Tissue-regenerative potential of the secretome of γ-irradiated peripheral blood mononuclear cells is mediated via TNFRSF1B-induced necroptosis.

Peripheral blood mononuclear cells (PBMCs) have been shown to produce and release a plethora of pro-angiogenetic factors in response to γ-irradiation, partially accounting for their tissue-regenerative capacity. Here, we investigated whether a certain cell subtype of PBMCs is responsible for this effect, and whether the type of cell death affects the pro-angiogenic potential of bioactive molecules released by γ-irradiated PBMCs. PBMCs and PBMC subpopulations, including CD4+ and CD8+ T cells, B cells, monocytes, and natural killer cells, were isolated and subjected to high-dose γ-irradiation. Transcriptome analysis revealed subpopulation-specific responses to γ-irradiation with distinct activation of pro-angiogenic pathways, cytokine production, and death receptor signalling. Analysis of the proteins released showed that interactions of the subsets are important for the generation of a pro-angiogenic secretome. This result was confirmed at the functional level by the finding that the secretome of γ-irradiated PBMCs displayed higher pro-angiogenic activity in an aortic ring assay. Scanning electron microscopy and image stream analysis of γ-irradiated PBMCs revealed distinct morphological changes, indicative for apoptotic and necroptotic cell death. While inhibition of apoptosis had no effect on the pro-angiogenic activity of the secretome, inhibiting necroptosis in stressed PBMCs abolished blood vessel sprouting. Mechanistically, we identified tumor necrosis factor (TNF) receptor superfamily member 1B as the main driver of necroptosis in response to γ-irradiation in PBMCs, which was most likely mediated via membrane-bound TNF-α. In conclusion, our study demonstrates that the pro-angiogenic activity of the secretome of γ-irradiated PBMCs requires interplay of different PBMC subpopulations. Furthermore, we show that TNF-dependent necroptosis is an indispensable molecular process for conferring tissue-regenerative activity and for the pro-angiogenic potential of the PBMC secretome. These findings contribute to a better understanding of secretome-based therapies in regenerative medicine

Effect of Ischemic Preconditioning and Postconditioning on Exosome-Rich Fraction microRNA Levels, in Relation with Electrophysiological Parameters and Ventricular Arrhythmia in Experimental Closed-Chest Reperfused Myocardial Infarction

We investigated the antiarrhythmic effects of ischemic preconditioning (IPC) and postconditioning (PostC) by intracardiac electrocardiogram (ECG) and measured circulating microRNAs (miRs) that are related to cardiac conduction. Domestic pigs underwent 90-min. percutaneous occlusion of the mid left anterior coronary artery, followed by reperfusion. The animals were divided into three groups: acute myocardial infarction (AMI, n = 7), ischemic preconditioning-acute myocardial infarction (IPC-AMI) (n = 9), or AMI-PostC (n = 5). IPC was induced by three 5-min. episodes of repetitive ischemia/reperfusion cycles (rI/R) before AMI. PostC was induced by six 30-s rI/R immediately after induction of reperfusion 90 min after occlusion. Before the angiographic procedure, a NOGA endocardial mapping catheter was placed again the distal anterior ventricular endocardium to record the intracardiac electrogram (R-amplitude, ST-Elevation, ST-area under the curve (AUC), QRS width, and corrected QT time (QTc)) during the entire procedure. An arrhythmia score was calculated. Cardiac MRI was performed after one-month. IPC led to significantly lower ST-elevation, heart rate, and arrhythmia score during ischemia. PostC induced a rapid recovery of R-amplitude, decrease in QTc, and lower arrhythmia score during reperfusion. Slightly higher levels of miR-26 and miR-133 were observed in AMI compared to groups IPC-AMI and AMI-PostC. Significantly lower levels of miR-1, miR-208, and miR-328 were measured in the AMI-PostC group as compared to animals in group AMI and IPC-AMI. The arrhythmia score was not significantly associated with miRNA plasma levels. Cardiac MRI showed significantly smaller infarct size in the IPC-AMI group when compared to the AMI and AMI-PostC groups. Thus, IPC led to better left ventricular ejection fraction at one-month and it exerted antiarrhythmic effects during ischemia, whereas PostC exhibited antiarrhythmic properties after reperfusion, with significant downregulaton of ischemia-related miRNAs

Transcriptional Alterations by Ischaemic Postconditioning in a Pig Infarction Model: Impact on Microvascular Protection

Although the application of cardioprotective ischaemia/reperfusion (I/R) stimuli after myocardial infarction (MI) is a promising concept for salvaging the myocardium, translation to a clinical scenario has not fulfilled expectations. We have previously shown that in pigs, ischaemic postconditioning (IPostC) reduces myocardial oedema and microvascular obstruction (MVO), without influencing myocardial infarct size. In the present study, we analyzed the mechanisms underlying the IPostC-induced microvascular protection by transcriptomic analysis, followed by pathway analysis. Closed-chest reperfused MI was induced by 90 min percutaneous balloon occlusion of the left anterior descending coronary artery, followed by balloon deflation in anaesthetised pigs. Animals were randomised to IPostC (n = 8), MI (non-conditioned, n = 8), or Control (sham-operated, n = 4) groups. After three hours or three days follow-up, myocardial tissue samples were harvested and subjected to RNA-seq analysis. Although the transcriptome analysis revealed similar expression between IPostC and MI in transcripts involved in cardioprotective pathways, we identified gene expression changes responding to IPostC at the three days follow-up. Focal adhesion signaling, downregulated genes participating in cardiomyopathy and activation of blood cells may have critical consequences for microvascular protection. Specific analyses of the gene subsets enriched in the endothelium of the infarcted area, revealed strong deregulation of transcriptional functional clusters, DNA processing, replication and repair, cell proliferation, and focal adhesion, suggesting sustentative function in the endothelial cell layer protection and integrity. The spatial and time-dependent transcriptome analysis of porcine myocardium supports a protective effect of IPostC on coronary microvasculature post-MI

Characterization of the molecular mechanisms underlying interaction of cells with the fibrin sealant matrix

Wundheilung ist ein komplexer Prozess mit streng regulierten Mechanismen zur Minimierung des Blutverlusts und zur Rekonstruktion des beschädigten Gewebes. In diesem Prozess spielen verschiedene Zelltypen eine Rolle, wie Endothelzellen, Fibroblasten oder Keratinozyten. Diese Zellen interagieren mit dem primären Thrombus, eine Matrix aus großteils quervernetzten Fibrinfasern, der als erstes Grundgerüst für die einwandernden Zellen dient. Die Interaktionen der Zellen mit diesem Gerüst sowie die Einwanderung der Zellen aus der Gewebeumgebung in diese Matrix stellen eine essentielle Phase der Wundheilung dar. Während dieses Schrittes wird die Fibrinmatrix abgebaut und durch gesundes, körpereigenes Gewebe ersetzt. Fibrinkleber sind als Hämostat in unterschiedlichen chirurgischen Indikationen sowie als Kleber für weiches Gewebe (soft tissue) etabliert. Sie haben gezeigt, dass sie verschiedene Aspekte der Geweberegeneration unterstützen wie etwa Zellmigration oder Reepithelialisierung – ähnlich wie der physiologisch gebildete Blutklot. Fibrinkleber bestehen großteils aus konzentriertem Fibrinogen und Thrombin humanen Ursprungs. Diese beiden Komponenten werden kurz vor der Applikation vermischt. Wegen unterschiedlicher Herstellungsverfahren besitzt jedes Produkt seine eigene charakteristische Formulierung. Unterschiede gibt es z.B. in Konzentrationen von Thrombin, Fibrinogen, Salzen oder Molekülen der extrazellulären Matrix (ECM). In aktuellen klinischen Studien wurden Fibrinkleber auch als Matrix zur Unterstützung der Wundheilung verwendet. Daher untersuchten wir, welche die wichtigsten Komponenten der Fibrinmatrix für optimale Interaktion mit Zellen sind. In dieser Arbeit wurde untersucht, wie sich in der Fibrinmatrix vorhandene Proteine auf Zell-Matrix-Interaktionen auswirken. Zuerst wurden zwei kommerziell erhältliche Fibrinkleber (Artiss und Evicel) mittels in vitro Zelltests auf deren Kompatibilität mit humanen epithelialen Keratinozyten (NHEK) untersucht. NHEK, die mit Evicel und dessen höherer Thrombinkonzentration in Kontakt gebracht wurden, zeigten weniger Adhäsion und Viabilität, eine degenerierte Morphologie, sowie einen höheren Prozentsatz an toten Zellen. Aufgrund der nicht-kovalenten Bindung von Thrombin an Fibrin während der Klotentstehung, wurden die Auswirkungen von fibringebundenem Thrombin auf die Zellviabilität getestet. Anfangs wurde die Aktivität von Thrombin in drei verschiedenen, kommerziell erhältlichen Fibrinklebern gemessen. Mit dieser Information wurden Fibrinklots mit Thrombinkonzentrationen von 4 IU/ml bis 820 IU/ml hergestellt, die sich aber in allen anderen Zusammensetzungen nicht unterschieden. Diese Klots wiesen keine Unterschiede in der Ultrastruktur auf. Bei jenen mit hoher Thrombinkonzentration (820 IU/ml) zeigten NHEK eine schlechtere Adhäsion und weniger Zellspreitung. Die Anzahl der anhaftenden Zellen an den Hochthrombinklot war signifikant niedriger. Der Grund für diese Beobachtungen war nicht nur weniger Proliferation der Zellen, sondern auch erhöhte Apoptose durch maßgeblich verstärkte Expression von Caspase 3 und 7 in Zellen, die auf den Klots mit 820 IU/ml wuchsen. Dies ging einher mit der Induktion der Expression von Trail-R2, ein wichtiger Rezeptor im Apoptosesignalweg. Wurde Thrombin mittels Hirudin geblockt, verbesserte sich die Zellmorphologie und eine größere Zahl an Keratinozyten adhärierte am Fibrinkleber. Weiters war die Expression von Caspase 3 und 7 reduziert. Hier wurde zum ersten Mal beschrieben, dass hohe Konzentrationen von Thrombin im Fibrinkleber nicht nur die Proliferation von Keratinozyten hemmt, sondern auch, dass dies zu vermehrter Apoptose von NHEK führt. Darauf aufbauend wurden Fibrinkleber in einem Exzisionswundheilungsmodell in Ratten getestet. Das Ziel der Studie war, verschiedene Fibrinkleber, sowie unterschiedliche Formulierungen, auf deren Effekt auf die Wundheilung von Ratten zu untersuchen. Wunden, die mit einer geringeren Thrombinkonzentration behandelt wurden, zeigten schnelleren Wundverschluss (an den Tagen 3 und 7) und eine geringere Schwere der Wunden (am Tag 7) verglichen mit Wunden, die mit hohen Thrombinkonzentrationen behandelt wurden. Weiters konnte eine geringere Anzahl an funktionellen Gefäßen in Wunden mit Evicel-Kleber gezählt werden. Diese Beobachtung passt zu den Ergebnissen, dass der Level an VEGF in Artiss-behandelten Wunden am Tag 2 höher war. Diese in vivo Resultate können teilweise mit den Beobachtungen zur Zellkompatibilität in vitro erklärt werden. Darüber hinaus zeigte eine Untersuchung des Fibrinabbaus, dass der Artiss-Klot nach zehn Tagen resorbiert wurde, der Evicel-Klot nach 15 Tagen. Als weiteren Aspekt wurde die Auswirkung von Fibronektin auf Zell-Matrix-Interaktionen untersucht. Da Keratinozyten keinen Integrinrezeptor ανβ3 exprimieren und dadurch nicht direkt an Fibrin binden können, spielen andere ECM-Moleküle wie Fibronektin eine wichtige Rolle in der Zelladhäsion. NHEK zeigten eine schlechtere Anhaftung an den Klot und eine degenerierte Morphologie am Fibrinkleber mit reduzierter Fibronektinkonzentration. Diese Resultate konnten durch Zugabe von Fibronektin in den Klot verbessert werden. Darüber hinaus wurde die Auswirkung von anderen Bestandteilen des Fibrinklebers auf die Klotstruktur und Zellkompatibilität untersucht. Durch Veränderung der Salzkonzentration oder die Verwendung von Fucoidanen anstelle von Thrombin konnte die Struktur des Klots signifikant modifiziert werden. Trotzdem blieb die Zellkompatibilität unverändert. Dies legt nahe, dass die Struktur nicht so wichtig für Zell-Matrix-Interaktionen ist wie die Zusammensetzung bestimmter Inhaltsstoffe der Matrix. Fibrinkleber sind wichtige und gut etablierte Werkzeuge in der Chirurgie. Das Ziel dieser Doktorarbeit ist, die Prinzipien und Signalwege der Zell-Matrix-Interaktionen besser zu verstehen. Eine Anzahl an Studien wurde durchgeführt um die Zusammensetzung und Charakteristik von Fibrinklebern zu optimieren und dadurch ein Produkt zu entwickeln, das chirurgischen Ansprüchen entspricht und Produktsicherheit gewährleistet. Die Ergebnisse dieses Projekts sollen zu einer weiteren Verbesserung der Fibrinmatrix führen- zur Unterstützung der Geweberegeneration.Wound healing is a complex process with strictly regulated mechanisms to minimize blood loss and to reconstruct the damaged tissue. In this process several cell types such as endothelial cells, fibroblasts, and keratinocytes interact with the primary thrombus that basically consists of a matrix of cross-linked fibrin fibers serving as scaffold for invading cells. Following the initial hemostasis, interaction with this scaffold and ingrowth of cells into the fibrin matrix from tissue surrounding the wound bed is essential for the subsequent stages of the wound healing process. By this process fibrin matrix is degraded and finally replaced by healthy tissue. Fibrin sealants are widely used as hemostats in various surgical indications and for sealing of soft tissue. Fibrin sealants have also been shown to promote different aspects of tissue regeneration such as cell migration and re-epithelialization in a similar way as their physiological counterpart, the blood clot. Fibrin sealants mostly consist of plasma derived concentrated fibrinogen and thrombin solutions that are mixed immediately before application. Due to different purification procedures each product has its characteristic formulation. Differences exist e.g. in the concentration of thrombin, fibrinogen, salts or ECM molecules. In recent clinical studies fibrin sealants have been used as a matrix to promote wound healing. This raised our interest to examine what are the most important components of a fibrin matrix for optimal interaction of the extracellular matrix with cells. In this thesis, we investigated how the presence of proteins present in fibrin sealant affects cell-matrix interaction. First we characterized two commercially available fibrin sealants Artiss and Evicel in their compatibility with normal human epithelial keratinocytes by several in vitro cell assays. NHEK grown on Evicel with its higher thrombin concentration showed less cell adhesion and viability, deteriorated cell morphology and a higher percentage of dead cells. Due to the non-covalent bonding of thrombin to fibrin during fibrin clot formation, we wanted to evaluate the impact of fibrin bound thrombin on cell viability. Initially, we quantified the activity of thrombin in 3 different, commercially available fibrin sealants. This information was used to prepare fibrin clots covering a range of thrombin concentrations from 4 IU/ml to 820 IU/ml, but which were identical with respect to all other constituents. Although these fibrin clots did not differ in their three-dimensional structure, clots prepared with highly concentrated thrombin (820 IU/ml) failed to support adhesion and spreading of primary human keratinocytes (NHEK). The number of attached cells was also significantly reduced on high thrombin activity clots. We hypothesized that these observations are not only the consequence of decreased proliferation but of apoptotic mechanisms, since the expression of cleaved caspase 3 and 7 was strongly enhanced on fibrin clots with high thrombin activity. This was accompanied by an induction of expression of Trail-R2 which is a receptor known to mediate apoptosis signals. Blocking of thrombin activity by hirudin led to an improvement of cell morphology and to an increase in number of attached cells. In addition, the induction of caspase 3 and 7 was also reduced. Thus, here we report for the first time that fibrin bound thrombin does not only decrease proliferation, it also does induce NHEK apoptosis when present at high concentrations. As a consequence and to confirm our in vitro data, fibrin sealants were tested in an excisional wound healing model in rats. The objective of this study was to compare the different fibrin sealants and formations regarding their effects on cells and wound healing in vivo. We found a more rapid wound closure (day3 and day7) and less wound severity (day 7) with sealants containing a lower concentration of thrombin compared to wound healing with high thrombin formulations. Furthermore, less new built functional vessels were counted in Evicel treated wounds after seven days which fits to the result that a lower level of VEGF was expressed after two days in such treated wounds. These in vivo results may be partially explained by the biocompatibility data found in vitro. At last, also fibrin degradation was observed: in animals treated with Artiss, the sealant was lysed after ten days compared to Evicel that needed 15 days. In addition to that, we analyzed the influence of fibronectin on cell-matrix interaction. Since keratinocytes lack of integrin αvβ3 and therefore are not able to bind directly fibrin, other ECM molecules like fibronectin are important for keratinocyte adhesion. We could show that on fibrin sealants depleted of fibronectin, NHEK had less adherence and poor cell morphology and these observations could be improved by addition fibronectin. Moreover, the influences of constituents on clot structure and further consequences on cell compatibility were observed. By changing the salt concentrations or other constituents (fucoidans instead of thrombin) the clot structure could be modified significantly. Nevertheless, cell compatibility remained unchanged upon structure is not so important for cell-matrix interaction as ingredients and constituents of the matrix. Fibrin sealants are important and well established tools in surgery. The aim of this thesis is to help to further understand the principles and pathways of cell-matrix interaction. Numerous studies have been performed to optimize compositions and characteristics of fibrin clots to develop a product which serves clinical requirements and product security guidelines. The results of this project may lead to create a further improved fibrin matrix which activates and promotes tissue regeneration

BioMed Research International / Generation of a Fibrin Based Three-Layered Skin Substitute

A variety of skin substitutes that restore epidermal and dermal structures are currently available on the market. However, the main focus in research and clinical application lies on dermal and epidermal substitutes whereas the development of a subcutaneous replacement (hypodermis) is often disregarded. In this study we used fibrin sealant as hydrogel scaffold to generate a three-layered skin substitute. For the hypodermal layer adipose-derived stem cells (ASCs) and mature adipocytes were embedded in the fibrin hydrogel and were combined with another fibrin clot with fibroblasts for the construction of the dermal layer. Keratinocytes were added on top of the two-layered construct to form the epidermal layer. The three-layered construct was cultivated for up to 3 weeks. Our results show that ASCs and fibroblasts were viable, proliferated normally, and showed physiological morphology in the skin substitute. ASCs were able to differentiate into mature adipocytes during the course of four weeks and showed morphological resemblance to native adipose tissue. On the surface keratinocytes formed an epithelial-like layer. For the first time we were able to generate a three-layered skin substitute based on a fibrin hydrogel not only serving as a dermal and epidermal substitute but also including the hypodermis(VLID)489611

Primary Human Fibroblasts in Culture Switch to a Myofibroblast-Like Phenotype Independently of TGF Beta

Fibroblasts are the prevalent cell type and main source for extracellular matrix (ECM) in connective tissue. Depending on their origin, fibroblasts play a central role in non-pathological tissue remodeling and disease like fibrosis. This study examined the effect of established culture conditions of primary human fibroblasts, from different origins on the myofibroblast-like phenotype formation. We isolated primary human fibroblasts from aortic adventitia, lung, juvenile- and adult skin and investigated the expression levels of CD90, alpha smooth muscle actin (αSMA) and procollagen I under different concentrations of fetal calf serum (FCS) and ascorbic acid (AA) in culture media by immunoblot and immunofluorescence assays. Furthermore, we determined the viability using XTT and migration/wound healing in scratch assays. Collagen 1 secretion was quantified by specific ELISA. Primary human fibroblasts show in part a myofibroblast-like phenotype even without addition of FCS. Supplemented AA reduces migration of cultured fibroblasts with no or low concentrations of FCS. Furthermore, AA and higher concentrations of FCS in culture media lead to higher levels of collagen 1 secretion instead of procollagen I accumulation. This study provides evidence for a partial switch of primary human fibroblasts of different origin to a myofibroblast-like phenotype under common culture conditions

MiR-21, MiR-29a, GATA4, and MEF2c Expression Changes in Endothelin-1 and Angiotensin II Cardiac Hypertrophy Stimulated Isl-1+Sca-1+c-kit+ Porcine Cardiac Progenitor Cells In Vitro

Cost- and time-intensive porcine translational disease models offer great opportunities to test drugs and therapies for pathological cardiac hypertrophy and can be supported by porcine cell culture models that provide further insights into basic disease mechanisms. Cardiac progenitor cells (CPCs) residing in the adult heart have been shown to differentiate in vitro into cardiomyocytes and could contribute to cardiac regeneration. Therefore, it is important to evaluate their changes on the cellular level caused by disease. We successfully isolated Isl1+Sca1+cKit+ porcine CPCs (pCPCs) from pig hearts and stimulated them with endothelin-1 (ET-1) and angiotensin II (Ang II) in vitro. We also performed a cardiac reprogramming transfection and tested the same conditions. Our results show that undifferentiated Isl1+Sca1+cKit+ pCPCs were significantly upregulated in GATA4, MEF2c, and miR-29a gene expressions and in BNP and MCP-1 protein expressions with Ang II stimulation, but they showed no significant changes in miR-29a and MCP-1 when stimulated with ET-1. Differentiated Isl1+Sca1+cKit+ pCPCs exhibited significantly higher levels of MEF2c, GATA4, miR-29a, and miR-21 as well as Cx43 and BNP with Ang II stimulation. pMx-MGT-transfected Isl1+Sca1+cKit+ pCPCs showed significant elevations in MEF2c, GATA4, and BNP expressions when stimulated with ET-1. Our model demonstrates that in vitro stimulation leads to successful Isl1+Sca1+cKit+ pCPC hypertrophy with upregulation of cardiac remodeling associated genes and profibrotic miRNAs and offers great possibilities for further investigations of disease mechanisms and treatment

Electrospun poly(ester-Urethane)- and poly(ester-Urethane-Urea) fleeces as promising tissue engineering scaffolds for adipose-derived stem cells.

An irreversible loss of subcutaneous adipose tissue in patients after tumor removal or deep dermal burns makes soft tissue engineering one of the most important challenges in biomedical research. The ideal scaffold for adipose tissue engineering has yet not been identified though biodegradable polymers gained an increasing interest during the last years. In the present study we synthesized two novel biodegradable polymers, poly(ε-caprolactone-co-urethane-co-urea) (PEUU) and poly[(L-lactide-co-ε-caprolactone)-co-(L-lysine ethyl ester diisocyanate)-block-oligo(ethylene glycol)-urethane] (PEU), containing different types of hydrolytically cleavable bondings. Solutions of the polymers at appropriate concentrations were used to fabricate fleeces by electrospinning. Ultrastructure, tensile properties, and degradation of the produced fleeces were evaluated. Adipose-derived stem cells (ASCs) were seeded on fleeces and morphology, viability, proliferation and differentiation were assessed. The biomaterials show fine micro- and nanostructures composed of fibers with diameters of about 0.5 to 1.3 µm. PEUU fleeces were more elastic, which might be favourable in soft tissue engineering, and degraded significantly slower compared to PEU. ASCs were able to adhere, proliferate and differentiate on both scaffolds. Morphology of the cells was slightly better on PEUU than on PEU showing a more physiological appearance. ASCs differentiated into the adipogenic lineage. Gene analysis of differentiated ASCs showed typical expression of adipogenetic markers such as PPARgamma and FABP4. Based on these results, PEUU and PEU meshes show a promising potential as scaffold materials in adipose tissue engineering

Reduced histologic neo in-stent restenosis after use of a paclitaxel-coated cutting balloon in porcine coronary arteries

The incidence of in-stent restenosis (ISR) has declined dramatically, but once it develops, no current treatment option, such as drug-eluting stents, drug-coated balloons, or cutting balloons (CBs), prevents re-narrowing of the stented atherosclerotic artery. In this preclinical study, we aimed to improve the efficacy of ISR treatment by coating CBs with paclitaxel (paclitaxel-eluting cutting balloon; PECB) and to characterize the histological features of neo-ISRs that arise after ISR treatment. ISR was induced by bare metal stent (BMS) implantation in coronary arteries in pigs. After one month of follow-up, BMS-induced ISR was treated with either CB or PECB. After another month, we performed quantitative coronary angiography, explanted the treated arteries and assessed histopathological and histomorphometric parameters. In addition, we compared histological features of neo-ISRs with pre-treatment ISRs. Injury, inflammation, fibrin deposition, and endothelialization scores were similar between the CB and PECB groups at one month after ISR treatment. Neointimal area (0.87±0.61 vs. 1.95±1.14 mm 2, p=0.02), mean neointimal thickness (0.40±0.39 vs. 0.99±0.56 mm, p=0.01), and percent area stenosis (27.3±20.4 vs. 48.3±22.9%, p=0.04) were decreased in PECB-treated coronary arteries compared to CB-treated arteries, respectively. Density of cells (predominantly smooth muscle cells; SMCs) was increased in neo-ISRs (3.51±3.05×10 3 vs. 6.35±2.57×10 3 cells/mm 2 , p<0.01),but significantly more CD68 + and CD20 + cells were found in pre-treatment ISRs. In conclusion, PECB treatment of ISRs led to better results in terms of smaller neointimal area and %area stenosis of neo-ISR. SMC density was increased in neo-ISRs in contrast with higher percentage of CD68 + and CD20 + cells in pre- treatment ISRs

Quantitative hybrid cardiac [18F] FDG-PET-MRI images for assessment of cardiac repair by preconditioned cardiosphere-derived cells

Cardiosphere-derived cells (CDCs) are progenitor cells derived from heart tissue and have shown promising results in preclinical models. APOSEC, the secretome of irradiated peripheral blood mononuclear cells, has decreased infarct size in acute and chronic experimental myocardial infarction (MI). We enhanced the effect of CDCs with APOSEC preconditioning (apoCDC) and investigated the reparative effect in a translational pig model of reperfused MI. Supernatants of CDCs, assessed by proteomic analysis, revealed reduced production of extracellular matrix proteins after in vitro APOSEC preconditioning. In a porcine model of catheter-based reperfused anterior acute MI (AMI), CDCs with (apoCDC, n = 8) or without APOSEC preconditioning (CDC, n = 6) were infused intracoronary, 15 min after the start of reperfusion. Untreated AMI animals (n = 7) and sham procedures (n = 5) functioned as controls. 2-deoxy-2-(18 F)-fluoro-D-glucose-positron emission tomography-magnetic resonance imaging ([$^{18}$F]FDG-PET-MRI), with late enhancement after 1 month, showed reduced scar volume and lower transmurality of the infarcted area in CDC and apoCDC compared to AMI controls. Segmental quantitative PET images displayed indicated more residual viability in apoCDC. The left-ventricle (LV) ejection fraction was improved nonsignificantly to 45.8% ± 8.6% for apoCDC and 43.5% ± 7.1% for CDCs compared to 38.5% ± 4.4% for untreated AMI. Quantitative hybrid [$^{18}$F]FDG-PET-MRI demonstrated improved metabolic and functional recovery after CDC administration, whereas apoCDCs induced preservation of viability of the infarcted area