Cysteine-rich angiogenic inducer 61 (Cyr61): a novel soluble biomarker of acute myocardial injury improves risk stratification after acute coronary syndromes.

AIMS: We aimed to identify a novel biomarker involved in the early events leading to an acute coronary syndrome (ACS) and evaluate its role in diagnosis and risk stratification. METHODS AND RESULTS: Biomarker identification was based on gene expression profiling. In coronary thrombi of ACS patients, cysteine-rich angiogenic inducer 61 (Cyr61, CCN1) gene transcripts were highly up-regulated compared with peripheral mononuclear cells. In a murine ischaemia-reperfusion model (I/R), myocardial Cyr61 expression was markedly increased compared with the controls. Cyr61 levels were determined in human serum using an enzyme-linked immunosorbent assay. Cohorts of ACS (n = 2168) referred for coronary angiography, stable coronary artery disease (CAD) (n = 53), and hypertrophic obstructive cardiomyopathy (HOCM) patients (n = 15) served to identify and evaluate the diagnostic and prognostic performance of the biomarker. Cyr61 was markedly elevated in ST-elevation myocardial infarction patients compared with non-ST-elevation myocardial infarction/unstable angina or stable CAD patients, irrespective of whether coronary thrombi were present. Cyr61 was rapidly released after occlusion of a septal branch in HOCM patients undergoing transcoronary ablation of septal hypertrophy. Cyr61 improved risk stratification for all-cause mortality when added to the reference GRACE risk score at 30 days (C-statistic 0.88 to 0.89, P = 0.001) and 1 year (C-statistic 0.77 to 0.80, P < 0.001) comparable to high-sensitivity troponin T (30 days: 0.88 to 0.89, P < 0.001; 1 year: 0.77 to 0.79, P < 0.001). Similar results were obtained for the composite endpoint of all-cause mortality or myocardial infarction. Conversely, in a population-based case-control cohort (n = 362), Cyr61 was not associated with adverse outcome. CONCLUSION: Cyr61 is a novel early biomarker reflecting myocardial injury that improves risk stratification in ACS patients.

Safety and efficacy of cardiopoietic stem cells in the treatment of post-infarction left-ventricular dysfunction - From cardioprotection to functional repair in a translational pig infarction model.

To date, clinical success of cardiac cell-therapies remains limited. To enhance the cardioreparative properties of stem cells, the concept of lineage-specification through cardiopoietic-guidance has been recently suggested. However, so far, only results from murine studies and from a clinical pilot-trial in chronic heart-failure (CHF) are available, while systematic evidence of its therapeutic-efficacy is still lacking. Importantly, also no data from large animals or for other indications are available. Therefore, we here investigate the therapeutic-efficacy of human cardiopoietic stem cells in the treatment of post-infarction LV-dysfunction using a translational pig-model. Using growth-factor priming, lineage-specification of human bone-marrow derived MSCs was achieved to generate cardiopoietic stem cells according to GMP-compliant protocols. Thereafter, pigs with post-infarction LV-dysfunction (sub-acute phase;1-month) were randomized to either receive transcatheter NOGA 3D electromechanical-mapping guided intramyocardial transplantation of cardiopoietic cells or saline (control). After 30days, cardiac MRI (cMRI) was performed for functional evaluation and in-vivo cell-tracking. This approach was coupled with a comprehensive post-mortem cell-fate and mode-of-repair analysis. Cardiopoietic cell therapy was safe and ejection-fraction was significantly higher when compared to controls (p = 0.012). It further prevented maladaptive LV-remodeling and revealed a significantly lower relative and total infarct-size (p = 0.043 and p = 0.012). As in-vivo tracking and post-mortem analysis displayed only limited intramyocardial cardiopoietic cell-integration, the significant induction of neo-angiogenesis (∼40% higher; p = 0.003) and recruitment of endogenous progenitors (∼2.5x higher; p = 0.008) to the infarct border-zone appeared to be the major modes-of-repair. This is the first report using a pre-clinical large animal-model to demonstrate the safety and efficacy of cardiopoietic stem cells for the treatment of post-infarction LV-dysfunction to prevent negative LV-remodeling and subsequent CHF. It further provides insight into post-delivery cardiopoietic cell-fate and suggests the mechanisms of cardiopoietic cell-induced cardiac-repair. The adoption of GMP-/GLP-compliant methodologies may accelerate the translation into a phase-I clinical-trial in patients with post-ischemic LV-dysfunction broadening the current indication of this interesting cell-type.

In vitro fabrication of autologous living tissue-engineered vascular grafts based on prenatally harvested ovine amniotic fluid-derived stem cells.

Amniotic fluid cells (AFCs) have been proposed as a valuable source for tissue engineering and regenerative medicine. However, before clinical implementation, rigorous evaluation of this cell source in clinically relevant animal models accepted by regulatory authorities is indispensable. Today, the ovine model represents one of the most accepted preclinical animal models, in particular for cardiovascular applications. Here, we investigate the isolation and use of autologous ovine AFCs as cell source for cardiovascular tissue engineering applications. Fetal fluids were aspirated in vivo from pregnant ewes (n = 9) and from explanted uteri post mortem at different gestational ages (n = 91). Amniotic non-allantoic fluid nature was evaluated biochemically and in vivo samples were compared with post mortem reference samples. Isolated cells revealed an immunohistochemical phenotype similar to ovine bone marrow-derived mesenchymal stem cells (MSCs) and showed expression of stem cell factors described for embryonic stem cells, such as NANOG and STAT-3. Isolated ovine amniotic fluid-derived MSCs were screened for numeric chromosomal aberrations and successfully differentiated into several mesodermal phenotypes. Myofibroblastic ovine AFC lineages were then successfully used for the in vitro fabrication of small- and large-diameter tissue-engineered vascular grafts (n = 10) and cardiovascular patches (n = 34), laying the foundation for the use of this relevant pre-clinical in vivo assessment model for future amniotic fluid cell-based therapeutic applications.

Clonal restriction and predominance of regulatory T cells in coronary thrombi of patients with acute coronary syndromes.

AIMS: Regulatory T cells (Treg) exert anti-inflammatory and atheroprotective effects in experimental atherosclerosis. Treg can be induced against specific antigens using immunization strategies associated with clonal restriction. No data exist on Treg in combination with clonal restriction of T cells in patients with acute coronary syndromes (ACS). METHODS AND RESULTS: Among T cell subsets characterized by flow cytometry, Treg (CD4(+) CD25(+) CD127(low)) were twice as frequent in coronary thrombi compared with peripheral blood. Treg prevailed among T cell subsets identified in coronary thrombi. To evaluate clonal restriction, genomic DNA was extracted from coronary thrombi and peripheral blood in order to evaluate T cell receptor (TCR) ß chain diversity by means of Multi-N-plex PCR using a primer specific for all TCR ß V gene segments and another primer specific for TCR ß J gene segments. T cell receptor diversity was reduced in thrombi compared with peripheral blood (intra-individual comparisons in 16 patients) with 8 gene rearrangements in the TCR common in at least 6 out of 16 analysed coronary thrombi. Compared with age-matched healthy controls (n = 16), TCR diversity was also reduced in peripheral blood of patients with ACS; these findings were independent of peripheral T cell numbers. CONCLUSION: We provide novel evidence for a perturbed T cell compartment characterized by clonal restriction in peripheral blood and coronary thrombi from patients with ACS. Our findings warrant further studies on Treg as novel therapeutic targets aimed at enhancing this anti-inflammatory component of adaptive immunity in human atherothrombosis.

Deletion of Sirt3 does not affect atherosclerosis but accelerates weight gain and impairs rapid metabolic adaptation in LDL receptor knockout mice: implications for cardiovascular risk factor development.

Sirt3 is a mitochondrial NAD(+)-dependent deacetylase that governs mitochondrial metabolism and reactive oxygen species homeostasis. Sirt3 deficiency has been reported to accelerate the development of the metabolic syndrome. However, the role of Sirt3 in atherosclerosis remains enigmatic. We aimed to investigate whether Sirt3 deficiency affects atherosclerosis, plaque vulnerability, and metabolic homeostasis. Low-density lipoprotein receptor knockout (LDLR(-/-)) and LDLR/Sirt3 double-knockout (Sirt3(-/-)LDLR(-/-)) mice were fed a high-cholesterol diet (1.25 % w/w) for 12 weeks. Atherosclerosis was assessed en face in thoraco-abdominal aortae and in cross sections of aortic roots. Sirt3 deletion led to hepatic mitochondrial protein hyperacetylation. Unexpectedly, though plasma malondialdehyde levels were elevated in Sirt3-deficient mice, Sirt3 deletion affected neither plaque burden nor features of plaque vulnerability (i.e., fibrous cap thickness and necrotic core diameter). Likewise, plaque macrophage and T cell infiltration as well as endothelial activation remained unaltered. Electron microscopy of aortic walls revealed no difference in mitochondrial microarchitecture between both groups. Interestingly, loss of Sirt3 was associated with accelerated weight gain and an impaired capacity to cope with rapid changes in nutrient supply as assessed by indirect calorimetry. Serum lipid levels and glucose tolerance were unaffected by Sirt3 deletion in LDLR(-/-) mice. Sirt3 deficiency does not affect atherosclerosis in LDLR(-/-) mice. However, Sirt3 controls systemic levels of oxidative stress, limits expedited weight gain, and allows rapid metabolic adaptation. Thus, Sirt3 may contribute to postponing cardiovascular risk factor development.

Off-the-shelf human decellularized tissue-engineered heart valves in a non-human primate model.

Heart valve tissue engineering based on decellularized xenogenic or allogenic starter matrices has shown promising first clinical results. However, the availability of healthy homologous donor valves is limited and xenogenic materials are associated with infectious and immunologic risks. To address such limitations, biodegradable synthetic materials have been successfully used for the creation of living autologous tissue-engineered heart valves (TEHVs) in vitro. Since these classical tissue engineering technologies necessitate substantial infrastructure and logistics, we recently introduced decellularized TEHVs (dTEHVs), based on biodegradable synthetic materials and vascular-derived cells, and successfully created a potential off-the-shelf starter matrix for guided tissue regeneration. Here, we investigate the host repopulation capacity of such dTEHVs in a non-human primate model with up to 8 weeks follow-up. After minimally invasive delivery into the orthotopic pulmonary position, dTEHVs revealed mobile and thin leaflets after 8 weeks of follow-up. Furthermore, mild-moderate valvular insufficiency and relative leaflet shortening were detected. However, in comparison to the decellularized human native heart valve control - representing currently used homografts - dTEHVs showed remarkable rapid cellular repopulation. Given this substantial in situ remodeling capacity, these results suggest that human cell-derived bioengineered decellularized materials represent a promising and clinically relevant starter matrix for heart valve tissue engineering. These biomaterials may ultimately overcome the limitations of currently used valve replacements by providing homologous, non-immunogenic, off-the-shelf replacement constructs.

Human stem cell-based three-dimensional microtissues for advanced cardiac cell therapies.

Cardiac stem cell therapy has been proposed as a therapy option to treat the diseased myocardium. However, the low retention rate of transplanted single-cell suspensions remains a major issue of current therapy strategies. Therefore, the concept of scaffold-free cellular self-assembly into three-dimensional microtissues (3D-MTs) prior to transplantation may be beneficial to enhance retention and survival. We compared clinically relevant, human stem cell sources for their ability to generate 3D-MTs with particular regards to formation characteristics, proliferation-activity, viability and extracellular-matrix production. Single-cell suspensions of human bone marrow- and adipose tissue-derived mesenchymal stem cells (hBMMSCs and hATMSCs), Isl1(+) cardiac progenitors derived from human embryonic stem cells (hESC-Isl1(+) cells), and undifferentiated human induced pluripotent cells (hiPSCs) were characterized before to generate 3D-MTs using a hanging-drop culture. Besides the principal feasibility of cell-specific 3D-MT formation, a detailed head-to-head comparison between cell sources was performed using histology, immunocyto- and histo-chemistry as well as flow cytometry. Round-oval shaped and uniform 3D-MTs could be successfully generated from all cell types starting with a loose formation within the first 24 h that fully stabilized after 3 days and resulting in a mean 3D-MT diameter of 194.56 ± 18.01 µm (hBMMSCs), 194.56 ± 16.30 µm (hATMSCs), 159.73 ± 19.20 µm (hESC-Isl1(+) cells) and 120.95 ± 7.97 µm (hiPSCs). While all 3D-MTs showed a homogenous cell distribution, hiPSC-derived 3D-MTs displayed a compact cell formation primarily located at the outer margin. hESC-Isl1(+) and hiPSC-derived 3D-MTs maintained their proliferation-activity which was rather limited in the MSC-based 3D-MTs. All four 3D-MT types revealed a comparable viability in excess of 70% and showed a cell-specific expression profile being comparable to their single-cell counterparts. Extracellular matrix (ECM) production during 3D-MT formation was observed for all cell-specific 3D-MTs, with hiPSC-derived 3D-MTs being the fastest one. Interestingly, ECM distribution was homogenous for hATMSC- and hiPSC-based 3D-MTs, while it appeared to be primarily concentrated within in the center of hESC-Isl1(+) and hBMMSC-based 3D-MTs. The results of this head-to-head comparative study indicated that 3D-MTs can be successfully generated from hESC-derived Isl1(+) cells, hiPSCs and MSC lines upon hanging drop culture. Cell-specific 3D-MTs displayed sufficient viability and instant ECM formation. The concept of 3D-MT in vitro generation prior to cell transplantation may represent a promising delivery format for future strategies to enhance cellular engraftment and survival.

Systemic VEGF inhibition accelerates experimental atherosclerosis and disrupts endothelial homeostasis--implications for cardiovascular safety.

OBJECTIVES: This study sought to examine the effects and underlying mechanisms of systemic VEGF inhibition in experimental atherosclerosis and aortic endothelial cells. BACKGROUND: Pharmacological inhibition of vascular endothelial growth factor (VEGF), a major mediator of angiogenesis, has become a widely applied treatment of certain cancers and multiple ocular diseases including age-related macular degeneration. However, recent clinical trials raise concern for systemic vascular adverse effects, prompting the Food and Drug Administration to revoke the approval of bevacizumab for metastatic breast cancer. METHODS: Eight-week old apolipoprotein E knockout mice received a high-cholesterol diet (1.25% cholesterol) for 24 weeks and were exposed to a systemic pan-VEGF receptor inhibitor (PTK787/ZK222584, 50mg/kg/d) or placebo (gavage) for the last 10 weeks. Atherosclerotic lesions were characterized in thoraco-abdominal aortae and aortic arches. Mechanistic analyses were performed in cultured human aortic endothelial cells. RESULTS: Systemic VEGF inhibition increased atherosclerotic lesions by 33% whereas features of plaque vulnerability (i.e. necrotic core size, fibrous cap thickness) remained unchanged compared with controls. Aortic eNOS expression was decreased (trend). In human endothelial cells VEGF inhibition induced a dose-dependent increase in mitochondrial superoxide generation with an uncoupling of eNOS, resulting in reduced NO availability and decreased proliferation. CONCLUSION: Systemic VEGF inhibition disrupts endothelial homeostasis and accelerates atherogenesis, suggesting that these events contribute to the clinical cardiovascular adverse events of VEGF-inhibiting therapies. Cardiovascular safety profiles of currently applied anti-angiogenic regimens should be determined to improve patient selection for therapy and allow close monitoring of patients at increased cardiovascular risk.

Intramyocardial transplantation and tracking of human mesenchymal stem cells in a novel intra-uterine pre-immune fetal sheep myocardial infarction model: a proof of concept study.

Although stem-cell therapies have been suggested for cardiac-regeneration after myocardial-infarction (MI), key-questions regarding the in-vivo cell-fate remain unknown. While most available animal-models require immunosuppressive-therapy when applying human cells, the fetal-sheep being pre-immune until day 75 of gestation has been proposed for the in-vivo tracking of human cells after intra-peritoneal transplantation. We introduce a novel intra-uterine myocardial-infarction model to track human mesenchymal stem cells after direct intra-myocardial transplantation into the pre-immune fetal-sheep. Thirteen fetal-sheep (gestation age: 70-75 days) were included. Ten animals either received an intra-uterine induction of MI only (n = 4) or MI+intra-myocardial injection (IMI;n = 6) using micron-sized, iron-oxide (MPIO) labeled human mesenchymal stem cells either derived from the adipose-tissue (ATMSCs;n = 3) or the bone-marrow (BMMSCs;n = 3). Three animals received an intra-peritoneal injection (IPI;n = 3; ATMSCs;n = 2/BMMSCs;n = 1). All procedures were performed successfully and follow-up was 7-9 days. To assess human cell-fate, multimodal cell-tracking was performed via MRI and/or Micro-CT, Flow-Cytometry, PCR and immunohistochemistry. After IMI, MRI displayed an estimated amount of 1×10(5)-5×10(5) human cells within ventricular-wall corresponding to the injection-sites which was further confirmed on Micro-CT. PCR and IHC verified intra-myocardial presence via detection of human-specific ß-2-microglobulin, MHC-1, ALU-Sequence and anti-FITC targeting the fluorochrome-labeled part of the MPIOs. The cells appeared viable, integrated and were found in clusters or in the interstitial-spaces. Flow-Cytometry confirmed intra-myocardial presence, and showed further distribution within the spleen, lungs, kidneys and brain. Following IPI, MRI indicated the cells within the intra-peritoneal-cavity involving the liver and kidneys. Flow-Cytometry detected the cells within spleen, lungs, kidneys, thymus, bone-marrow and intra-peritoneal lavage, but not within the heart. For the first time we demonstrate the feasibility of intra-uterine, intra-myocardial stem-cell transplantation into the pre-immune fetal-sheep after MI. Utilizing cell-tracking strategies comprising advanced imaging-technologies and in-vitro tracking-tools, this novel model may serve as a unique platform to assess human cell-fate after intra-myocardial transplantation without the necessity of immunosuppressive-therapy.

Stem cell-based transcatheter aortic valve implantation: first experiences in a pre-clinical model.

OBJECTIVES: This study sought to investigate the combination of transcatheter aortic valve implantation and a novel concept of stem cell-based, tissue-engineered heart valves (TEHV) comprising minimally invasive techniques for both cell harvest and valve delivery. BACKGROUND: TAVI represents an emerging technology for the treatment of aortic valve disease. The used bioprostheses are inherently prone to calcific degeneration and recent evidence suggests even accelerated degeneration resulting from structural damage due to the crimping procedures. An autologous, living heart valve prosthesis with regeneration and repair capacities would overcome such limitations. METHODS: Within a 1-step intervention, trileaflet 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). Planned follow-up was 4 h (Group A, n = 4), 48 h (Group B, n = 5) or 1 and 2 weeks (Group C, n = 3). TEHV functionality was assessed by fluoroscopy, echocardiography, and computed tomography. Post-mortem analysis was performed using histology, extracellular matrix analysis, and electron microscopy. RESULTS: Transapical implantation of TEHV was successful in all animals (n = 12). Follow-up was complete in all animals of Group A, three-fifths of Group B, and two-thirds of Group C (1 week, n = 1; 2 weeks, n = 1). Fluoroscopy and echocardiography displayed TEHV functionality demonstrating adequate leaflet mobility and coaptation. TEHV showed intact leaflet structures with well-defined cusps without signs of thrombus formation or structural damage. Histology and extracellular matrix displayed a high cellularity indicative for an early cellular remodeling and in-growth after 2 weeks. CONCLUSIONS: We demonstrate the principal feasibility of a transcatheter, stem cell-based TEHV implantation into the aortic valve position within a 1-step intervention. Its long-term functionality proven, a stem cell-based TEHV approach may represent a next-generation heart valve concept.

Functionality, growth and accelerated aging of tissue engineered living autologous vascular grafts.

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.

Prenatally engineered autologous amniotic fluid stem cell-based heart valves in the fetal circulation.

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.

Fetal trans-apical stent delivery into the pulmonary artery: prospects for prenatal heart-valve implantation.

OBJECTIVE: The purpose of this study was to assess the technical feasibility of a fetal trans-apical stent delivery into the pulmonary artery using a novel hybrid-intervention technique as a possible route for prenatal minimally invasive heart-valve-implantation approaches. METHODS: Pregnant Pre-Alp sheep between 122 and 128 days' gestation (n = 3) underwent a midline laparotomy. The fetus was left in utero or partially externalized and its chest was opened via a left-sided minithoracotomy. The fetal heart was cannulated and a guide wire was introduced through the ductus arteriosus into the aorta. A 14-French delivery system was then mounted onto the guide wire and advanced to the landing zone in the pulmonary artery, where the stent was deployed. The position of the stent was confirmed using echocardiography, angiography as well as computed tomography. RESULTS: The trans-apical implantation was successful in all animals. However, at necropsy in one animal, the stent was found to partly occlude one of the pulmonary valvular leaflets. Bleeding at the antero-apical incision occurred in all animals but could be managed without fetal demise. No fetal cardiopulmonary bypass was performed. In all animals, contrast angiography displayed normal perfusion of the pulmonary vasculature as well as the ductus arteriosus. CONCLUSIONS: Our study demonstrates the principal technical feasibility of a prenatal stent delivery into the pulmonary artery using a novel trans-apical hybrid-intervention technique. This approach demonstrates the first step towards possible future minimally invasive prenatal heart-valve-implantation procedures.

Injectable living marrow stromal cell-based autologous tissue engineered heart valves: first experiences with a one-step intervention in primates.

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.

Fibroblast activation protein is induced by inflammation and degrades type I collagen in thin-cap fibroatheromata.

AIMS: Collagen degradation in atherosclerotic plaques with thin fibrous caps renders them more prone to rupture. Fibroblast activation protein (FAP) plays a role in arthritis and tumour formation through its collagenase activity. However, the significance of FAP in thin-cap human fibroatheromata remains unknown. METHODS AND RESULTS: We detected enhanced FAP expression in type IV-V human aortic atheromata (n = 12), compared with type II-III lesions (n = 9; P < 0.01) and healthy aortae (n = 8; P < 0.01) by immunostaining and western blot analyses. Fibroblast activation protein was also increased in thin-cap (<65 µm) vs. thick-cap (≥ 65 µm) human coronary fibroatheromata (n = 12; P < 0.01). Fibroblast activation protein was expressed by human aortic smooth muscle cells (HASMC) as shown by colocalization on immunofluorescent aortic plaque stainings (n = 10; P < 0.01) and by flow cytometry in cell culture. Although macrophages did not express FAP, macrophage burden in human aortic plaques correlated with FAP expression (n = 12; R(2)= 0.763; P < 0.05). Enzyme-linked immunosorbent assays showed a time- and dose-dependent up-regulation of FAP in response to human tumour necrosis factor α (TNFα) in HASMC (n = 6; P < 0.01). Moreover, supernatants from peripheral blood-derived macrophages induced FAP expression in cultured HASMC (n = 6; P < 0.01), an effect abolished by blocking TNFα (n = 6; P < 0.01). Fibroblast activation protein associated with collagen-poor regions in human coronary fibrous caps and digested type I collagen and gelatin in vitro (n = 6; P < 0.01). Zymography revealed that FAP-mediated collagenase activity was neutralized by an antibody directed against the FAP catalytic domain both in HASMC (n = 6; P < 0.01) and in fibrous caps of atherosclerotic plaques (n = 10; P < 0.01). CONCLUSION: Fibroblast activation protein expression in HASMC is induced by macrophage-derived TNFα. Fibroblast activation protein associates with thin-cap human coronary fibroatheromata and contributes to type I collagen breakdown in fibrous caps.

Tissue engineering on matrix: future of autologous tissue replacement.

Tissue engineering aims at the creation of living neo-tissues identical or close to their native human counterparts. As basis of this approach, temporary biodegradable supporter matrices are fabricated in the shape of a desired construct, which promote tissue strength and provide functionality until sufficient neo-tissue is formed. Besides fully synthetic polymer-based scaffolds, decellularized biological tissue of xenogenic or homogenic origin can be used. In a second step, these scaffolds are seeded with autologous cells attaching to the scaffold microstructure. In order to promote neo-tissue formation and maturation, the seeded scaffolds are exposed to different forms of stimulation. In cardiovascular tissue engineering, this "conditioning" can be achieved via culture media and biomimetic in vitro exposure, e.g., using flow bioreactors. This aims at adequate cellular differentiation, proliferation, and extracellular matrix production to form a living tissue called the construct. These living autologous constructs, such as heart valves or vascular grafts, are created in vitro, comprising a viable interstitium with repair and remodeling capabilities already prior to implantation. In situ further in vivo remodeling is intended to recapitulate physiological vascular architecture and function. The remodeling mechanisms were shown to be dominated by monocytic infiltration and chemotactic host-cell attraction leading into a multifaceted inflammatory process and neo-tissue formation. Key molecules of these processes can be integrated into the scaffold matrix to direct cell and tissue fate in vivo.

EH-myomesin splice isoform is a novel marker for dilated cardiomyopathy.

The M-band is the prominent cytoskeletal structure that cross-links the myosin and titin filaments in the middle of the sarcomere. To investigate M-band alterations in heart disease, we analyzed the expression of its main components, proteins of the myomesin family, in mouse and human cardiomyopathy. Cardiac function was assessed by echocardiography and compared to the expression pattern of myomesins evaluated with RT-PCR, Western blot, and immunofluorescent analysis. Disease progression in transgenic mouse models for dilated cardiomyopathy (DCM) was accompanied by specific M-band alterations. The dominant splice isoform in the embryonic heart, EH-myomesin, was strongly up-regulated in the failing heart and correlated with a decrease in cardiac function (R = -0.86). In addition, we have analyzed the expressions of myomesins in human myocardial biopsies (N = 40) obtained from DCM patients, DCM patients supported by a left ventricular assist device (LVAD), hypertrophic cardiomyopathy (HCM) patients and controls. Quantitative RT-PCR revealed that the EH-myomesin isoform was up-regulated 41-fold (P < 0.001) in the DCM patients compared to control patients. In DCM hearts supported by a LVAD and HCM hearts, the EH-myomesin expression was comparable to controls. Immunofluorescent analyses indicate that EH-myomesin was enhanced in a cell-specific manner, leading to a higher heterogeneity of the myocytes' cytoskeleton through the myocardial wall. We suggest that the up-regulation of EH-myomesin denotes an adaptive remodeling of the sarcomere cytoskeleton in the dilated heart and might serve as a marker for DCM in mouse and human myocardium.

Upregulation of transmembrane endothelial junction proteins in human cerebral cavernous malformations.

OBJECT: Cerebral cavernous malformations (CCMs) are among the most prevalent cerebrovascular malformations, and endothelial cells seem to play a major role in the disease. However, the underlying mechanisms, including endothelial intercellular communication, have not yet been fully elucidated. In this article, the authors focus on the endothelial junction proteins CD31, VE-cadherin, and occludin as important factors for functional cell-cell contacts known as vascular adhesion molecules and adherence and tight junctions. METHODS: Thirteen human CCM specimens and 6 control tissue specimens were cryopreserved and examined for the presence of VE-cadherin, occludin, and CD31 by immunofluorescence staining. Protein quantification was performed by triplicate measurements using western blot analysis. RESULTS: Immunofluorescent analyses of the CCM sections revealed a discontinuous pattern of dilated microvessels and capillaries as well as increased expression of occludin, VE-cadherin, and CD31 in the intima and in the enclosed parenchymal tissue compared with controls. Protein quantification confirmed these findings by showing upregulation of the levels of these proteins up to 2-6 times. CONCLUSIONS: A protocol enabling the molecular and morphological examination of the intercellular contact proteins in human CCM was validated. The abnormal and discontinuous pattern in these endothelial cell-contact proteins compared with control tissue explains the loose intercellular junctions that are considered to be one of the causes of CCM-associated bleeding or transendothelial oozing of erythrocytes. Despite the small number of specimens, this study demonstrates for the first time a quantitative analysis of endothelial junction proteins in human CCM.

Effects of small pulsed nanocurrents on cell viability in vitro and in vivo: implications for biomedical electrodes.

Using a custom-built, implantable pulse generator, we studied the effects of small pulsed currents on the viability on rat aortic-derived cells (RAOC) in vitro. The pulsed currents (0.37A/m(2)) underwent apoptosis within 24h as shown by the positive staining for cleaved caspase-3 and classically apoptotic morphology. Based on these findings, we examined the effects of nanocurrents in vivo. The pulse generator was implanted subcutaneously in the rat model. The electrode|tissue interface histology revealed no difference between the active platinum surface and the neighboring control surface, however we found a large difference between electrodes that were functional during the entire experiment and non-active electrodes. These non-active electrodes showed an increase in impedance at higher frequencies 21 days post-implantation, whereas working electrodes retained their impedance value for the entire experiment. These results indicate that applied currents can reduce the impedance of implanted electrodes.