RESUMO
OBJECTIVES: Sphingosine 1-phosphate (S1P) is a bioactive phospholipid acting both as a ligand for the G protein-coupled receptors S1P1-5 and as a second messenger. Because S1P1 knockout is lethal in the transgenic mouse, an alternative approach to study the function of S1P1 in endothelial cells is needed. METHODS AND RESULTS: All human endothelial cells analyzed expressed abundant S1P1 transcripts. We permanently silenced (by RNA interference) the expression of S1P1 in the human endothelial cell lines AS-M.5 and ISO-HAS.1. The S1P1 knock-down cells manifested a distinct morphology and showed neither actin ruffles in response to S1P nor an angiogenic reaction. In addition, these cells were more sensitive to oxidant stress-mediated injury. New S1P1-dependent gene targets were identified in human endothelial cells. S1P1 silencing decreased the expression of platelet-endothelial cell adhesion molecule-1 and VE-cadherin and abolished the induction of E-selectin after cell stimulation with lipopolysaccharide or tumor necrosis factor-alpha. Microarray analysis revealed downregulation of further endothelial specific transcripts after S1P1 silencing. CONCLUSIONS: Long-term silencing of S1P1 enabled us for the first time to demonstrate the involvement of S1P1 in key functions of endothelial cells and to identify new S1P1-dependent gene targets.
Assuntos
Endotélio Vascular/fisiologia , Biologia Molecular/métodos , RNA Interferente Pequeno/genética , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/metabolismo , Antígenos CD , Caderinas/genética , Linhagem Celular , Endotélio Vascular/citologia , Inativação Gênica , Hemangiossarcoma , Humanos , Microcirculação , Neovascularização Fisiológica/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Circulação Pulmonar , RNA Mensageiro/genética , Sistemas do Segundo Mensageiro/fisiologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais/citologia , Neoplasias Vasculares , Vasculite/genética , Vasculite/imunologia , Vasculite/fisiopatologiaRESUMO
n-3 polyunsaturated fatty acids (PUFAs) have been shown to exert beneficial effects in the prevention of cardiovascular disease, inflammation, and on tumor growth. To investigate effects of PUFAs on proliferation and apoptosis in endothelial cells, we tested the n-3 PUFA docosahexaenoic acid (DHA) and the n-6 PUFA arachidonic acid (AA) in human umbilical vein endothelial cells (HUVEC). The mitochondrial membrane potential (MMP) and the production of reactive oxygen species were examined by flow cytometry. Phosphorylation of p53 or p38 MAP kinase, and total levels of p53 were measured by Western blot. DNA binding activity of p53 was analyzed with a TransAM transcription factor assay kit. Tube formation was assessed on Matrigel. In proliferating HUVEC, but not in confluent cells, DHA reduced cell viability and induced apoptosis, as demonstrated by increases in membrane leakage (propidium iodide (PI) staining), Annexin-V binding, sub G(1) phase in the cell cycle, and TUNEL-positive cells. AA had no effect on these parameters. In addition to a reduced MMP and increased reactive oxygen species, phosphorylation of p38 and p53 (serine 15) and impaired DNA binding of p53 were observed. There was no change in total levels of p53. The p38 inhibitor SB203580 had no effect on Annexin V binding. DHA also attenuated HUVEC tube formation. Taken together, DHA induces apoptosis in proliferating, but not in resting HUVEC, potentially via the phosphorylation of p53, resulting in decreased p53 DNA binding. The results suggest that anti-angiogenic effects of DHA may be due to induction of apoptosis in proliferating endothelial cells.
Assuntos
Apoptose/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Ácido Araquidônico/farmacologia , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , DNA/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Células Endoteliais/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosfosserina/metabolismo , Fosfotirosina/metabolismo , Superóxidos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Cordão Umbilical/citologia , Cordão Umbilical/efeitos dos fármacos , Cordão Umbilical/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Prostaglandins are a family of structurally related molecules formed by many cells in response to extrinsic stimuli. A member of this family, 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), shows unique biological properties including anti-inflammatory, anti-viral, and anti-tumour activity, and has attracted much attention as a high affinity ligand for the peroxisome proliferator-activated receptor gamma. Increasing evidence points to additional effects. We investigated several structurally related prostaglandins in comparison to 15d-PGJ(2) with respect to their apoptosis-inducing capacity in human umbilical endothelial cells (HUVEC). Cell viability was tested with a modified MTT assay and apoptosis was detected by Annexin V staining and cell cycle analysis by flow cytometry. Incubation of confluent HUVECs with 15d-PGJ(2) markedly reduced endothelial cell viability which was due to apoptosis. In contrast, none of the other PGs tested affected cell viability. Interestingly, the cyclopentenone ring alone dose-dependently reduced cell viability and significantly induced apoptosis in HUVECs with as low a concentration as 0.25 microM. In conclusion, we report that the cyclopentenone moiety of cyPGs is an essential component for the apoptosis-inducing properties of 15d-PGJ(2). For 15d-PGJ(2) the position of the cyclopentenone ring in conjunction with the side chains yields a molecule with unique biological properties.
Assuntos
Apoptose , Ciclopentanos/química , Ciclopentanos/farmacologia , Endotélio Vascular/citologia , Prostaglandinas/química , Prostaglandinas/farmacologia , Linhagem Celular , Humanos , Estrutura Molecular , Prostaglandina D2/análogos & derivados , Prostaglandina D2/química , Prostaglandina D2/farmacologiaRESUMO
Cyclopentenone prostaglandins (CP-PG), such as prostaglandin A1 (PGA1) or 15-deoxy-Delta(12,14)-prostaglandin J2 (PGJ2), induce apoptosis in different cell types. PGJ2 is also a potent activator of the peroxisome proliferator-activated receptor-gamma (PPARgamma). We investigated whether PPARgamma regulates CP-PG-induced apoptosis in endothelial cells (EC). We show that CP-PG induce apoptosis in human umbilical vein EC (HUVEC). Incubation with PGA1 or PGJ2 for 24 h reduced HUVEC number and viability, while the synthetic activators Wy14643 or rosiglitazone had no effect. Flow cytometry and cell cycle analysis revealed externalized phosphatidylserine, caspase-3 activation, and an increased percentage of cells with a reduced DNA content by CP-PG treatment. EMSA demonstrated an activation of PPARgamma by PGJ2 and rosiglitazone. Immunohistochemistry of HUVEC and immunoblot analyses of protein extracts showed that PPARgamma was localized in the nuclei of HUVEC, and that CP-PG treatment decreased the amount of PPARgamma protein. This degradation was prevented by a pan-caspase inhibitor. Treatment of differentiated, endothelial-like PPARgamma-deficient stem cells, or of HUVEC transfected with dominant-negative PPARgamma with CP-PG, induced cell death and apoptosis. Our findings show that PGA1 and PGJ2 induce apoptosis in endothelial cells independent of PPARgamma. As the synthesis of PGJ2 is increased at sites of inflammation, our results may suggest a possible mechanism for endothelial damage.