RESUMO
The limited availability of human vascular endothelial cells (ECs) hampers research into EC function whilst the lack of precisely defined culture conditions for this cell type presents problems for addressing basic questions surrounding EC physiology. We aimed to generate endothelial progenitors from human pluripotent stem cells to facilitate the study of human EC physiology, using a defined serum-free protocol. Human embryonic stem cells (hESC-ECs) differentiated under serum-free conditions generated CD34(+)KDR(+) endothelial progenitor cells after 6days that could be further expanded in the presence of vascular endothelial growth factor (VEGF). The resultant EC population expressed CD31 and TIE2/TEK, took up acetylated low-density lipoprotein (LDL) and up-regulated expression of ICAM-1, PAI-1 and ET-1 following treatment with TNFα. Immunofluorescence studies indicated that a key mediator of vascular tone, endothelial nitric oxide synthase (eNOS), was localised to a perinuclear compartment of hESC-ECs, in contrast with the pan-cellular distribution of this enzyme within human umbilical vein ECs (HUVECs). Further investigation revealed that that the serum-associated lipids, lysophosphatidic acid (LPA) and platelet activating factor (PAF), were the key molecules that affected eNOS localisation in hESC-ECs cultures. These studies illustrate the feasibility of EC generation from hESCs and the utility of these cells for investigating environmental cues that impact on EC phenotype. We have demonstrated a hitherto unrecognized role for LPA and PAF in the regulation of eNOS subcellular localization.
Assuntos
Meios de Cultura/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células Endoteliais/citologia , Lisofosfolipídeos/farmacologia , Óxido Nítrico Sintase Tipo III/análise , Fator de Ativação de Plaquetas/farmacologia , Antígenos CD34/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Colágeno/química , Combinação de Medicamentos , Células-Tronco Embrionárias/citologia , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Laminina/química , Óxido Nítrico Sintase Tipo III/metabolismo , Proteoglicanas/química , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
We investigated the ability of several novel class I histone deacetylase inhibitors to activate HIV-1 transcription in latently infected cell lines. Oxamflatin, metacept-1 and metacept-3 induced high levels of HIV-1 transcription in latently infected T cell and monocytic cells lines, were potent inhibitors of histone deacetylase activity and caused preferential cell death in transcriptionally active cells. Although these compounds had potent in-vitro activity, their cytotoxicity may limit their use in patients.
Assuntos
HIV-1/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Sulfonamidas/farmacologia , Linfócitos T/virologia , Ativação Transcricional/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , HIV-1/genética , HIV-1/fisiologia , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Humanos , Latência Viral/efeitos dos fármacosRESUMO
Conformational analogues of the hydroxamic acid Oxamflatin compounds, have been synthesised to enable evaluation of the impact of varying the linking section on histone deacetylase inhibition. Preliminary testing indicates treatment of leukaemia cells with each of the analogues leads to significant inhibition of histone deacetylase and reduction in cell growth and proliferation.
