Suite à une lésion, la réparation d’un tissu à l’identique est l’objectif clinique recherché. Ainsi un hémostatique, au-delà de sa capacité à arrêter un saignement, se doit au minimum de ne pas être délétère pour la réparation tissulaire. Or, malgré une efficacité hémostatique démontrée Tachosil® et Surgicel®, couramment utilisés en chirurgie, peuvent générer en raison de leur persistance in situ associée à une résorption incomplète, des évènements indésirables graves (nécrose, hypersensibilité, allergie, sténose, complication thromboembolique, sepsis…). Dans ce contexte, nous avons étudié les effets d’un nouvel hémostatique non implantable, HÉMO-IONIC®, sur toutes les étapes de la réparation tissulaire : de l’hémostase à la phase de maturation. HÉMO-IONIC®, Tachosil® et Surgicel® ont été évalués in vitro sur la fonctionnalité des Endothelial Colony Forming Cells (ECFCs) ; et in vivo dans deux modèles murins, un modèle d’hémostase et un de réparation tissulaire. Grâce à ces travaux, constituant une approche originale de l’évaluation des hémostatiques, nous avons démontré que seul HÉMO-IONIC®, en plus d’être un hémostatique efficace, maintient l’ensemble des propriétés fonctionnelles des cellules endothéliales et agit, à plus long terme, positivement sur l’ensemble des étapes de la réparation tissulaire. Ces données ouvrent des perspectives particulièrement intéressantes, notamment dans la prise en charge des brûlures nécessitant la restitution d’un tissu cutané de qualité (souple et élastique). Ainsi, l’association d’HÉMO-IONIC® à l’expertise du Service de Santé des Armées (SSA) dans l’utilisation des cellules stromales mésenchymateuses comme Médicament de Thérapie Innovante (MTI) pourrait représenter, dans la prise en charge des brûlures, une avancée thérapeutique en améliorant les résultats fonctionnels et esthétiques. / After an injury, the clinical objective is to faithfully repair tissues. Thus, a hemostatic agent, beyond its ability to stop bleeding, must at least not be deleterious for tissue repair. However, despite proven hemostatic efficacy, Tachosil® and Surgicel®, which are commonly used in surgery, can lead to serious adverse events (necrosis, hypersensitivity, allergy, stenosis, thromboembolic complication, sepsis…) due to their in situ persistence associated with their incomplete resorption. In this context, we studied the effects of a new non-implantable hemostatic agent, HÉMO-IONIC®, on all stages of tissue repair : from hemostasis to maturation phase. The effects of HÉMO-IONIC®, Tachosil® and Surgicel® were assessed in vitro on Endothelial Colony Forming Cell function (ECFCs) and in vivo in two mouse models of hemostasis and tissue repair. Using an original approach to the assessment of hemostatic agents, we showed that only HÉMO-IONIC®, in addition to being effective, maintained all the functional properties of endothelial cells and acted positively on all stages of tissue repair in the longer term. These data open up very interesting perspectives, in particular in the management of burns where restoring a quality cutaneous tissue (supple and elastic) is required. Thus, in burn management, combining HÉMO-IONIC® with the French Defence Health service expertise in the use of mesenchymatous stromal cells as an advanced therapy medicinal product could be a therapeutic advance by improving the functional and esthetic outcomes.

No description available.

Disruptions in the response of endothelial progenitor cells to changes in oxygen environment may present a possible mechanism behind multiple pediatric pulmonary disease models, such as bronchopulmonary dysplasia. Using high-throughput fixed single-cell protein and RNA imaging, we have created "stop-motion" movies of Thymosin. 4 (T beta 4) and Hypoxia Inducible Factor 1 alpha (HIF-1 alpha) protein expression and vascular endothelial growth factor (vegf) and endothelial nitric oxide synthase (eNOS) mRNA in human umbilical cord-derived endothelial colony-forming cells (ECFC). ECFC were grown in vitro under both room air and hyperoxia (50% O-2). We find elevated basal T beta 4 protein expression in ECFC derived from prematurely born infants versus full term infants. T beta 4 is a potent growth hormone that additionally acts as an actin sequestration protein and regulates the stability of HIF-1 alpha. This basal level increase of T beta 4 is associated with lower HIF1 alpha nuclear localization in preterm versus term ECFC upon exposure to hyperoxia. We find altered expression in the pro-angiogenic genes vegf and eNOS, two genes that HIF-1 alpha acts as a transcription factor for. This provides a potential link between a developmentally regulated protein and previously observed impaired function of preterm ECFC in response to hyperoxia.

Indiana University-Purdue University Indianapolis (IUPUI) / In the United States, 10% of pregnancies are complicated by diabetes mellitus (DM). Intrauterine DM exposure can have long-lasting implications for the fetus, including cardiovascular morbidity. Previously, we showed that fetal endothelial colony forming cells (ECFCs) from DM pregnancies have decreased vessel-forming ability and increased senescence. However, the molecular mechanisms responsible for this dysfunction remain largely unknown. The objective of this thesis was to understand how Mesenchyme Homeobox 2 (MEOX2) interacts with pathways that regulate cell cycle progression and migration, and how this interaction results in impaired vasculogenesis in DM exposed ECFCs. We tested the hypothesis that upregulated MEOX2 in DM-exposed ECFCs decreases network formation through impairments in senescence, cell cycle progression, migration, and adhesion. MEOX2 is a transcription factor which inhibits angiogenesis by upregulating cyclin dependent kinase inhibitors. Here, data show that nuclear MEOX2 is increased in DM-exposed ECFCs. Lentiviral-mediated overexpression of MEOX2 in control ECFCs increased network formation, altered cell cycle progression, increased senescence, and enhanced migration. In contrast, MEOX2-knockdown in DM-exposed ECFCs decreased network formation and migration, while cell cycle progression and senescence were unchanged. Adhesion and integrin expression defects were evaluated as mechanisms by which MEOX2 altered ECFC migration. While MEOX2-overexpression did not alter adhesion or cell surface integrin levels in control cells, MEOX2 overexpression in DM-exposed ECFCs resulted in an increase in α6 integrin surface expression. Similarly, MEOX2-knockdown in DM-exposed ECFCs did not alter adhesion, though did reduce α6 integrin surface expression and total cellular α6 mRNA and protein levels. Together, these data suggest that alterations in cell cycle progression and senescence are not responsible for the disrupted vasculogenesis of DM-exposed ECFCs. Importantly, these data suggest that altered migration may be a key mechanism involved and that altered cell surface levels of the α6 integrin may modify migratory capacity. Moreover, these data suggest that the α6 integrin may be a previously unrecognized transcriptional target of MEOX2. Ultimately, while initially believed to be maladaptive, these data suggest that increased nuclear MEOX2 in DM-exposed ECFCs may serve a protective role, enabling vessel formation despite exposure to a DM intrauterine environment.

No description available.

Thesis (Ph.D.)--Indiana University, 2009. / Title from screen (viewed on February 2, 2010). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Mervin C. Yoder, Jr., David A. Ingram, Jr., Lawrence A. Quilliam, Mark D. Pescovitz. Includes vitae. Includes bibliographical references (leaves 171-194).

L’hypoxo-ischémie (HI) cérébrale néonatale représente une des principales causes de mortalité et de morbidité chez les nouveau-nés. Sa physiopathologie implique différents processus délétères menant vers la perte neuronale et responsables de séquelles neuro-cognitives. L'hypothermie thérapeutique est le seul traitement actuel mais est insuffisant. Cette étude a caractérisé et comparé l’effet de deux types de cellules issues du sang de cordon humain, les cellules mononuclées (HUCBCs) et les progéniteurs endothéliaux tardifs (ECFCs) sur l’amélioration des scores neuro-comportementaux mais aussi à l’échelle moléculaire et fonctionnelle dans le modèle d’hypoxo-ischémie néonatale à court (7 jours après l’épisode ischémique) et long terme (12 semaines après l’épisode ischémique).L’injection intrapéritonéale d'ECFCs ou de HUCBCs, 2 jours après HI, améliore les capacités de motricité et de mémorisation précoce et tardive des animaux à l’âge adulte, et diminue les comportements anxieux. Ces résultats sont associés à une augmentation de la densité capillaire en temps précoce et tardif. L’imagerie de perfusion cérébrale SPECT/CT a objectivé une restauration complète de la perfusion cérébrale de l’hémisphère lésé à l’âge adulte par les deux types cellulaires. Ces observations tardives sont associées à un effet protecteur précoce de ces cellules sur l’augmentation de la survie neuronale et la diminution de l’astrogliose réactionnelle ou encore sur la composante inflammatoire par diminution de l’activation microgliale pro-inflammatoire au niveau striatal. Les résultats de cette étude ouvrent ainsi de nouvelles perspectives pour l’usage des ECFCs dans le traitement de l’HI néonatale. / Neonatal hypoxic-ischemic encephalopathy (NHIE) is a dramatic perinatal complication, associated with poor neurological prognosis despite neuroprotection by therapeutic hypothermia, in the absence of an available curative therapy. We evaluated and compared ready-to-use human umbilical cord blood cells (HUCBCs) and bankable but allogeneic endothelial progenitors (ECFCs) as cell therapy candidate for NHIE. We compared benefits of HUCBC and ECFC transplantation 48 hours after injury in male rat NHIE model, based on the Rice-Vannucci approach. Based on behavioral tests, immune-histological assessment and metabolic imaging of brain perfusion using SPECT, HUCBC or ECFC administration provided equally early and sustained functional benefits, up to 8 weeks after injury. These results were associated with total normalization of injured hemisphere cerebral blood flow assessed by SPECT/CT imaging. In conclusion, even if ECFCs represent an efficient candidate, HUCBCs’ autologous criteria and easier availability make them the ideal candidate for hypoxic-ischemic cell therapy.

Indiana University-Purdue University Indianapolis (IUPUI) / A hierarchy of endothelial colony forming cells (ECFCs) with different levels of proliferative potential has been identified in human circulating blood and blood vessels. High proliferative potential ECFCs (HPP-ECFCs) display properties (robust proliferative potential in vitro and vessel-forming ability in vivo) consistent with stem/progenitor cells for the endothelial lineage. Corneal endothelial cells (CECs) are different from circulating and resident vascular endothelial cells (ECs). Whereas systemic vascular endothelium slowly proliferates throughout life, CECs fail to proliferate in situ and merely expand in size to accommodate areas of CEC loss due to injury or senescence. However, we have identified an entire hierarchy of ECFC resident in bovine CECs. Thus, this study provides a new conceptual framework for defining corneal endothelial progenitor cell potential. The identification of persistent corneal HPP-ECFCs in adult subjects might contribute to regenerative medicine in corneal transplantation. While human cord blood derived ECFCs are able to form vessels in vivo, it is unknown whether they are committed to an arterial or venous fate. We have demonstrated that human cord blood derived ECFCs heterogeneously express gene transcripts normally restricted to arterial or venous endothelium. They can be induced to display an arterial gene expression pattern after vascular endothelial growth factor 165 (VEGF165) or Notch ligand Dll1 (Delta1ext-IgG) stimulation in vitro. However, the in vitro Dll1 primed ECFCs fail to display significant skewing toward arterial EC phenotype and function in vivo upon implantation, suggesting that in vitro priming is not sufficient for in vivo specification. Future studies will determine whether ECFCs are amenable to specification in vivo by altering the properties of the implantation microenvironment. There is emerging evidence suggesting that the concentration of circulating ECFCs is closely related to the adverse progression of cardiovascular disorders. In a pig model of acute myocardial ischemia (AMI), we have demonstrated that AMI rapidly mobilizes ECFCs into the circulation, with a significant shift toward HPP-ECFCs. The exact role of the mobilized HPP-ECFCs in homing and participation in repair of the ischemic tissue remains unknown. In summary, these studies contribute to an improved understanding of ECFCs and suggest several possible therapeutic applications of ECFCs.

Endothelzell-basierte Therapien vermitteln regenerative Effekte hinsichtlich der Revaskularisierung von ischämischen Geweben. Doch ist die Verfügbarkeit von autologen Endothelzellen aufgrund einer krankheitsbedingt reduzierten Frequenz im peripheren Blut oder einer verminderten Integrität der endogenen Endothelzell-Populationen eingeschränkt. Hingegen ist es möglich, allogene endotheliale Vorläuferzellen aus der Nabelschnur in zelltherapeutisch relevanten Mengen zu isolieren. In der vorliegenden Arbeit wurden zunächst die Eigenschaften allogener humaner Nabelschnur (NS)-abgeleiteter sog. Endothelial Colony-Forming Cells (ECFCs) mit denen von venösen NS-abgeleiteten Endothelzellen verglichen. Aufgrund der nachgewiesenen Immunogenität von allogenen ECFCs wurde eine weiterführende Strategie zur Reduktion dieser immunogenen Eigenschaften durch die Koapplikation mit Mesenchymalen Vorläuferzellen (MSCs) verfolgt. Humane ECFCs wurden mit MSCs desselben Spenders kombiniert und in funktionellen in vitro- und in vivo-Assays untersucht. Dadurch konnte nachgewiesen werden, dass IFNγ-stimulierte ECFC/MSC-Kokulturen eine reduzierte Expression von HLA-Molekülen zeigen. Entsprechend induzierten spezifische CD8+ T-Zellen eine reduzierte Lyse der kokultivierten ECFCs und MSCs. Die Kokultur von ECFCs und MSCs mit allogenen Immunzellen führte zu einer nahezu vollständigen Inhibition der T-Zell-Proliferation. Um die reduzierte Immunogenität von ECFC und MSC in vivo zu verifizieren, wurden die Zellen in immundefiziente Mäuse injiziert, welche nachfolgend mit humanen PBMCs rekonstituiert wurden. So konnte nachgewiesen werden, dass die Koapplikation von ECFCs und MSCs nicht nur die Entstehung von stabilen Gefäßnetzwerken begünstigt, sondern zudem in den Transplantaten zu einer verringerten Immunzell-Infiltration führte. Die Koapplikation von ECFCs mit MSCs könnte daher eine klinische Nutzung dieser allogenen Quelle für die therapeutische Unterstützung der Vaskularisierung ermöglichen. / Endothelial cell-based therapies promote tissue regeneration and vascularization after ischemic damage. The availability of autologous endothelial progenitor cells is restricted in diseased patients, however therapeutically relevant numbers of allogeneic Endothelial Progenitor Cells can be isolated from an umbilical cord (UC). In the present study, the immunogenic properties of these Endothelial Colony Forming Cells (ECFCs) were first compared to human umbilical vein endothelial cells (HUVECs). Both cytokine-treated endothelial cells induced CD4+ and CD8+ T cell proliferation after coculture with allogeneic immune cells. So far, the potential interactions between ECFCs and Mesenchymal Stem/Progenitor Cells (MSCs) concerning their immunological features is poorly understood, but we hypothesize that MSCs might improve the immune compatibility and vessel building characteristics of ECFCs. Therefore, human UC-derived ECFC and MSC cocultures from the same donor were analyzed using various functional in vitro and in vivo assays. Stimulation of these cocultures with IFNγ caused strongly reduced expression levels of HLA-molecules compared to ECFC monocultures. The decreased molecular density on the cocultured ECFCs resulted in reduced cytotoxic CD8+ T cell-mediated lysis. Further, during IFNγ stimulation, the combination of ECFCs with MSCs prevented initiation of allogeneic T cell proliferation. To verify this concept in vivo, ECFCs and MSCs were co-transplanted in a humanized allograft mouse model in immunodeficient mice in order to effectively induce stable microvessels. These experiments demonstrate that when MSCs are co-applied with ECFCs, they not only support the formation of stable blood vessels, but also lead to fewer HLA-DR+ human vascular structures and fewer infiltrating human leukocytes. The data presented indicate that crosstalk between UC-derived ECFCs and MSCs might lower the risk of allogeneic ECFC rejection.

Indiana University-Purdue University Indianapolis (IUPUI) / Endothelial Colony Forming Cells (ECFCs) are highly proliferative endothelial progenitor cells with clonal proliferative potential and in vivo vessel forming ability. While endothelial cells have been derived from human induced pluripotent stem cells (hiPS) or human embryonic stem cells (hES), they are not highly proliferative and require ectopic expression of a TGFβ inhibitor to restrict plasticity. Neuropilin-1 (NRP-1) has been reported to identify the emergence of endothelial precursor cells from human and mouse ES cells undergoing endothelial differentiation. However, the protocol used in that study was not well defined, used uncharacterized neuronal induction reagents in the culture medium, and failed to fully characterize the endothelial cells derived. We hypothesize that NRP-1 expression is critical for the emergence of stable endothelial cells with ECFC properties from hES cells. We developed a novel serum and feeder free defined endothelial differentiation protocol to induce stable endothelial cells possessing cells with cord blood ECFC-like properties from hES cells. We have shown that Day 12 hES cell-derived endothelial cells express the endothelial markers CD31+ NRP-1+, exhibit high proliferative potential at a single cell level, and display robust in vivo vessel forming ability similar to that of cord blood-derived ECFCs. The efficient production of the ECFCs from hES cells is 6 logs higher with this protocol than any previously published method. These results demonstrate progress towards differentiating ECFC from hES and may provide patients with stable autologous cells capable of repairing injured, dysfunctional, or senescent vasculature if these findings can be repeated with hiPS.