L’implication de SHP-1 en condition élevée de glucose inhibe la signalisation de l’insuline et du PDGF-BB dans les cellules musculaires lisses vasculaires hypoxiques

Résumé : Bien que l’hypoxie soit un puissant inducteur de l’angiogenèse, l’activation des facteurs de croissance est perturbée en hyperglycémie au niveau du pied et du cœur. Cette perturbation entraîne la perte de prolifération et de migration chez les cellules endothéliales, musculaires lisses vasculaires et péricytes empêchant la formation de nouveaux vaisseaux qui mènera à l’amputation des membres inférieurs chez les patients diabétiques. Une étude a démontré qu’une augmentation de la protéine tyrosine phosphatase Src homology-2 domain-containing phosphatase-1 (SHP-1) en condition hyperglycémique chez les péricytes entraînait l’inhibition de la signalisation du PDGF-BB, ce qui résultait en le développement d’une rétinopathie diabétique. Nous avons alors soulevé l’hypothèse que l’expression de SHP-1 dans les cellules musculaires lisses vasculaires affecte la prolifération et la migration cellulaire par l’inhibition de la signalisation de l’insuline et du PDGF-BB en condition diabétique. Nos expérimentations ont été effectuées principalement à l’aide d’une culture primaire de cellules musculaires lisses primaires provenant d’aortes bovines. Comparativement aux concentrations normales de glucose (NG : 5,6 mM), l’exposition à des concentrations élevées de glucose (HG : 25 mM) pendant 48 h a résulté en l’inhibition de la prolifération cellulaire par l’insuline et le PDGF-BB autant en normoxie (20% O2) qu’en hypoxie (24 dernières heures à 1% O2). Lors des essais de migration cellulaire, aucun effet de l’insuline n’a été observé alors que la migration par le PDGF-BB fut inhibée en HG autant en normoxie qu’en hypoxie. L’exposition en HG à mener à l’inhibition de la signalisation de la voie PI3K/Akt de l’insuline et du PDGF-BB en hypoxie. Aucune variation de l’expression de SHP-1 n’a été observée mais son activité phosphatase en hypoxie était fortement inhibée en NG contrairement en HG où on observait une augmentation de cette activité. Finalement, une association a été constatée entre SHP-1 et la sous-unité bêta du récepteur au PDGF. En conclusion, nous avons démontré que l’augmentation de l’activité phosphatase de SHP-1 en hypoxie cause l’inhibition des voies de l’insuline et du PDGF-BB réduisant les processus angiogéniques des cellules musculaires lisses vasculaires dans la maladie des artères périphériques.

Eph kinases and their ligands ephrins act in concert with sex hormones in regulating blood pressure

Les Erythropoietin-producing hepatocyte (EPH) sont la plus grande famille de récepteurs tyrosine kinase. Leurs ligands, les éphrines (EFNs), sont aussi des molécules exprimées à la surface cellulaire. Les EPH/EFNs sont impliqués dans de nombreux processus biologiques. L'hypertension artérielle (PA) est une maladie chronique qui, aujourd'hui, est devenue un problème médical critique dans le monde entier et un enjeu de santé publique. La découverte de nouvelles thérapeutiques de l'hypertension sont d'une grande importance pour la santé publique. Jusqu’à tout récemment, il existe seulement quelques études concernant le rôle de l’axe EPH/EFNs sur la fonction des cellules musculaires lisses vasculaires (CMLV). Dans nos études précédentes, nous avons montré qu'EPHB6 et EFNB1, de concert avec les hormones sexuelles, régulent la PA. Dans la présente étude, nous avons constaté que les différents membres de la famille EPH/EFN peuvent réguler soit positivement, soit négativement, la contractilité des CMLV et la PA: tandis que EPHB4 et EFNB2 appartiennent à la première catégorie, EFNB1, EFNB3 et EPHB6 appartiennent à la deuxième. In vivo, des souris males, mais non pas des femelles, porteuses d’une mutation EPHB4 (KO) spécifique du muscle lisse présentent une PA diminuée, comparée aux souris témoins (WT). Les CMLV de souris EPHB4 KO, en présence de testostérone, ont montré une contractilité réduite lors de la stimulation par la phényléphrine (PE). Au niveau moléculaire, la phosphorylation de la protéine kinase II dépendante de Ca2+/calmoduline et de la kinase de la chaine légère de la myosine (CLM) est augmentée, tandis que la phosphorylation de la kinase de la CLM est réduite dans les CMLV KO lors de la stimulation par PE, par rapport au WT CMLV. Cela fournit une base moléculaire à la réduction de la PA et de la contractilité des CMLV chez les souris EPHB4 KO. EFNB2 est le ligand majeur de l’EPHB4. Comme attendu, les souris EFNB2 KO spécifique du muscle lisse avaient un phénotype de PA semblable, quoique non identique, aux souris EPHB4 KO. Les souris mâles EFNB2 KO, mais pas femelles, sous régime régulier ou riche en sel, présentent une PA réduite, par rapport à leurs homologues WT. Au niveau cellulaire, les CMLV des souris KO ont montré une contractilité réduite lors de la stimulation par PE par rapport aux témoins WT. Une région de l’acide aminé (aa) 313 à l’aa 331 dans la partie intracellulaire d’EFNB2 est essentielle pour la signalisation inverse qui régule la contractilité des CMLV, selon des études de mutation-délétion. Dans une étude de génétique humaine, nous avons identifié, dans le gène EFNB2, six SNP qui étaient associées significativement au risque d'hypertension artérielle, de façon dépendante du sexe, ce qui corrobore nos résultats chez les souris. En revanche, la délétion du gène EFNB3 (KO) chez les souris femelles aboutit à une PA élevée et à une augmentation des résistances des petites artères in vivo, améliore la contractilité des petites artères ex-vivo et augmente la contractilité des CMLV in vitro. Les souris mâles KO ont une PA normale, mais la castration conduit à une augmentation significative de la PA dans les souris KO, mais pas dans les souris WT. Les CMLV des souris KO femelles ont montré une phosphorylation accrue de la CLM et une phosphorylation réduite de la kinase de la CLM, ce qui fournit à nouveau une base moléculaire aux phénotypes de PA et de contractilité des CMLV observés. Ce changement de signalisation est attribuable à une protéine adaptatrice Grip1. En effet, dans une étude d'association pan génomique par le Consortium International pour la Pression Sanguine, un SNP dans le gène GRIP1 a approché le seuil de significativité de la valeur p pour son association avec la pression diastolique. Nos recherches, pour la première fois, ont révélé que EPH/EFNs sont de nouveaux composants dans le système de régulation de la PA. Les membres de la famille EPH/EFN peuvent agir comme des forces Yin et Yang pour régler finement le tonus des vaisseaux pour assurer l'homéostasie de la PA et de sa régulation. Ces effets de EPH/EFNs dépendent du sexe et des niveaux d’hormones sexuelles. À partir de ces nouvelles connaissances, nous pourrions développer une nouvelle thérapie personnalisée pour l’hypertension artérielle, utilisant des antagonistes d'hormones sexuelles ou des thérapies de remplacement d'hormones sexuelles, selon les niveaux d'hormones sexuelles des patients et les mutations dans les gènes de l'EPH/EFN.

Reguladores da expressão do gene da proteía Gla da matriz (MGP) numa linha celular derivada de peixe

Dissertação de mest. em Biotecnologia, Faculdade de Engenharia de Recursos Naturais, Univ. do Algarve, 2004

Régulation de la prolifération des cellules musculaires lisses vasculaires par l’activation in vivo du récepteur natriurétique de type C.

Le remodelage vasculaire dû à l’hyper-prolifération cellulaire des cellules musculaires lisses vasculaires (CMLVs) observé chez les rats spontanément hypertendus (RSH) est associé à l’hypertension artérielle. Nous avons précédemment démontré que le traitement in vivo des RSH par l’agoniste spécifique du récepteur du peptide natriurétique de type C (NPR-C), le C-ANP4-23 atténue l’hyper-prolifération des CMLVs. Nous avons entrepris cette étude afin d’investiguer si l’effet antiprolifératif du C-ANP4-23 agit par l’entremise de l’inhibition de la surexpression des protéines du cycle cellulaire, et afin d’en explorer les mécanismes sous-jacents. Pour cette étude, des RSH et des rats Wistar Kyoto (WKYs) âgés de deux semaines ont été injectés en intra-péritonéale par le C-ANP4-23 de 2 jusqu’à 8 semaines d’âge, deux fois par semaine et sacrifiés à la 9ème semaine. La pression artérielle a été mesurée par méthode Queue-coiffe, la prolifération des CMLVs a été déterminée par incorporation de thymidine et par test MTT, et l’expression des protéines a été quant à elle déterminée par technique d’immunobuvardage de type Western. Les CMLVs des RSH ont démontré une prolifération élevée en comparaison avec celles des WKYs, et le traitement par le C-ANP4-23 a atténué l’hyperprolifération à un niveau de contrôle. De plus, la surexpression des cyclines D1/A/E, des kinases cyclines dépendantes 2 et 4 (cdk2, cdk4), de la forme phosphorylée de la protéine du rétinoblastome et des protéines Gαi des CMLV des RSH a été atténuée à un niveau de contrôle. Par ailleurs, l’hyperphosphorylation d’ERK1/2, AKT, EGF-R, PDGF-R, IGF-R et de c-Src a significativement diminué par le traitement au C-ANP4-23. En outre, le niveau élevé de l’anion superoxyde (O2-), l’activité de la NADP(H) oxydase et de ses sous unités chez les RSH ont été atténués par le C-ANP4-23 .Ces résultats indiquent que l’activation in vivo de NPR-C atténue la surexpression des protéines du cycle cellulaire via l’inhibition de l’activité élevée du stress oxydatif, de c-Src et de l’activation de EGF-R, PDGF- R, IGF-R, de la signalisation de MAPK et la surexpression des protéines Gαi résultant ainsi en l’inhibition de l’hyperprolifération des CMLVs des RSH. Ainsi, il peut être suggéré que le C-ANP4-23 pourrait être utilisé comme agent thérapeutique pour le traitement des complications vasculaires associées à l’hypertension et à l’athérosclérose.

Régulation de la prolifération des cellules musculaires lisses vasculaires par l’activation in vivo du récepteur natriurétique de type C

Le remodelage vasculaire dû à l’hyper-prolifération cellulaire des cellules musculaires lisses vasculaires (CMLVs) observé chez les rats spontanément hypertendus (RSH) est associé à l’hypertension artérielle. Nous avons précédemment démontré que le traitement in vivo des RSH par l’agoniste spécifique du récepteur du peptide natriurétique de type C (NPR-C), le C-ANP4-23 atténue l’hyper-prolifération des CMLVs. Nous avons entrepris cette étude afin d’investiguer si l’effet antiprolifératif du C-ANP4-23 agit par l’entremise de l’inhibition de la surexpression des protéines du cycle cellulaire, et afin d’en explorer les mécanismes sous-jacents. Pour cette étude, des RSH et des rats Wistar Kyoto (WKYs) âgés de deux semaines ont été injectés en intra-péritonéale par le C-ANP4-23 de 2 jusqu’à 8 semaines d’âge, deux fois par semaine et sacrifiés à la 9ème semaine. La pression artérielle a été mesurée par méthode Queue-coiffe, la prolifération des CMLVs a été déterminée par incorporation de thymidine et par test MTT, et l’expression des protéines a été quant à elle déterminée par technique d’immunobuvardage de type Western. Les CMLVs des RSH ont démontré une prolifération élevée en comparaison avec celles des WKYs, et le traitement par le C-ANP4-23 a atténué l’hyperprolifération à un niveau de contrôle. De plus, la surexpression des cyclines D1/A/E, des kinases cyclines dépendantes 2 et 4 (cdk2, cdk4), de la forme phosphorylée de la protéine du rétinoblastome et des protéines Gαi des CMLV des RSH a été atténuée à un niveau de contrôle. Par ailleurs, l’hyperphosphorylation d’ERK1/2, AKT, EGF-R, PDGF-R, IGF-R et de c-Src a significativement diminué par le traitement au C-ANP4-23. En outre, le niveau élevé de l’anion superoxyde (O2-), l’activité de la NADP(H) oxydase et de ses sous unités chez les RSH ont été atténués par le C-ANP4-23 .Ces résultats indiquent que l’activation in vivo de NPR-C atténue la surexpression des protéines du cycle cellulaire via l’inhibition de l’activité élevée du stress oxydatif, de c-Src et de l’activation de EGF-R, PDGF- R, IGF-R, de la signalisation de MAPK et la surexpression des protéines Gαi résultant ainsi en l’inhibition de l’hyperprolifération des CMLVs des RSH. Ainsi, il peut être suggéré que le C-ANP4-23 pourrait être utilisé comme agent thérapeutique pour le traitement des complications vasculaires associées à l’hypertension et à l’athérosclérose.

New molecular strategies for prevention of fibroproliferative vascular disease : An experimental approach to restenosis

Vascular intimal hyperplasia is a major complication following angioplasty. The hallmark feature of this disorder is accumulation of dedifferentiated smooth muscle cells (SMCs) to the luminal side of the injured artery, cellular proliferation, migration, and synthesis of extracellular matrix. This finally results in intimal hyperplasia, which is currently considered an untreatable condition. According to current knowledge, a major part of neointimal cells derive from circulating precursor cells. This has outdated the traditional in vitro cell culture methods of studying neointimal cell migration and proliferation using cultured medial SMCs. Somatostatin and some of its analogs with different selectivity for the five somatostatin receptors (sst1 through sst5) have been shown to have vasculoprotective properties in animal studies. However, clinical trials using analogs selective for sst2/sst3/sst5 to prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA) have failed to show any major benefits. Sirolimus is a cell cycle inhibitor that has been suggested to act synergistically with the protein-tyrosine kinase inhibitor imatinib to inhibit intimal hyperplasia in rat already at well-tolerated submaximal oral doses. The mechanisms behind this synergy and its long-term efficacy are not known. The aim of this study was to set up an ex vivo vascular explant culture model to measure neointimal cell activity without excluding the participation of circulating progenitor cells. Furthermore, two novel potential vasculoprotective treatment strategies were evaluated in detail in rat models of intimal hyperplasia and in the ex vivo explant model: sst1/sst4-selective somatostatin receptor analogs and combination treatment with sirolimus and imatinib. This study shows how whole vessel explants can be used to study the kinetics of neointimal cells and their progenitors, and to evaluate the anti-migratory and anti-proliferative properties of potential vasculoprotective compounds. It also shows how the influx of neointimal progenitor cells occurs already during the first days after vascular injury, how the contribution of cell migration is more important in the injury response than cell proliferation, and how the adventitia actively contribute in vascular repair. The vasculoprotective effect of somatostatin is mediated preferentially through sst4, and through inhibition of cell migration rather than of proliferation, which may explain why sst2/sst3/sst5-selective analogs have failed in clinical trials. Furthermore, a brief early oral treatment with the combination of sirolimus and imatinib at submaximal doses results in long-term synergistic suppression of intimal hyperplasia. The synergy is a result of inhibition of post-operative thrombocytosis and leukocytosis, inhibition of neointimal cell migration to the injury-site, and maintenance of cell integrity by inhibition of apoptosis and SMC dedifferentiation. In conclusion, the influx of progenitor cells already during the first days after injury and the high neointimal cell migratory activity underlines the importance of early therapeutic intervention with anti-migratory compounds to prevent neointimal hyperplasia. Sst4-selective analogs and the combination therapy with sirolimus and imatinib represent potential targets for the development of such vasculoprotective therapies.

Vascular growth factors and progenitor cells in cardiac allograft arteriosclerosis

Heart transplantation is the only therapeutic modality for many end-stage heart diseases but poor long-term survival remains a challenging problem. This is mainly due to the development of cardiac allograft arteriosclerosis (TxCAD) that is an accelerated form of coronary artery disease. Both traditional cardiovascular and transplantation-related risk factors for TxCAD have been identified but options for therapy are limited. TxCAD involves dysfunction of cardiac allograft vascular cells. Activated endothelial cells (EC) regulate allograft inflammation and secrete smooth muscle cell (SMC) growth factors. In turn, SMC and their progenitors invade the intima of the injured vessels and occlude the affected coronary arteries. Different vascular growth factors have to be delicately regulated in normal vascular development. In the present study, experimental heterotopic transplantation models were used to study the role of angiogenic and pro-inflammatory vascular endothelial growth factor (VEGF), EC growth factor angiopoietin (Ang), and SMC mitogen platelet-derived growth factor (PDGF) in the development of TxCAD. Pharmacological and gene transfer approaches were used to target these growth factors and to assess their therapeutic potential. This study shows that alloimmune response in heart transplants upregulates VEGF expression, and induces allograft angiogenesis that involves donor-derived primitive EC. Intracoronary adenoviral VEGF gene transfer increased macrophage infiltration, intimal angiogenesis and TxCAD. VEGF inhibition with PTK787 decreased allograft inflammation and TxCAD, and simultaneous PDGF inhibition with imatinib further decreased TxCAD. Specific inhibition of two VEGF-receptors (VEGFR) decreased allograft inflammation and TxCAD, and VEGFR-2 inhibition normalized the density of primitive and mature capillaries in the allografts. Adenovirus-mediated transient Ang1 expression in the allograft had anti-inflammatory and anti-arteriosclerotic effects. Adeno-associated virus (AAV)-mediated prolonged Ang1 or Ang2 expression had similar anti-inflammatory effects. However, AAV-Ang1 activated allograft SMC whereas AAV-Ang2 had no effects on SMC activation and decreased the development of TxCAD. These studies indicate an interplay of inflammation, angiogenesis and arteriosclerosis in cardiac allografts, and show that vascular growth factors are important regulators in the process. Also, VEGF inhibition, PDGF inhibition and angiopoietin therapy with clinically-relevant pharmacological agents or novel gene therapy approaches may counteract vascular dysfunction in cardiac allografts, and have beneficial effects on the survival of heart transplant patients in the future.

Lymphatic vascular maturation : Roles of FOXC2, NFATc1 and liprin ß1

The blood vascular system is a closed circulatory system, responsible for delivering oxygen and nutrients to the tissues. In contrast, the lymphatic vascular system is a blind-ended transport system that collects the extravasated tissue fluid from the capillary beds, and transports it back to the blood circulation. Failure in collecting or transporting the lymph, due to defects in the lymphatic vasculature, leads to accumulation of extra fluid in the tissues, and consequently to tissue swelling lymphedema. The two vascular systems function in concert. They are structurally related, but their development is regulated by separate, however overlapping, molecular mechanisms. During embryonic development, blood vessels are formed by vasculogenesis and angiogenesis, processes largely mediated by members of the vascular endothelial growth factor (VEGF) family and their tyrosine kinase receptors. The lymphatic vessels are formed after the cardiovascular system is already functional. This process, called lymphangiogenesis, is controlled by distinct members of the VEGF family, together with the transcription factors Prox1 and Sox18. After the primary formation of the vessels, the vasculature needs to mature and remodel into a functional network of hierarchically organized vessels: the blood vasculature into arteries, capillaries and veins; and the lymphatic vasculature into lymphatic capillaries, responsible for the uptake of the extravasated fluid from the tissues, and collecting vessels, responsible for the transport of the lymph back to the blood circulation. A major event in the maturation of the lymphatic vasculature is the formation of collecting lymphatic vessels. These vessels are characterized by the presence of intraluminal valves, preventing backflow of the lymph, and a sparse coverage of smooth muscle cells, which help in pumping the lymph forward. In our study, we have characterized the molecular and morphological events leading to formation of collecting lymphatic vessels. We found that this process is regulated cooperatively by the transcription factors Foxc2 and NFATc1. Mice lacking either Foxc2 or active NFATc1 fail to remodel the primary lymphatic plexus into functional lymphatic capillaries and collecting vessels. The resulting vessels lack valves, display abnormal expression of lymphatic molecules, and are hyperplastic. Moreover, the lymphatic capillaries show aberrant sprouting, and are abnormally covered with smooth muscle cells. In humans, mutations in FOXC2 lead to Lymphedema-Distichiasis (LD), a disabling disease characterized by swelling of the limbs due to insufficient lymphatic function. Our results from Foxc2 mutant mice and LD patients indicate that the underlying cause for lymphatic failure in LD is agenesis of collecting lymphatic valves and aberrant recruitment of periendothelial cells and basal lamina components to lymphatic capillaries. Furthermore, we show that liprin β1, a poorly characterized member of the liprin family of cytoplasmic proteins, is highly expressed in lymphatic endothelial cells in vivo, and is required for lymphatic vessel integrity. These data highlight the important role of FOXC2, NFATc1 and liprin β1 in the regulation of lymphatic development, specifically in the maturation and formation of the collecting lymphatic vessels. As damage to collecting vessels is a major cause of lymphatic dysfunction in humans, our results also suggest that FOXC2 and NFATc1 are potential targets for therapeutic intervention.

Isolation of mineralizing Nestin+ Nkx6.1+ vascular muscular cells from the adult human spinal cord

Background: The adult central nervous system (CNS) contains different populations of immature cells that could possibly be used to repair brain and spinal cord lesions. The diversity and the properties of these cells in the human adult CNS remain to be fully explored. We previously isolated Nestin(+) Sox2(+) neural multipotential cells from the adult human spinal cord using the neurosphere method (i.e. non adherent conditions and defined medium). -- Results: Here we report the isolation and long term propagation of another population of Nestin(+) cells from this tissue using adherent culture conditions and serum. QPCR and immunofluorescence indicated that these cells had mesenchymal features as evidenced by the expression of Snai2 and Twist1 and lack of expression of neural markers such as Sox2, Olig2 or GFAP. Indeed, these cells expressed markers typical of smooth muscle vascular cells such as Calponin, Caldesmone and Acta2 (Smooth muscle actin). These cells could not differentiate into chondrocytes, adipocytes, neuronal and glial cells, however they readily mineralized when placed in osteogenic conditions. Further characterization allowed us to identify the Nkx6.1 transcription factor as a marker for these cells. Nkx6.1 was expressed in vivo by CNS vascular muscular cells located in the parenchyma and the meninges. -- Conclusion: Smooth muscle cells expressing Nestin and Nkx6.1 is the main cell population derived from culturing human spinal cord cells in adherent conditions with serum. Mineralization of these cells in vitro could represent a valuable model for studying calcifications of CNS vessels which are observed in pathological situations or as part of the normal aging. In addition, long term propagation of these cells will allow the study of their interaction with other CNS cells and their implication in scar formation during spinal cord injury.

Avaliação dos efeitos da hiperglicemia sobre a estrutura vascular peniana em coelhos Nova Zelândia

Pacientes com diabetes mellitus (DM) têm elevadas taxas de disfunção erétil (ED). Diversos estudos examinaram esta associação. A associação entre o processo de hipertrofia vascular diabética e o grau de comprometimento dos seios cavernosos é pouco estudada. A proposta do presente estudo foi avaliar a estrutura vascular do tecido erétil de coelhos diabéticos e coelhos normais, através de histomorfometria computadorizada da artéria dorsal do pênis e dos seios vasculares cavernosos em ambos os grupos. Foram utilizados 20 coelhos adultos machos da raça Nova Zelândia, divididos em dois grupos com 10 animais cada, o grupo diabético (GD) e o grupo controle (GC). Os animais foram previamente anestesiados e os coelhos do grupo GD receberam aloxano na dose de 100mg/kg, via endovenosa para indução da diabetes. Após 10 semanas, os animais foram mortos e os pênis retirados. Fragmentos do pênis foram fixados em formalina tamponada durante 24-48 horas e processados para parafina. Para análise imunohistoquímica e identificação das fibras musculares lisas, foi utilizado o anticorpo anti alfa-actina. Foram feitas análises da espessura média das túnicas íntima e média da parede da artéria dorsal do pênis (ADP), densidade nuclear na túnica média e avaliação por microscopia de polarização do conteúdo colágeno na túnica adventícia. Na túnica íntima foram observadas vacuolizações no endotélio. Os valores encontrados para espessura de GC e GD foram respectivamente (em m): 35,0123,177 e 44,3308,434 (P=0,0350). Foi encontrada diferença na área média da parede da ADP (P=0,0179). Para densidade nuclear GC 0,0071540,001954 núcleos/μm e GD 0,0048080,002069 núcleos/μm (P=0,0855). Foram observadas mudanças na birrefringência das fibras colágenas na túnica adventícia, passando de alaranjado no grupo GC para esverdeado no grupo GD, indicando a mudança em sua espessura. A área ocupada pelos seios cavernosos apresentou diminuição significativa de 37 % no grupo diabético (P=0,0013). Este conjunto de alterações sugere que a hiperglicemia crônica provocada pelo diabetes levou a um processo de hipertrofia da musculatura lisa na parede vascular e nas trabéculas do corpo cavernoso, com diminuição da área dos seios, o que possivelmente altera as propriedades hemodinâmicas do órgão

Utility of telomerase-pot1 fusion protein in vascular tissue engineering.

While advances in regenerative medicine and vascular tissue engineering have been substantial in recent years, important stumbling blocks remain. In particular, the limited life span of differentiated cells that are harvested from elderly human donors is an important limitation in many areas of regenerative medicine. Recently, a mutant of the human telomerase reverse transcriptase enzyme (TERT) was described, which is highly processive and elongates telomeres more rapidly than conventional telomerase. This mutant, called pot1-TERT, is a chimeric fusion between the DNA binding protein pot1 and TERT. Because pot1-TERT is highly processive, it is possible that transient delivery of this transgene to cells that are utilized in regenerative medicine applications may elongate telomeres and extend cellular life span while avoiding risks that are associated with retroviral or lentiviral vectors. In the present study, adenoviral delivery of pot1-TERT resulted in transient reconstitution of telomerase activity in human smooth muscle cells, as demonstrated by telomeric repeat amplification protocol (TRAP). In addition, human engineered vessels that were cultured using pot1-TERT-expressing cells had greater collagen content and somewhat better performance in vivo than control grafts. Hence, transient delivery of pot1-TERT to elderly human cells may be useful for increasing cellular life span and improving the functional characteristics of resultant tissue-engineered constructs.

A Tissue-Engineered Microvascular System to Evaluate Vascular Progenitor Cells for Angiogenic Therapies

The ability of tissue engineered constructs to replace diseased or damaged organs is limited without the incorporation of a functional vascular system. To design microvasculature that recapitulates the vascular niche functions for each tissue in the body, we investigated the following hypotheses: (1) cocultures of human umbilical cord blood-derived endothelial progenitor cells (hCB-EPCs) with mural cells can produce the microenvironmental cues necessary to support physiological microvessel formation in vitro; (2) poly(ethylene glycol) (PEG) hydrogel systems can support 3D microvessel formation by hCB-EPCs in coculture with mural cells; (3) mesenchymal cells, derived from either umbilical cord blood (MPCs) or bone marrow (MSCs), can serve as mural cells upon coculture with hCB-EPCs. Coculture ratios between 0.2 (16,000 cells/cm2) and 0.6 (48,000 cells/cm2) of hCB-EPCs plated upon 3.3 µg/ml of fibronectin-coated tissue culture plastic with (80,000 cells/cm2) of human aortic smooth muscle cells (SMCs), results in robust microvessel structures observable for several weeks in vitro. Endothelial basal media (EBM-2, Lonza) with 9% v/v fetal bovine serum (FBS) could support viability of both hCB-EPCs and SMCs. Coculture spatial arrangement of hCB-EPCs and SMCs significantly affected network formation with mixed systems showing greater connectivity and increased solution levels of angiogenic cytokines than lamellar systems. We extended this model into a 3D system by encapsulation of a 1 to 1 ratio of hCB-EPC and SMCs (30,000 cells/µl) within hydrogels of PEG-conjugated RGDS adhesive peptide (3.5 mM) and PEG-conjugated protease sensitive peptide (6 mM). Robust hCB-EPC microvessels formed within the gel with invasion up to 150 µm depths and parameters of total tubule length (12 mm/mm2), branch points (127/mm2), and average tubule thickness (27 µm). 3D hCB-EPC microvessels showed quiescence of hCB-EPCs (<1% proliferating cells), lumen formation, expression of EC proteins connexin 32 and VE-cadherin, eNOS, basement membrane formation by collagen IV and laminin, and perivascular investment of PDGFR-β+/α-SMA+ cells. MPCs present in <15% of isolations displayed >98% expression for mural markers PDGFR-β, α-SMA, NG2 and supported hCB-EPC by day 14 of coculture with total tubule lengths near 12 mm/mm2. hCB-EPCs cocultured with MSCs underwent cell loss by day 10 with a 4-fold reduction in CD31/PECAM+ cells, in comparison to controls of hCB-EPCs in SMC coculture. Changing the coculture media to endothelial growth media (EBM-2 + 2% v/v FBS + EGM-2 supplement containing VEGF, FGF-2, EGF, hydrocortisone, IGF-1, ascorbic acid, and heparin), promoted stable hCB-EPC network formation in MSC cocultures over 2 weeks in vitro, with total segment length per image area of 9 mm/mm2. Taken together, these findings demonstrate a tissue engineered system that can be utilized to evaluate vascular progenitor cells for angiogenic therapies.

AM1- receptor dependent protection by intermedin of human vascular and cardiac non-vascular cells from ischaemia-reperfusion injury

Intermedin (IMD) protects rodent heart and vasculature from oxidative stress and ischaemia. Less is known about distribution of IMD and its receptors and the potential for similar protection in man. Expression of IMD and receptor components were studied in human aortic endothelium cells (HAECs), smooth muscle cells (HASMCs), cardiac microvascular endothelium cells (HMVECs) and fibroblasts (v-HCFs). Receptor subtype involvement in protection by IMD against injury by hydrogen peroxide (H2O2, 1 mmol l?¹) and simulated ischaemia and reperfusion were investigated using receptor component-specific siRNAs. IMD and CRLR, RAMP1, RAMP2 and RAMP3 were expressed in all cell types.When cells were treated with 1 nmol l?¹ IMD during exposure to 1 mmol l?¹ H2O2 for 4 h, viability was greater vs. H2O2 alone (P<0.05 for all cell types). Viabilities under 6 h simulated ischaemia differed (P<0.05) in the absence and presence of 1 nmol l?¹ IMD: HAECs 63% and 85%; HMVECs 51% and 68%; v-HCFs 42% and 96%. IMD 1 nmol l?¹ present throughout ischaemia (3 h) and reperfusion (1 h) attenuated injury (P<0.05): viabilities were 95%, 74% and 82% for HAECs, HMVECs and v-HCFs, respectively, relative to those in the absence of IMD (62%, 35%, 32%, respectively). When IMD 1 nmol l?¹ was present during reperfusion only, protection was still evident (P<0.05, 79%, 55%, 48%, respectively). Cytoskeletal disruption and protein carbonyl formation followed similar patterns. Pre-treatment (4 days) of HAECs with CRLR or RAMP2, but not RAMP1 or RAMP3, siRNAs abolished protection by IMD (1 nmol l?¹) against ischaemia-reperfusion injury. IMD protects human vascular and cardiac non-vascular cells from oxidative stress and ischaemia-reperfusion,predominantly via AM1 receptors.

Epigenetics in Vascular Disease - Therapeutic Potential of New Agents

Vascular diseases, including atherosclerosis, angioplasty-induced restenosis, vessel graft arteriosclerosis and hypertension-related stenosis, remain the most prevalent cause of death in the developed world. The aetiology of vascular diseases is multifactorial with both genetic and environmental factors. Recently, some of the most promising research identifies the epigenetic modification of the genome to play a major role in the disease development, linking the environmental insults with gene regulation. In this process, modification of DNA by methylation, and histone modification by acetylation, methylation, phosphorylation and/or SUMOylation are reported. Importantly, recent studies demonstrated that histone deacetylase (HDAC) enzymes are crucial in endothelial integrity, smooth muscle proliferation and in the formation of arteriosclerosis in animal models. The study of HDACs has shown remarkable specificity of HDAC family members in vascular cell growth/death that influences the disease process. Interestingly, the effects of HDACs on arteriosclerosis development in animal models have been observed after HDAC inhibition using specific inhibitors. This provides a new approach for the treatment of vascular disease using the agents that influence the epigenetic process in vascular cells. This review updates the rapid advances in epigenetics of vascular diseases focusing on the role of HDAC family in atherosclerosis. It will also discuss the underlying mechanisms of histone acetylation in vascular cells and highlight the therapeutic potential of such agents.

Chromobox protein homolog 3 is essential for stem cell differentiation to smooth muscles in vitro and in embryonic arteriogenesis

Smooth muscle cell (SMC) differentiation is a critical process during cardiovascular formation and development, but the underlying molecular mechanism remains unclear.