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+ CD127low) 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) β chain diversity by means of Multi-N-plex PCR using a primer specific for all TCR β V gene segments and another primer specific for TCR β 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 atherothrombosi

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; R2= 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 cap

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

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

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

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 invitro production of TEVGs requires extensive exvivo 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 invitro 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

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

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