arterial leukocyte recruitment. With the ability of Ac2-26 to counteract integrin activation exerted by various chemokines, delivery of Ac2-26 may be superior in inhibition of arterial leukocyte recruitment when compared with blocking individual chemokine receptors.

Diabetes and hypercholesterolemia are two of the major risk factors for developing cardiovascular disease, especially in combination with chronic myocardial ischemia it represents one of the most common causes of disability or death. New pro-angiogenic factors like Thymosin ß4 (Tß4), a small 4.9 kDa peptide influences cell motility and migration might be suitable for inducing therapeutic neovascularization in chronic myocardial ischemia a cardiovascular risk factors. Methods: Chronic ischemia was induced via reduction stent graft in the circumflex artery, leading to a total occlusion on day 28 (d28). Beside wildtyp animals (wt) ± high fat diet (hypercholesteric (hyp), choselsterol 78±1 wt vs 90±2 hyp), d iabetic transgenic pigs were used (increased blood glucose 305 ±12 mg/dL) . Regional application of rAAV Tß4 (5x10E12 viral particles, d28) was performed in wt, hyp and diabetic pigs respectively. Global myocardial function was obtained at day 28 and 56. Regional myocardial function and post mortem angiography were examined on day 56. Histological analysis of capillary density (capillaries/field (C/F)) was performed in the ischemic tissue. Results: rAAV.Tß4 transduction significantly enhanced capillaries (278±6 vs. 148±6 c/hpf) and collaterals (9±1 vs. 3±1) compared to control wt animals. Furthermore, global (EF: 47±4 vs. 29±3 %); and regional myocardial function (SES at 150 b/min: 73±5 vs. 10±6 % of non-ischemic area) were increased after Tß4 overexpression. In the models of cardiovascular risk factors and chronic myocardial ischemia increased neovascularization and improved myocardial function could be achieved after rAAV Tß4 application, albeit at a lower level (capillaries: diabetic: 190±4 (Tß4) vs. 120±5 (control) c/hpf hyp: 188±6 (Tß4) vs 130±12 (control) c/hpf; collaterals: diabetic: 4±1 (Tß4) vs. 2±1 (control) hyp: 6±1 (Tß4) vs 3±1 (control); EF: diabetic: 32±2 % (Tß4) vs. 27±1 % (control) hyp: 42±4 % (Tß4) vs 28±3 % (control)). Conclusion: Long term Thymosin ß4 expression induced neovascularization and improved myocardial perfusion and function even in pigs with additional risk factors. Therefore, rAAV.Tß4 appears suitable for treatment of ischemic cardiomyopathy associated with these cardiovascular risk factors.

Hif-1α, a master regulator of ischemia-responsive gene induction, controls pro-angiogenic gene expression of VEGF-A, flt-1, IGF-1 and erythropoietin, rendering its overexpression an attractive tool for therapeutic neovascularization. Utilizing an adenoviral vector system, we investigated the efficacy of selective pressure-regulated venous retroinfusion of an enhanced Hif-1α mutant (Hif-1α/VP16) in a randomized investigator-blinded study. Methods: Pigs were subjected to percutaneous implantation of a reduction-stent into the circumflex artery, leading to progressive stenosis and complete occlusion at day 28. Selective pressure-regulated retroinfusion of the great cardiac vein was performed at day 28 for regional delivery of either saline or empty vector or Ad2/Hif-1α/VP16. Collateral growth and global myocardial function were obtained by fluoroscopy, whereas regional blood flow and regional myocardial function were assessed by fluorescent microsphere analysis and sonomicrometry, respectively. Capillary density in the ischemic myocardium was analyzed by PECAM-1 staining. Results: Compared to saline and Ad empty vector controls, overexpression of Hif-1α in the ischemic region induced an increase of small (capillary) and large (collateral) vessels, resulting in an improved perfusion of the ischemic myocardium. Concomitantly, an ischemia induced loss of myocardial function (hibernating myocardium) was resolved only after transfection with the Hif 1-α transgene, but not the empty vector or saline control. Conclusion: Retroinfusion of Ad2/Hif-1α/VP16, combining a master pro-angiogenic protein with regional myocardial application, may offer an efficient approach to cardiac gene therapy of chronic ischemic cardiomyopathy.

Background— Excessive formation of reactive oxygen species contributes to tissue injury and functional deterioration after myocardial ischemia/reperfusion. Especially, mitochondrial reactive oxygen species are capable of opening the mitochondrial permeability transition pore, a harmful event in cardiac ischemia/reperfusion. Thioredoxins are key players in the cardiac defense against oxidative stress. Mutations in the mitochondrial thioredoxin reductase (thioredoxin reductase-2, Txnrd2) gene have been recently identified to cause dilated cardiomyopathy in patients. Here, we investigated whether mitochondrial thioredoxin reductase is protective against myocardial ischemia/reperfusion injury. Methods and Results— In mice, α-MHC-restricted Cre-mediated Txnrd2 deficiency, induced by tamoxifen ( Txnrd2-/-ic ), aggravated systolic dysfunction and cardiomyocyte cell death after ischemia (90 minutes) and reperfusion (24 hours). Txnrd2-/-ic was accompanied by a loss of mitochondrial integrity and function, which was resolved on pretreatment with the reactive oxygen species scavenger N-acetylcysteine and the mitochondrial permeability transition pore blocker cyclosporin A. Likewise, Txnrd2 deletion in embryonic endothelial precursor cells and embryonic stem cell-derived cardiomyocytes, as well as introduction of Txnrd2-shRNA into adult HL-1 cardiomyocytes, increased cell death on hypoxia and reoxygenation, unless N-acetylcysteine was coadministered. Conclusions— We report that Txnrd2 exerts a crucial function during postischemic reperfusion via thiol regeneration. The efficacy of cyclosporin A in cardiac Txnrd2 deficiency may indicate a role for Txnrd2 in reducing mitochondrial reactive oxygen species, thereby preventing opening of the mitochondrial permeability transition pore.

Therapeutic neovascularization by several growth factors is a concept well established in multiple animal models, albeit lacking clearcut clinical success to date. Therefore, we modified our therapeutic approach by combining the potent angiogenic agent hVEGF-A with hPDGF, a growth factor known for attracting pericytes and smooth muscle cells for arterial vessel maturation. In order to enhance efficacy, we utilized adeno-associated virus (AAV) 2/9, allowing for prolonged transgene expression. A lower hVEGF-AAV-concentration was used to avoid hemangiomaformation. 7 days after femoral artery excision, 1×10e11 hVEGF-A-AAV alone or in combination with 1×10e12 hPDGF-AAV particles were retroinfused into the anterior tibial vein of rabbits (n=5/group, p

Recruitment of endothelial progenitor cells to the sites of ischemia is a prerequisite for efficient therapeutic neovascularization via vasculogenesis. Chemokines play a major role in the homing of EPCs at the ischemic vasculature, a mechanism fading in chronic ischemia. To overcome this limitation, we constructed an artificial adhesion molecule consisting of a GPI-anchor, a fractalkine-backbone and an SDF-1 head (SDF-1-fra-GPI), which was applied for enhanced recruitment of embryonic EPCs (eEPCs: CXCR4++, Tie2++, Thrombomodulin++, CD34-, MHCI-, vWF inducible, eNOS inducible) in vitro and in vivo . Methods: In a flow chamber adhesion assay, Control plasmids (pcDNA or GPI-SDF-1 cDNA) were compared to the SDF-1-fra-GPI construct for eEPC recruitment 24h after liposomal transfection of rat endothelial cells. In vivo, in rabbits (n=5 per group) at day 7 (d7) after femoral artery excision, 1 mg of the SDF-1-fra-GPI or eGFP cDNA was transfected into the ischemic limb. At d9, ischemic hindlimbs were retroinfused with 5x10 6 eEPCs. Angiography was performed for collateral quantification and frame count score at d9 and d37 (% of d9), capillary density was assessed via PECAM-1-staining (capillaries/muscle fiber = c/mf). Results: In vitro, eEPC adhesion (16±12 cells/field) was increased to a higher extent by SDF-1-fra-GPI (79±13) than SDF1-GPI (54±8) or control vector (37±8). In vivo , eEPC adhesion in the ischemic hindlimb after SDF-1-fra-GPI transfection compared to mock transfection (30±3 vs. 9±1 cells/field). Whereas capillary density was unaffected (1.66±0.30 SDF-1-Fra-GPI vs. 1.56±0.29 eEPCs), collateral growth (152±10% SDF-1-fra-GPI vs. 124±13%) as well as perfusion score (198±17% SDF-1-fra-GPI vs.160±6% eEPCs) further increased after SDF-1-fra-GPI transfection (controls: 1.24±0.12 c/mf, collaterals 105±8%, perfusion score 112±11%). We conclude that recruitment of EPCs expressing CXCR4 (the SDF-1 receptor) may benefit from pre-treatment of the recipient vasculature with SDF-1-Fra-GPI, an artificial adhesion molecule. This approach might be valuable for enhancing EPC recruitment in the scenario of therapeutic neovascular-ization of chronic ischemic syndromes.

Background— Adult endothelial progenitor cells (EPCs) reduce myocardial infarct size and improve postischemic myocardial function. We have recently shown that clonal embryonic EPCs (eEPCs), derived from 7.5-day-old mice, home specifically to hypoxic areas in tumor metastasis mouse models but spare normal organs and do not form carcinomas. Here, we assessed the potential of eEPCs to limit organ dysfunction after ischemia and reperfusion in a preclinical pig model. Methods and Results— Pigs were subjected to ischemia (60-minute left anterior descending [LAD] artery occlusion) and reperfusion (7 days). At the end of ischemia, we applied medium with or without 5×10 6 eEPCs by either pressure-regulated retroinfusion or intravenous transfusion. One hour after reperfusion, 99 Tc-labeled eEPCs engrafted to a 6-fold higher extent in the ischemic myocardium after retroinfusion than after intravenous application. Regional myocardial function (subendocardial segment shortening [SES] at 150/min, given in percent of nonischemic circumflex region) and infarct size (TTC viability and Methylene-blue exclusion) were determined 24 hours and 7 days later. Compared with medium-treated animals, retroinfusion of eEPCs decreased infarct size (35±4% versus 51±6%) and improved regional myocardial reserve of the apical LAD region (SES 31±4% versus 6±8%), whereas intravenous application displayed a less pronounced effect (infarct size 44±4%; SES 12±3%). Retroinfusion of an equal amount of neonatal coronary endothelial cells (rat) did not affect infarct size (49±5%) nor regional myocardial reserve (16±7%). The eEPC-dependent effect was detected at 24 hours of reperfusion (infarct size 34±7% versus 58±6%) and was sensitive to Wortmannin coapplication (50±5%). Conclusion— Our findings show that eEPCs reduce ischemia-reperfusion injury in a preclinical pig model. The rapid effect (as early as 24 hours) indicates a role for enzyme-mediated cardioprotection, which involves, at least in part, the phosphatidylinositol 3-kinase/AKT pathway.

Background— The hemodynamic and vascular effects of ventricular sourcing by a stent-based (VSTENT, Percardia) left ventricle-to-coronary artery bypass were studied in a patient subgroup of the European multicenter ADVANTAGE study (ADjunctive treatment with the VCAB/VSTENT myocardial implANT system in coronary Artery bypass Graft patiEnts). Methods and Results— Twelve patients who underwent VSTENT implantation in addition to coronary artery bypass surgery were studied up to 12 months after the procedure. The VSTENT was implanted distal to a hemodynamically relevant coronary artery stenosis. Coronary flow velocity was assessed at rest and during dobutamine stress. Intraoperative VSTENT implantation was successful in 11 of 12 patients. Early postoperative angiograms showed patent VSTENT in 8 of 11 patients, with predominantly systolic flow distal to the VSTENT. Coronary flow velocity reserve induced by dobutamine stress was 1.7±0.1 ( P =0.006). VSTENT patency at 2- to 6-month follow-up was present in 5 of 11 patients, with concomitant VSTENT stenosis in 4 of those 5. In all patients, coronary flow velocity increased 3- to 4-fold proximal to the VSTENT, which was associated with a moderate degree of arterial remodeling. Except for target vessel reintervention (n=5), no other major adverse events were observed in 11 of 12 patients. One patient died on the second postoperative day, though the cause was probably not related to the VSTENT implantation. Conclusions— VSTENT implantation seems to be safe in the mid-term follow-up and leads to a predominantly systolic coronary flow pattern in the vessel supplied by the VSTENT, with a flow reserve similar or close to that seen with conventional bypass. VSTENT patency rate, however, was unacceptably low at 3- to 6-month follow-up and needs to be improved.

Background Stent-based left ventricle-to-coronary artery stent (VSTENT) is a newly developed, alternative surgical revascularization procedure (VCAB). We present here our initial experience using this technique. Methods Twelve patients (10 male and 2 female, mean age 61 ± 13 years) underwent a VCAB concomitant to coronary artery bypass surgery. Myocardial revascularization was performed on-pump with arrested heart in 4 patients, on-pump with beating heart in 6 patients, and off-pump in 2 patients. Average number of coronary anastomoses per patient was 2.4 ± 0.8. In all cases left internal thoracic artery-to-left anterior descending was used. In each patient only one VSTENT was implanted. Target artery for the VCAB was a diagonal branch in 5 patients, an intermediate branch in one patient and a marginal branch in 6 patients. Mean time for the VCAB was 23 ± 5 minutes (17 to 30 minutes). Results An immediate procedural success was observed in 11 of 12 cases. In one case VCAB was not successful and conventional aortocoronary bypass was performed. One patient died on the second postoperative day due to a systemic inflammatory response syndrome. Autopsy demonstrated a patent VSTENT. Angiography was performed in 10 patients 2 to16 days (9 ± 5 days) postoperatively showing a patent VSTENT in 8 patients. Conclusions The VCAB was feasible and potentially safe in the short-term postoperative follow-up, particularly with increasing experience after the first patients. Though the VSTENT is a promising tool for myocardial revascularization, long-term safety, patency, and performance of the device needs to be determined.

Objectives— The interaction of the heat shock protein 90 (Hsp90) with the endothelial NO synthase (eNOS) has been shown to account for a sustained production of NO in vitro. Here, we examined whether overexpression of Hsp90 in a pig model of cardiac infarct could preserve the myocardium from the deleterious effects of ischemia–reperfusion. Methods and Results— Percutaneous liposome-based gene transfer was performed by retroinfusion of the anterior interventricular vein before left anterior descending occlusion and reperfusion. We found that recombinant Hsp90 expression in the ischemic region of the heart led to a 33% reduction in infarct size and prevented the increase in postischemic left ventricular end diastolic pressure observed in mock-transfected animals. Regional myocardial function, assessed by subendocardial segment shortening in the infarct region, was increased in Hsp90-transfected animals at baseline and after pacing. All these effects were completely abrogated by administration of the NOS inhibitor N G -nitro- l -arginine methyl ester. We further documented in vivo and in cultured endothelial cells that the cardioprotective effects of Hsp90 were associated to its capacity to act as an adaptor for both the kinase Akt and the phosphatase calcineurin, thereby promoting eNOS serine 1177 phosphorylation and threonine 495 dephosphorylation, respectively. Conclusions— Hsp90 is a promising target to enhance NO formation in vivo, which may efficiently reduce myocardial reperfusion injury.

Objective: Reperfusion after ischemia may contribute to loss of myocardial function and increase in infarct size. Scavenging of reactive oxygen species (ROS) by glutathione (GSH) and inhibition of the sodium-proton-exchanger by cariporide are both capable of reducing myocardial reperfusion injury. We tested the efficacy of both agents applied regionally into the myocardium immediately before reperfusion. Methods: Neonatal rat cardiomyocytes (NRCMs) were exposed to either hypoxia (H, 8 h)/reoxygenation (R, 1 h) or H2O2 (300 μM) in the presence or absence of GSH (10 mg/ml). In pigs ( n =5 per group), percutaneous LAD occlusion was performed for 60 min. Application of GSH (250 mg/kg) and/or cariporide (1 mg/kg) was achieved by pressure-regulated retroinfusion of the anterior cardiac vein draining the ischemic area starting 5 min before reopening of the occluded LAD. Seven days later, subendocardial segment shortening (SES) was analyzed by sonomicrometry. Infarct size was determined by methylene-blue staining of the non-ischemic area and tetrazolium red staining of the viable myocardium in the area at risk (AAR). Results: NRCM incubated with GSH (10 mg/ml) survived H/R or H2O2 (0.3 mM) to a larger extent than untreated cells. In pigs, infarct size of untreated hearts (51±6% of the AAR) was not significantly altered by GSH or cariporide retroinfusion alone (41±3% and 42±6%). In contrast, combined retroinfusion of cariporide and GSH significantly reduced infarct size (29±3%). SES of the infarcted area was improved only after cariporide/GSH retroinfusion as compared to untreated hearts. Additional systemic application of CD18-antibody IB4 (1.5 mg/kg) did not alter infarct size or SES in comparison to GSH/cariporide retroinfusion alone. Conclusion: Timely application of GSH scavenging ROS and cariporide targeting ion imbalance provides cardioprotection to the postischemic heart, which is superior to either treatment alone. The lack of an effect of additional IB4 treatment may indicate that GSH/cariporide retroinfusion itself affects leukocyte-dependent reperfusion injury.

Background— Ventricle-to–coronary artery bypass (VCAB) is an experimental revascularization procedure that provides predominantly systolic instead of diastolic blood flow to a coronary artery. Methods and Results— In a pig model, a stent-based procedure (VSTENT) was developed to create a VCAB. After thoracotomy, a covered VSTENT was implanted between the left ventricle and the left anterior descending coronary artery (LAD). Distal LAD flow, regional myocardial function, and intracoronary pressures were determined at different degrees of LAD stenosis and during complete LAD occlusion. During 3 hours of LAD occlusion, VSTENT preserved net forward flow at 70±6% and regional myocardial function at 71±8% of baseline. Preservation of net flow was influenced by the positioning of the VSTENT, with higher preservation also under conditions of increased oxygen demand if a “valve-like mechanism” was present during diastole. At a hemodynamically relevant level of LAD stenosis (>70%), systolic inflow was predominant after VSTENT implantation. Changes in mean diastolic intracoronary pressure that resulted from different degrees of LAD stenosis were linearly correlated to net flow after VSTENT implantation ( r =0.88; P Conclusions— VSTENT for ventricle-to–coronary artery bypass was feasible and preserved 70±6% of baseline flow during complete LAD occlusion. The degree of preservation was dependent on the position of the VSTENT creating a valve-like mechanism during diastole. Residual diastolic blood flow through a high-grade LAD stenosis influenced net flow favorably, because diastolic backflow decreased with increasing mean diastolic intracoronary pressure.

ObjectivesWe sought to improve regional myocardial delivery and subsequent collateral perfusion induced by basic fibroblast growth factor-2 (FGF-2) using selective pressure-regulated retroinfusion of coronary veins for delivery. This hypothesis was tested in a newly developed pig model with percutaneous induction of chronic ischemia.BackgroundSelective pressure-regulated retroinfusion of coronary veins is a catheter-based procedure that has been shown to provide effective regional delivery of drugs and gene vectors into ischemic myocardium.MethodsA high-grade stenosis with subsequent progression to total occlusion within 28 days was induced by implanting a reduction stent graft into the left anterior descending artery (LAD). After seven days, a 30-min retroinfusion (anterior cardiac vein) was performed with (n = 7) or without (n = 7) 150 μg FGF-2 and compared with a 30-min antegrade infusion of 150 μg FGF-2 into the LAD (n = 7). Sonomicrometry to assess regional myocardial function at rest and during pacing, and microspheres to assess regional myocardial blood flow, were performed 28 days after implantation of the reduction stent.ResultsRetroinfusion of FGF-2 compared favorably with controls and with antegrade infusion of FGF-2 with regard to regional myocardial function at rest (18.5 ± 4.1% vs. 5.7 ± 2.9% vs. 7.9 ± 1.8%, respectively, p < 0.05) and during pacing. Regional myocardial blood flow was also higher in the LAD territory after retroinfusion of FGF-2 (1.07 ± 0.14 vs. 0.66 ± 0.07 vs. 0.72 ± 0.17 ml·min−1·g−1, p < 0.05).ConclusionsSelective pressure-regulated retroinfusion increased tissue binding of FGF-2 and enhanced functionally relevant collateral perfusion compared with antegrade intracoronary delivery in pigs with chronic myocardial ischemia.