RESUMO
Aging is a major factor predisposing for multiple diseases. Telomeres at the ends of chromosomes protect the integrity of chromosomal DNA. A specialized six-protein complex termed shelterin protects the telomere from unwanted interaction with DNA damage pathways. The aim of our study was to evaluate the integrity of telomeres and the stability of telomere protection during aging in endothelial cells (EC). We describe that aging EC can be characterized by an increased cell size (40%, p=0.02) and increased expression of PAI 1 (4 fold, p=0.02), MCP1 (10 fold, p=0.001) and GMCSF (15 fold, p=0.004). Telomeric state in aging cells is defined by an increased telomere oxidation (27%, p=0.01), reduced telomere length (62%, p=0.02), and increased DNA damage foci formation (5% in young EC versus 16% in aged EC, p=0.003). This telomeric dysfunction is accompanied by a reduction in the shelterin component TRF1 (33% mRNA, p=0.001; 24% protein, p=0.007). Overexpression of TRF1 in aging EC reduced telomere-associated DNA damage foci to 5% (p=0.02) and reduced expression levels of MCP1 (18% reduction, p=0.008). Aged EC have increased telomere damage and an intrinsic loss of telomere protection. Reestablishing telomere integrity could therefore be a target for rejuvenating endothelial cell function.
Assuntos
Senescência Celular/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Telômero/genética , Proteína 1 de Ligação a Repetições Teloméricas/genética , Western Blotting , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Dano ao DNA , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Hibridização in Situ Fluorescente , Microscopia Confocal , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telômero/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/metabolismoRESUMO
BACKGROUND: Periodontitis represents a chronic infection of supportive dental tissues by distinct gram-negative bacteria. It is characterized by chronic and local inflammation as well as transient bacteremia with frequently occurring infections at distant sites. OBJECTIVES: The present work aimed to clarify the role of platelets and plasma factors in neutrophil interactions with the periodontopathogens A. actinomycetemcomitans and P. gingivalis. METHODS: Phagocytosis, cell-cell interactions and activation of platelets and neutrophils in response to periodontopathogens were analyzed by flow cytometry, confocal microscopy and bacteria survival assay. Plasma factors, platelet signaling pathways and receptors involved in platelet-neutrophil-bacteria interactions were determined. The role of platelet and neutrophil TLR2 in phagocytosis was further evaluated in a murine TLR2 knockout model. RESULTS: In the presence of plasma neutrophil-mediated clearance of periodontopathogens is doubled due to opsonisation of bacteria. Platelets, which become activated by periodontopathogens, further enhance clearance of bacteria by 20%, via direct interaction with neutrophils. Plasma factors (e.g. CD14) are required for platelet activation, which is mainly TLR2 dependent and results in PI3K/Akt activation. In a murine TLR2 knockout model we prove that platelet TLR2 is important for formation of platelet-neutrophil aggregates and enhanced phagocytosis of periodontopathogens. In contrast, neutrophil TLR2 is not involved in platelet-neutrophil aggregate formation but is required for efficient phagocytosis. CONCLUSIONS: These data indicate that efficient elimination of periodontopathogens by neutrophils involves a complex interplay of plasma factors as well as platelets and requires functional TLR2. By enhancing neutrophil activation platelets contribute to immune defense but may also foster inflammation.