Kardioprotektion nach Ischämie/Reperfusion durch embryonale endotheliale Progenitorzellen

Der akute Myokardinfarkt stellt in den Industrienationen immer noch eine der häufigsten Todesursachen dar. Auch nach Wiedereröffnen des Gefäßes führt eine prolongierte myokardiale Ischämie zur Ausbildung eines Infarktareals. Neben der irreversiblen Schädigung der Myozyten während der Ischämie kommt es auch zu dem so genannten Reperfusionsschaden, dieser kann aber, zumindest tierexperimentell, durch eine entsprechende Therapie verringert werden. Wir konnten bereits zeigen, dass die retrograde Applikation von embryonalen endothelialen Vorläuferzellen, von murinen Embryonen Tag 7,5 (Tie-2+, c-Kit+, Sca-1+, flk-1 low, MHC-1-) eine Kardioprotektion über lösliche Faktoren vermittelt. Diese Reduktion der Infarktgöße war über einen PI3K-AKT Signaltransduktionsweg vermittet. In der hier vorliegenden Studie haben wir uns mit dem Einfluss von Thymosin β4 auf die eEPC vermittelte Kardioprotektion beschäftigt. Methoden: In vitro wurden neonatale ventrikuläre Myozyten der Ratte einer Hypoxie (4 h) und Reoxygenation (1 h) ausgesetzt. Die überlebenden Zellen wurden mittels Trypan-Blau-Exklusion identifiziert. Des Weiteren wurden neonatale ventrikuläre Endothelzellen der Ratte auch einer Hypoxie (18 h) und Reoxygenation (4 h) ausgesetzt und die Apoptoserate mittles TUNEL-Färbung analysiert. Embryonale EPCs mit/ohne Thymosin β4 shRNA Transfektion wurden während Hypoxie kokultiviert oder Thymosin β4 Protein wurde dem Medium zugesetzt. In Schweinen (n= 9 pro Gruppe) wurde am Tag 1 mittels LAD-Verschluß (1 h) ein Infarkt induziert. 5x106 eEPCs mit/ohne Thymosin β4 shRNA Transfektion oder Thymosin β4 Protein wurden nach 55 min Ischämie in die anteriore interventrikulare Herzvene retroinfundiert. Nach 24 h Reperfusion wurden die globale und regionale Myokardfunktion (Sonomikrometrie) sowie die Infarktgröße bestimmt. Darüber hinaus wurde die Inflammation mittels Myeloperoxidase Analyse im Gewebe untersucht. Ergebnisse: Die „short hairpin“ Ribonukleinsäure (shRNA) Transfektion führte zu einer verringerten Thymosin „messanger“ RNA Expression in „real time“ Polimerase Kettenreaktions-Untersuchungen (rt-PCR). In Zellkultur war der Anteil überlebender neonataler Kardiomyozyten in Anwesenheit von eEPCs signifikant erhöht, wenn diese Zellen Thymosin β4 exprimierten. Die Analyse der TUNEL-Färbung zeigte eine deutlich geringere Apoptoserate der neonatalen Endothelzellen, die mit eEPCs kokultiviert wurden, es sei denn die Thymosin β4 Expression wurde durch Transfektion der shRNA reduziert. Die Applikation von Thymosin β4 Protein zeigte bei beiden Zellarten ein ähnliches Ergebnis wie die Kokultivierung mit den eEPCs. In vivo waren nach 24 h zahlreiche Zellen im ischämischen Areal nachweisbar. Die Anzahl der Zellen war durch die Reduktion der Thymosin β4 Expression nicht beeinträchtigt. Die regionale Applikation der eEPCs reduzierte die Infarktgröße signifikant gegenüber der Kontrollgruppe, wohingegen die Thymosin β4 shRNA Transfektion der eEPCs diesen Effekt inhibierte. Auch hier zeigte die retrograde Applikation des Thymosin β4 Proteins eine kardioprotektive Wirkung, die ähnlich ausgeprägt war wie die der eEPCs. Die Analyse der TUNEL-positiven Zellen zeigte eine deutliche Reduktion der Apoptoserate nach Retroinfusion der eEPCs oder des Thymosin β4 Protein, auch hier verloren die eEPCs ihre protektiven Eigenschaften nach der Transfektion mit Thymosin β4 shRNA. Die Inflammation im Ischämieareal, ein wichtiges Kennzeichen für die Ausprägung des Ischämie/Reperfusionsschadens, konnte durch die Verabreichung von eEPCs und auch Thymosin β4 Protein signifikant reduziert werden. Die Reduktion der Thymosin β4 Expression verhinderte wiederum diesen kardioprotektiven Effekt. Diese Untersuchungen zeigen, dass embryonale endotheliale Vorläuferzellen den Ischämie/Reperfusionsschaden zu einem frühen postischämischen Zeitpunkt verringern. Der kardioprotektive Effekt dieser Zellen ist zumindest teilweise Thymosin β4 abhängig, da eine analoge Protektion durch die lokale Applikation von Thymosin β4 Protein erreicht werden kann. Generell zeigt diese Arbeit, dass neben dem direkten Einsatz von Vorläuferzellen und Stammzellen zur Behandlung des Reperfusionsschadens diese Zellen auch genutzt werden können, um mögliche Kandidatenproteine zur Kardioprotektion nach akutem Myokardinfarkt zu identifizieren und somit eine effektive Therapie des Reperfusions-schadens beim Menschen zu ermöglichen.The acute myocardial infarction is still one of the major causes of death in the industrial nations. Also after reopening of the vessel, a prolonged myocardial ischemia still leads to an infarcted area. Beside the irreversible damage of the myocytes during ischemia, a so called reperfusion injury occurs, which may be reduced through an adequate therapy, at least in animal experiments. We could demonstrate before, that the retrograde application of embryonic endothelial progneitor cells, derived from murine embryos day 7.5 (Tie-2+, c-Kit+, Sca-1+, flk-1 low, MHC-1-) is cardioprotective, mediated through paracrine factors. This reduction of the infarct size is PI3K-AKT pathway mediated. In the here presented study we investigated the influence of thymosin β4 on the eEPCs mediated cardioprotection. Methods: In vitro, neonatal rat cardiomyocytes were subjected to hypoxia (4 h) and reoxygenation (1 h), cell survival was analysed via trypan blue staining. Furthermore rat neonatal ventricular endothelial cells were subjected to hypoxia (18 h) and reoxygenation (4 h), apoptosis rate was investigated using TUNEL-staining. Embryonic EPCs with/without thymosin β4 shRNA transfection were during hypoxia and reoxygenation cocultivated, or thymosin β4 protein was added to the media. In pigs (n=9 per group) percutaneous LAD occlusion (1 h) at day 1 was performed to induce an infarction. 5x106 eEPCs with/without thymosin β4 shRNA transfection or thymosin β4 protein were applied after 55 min of ischemia through retroinfusion into the anterior interventricular vene. After 24 h of reperfusion regional and global myocardial function were analysed and infarct size was determined. In addition, the inflammation via Myeloperoxidase analysis was examined in the tissue. Results: The transfection of the short hairpin ribonucleotid acid (shRNA) led to a reduction of the messenger ribonucleotid acid (mRNA) expression of the thymosin in real time polymerase chain reaction (rt-PCR) analysis. In cell culture the survival of the neonatal cardiomyocytes was significantly increased after eEPC application, if the cells express thymosin β4. The anaysis of the TUNEL-staining displayed a considerable lower apoptosis rate of the neonatal endothelial cells, if cocultivated with eEPCs, unless thymosin β4 expression was reduced via shRNA transfection. The application of thymosin β4 protein showed similar effects to the eEPC cocultivation in both celltypes. In vivo, numerous eEPCs were detected after 24 h in the ischemic area. The number of cells was not influenced through the expression of thymosin β4. The regional application of the eEPCs significantly decreased the infarct size compared to the control group, whereas thymosin β4 shRNA transfected eEPCs blunted this effect. Once again, the retrograde application of thymison β4 protein showed a cardioprotective effect, analog to the effect of the eEPCs. The analysis of the TUNEL-positve cells displayed a clear reduction of the apoptosis rate after retroinfusion of the eEPCs or thymosin β4 protein. Once more, the eEPCs lost their cardioprotective potential when thymosin β4 shRNA was transfected. The inflammation in the ischemic area, an important marker for the value of the ischemia/reperfusion injury, was significantly reduced through the administration of the eEPCs or thymosin β4 protein. Again the reduction of the thymosin β4 expression abrogated this cardioprotective effect. These investigations show that embryonic endothelial progenitor cells reduce the ischämia/reperfusion injury at an early postischemic timepoint. The cardioprotective effect of the cells is at least partially dependent on thymosin β4, since an analog protection may be achieved by local delivery of thymosin β4 protein. In general this work shows, that beside a direct use of the progenitor cells and stem cells for the treatment of the reperfusion injury, these cells may be used to identify potential candidate proteins for cardioprotection after acute myocardial infarction and to enable a more effective treatment of the reperfusion injury in humans

Kardioprotektion nach Ischämie/Reperfusion durch embryonale endotheliale Progenitorzellen

Der akute Myokardinfarkt stellt in den Industrienationen immer noch eine der häufigsten Todesursachen dar. Auch nach Wiedereröffnen des Gefäßes führt eine prolongierte myokardiale Ischämie zur Ausbildung eines Infarktareals. Neben der irreversiblen Schädigung der Myozyten während der Ischämie kommt es auch zu dem so genannten Reperfusionsschaden, dieser kann aber, zumindest tierexperimentell, durch eine entsprechende Therapie verringert werden. Wir konnten bereits zeigen, dass die retrograde Applikation von embryonalen endothelialen Vorläuferzellen, von murinen Embryonen Tag 7,5 (Tie-2+, c-Kit+, Sca-1+, flk-1 low, MHC-1-) eine Kardioprotektion über lösliche Faktoren vermittelt. Diese Reduktion der Infarktgöße war über einen PI3K-AKT Signaltransduktionsweg vermittet. In der hier vorliegenden Studie haben wir uns mit dem Einfluss von Thymosin β4 auf die eEPC vermittelte Kardioprotektion beschäftigt. Methoden: In vitro wurden neonatale ventrikuläre Myozyten der Ratte einer Hypoxie (4 h) und Reoxygenation (1 h) ausgesetzt. Die überlebenden Zellen wurden mittels Trypan-Blau-Exklusion identifiziert. Des Weiteren wurden neonatale ventrikuläre Endothelzellen der Ratte auch einer Hypoxie (18 h) und Reoxygenation (4 h) ausgesetzt und die Apoptoserate mittles TUNEL-Färbung analysiert. Embryonale EPCs mit/ohne Thymosin β4 shRNA Transfektion wurden während Hypoxie kokultiviert oder Thymosin β4 Protein wurde dem Medium zugesetzt. In Schweinen (n= 9 pro Gruppe) wurde am Tag 1 mittels LAD-Verschluß (1 h) ein Infarkt induziert. 5x106 eEPCs mit/ohne Thymosin β4 shRNA Transfektion oder Thymosin β4 Protein wurden nach 55 min Ischämie in die anteriore interventrikulare Herzvene retroinfundiert. Nach 24 h Reperfusion wurden die globale und regionale Myokardfunktion (Sonomikrometrie) sowie die Infarktgröße bestimmt. Darüber hinaus wurde die Inflammation mittels Myeloperoxidase Analyse im Gewebe untersucht. Ergebnisse: Die „short hairpin“ Ribonukleinsäure (shRNA) Transfektion führte zu einer verringerten Thymosin „messanger“ RNA Expression in „real time“ Polimerase Kettenreaktions-Untersuchungen (rt-PCR). In Zellkultur war der Anteil überlebender neonataler Kardiomyozyten in Anwesenheit von eEPCs signifikant erhöht, wenn diese Zellen Thymosin β4 exprimierten. Die Analyse der TUNEL-Färbung zeigte eine deutlich geringere Apoptoserate der neonatalen Endothelzellen, die mit eEPCs kokultiviert wurden, es sei denn die Thymosin β4 Expression wurde durch Transfektion der shRNA reduziert. Die Applikation von Thymosin β4 Protein zeigte bei beiden Zellarten ein ähnliches Ergebnis wie die Kokultivierung mit den eEPCs. In vivo waren nach 24 h zahlreiche Zellen im ischämischen Areal nachweisbar. Die Anzahl der Zellen war durch die Reduktion der Thymosin β4 Expression nicht beeinträchtigt. Die regionale Applikation der eEPCs reduzierte die Infarktgröße signifikant gegenüber der Kontrollgruppe, wohingegen die Thymosin β4 shRNA Transfektion der eEPCs diesen Effekt inhibierte. Auch hier zeigte die retrograde Applikation des Thymosin β4 Proteins eine kardioprotektive Wirkung, die ähnlich ausgeprägt war wie die der eEPCs. Die Analyse der TUNEL-positiven Zellen zeigte eine deutliche Reduktion der Apoptoserate nach Retroinfusion der eEPCs oder des Thymosin β4 Protein, auch hier verloren die eEPCs ihre protektiven Eigenschaften nach der Transfektion mit Thymosin β4 shRNA. Die Inflammation im Ischämieareal, ein wichtiges Kennzeichen für die Ausprägung des Ischämie/Reperfusionsschadens, konnte durch die Verabreichung von eEPCs und auch Thymosin β4 Protein signifikant reduziert werden. Die Reduktion der Thymosin β4 Expression verhinderte wiederum diesen kardioprotektiven Effekt. Diese Untersuchungen zeigen, dass embryonale endotheliale Vorläuferzellen den Ischämie/Reperfusionsschaden zu einem frühen postischämischen Zeitpunkt verringern. Der kardioprotektive Effekt dieser Zellen ist zumindest teilweise Thymosin β4 abhängig, da eine analoge Protektion durch die lokale Applikation von Thymosin β4 Protein erreicht werden kann. Generell zeigt diese Arbeit, dass neben dem direkten Einsatz von Vorläuferzellen und Stammzellen zur Behandlung des Reperfusionsschadens diese Zellen auch genutzt werden können, um mögliche Kandidatenproteine zur Kardioprotektion nach akutem Myokardinfarkt zu identifizieren und somit eine effektive Therapie des Reperfusions-schadens beim Menschen zu ermöglichen.The acute myocardial infarction is still one of the major causes of death in the industrial nations. Also after reopening of the vessel, a prolonged myocardial ischemia still leads to an infarcted area. Beside the irreversible damage of the myocytes during ischemia, a so called reperfusion injury occurs, which may be reduced through an adequate therapy, at least in animal experiments. We could demonstrate before, that the retrograde application of embryonic endothelial progneitor cells, derived from murine embryos day 7.5 (Tie-2+, c-Kit+, Sca-1+, flk-1 low, MHC-1-) is cardioprotective, mediated through paracrine factors. This reduction of the infarct size is PI3K-AKT pathway mediated. In the here presented study we investigated the influence of thymosin β4 on the eEPCs mediated cardioprotection. Methods: In vitro, neonatal rat cardiomyocytes were subjected to hypoxia (4 h) and reoxygenation (1 h), cell survival was analysed via trypan blue staining. Furthermore rat neonatal ventricular endothelial cells were subjected to hypoxia (18 h) and reoxygenation (4 h), apoptosis rate was investigated using TUNEL-staining. Embryonic EPCs with/without thymosin β4 shRNA transfection were during hypoxia and reoxygenation cocultivated, or thymosin β4 protein was added to the media. In pigs (n=9 per group) percutaneous LAD occlusion (1 h) at day 1 was performed to induce an infarction. 5x106 eEPCs with/without thymosin β4 shRNA transfection or thymosin β4 protein were applied after 55 min of ischemia through retroinfusion into the anterior interventricular vene. After 24 h of reperfusion regional and global myocardial function were analysed and infarct size was determined. In addition, the inflammation via Myeloperoxidase analysis was examined in the tissue. Results: The transfection of the short hairpin ribonucleotid acid (shRNA) led to a reduction of the messenger ribonucleotid acid (mRNA) expression of the thymosin in real time polymerase chain reaction (rt-PCR) analysis. In cell culture the survival of the neonatal cardiomyocytes was significantly increased after eEPC application, if the cells express thymosin β4. The anaysis of the TUNEL-staining displayed a considerable lower apoptosis rate of the neonatal endothelial cells, if cocultivated with eEPCs, unless thymosin β4 expression was reduced via shRNA transfection. The application of thymosin β4 protein showed similar effects to the eEPC cocultivation in both celltypes. In vivo, numerous eEPCs were detected after 24 h in the ischemic area. The number of cells was not influenced through the expression of thymosin β4. The regional application of the eEPCs significantly decreased the infarct size compared to the control group, whereas thymosin β4 shRNA transfected eEPCs blunted this effect. Once again, the retrograde application of thymison β4 protein showed a cardioprotective effect, analog to the effect of the eEPCs. The analysis of the TUNEL-positve cells displayed a clear reduction of the apoptosis rate after retroinfusion of the eEPCs or thymosin β4 protein. Once more, the eEPCs lost their cardioprotective potential when thymosin β4 shRNA was transfected. The inflammation in the ischemic area, an important marker for the value of the ischemia/reperfusion injury, was significantly reduced through the administration of the eEPCs or thymosin β4 protein. Again the reduction of the thymosin β4 expression abrogated this cardioprotective effect. These investigations show that embryonic endothelial progenitor cells reduce the ischämia/reperfusion injury at an early postischemic timepoint. The cardioprotective effect of the cells is at least partially dependent on thymosin β4, since an analog protection may be achieved by local delivery of thymosin β4 protein. In general this work shows, that beside a direct use of the progenitor cells and stem cells for the treatment of the reperfusion injury, these cells may be used to identify potential candidate proteins for cardioprotection after acute myocardial infarction and to enable a more effective treatment of the reperfusion injury in humans

Early vessel destabilization mediated by Angiopoietin-2 and subsequent vessel maturation via Angiopoietin-1 induce functional neovasculature after ischemia.

We assessed whether Angiopoietin-2 (Ang2), a Tie2 ligand and partial antagonist of Angiopoietin-1 (Ang1), is required for early vessel destabilization during postischemic angiogenesis, when combined with vascular growth factors. In vitro, matrigel co-cultures assessed endothelial-cell tube formation and pericyte recruitment after stimulation of VEGF-A, Apelin (APLN), Ang1 with or without Ang2. In a murine hindlimb ischemia model, adeno-associated virus (rAAV, 3×10(12) virusparticles) transduction of VEGF-A, APLN and Ang1 with or without Ang2 (continuous or early expression d0-3) was performed intramuscularly (d-14). Femoral artery ligation was performed at d0, followed by laser doppler perfusion meassurements (LDI) 7 and 14. At d7 (early timepoint) and d14 (late timepoint), histological analysis of capillary/muscle fiber ratio (CMF-R, PECAM-1) and pericyte/capillary ratio (PC-R, NG2) was performed. In vitro, VEGF-A, APLN and Ang1 induced ring formation, but only APLN and Ang1 recruited pericytes. Ang2 did not affect tube formation by APLN, but reduced pericyte recruitment after APLN or Ang1 overexpression. In vivo, rAAV.VEGF-A did not alter LDI-perfusion at d14, consistent with an impaired PC-R despite a rise in CMF-R. rAAV.APLN improved perfusion at d14, with or without continuous Ang2, increasing CMF-R and PC-R. rAAV.Ang1 improved perfusion at d14, when combined with rAAV.Ang2 (d0-3), accompanied by an increased CMF-R and PC-R. The combination of early vessel destabilization (Ang2 d0-3) and continuous Ang1 overexpression improves hindlimb perfusion, pointing to the importance of early vessel destabilization and subsequent vessel maturation for enhanced therapeutic neovascularization

Micro-RNA 92a as a Therapeutic Target for Cardiac Microvascular Dysfunction in Diabetes

Microvascular dysfunction is a pathological hallmark of diabetes, and is central to the ethology of diabetes-associated cardiac events. Herein, previous studies have highlighted the role of the vasoactive micro-RNA 92a (miR-92a) in small, as well as large, animal models. In this study, we explore the effects of miR-92a on mouse and human cardiac microvascular endothelial cells (MCMEC, HCMEC), and its underlying molecular mechanisms. Diabetic HCMEC displayed impaired angiogenesis and a pronounced inflammatory phenotype. Quantitative PCR (qPCR) showed an upregulation of miR-92a in primary diabetic HCMEC. Downregulation of miR-92a by antagomir transfection in diabetic HCMEC rescued angiogenesis and ameliorated diabetic endothelial bed inflammation. Furthermore, additional analysis of potential in silico-identified miR-92a targets in diabetic HCMEC revealed the miR-92a dependent downregulation of an essential metalloprotease, ADAM10. Accordingly, downregulation of ADAM10 impaired angiogenesis and wound healing in MCMEC. In myocardial tissue slices from diabetic pigs, ADAM10 dysregulation in micro- and macro-vasculature could be shown. Altogether, our data demonstrate the role of miR-92a in cardiac microvascular dysfunction and inflammation in diabetes. Moreover, we describe for the first time the metalloprotease ADAM10 as a novel miR-92a target, mediating its anti-angiogenic effect

Free-Breathing Myocardial T1 Mapping using Inversion-Recovery Radial FLASH and Motion-Resolved Model-Based Reconstruction

Purpose: To develop a free-breathing myocardial T1 mapping technique using inversion-recovery (IR) radial fast low-angle shot (FLASH) and calibrationless motion-resolved model-based reconstruction. Methods: Free-running (free-breathing, retrospective cardiac gating) IR radial FLASH is used for data acquisition at 3T. First, to reduce the waiting time between inversions, an analytical formula is derived that takes the incomplete T1 recovery into account for an accurate T1 calculation. Second, the respiratory motion signal is estimated from the k-space center of the contrast varying acquisition using an adapted singular spectrum analysis (SSA-FARY) technique. Third, a motion-resolved model-based reconstruction is used to estimate both parameter and coil sensitivity maps directly from the sorted k-space data. Thus, spatio-temporal total variation, in addition to the spatial sparsity constraints, can be directly applied to the parameter maps. Validations are performed on an experimental phantom, eleven human subjects, and a young landrace pig with myocardial infarction. Results: In comparison to an IR spin-echo reference, phantom results confirm good T1 accuracy, when reducing the waiting time from five seconds to one second using the new correction. The motion-resolved model-based reconstruction further improves T1 precision compared to the spatial regularization-only reconstruction. Aside from showing that a reliable respiratory motion signal can be estimated using modified SSA-FARY, in vivo studies demonstrate that dynamic myocardial T1 maps can be obtained within two minutes with good precision and repeatability. Conclusion: Motion-resolved myocardial T1 mapping during free-breathing with good accuracy, precision and repeatability can be achieved by combining inversion-recovery radial FLASH, self-gating and a calibrationless motion-resolved model-based reconstruction.Comment: Part of this work has been presented at the ISMRM Annual Conference 2021 (Virtual), submitted to Magnetic Resonance in Medicin

Individualized Biventricular Epicardial Augmentation Technology in a Drug-Induced Porcine Failing Heart Model

For treatment of advanced heart failure, current strategies include cardiac transplantation or blood-contacting pump technology associated with complications, including stroke and bleeding. This study investigated an individualized biventricular epicardial augmentation technology in a drug-induced porcine failing heart model. A total of 11 pigs were used, for the assessment of hemodynamics and cardiac function under various conditions of support pressures and support durations (n = 4), to assess device positioning and function by in vivo computer tomographic imaging (n = 3) and to investigate a minimally invasive implantation on the beating heart (n = 4). Support pressures of 20-80 mmHg gradually augmented cardiac function parameters in this animal model as indicated by increased left ventricular stroke volume, end-systolic pressures, and decreased end-diastolic pressures. Strong evidence was found regarding the necessity of mechanical synchronization of support end with the isovolumetric relaxation phase of the heart. In addition, the customized, self-expandable implant enabled a marker-guided minimally invasive implantation through a 4cm skin incision using fluoroscopy. Correct positioning was confirmed in computer tomographic images. Continued long-term survival investigations will deliver preclinical evidence for further development of this concept

Thymosin beta 4 Improves Differentiation and Vascularization of EHTs

Induced pluripotent stem cells (iPSC) constitute a powerful tool to study cardiac physiology and represents a promising treatment strategy to tackle cardiac disease. However, iPSCs remain relatively immature after differentiation. Additionally, engineered heart tissue (EHT) has been investigated as a therapy option in preclinical disease models with promising results, although their vascularization and functionality leave room for improvement. Thymosin beta 4 (T beta 4) has been shown to promote the differentiation of progenitor cell lines to cardiomyocytes while it also induces angiogenic sprouting and vascular maturation. We examined the potential impact of T beta 4 to enhance maturation of cardiomyocytes from iPSCs. Assessing the expression of transcription factors associated with cardiac differentiation, we were able to demonstrate the increased generation of cells displaying cardiomyocyte characteristics in vitro. Furthermore, we demonstrated, in a zebrafish model of embryonic vascular development, that T beta 4 is crucial for the proper execution of lymphatic and angiogenic vessel sprouting. Finally, utilizing T beta 4-transduced EHTs generated from mice genetically engineered to label endothelial cells in vitro, we show that treatment with T beta 4 promotes vascularization and contractility in EHTs, highlighting T beta 4 as a growth factor improving the formation of cardiomyocytes from iPSC and enhancing the performance of EHTs generated from neonatal cardiomyocytes

The GEF Trio controls endothelial cell size and arterial remodeling downstream of Vegf signaling in both zebrafish and cell models

Arterial networks enlarge in response to increase in tissue metabolism to facilitate flow and nutrient delivery. Typically, the transition of a growing artery with a small diameter into a large caliber artery with a sizeable diameter occurs upon the blood flow driven change in number and shape of endothelial cells lining the arterial lumen. Here, using zebrafish embryos and endothelial cell models, we describe an alternative, flow independent model, involving enlargement of arterial endothelial cells, which results in the formation of large diameter arteries. Endothelial enlargement requires the GEF1 domain of the guanine nucleotide exchange factor Trio and activation of Rho-GTPases Rac1 and RhoG in the cell periphery, inducing F-actin cytoskeleton remodeling, myosin based tension at junction regions and focal adhesions. Activation of Trio in developing arteries in vivo involves precise titration of the Vegf signaling strength in the arterial wall, which is controlled by the soluble Vegf receptor Flt1. Arterial flow regulates artery diameter but other mechanisms may also affect this. Here, the authors show that the guanine nucleotide exchange factor Trio and GTPases Rac1 and RhoG, triggers F-actin remodeling in arterial endothelial cells, independent of flow, to enhance lumen diameter in zebrafish and cell models.Peer reviewe