Left ventricular hypertrophy (LVH) is a potent risk factor for sudden death and congestive heart failure.

Hyperkalaemia due to potassium-increasing drug–drug interactions (DDIs) is a clinically important adverse drug event. The purpose of this study was to identify patient- and physician-related risk factors for the development of hyperkalaemia.

Prenatal heart valve interventions aiming at the early and systematic correction of congenital cardiac malformations represent a promising treatment option in maternal-fetal care. However, definite fetal valve replacements require growing implants adaptive to fetal and postnatal development. The presented study investigates the fetal implantation of prenatally engineered living autologous cell-based heart valves. Autologous amniotic fluid cells (AFCs) were isolated from pregnant sheep between 122 and 128 days of gestation via transuterine sonographic sampling. Stented trileaflet heart valves were fabricated from biodegradable PGA-P4HB composite matrices (n = 9) and seeded with AFCs in vitro. Within the same intervention, tissue engineered heart valves (TEHVs) and unseeded controls were implanted orthotopically into the pulmonary position using an in-utero closed-heart hybrid approach. The transapical valve deployments were successful in all animals with acute survival of 77.8% of fetuses. TEHV in-vivo functionality was assessed using echocardiography as well as angiography. Fetuses were harvested up to 1 week after implantation representing a birth-relevant gestational age. TEHVs showed in vivo functionality with intact valvular integrity and absence of thrombus formation. The presented approach may serve as an experimental basis for future human prenatal cardiac interventions using fully biodegradable autologous cell-based living materials.

AbstractWe investigate the combination of transcatheter aortic-valve implantation (TAVI) and a novel concept of stem cell-based, tissue-engineered heart-valves (TEHV) comprising minimally-invasive techniques for both, cell-harvest and valve-delivery. TAVI represents an emerging technology for the treatment of aortic-valve disease. The utilized bioprostheses are inherently prone to calcific-degeneration and recent evidence suggests even accelerated degeneration resulting from structural-damage due to the crimping-procedures. Autologous, living heart-valve prosthesis with regeneration and repair capacities would overcome such limitations. Methods: Within a one-step intervention, tri-leaflet TEHV, generated from biodegradable synthetic-scaffolds, were integrated into self-expanding nitinol-stents, seeded with autologous bone-marrow mononuclear cells, crimped and transapically delivered into adult sheep (n=12). The animals were followed up for up to 2 weeks. TEHV-functionality was assessed by fluoroscopy, echocardiography and computed-tomography. Post-mortem analysis was performed using histology, extracellular-matrix analysis and electron-microscopy. Transapical aortic implantation of TEHV was successful in all animals (n=12) and the entire procedure-time from cell-harvest to TEHV-delivery was 109±14min. Fluoroscopy and echocardiography displayed TEHV-functionality demonstrating an adequate leaflet-mobility and co-aptation. Explanted TEHV showed intact leaflet-structures with well defined cusps without signs of thrombus-formation or structural-damage. Histology and ECM analysis displayed a high cellularity indicative for an early cellular-remodelling and in-growth after 2weeks. For the first time, we demonstrate the principal feasibility of a transcatheter, stem cell-based TEHV implantation into the aortic-valve position within a one-step intervention. Its long term functionality proven, a stem cell-based TEHV approach may represent a next generation heart-valve concept extending the clinical indication of transcatheter valves beyond elderly high-risk patients.

Living autologous tissue engineered vascular-grafts (TEVGs) with growth-capacity may overcome the limitations of contemporary artificial-prostheses. However, the multi-step in vitro production of TEVGs requires extensive ex vivo cell-manipulations with unknown effects on functionality and quality of TEVGs due to an accelerated biological age of the cells. Here, the impact of biological cell-age and tissue-remodeling capacity of TEVGs in relation to their clinical long-term functionality are investigated. TEVGs were implanted as pulmonary-artery (PA) replacements in juvenile sheep and followed for up to 240 weeks (∼4.5years). Telomere length and telomerase activity were compared amongst TEVGs and adjacent native tissue. Telomerase-activity of in vitro expanded autologous vascular-cells prior to seeding was <5% as compared to a leukemic cell line, indicating biological-aging associated with decreasing telomere-length with each cellular-doubling. Up to 100 weeks, the cells in the TEVGs had consistently shorter telomeres compared to the native counterpart, whereas no significant differences were detectable at 240 weeks. Computed tomography (CT) analysis demonstrated physiological wall-pressures, shear-stresses, and flow-pattern comparable to the native PA. There were no signs of degeneration detectable and continuous native-analogous growth was confirmed by vessel-volumetry. TEVGs exhibit a higher biological age compared to their native counterparts. However, despite of this tissue engineering technology related accelerated biological-aging, growth-capacity and long-term functionality was not compromised. To the contrary, extensive in-vivo remodeling processes with substantial endogenous cellular turnover appears to result in "TEVG rejuvenation" and excellent clinical performance. As these large-animal results can be extrapolated to approximately 20 human years, this study suggests long-term clinical-safety of cardiovascular in vitro tissue engineering and may contribute to safety-criteria as to first-in-man clinical-trials.

AIMS:A living heart valve with regeneration capacity based on autologous cells and minimally invasive implantation technology would represent a substantial improvement upon contemporary heart valve prostheses. This study investigates the feasibility of injectable, marrow stromal cell-based, autologous, living tissue engineered heart valves (TEHV) generated and implanted in a one-step intervention in non-human primates.METHODS AND RESULTS:Trileaflet heart valves were fabricated from non-woven biodegradable synthetic composite scaffolds and integrated into self-expanding nitinol stents. During the same intervention autologous bone marrow-derived mononuclear cells were harvested, seeded onto the scaffold matrix, and implanted transapically as pulmonary valve replacements into non-human primates (n = 6). The transapical implantations were successful in all animals and the overall procedure time from cell harvest to TEHV implantation was 118 ± 17 min. In vivo functionality assessed by echocardiography revealed preserved valvular structures and adequate functionality up to 4 weeks post implantation. Substantial cellular remodelling and in-growth into the scaffold materials resulted in layered, endothelialized tissues as visualized by histology and immunohistochemistry. Biomechanical analysis showed non-linear stress-strain curves of the leaflets, indicating replacement of the initial biodegradable matrix by living tissue.CONCLUSION:Here, we provide a novel concept demonstrating that heart valve tissue engineering based on a minimally invasive technique for both cell harvest and valve delivery as a one-step intervention is feasible in non-human primates. This innovative approach may overcome the limitations of contemporary surgical and interventional bioprosthetic heart valve prostheses.

Current scientific attempts to generate in vitro tissue-engineered living blood vessels (TEBVs) show substantial limitations, thereby preventing routine clinical use. In the present report, we describe a novel biotechnology concept to create living small diameter TEBV based exclusively on microtissue self-assembly (living cellular re-aggregates). A novel bioreactor was designed to assemble microtissues in a vascular shape and apply pulsatile flow and circumferential mechanical stimulation. Microtissues composed of human artery-derived fibroblasts (HAFs) and endothelial cells (HUVECs) were accumulated and cultured for 7 and 14 days under pulsatile flow/mechanical stimulation or static culture conditions with a diameter of 3mm and a wall thickness of 1mm. The resulting vessels were analyzed by immunohistochemistry for extracellular matrix (ECM) and cell phenotype (von Willebrand factor, alpha-SMA, Ki67, VEGF). Self-assembled microtissues composed of fibroblasts displayed significantly accelerated ECM formation compared to monolayer cell sheets. Accumulation of vessel-like tissue occurred within 14 days under both, static and flow/mechanical stimulation conditions. A layered tissue formation was observed only in the dynamic group, as indicated by luminal aligned alpha-SMA positive fibroblasts. We could demonstrate that self-assembled cell-based microtissues can be used to generate small diameter TEBV. The significant enhancement of ECM expression and maturation, together with the pre-vascularization capacity makes this approach highly attractive in terms of generating functional small diameter TEBV devoid of any foreign material.

Objectives The aim of this study was to demonstrate the feasibility of combining the novel heart valve replacement technologies of: 1) tissue engineering; and 2) minimally-invasive implantation based on autologous cells and composite self-expandable biodegradable biomaterials.Background Minimally-invasive valve replacement procedures are rapidly evolving as alternative treatment option for patients with valvular heart disease. However, currently used valve substitutes are bioprosthetic and as such have limited durability. To overcome this limitation, tissue engineering technologies provide living autologous valve replacements with regeneration and growth potential.Methods Trileaflet heart valves fabricated from biodegradable synthetic scaffolds, integrated in self-expanding stents and seeded with autologous vascular or stem cells ( bone marrow and peripheral blood), were generated in vitro using dynamic bioreactors. Subsequently, the tissue engineered heart valves (TEHV) were minimally-invasively implanted as pulmonary valve replacements in sheep. In vivo functionality was assessed by echocardiography and angiography up to 8 weeks. The tissue composition of explanted TEHV and corresponding control valves was analyzed.Results The transapical implantations were successful in all animals. The TEHV demonstrated in vivo functionality with mobile but thickened leaflets. Histology revealed layered neotissues with endothelialized surfaces. Quantitative extracellular matrix analysis at 8 weeks showed higher values for deoxyribonucleic acid, collagen, and glycosaminoglycans compared to native valves. Mechanical profiles demonstrated sufficient tissue strength, but less pliability independent of the cell source.Conclusions This study demonstrates the principal feasibility of merging tissue engineering and minimally-invasive valve replacement technologies. Using adult stem cells is successful, enabling minimally-invasive cell harvest. Thus, this new technology may enable a valid alternative to current bioprosthetic devices. (J Am Coll Cardiol 2010; 56: 510-20) (C) 2010 by the American College of Cardiology Foundation

Objective: Cardioprotective properties of recombinant human Erythropoietin (rhEpo) have been shown in in vivo regional or ex vivo global models of ischemia-reperfusion (I/R) injury. The aim of this study was to characterize the cardioprotective potential of rhEPO in an in vivo experimental model of global I/R approximating the clinical cardiac surgical setting and to gain insights into the myocardial binding sites of rhEpo and the mechanism involved in its cardioprotective effect. Methods: Hearts of donor Lewis rats were arrested with cold crystalloid cardioplegia and after 45 min of cold global ischemia grafted heterotopically into the abdomen of recipient Lewis rats. Recipients were randomly assigned to control non-treated or Epo-treated group receiving 5000 U/kg of rhEpo intravenously 20 min prior to reperfusion. At 5 time points 5-1440 min after reperfusion, the recipients (n = 6-8 at each point) were sacrificed, blood and native and grafted hearts harvested for subsequent analysis. Results: Treatment with rhEpo resulted in a significant reduction in myocardial I/R injury (plasma troponin T) in correlation with preservation of the myocardial redox state (reduced glutathione). The extent of apoptosis (activity of caspase 3 and caspase 9, TUNEL test) in our model was very modest and not significantly affected by rhEpo. Immunostaining of the heart tissue with anti-Epo antibodies showed an exclusive binding of rhEpo to the coronary endothelium with no binding of rhEpo to cardiomyocytes. Administration of rhEpo resulted in a significant increase in nitric oxide (NO) production assessed by plasma nitrite levels. Immunostaining of heart tissue with anti-phospho-eNOS antibodies showed that after binding to the coronary endothelium, rhEpo increased the phosphorylation and thus activation of endothelial nitric oxide synthase (eNOS) in coronary vessels. There was no activation of eNOS in cardiomyocytes. Conclusions: Intravenous administration of rhEpo protects the heart against cold global I/R. Apoptosis does not seem to play a major role in the process of tissue injury in this model. After binding to the coronary endothelium, rhEpo enhances NO production by phosphorylation and thus activation of eNOS in coronary vessels. Our results suggest that cardioprotective properties of rhEpo are at least partially mediated by NO released by the coronary endothelium. (C) 2009 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved.

Objective: Monitoring of cardiac surgical patients after transfer from the intensive care unit to the normal ward is incomplete. Undetected hypoxia, however, is known to be a risk factor for occurrence of atrial fibrillation. We have utilized Auricall (R) for continuous wireless monitoring of oxygen saturation and heart rate until discharge. The object of the study was to analyze if oxygen therapy as a result of Auricall (R) alerts of hypoxia can decrease the incidence of postoperative atrial, fibrillation. Methods: Auricall (R) is a wireless portable pulse oximeter. An alert is generated depending on preset threshold values (heart rate, oxygen saturation). Over a period of 6 months, 119 patients were monitored with the Auricall (R) following coronary artery bypass graft and/or valve surgery. Oxygen therapy was started subsequent to an oxygen saturation below 90%. These patients were compared with a cohort of 238 patients from the time period before availability of Auricall (R). The patient characteristics were comparable in both groups. In a retrospective study, the incidence of atrial fibrillation was measured in both groups. Results: The postoperative AF was observed in 22/119 patients (18%) in group I and in 66/238 patients (28%) in group II. This difference between the two groups approached significance (p = 0.056). In the subgroup of patients with coronary artery bypass graft with our without simultaneous valve surgery (n = 312), Auricall (R) monitoring resulted in a significantly reduced incidence of atrial. fibrillation (14% vs 26%, p = 0.016). Conclusions: Continuous monitoring of oxygen saturation on the normal ward and subsequent oxygen therapy for hypoxia can reduce the incidence of atrial. fibrillation in a subgroup of patients after cardiac surgery. Prospective randomized trials are warranted to confirm these data. (C) 2008 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. Ail rights reserved.

Introduction: Tissue engineering (TE) technologies providing living, autologous heart valves with the capacity of regeneration and growth have shown functionality in chronic animal studies and first human applications. We have previously demonstrated the successful merging of tissue engineering and minimally invasive implantation technologies in a large animal model. Here we investigate the influence of a novel, biomimetic in vitro technology on valve performance. Methods: Trileaflet heart valves (n=4, 30mm) based on rapidly degrading polymer scaffolds integrated in self-expandable stents were engineered from sequentially seeded ovine vascular derived autologous cells. Valves were grown in-vitro for 19d utilizing combined strain-flow bioreactor systems. Thereafter, valves were crimped (12mm) and delivered minimally invasively (trans-apical) in sheep replacing the native pulmonary valves. Controls were analyzed directly after the crimping/delivery process for structural integrity. Post-operative follow-up (4w) comprised angiography and echocardiography. Neo-tissue analyses included histology, SEM, ECM quantification and biomechanical testing. Results: TE heart valves showed preserved structural integrity after the crimping and delivery process. The minimally invasive procedure was successful in all implanted valves and adequate functionality was observed up to 4 weeks. Morphological analyses of the leaflets demonstrated a thickened, layered tissue formation comparable to previous animal studies. The TE valve ring structures were integrated into the adjacent native tissue after 4w. Conclusions: This study demonstrates that based on a novel biomimetic in vitro technology living, tissue engineered heart valves can be generated and implanted by minimally invasive procedures. Process safety and short-term functionality were shown and long-term animal studies are initiated.

Background : A clinically relevant heart valve tissue engineering concept requires minimally invasive techniques for both cell harvest and valve implantation. Here, we present first experiences with autologous tissue engineered heart valves fabricated from composite self-expandable biodegradable scaffolds and adult stem cells implanted by minimally invasive procedures in a sheep model. Methods : Sandwich-structured heart valve scaffolds (n= 12) were fabricated from non-woven PLDLA meshes coated with electrospun PLDLA nanofibers and integrated in self-expanding nitinol stents. Scaffolds were seeded with either autologous ovine bone marrow (BMC; n= 4) or jugular vein-derived cells (JVC; n= 8) and cultured in bioreactors. After 9d, heart valves were endothelialized with autologous peripheral blood-derived endothelial progenitor cells and jugular vein-derived endothelial cells, respectively. After additional 3d, heart valves (n= 6) were implanted trans-apically in pulmonary position. Controls were analysed (n= 6) as to tissue formation and composition (histology, biochemical assays). Mechanical properties were determined by tensile tests. In vivo performance was assessed by echocardiography up to 4 weeks. Results : Histology revealed cell attachment and ingrowth into the scaffold material resulting in layered tissues with endothelialized, eNOS positive surfaces. Amounts of GAG and cell number were similar in all heart valves, comparable to native tissues. Collagen production was higher in BMC based heart valves compared to JVC-derived tissues (Hydroyproline amount 34% vs. 20% of native tissues). Mechanical profiles demonstrated physiological tissue strength (max. tensile stress 0.41± 0.21 MPa) but less elasticity (E-Moduli 1.89± 0.79 MPa) independent of the cell source. Echocardiography displayed in vivo functionality (transvalvular mean pressure gradient 10.36± 3.17 mm/Hg) with more flexibility of BMC based heart valves leaflets. Conclusions : These results demonstrate that heart valve tissue engineering based on a minimally invasive technique for both cell harvest and valve implantation is feasible. This clinically relevant approach is currently investigated in long-term animal studies.

Smooth muscle cell (SMC) migration contributes to vascular remodeling. Nitric oxide (NO) produced via endothelial NO synthase (eNOS) inhibits SMC migration. This study analyzes signal transduction mechanisms of SMC migration targeted by NO. SMCs were cultured from human saphenous veins, and cell migration was studied using Boyden chambers. PDGF-BB (0.1 to 10 ng/ml) stimulated SMC migration in a concentration-dependent manner, which was inhibited by adenoviral-mediated overexpression of eNOS and by the NO donor diethylentriamine NONOate (DETANO, 10 to 10 mol/L). NO release was enhanced in eNOS-transduced SMCs, and L-NAME blunted the effect of eNOS overexpression on migration. PDGF-BB (10 ng/ml) activated Rho A, which was inhibited by the overexpression of eNOS by DETANO and by 8 bromo-cGMP. The inhibitory effect of DETANO on Rho A activity was prevented by the cGMP-dependant kinase inhibitor. Furthermore, inhibition of Rho A by C3 exoenzyme and inhibition of ROCK by Y-27632 diminished cell migration stimulated by PDGF-BB. Finally, in the cells overexpressing constitutively active ROCK mutant (CAT), DETANO failed to prevent PDGF-BB-induced SMC migration. In conclusion, NO inhibits human SMC migration via blockade of the Rho A pathway.