Porphyromonas gingivalis infection causes umbilical vein endothelial barrier dysfunction in vitro by down-regulating ZO-1, occludin and VE-cadherin expression / 南方医科大学学报

OBJECTIVE@#To explore the molecular mechanisms of Porphyromonas gingivalis infection-induced umbilical vein endothelial barrier dysfunction in vitro.@*METHODS@#Human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and after the formation of the endothelial barrier, the cells were infected with P. gingivals at a multiplicity of infection (MOI). The transepithelial electrical resistance (TEER) of the cell barrier was measured, and FITC-dextran trans-endothelial permeability assay and bacterial translocation assay were performed to assess the endothelial barrier function. The expression levels of cell junction proteins including ZO-1, occludin and VE-cadherin in the cells were examined by qRT-PCR and Western blotting.@*RESULTS@#In freshly seeded HUVECs, TEER increased until reaching the maximum on Day 5 (94 Ωcm2), suggesting the formation of the endothelial barrier. P. gingivals infection caused an increase of the permeability of the endothelial barrier as early as 0.5 h after bacterial inoculation, and the barrier function further exacerbated with time, as shown by significantly lowered TEER, increased permeability of FITC-dextran (40 000/70 000), and increased translocation of SYTO9-E. coli cross the barrier. MTT assay suggested that P. gingivals infection did not significantly affect the proliferation of HUVECs (P>0.05), but in P. gingivalsinfected cells, the expressions of ZO-1, occludin and VE-cadherin increased significantly at 24 and 48 h after bacterial inoculation (P < 0.05).@*CONCLUSION@#P. gingivals may disrupt the endothelial barrier function by down-regulating the expressions of the cell junction proteins (ZO-1, occludin, VE-cadherin) and increasing the permeability of the endothelial barrier.

Dopplervelocimetria do ducto venoso na predicão da acidemia fetal / Ductus venosus Doppler velocimetry to predict acidemia at birth in pregnancies with placental insufficiency

OBJETIVOS: Investigar a possibilidade da predicão da acidemia no nascimento mediante dopplervelocimetria do ducto venoso e definir qual o melhor parâmetro e seus pontos de corte nessa predicão em gestacões com insuficiência placentária. MÉTODOS: Trata-se de estudo transversal e prospectivo que analisou 47 gestacões únicas com insuficiência placentária e idade gestacional superior a 26 semanas, realizado no Hospital São Paulo (UNIFESP) e na Maternidade-Escola Assis Chateaubriand (UFC). A insuficiência placentária foi diagnosticada quando o índice de pulsatilidade da artéria umbilical encontrava-se acima do percentil 95 para a idade gestacional estimada. Fetos com anomalias estruturais ou cromossômicas foram excluídos. O doppler foi realizado a menos de 24 horas do parto. A amostra de sangue da artéria umbilical foi coletada imediatamente após o nascimento para análise da gasometria. Diagnosticou-se acidemia quando o pH encontrava-se abaixo de 7,20 na ausência de trabalho de parto e abaixo de 7,15 quando parto vaginal. Foram consideradas patológicas as acidemias metabólicas ou mistas. Construiu-se curva ROC para as velocidades S, D e A e para o IPV e as relacões S/A e (S-A)/S do DV (variáveis independentes) e acidemia (variável dependente). O teste de MacNemar foi utilizado para comparar os parâmetros entre si. RESULTADOS: As velocidades absolutas S, D e A mostraram ser pobres preditoras da acidemia no nascimento. O IPV mostrou ser bom preditor de acidemia (área sob a curva ROC 0,79, p=0,003). As relacões S/A e (S-A)/S também mostraram ser boas preditoras da acidemia (área sob a curva ROC 0,818, p=0,001). Os pontos de corte calculados foram: IPV = 0,76, S/A = 2,67 e (S-A)/S = 0,63. CONCLUSÕES: Os índices ângulo-independentes do doppler do DV mostraram excelente correlacão com acidemia no nascimento nesta populacão. Não houve diferenca estatisticamene significativa entre estes parâmetros.

Potential role of leptin in angiogenesis: leptin induces endothelial cell proliferation and expression of matrix metalloproteinases in vivo and in vitro

Leptin, the product of ob gene, is an endocrine hormone that regulates adipose tissue mass. Recently, leptin has been found to generate a growth signal involving a tyrosine kinase-dependent intracellular pathway and promote angiogenic processes via activation of leptin receptor (Ob-R) in endothelial cells. However, it is not clear how leptin functions to promote multi-step processes involved in the neovascularization at the atherosclerotic plaque. We have examined the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) and Ob-R in human atherosclerotic lesions, leptin-mediated angiogenesis in vivo and in vitro. Immunohistochemical analysis of human atherosclerotic aorta revealed an increased expression of Ob-R in the intima of neorevascularized regions and of both MMPs and TIMPs predominantly in the endothelial lining of intimal neovessels and macrophages/foam cells. In the rat corneal angiogenesis assay, leptin elicited a comparable sensitivity of angiogenic activity to those of vascular endothelial growth factor (VEGF). The immunohistological analysis of the leptin-treated rat cornea showed definitive rises in Ob-R, MMPs and TIMPs expression as well as those of VEGF receptor (VEGFR-1). Leptin (10-40 ng/ml) induced proliferation of the human umbilical vein endothelial cells (HUVECs) and elevation of MMP-2, MMP-9, TIMP-1, and TIMP-2 expression in a dose-dependent manner. Leptin also induced increases of MMP-2, MMP-9, TIMP-1, and Up-regulated the human coronary artery smooth muscle cells (HCASMCs). These findings suggest that leptin, a hormone with pluralistic properties including a mitogenic activity on vascular endothelial cells, plays a role in matrix remodeling by regulating the expression of MMPs and TIMPs. Taken together, our findings further provide evidences for leptin's role as an angiogenesis inducer in the normal organ (rat cornea) and in aberrant vasculature under duress like atherosclerosis.

Endothelial cell oxidative stress and signal transduction

Endothelial dysfunction (ED) is an early event in atherosclerotic disease, preceding clinical manifestations and complications. Increased reactive oxygen species (ROS) have been implicated as important mechanisms that contribute to ED, and ROS's may function as intracellular messengers that modulate signaling pathways. Several intracellular signal events stimulated by ROS have been defined, including the identification of two members of the mitogen activated protein kinase family (ERK1/2 and big MAP kinase, BMK1), tyrosine kinases (Src and Syk) and different isoenzymes of PKC as redox-sensitive kinases. ROS regulation of signal transduction components include the modification in the activity of transcriptional factors such as NFkB and others that result in changes in gene expression and modifications in cellular responses. In order to understand the intracellular mechanisms induced by ROS in endothelial cells (EC), we are studying the response of human umbilical cord vein endothelial cells to increased ROS generation by different pro-atherogenic stimuli. Our results show that Homocysteine (Hcy) and oxidized LDL (oxLDL) enhance the activity and expression of oxidative stress markers, such as NFkB and heme oxygenase 1. These results suggest that these pro-atherogenic stimuli increase oxidative stress in EC, and thus explain the loss of endothelial function associated with the atherogenic process.