Microarray analyses of the effects of NF-kappaB or PI3K pathway inhibitors on the LPS-induced gene expression profile in RAW264.7 cells: synergistic effects of rapamycin on LPS-induced MMP9-overexpression.

Lipopolysaccharide (LPS) activates a broad range of signalling pathways including mainly NF-kappaB and the MAPK cascade, but recent evidence suggests that LPS stimulation also activates the PI3K pathway. To unravel the specific roles of both pathways in LPS signalling and gene expression profiling, we investigated the effects of different inhibitors of NF-kappaB (BAY 11-7082), PI3K (wortmannin and LY294002) but also of mTOR (rapamycin), a kinase acting downstream of PI3K/Akt, in LPS-stimulated RAW264.7 macrophages, analyzing their effects on the LPS-induced gene expression profile using a low density DNA microarray designed to monitor the expression of pro-inflammatory genes. After statistical and hierarchical cluster analyses, we determined five clusters of genes differentially affected by the four inhibitors used. In the fifth cluster corresponding to genes upregulated by LPS and mainly affected by BAY 11-7082, the gene encoding MMP9 displayed a particular expression profile, since rapamycin drastically enhanced the LPS-induced upregulation at both the mRNA and protein levels. Rapamycin also enhanced the LPS-induced NF-kappaB transactivation as determined by a reporter assay, phosphorylation of the p38 and Erk1/2 MAPKs, and counteracted PPAR activity. These results suggest that mTOR could negatively regulate the effects of LPS on the NF-kappaB and MAPK pathways. We also performed real-time RT-PCR assays on mmp9 expression using rosiglitazone (agonist of PPARgamma), PD98059 (inhibitor of Erk 1/2) and SB203580 (inhibitor of p38(MAPK)), that were able to counteract the rapamycin mediated overexpression of mmp9 in response to LPS. Our results suggest a new pathway involving mTOR for regulating specifically mmp9 in LPS-stimulated RAW264.7 cells.

Overexpression of the soluble vascular endothelial growth factor receptor in preeclamptic patients: pathophysiological consequences.

Several growth factors such as vascular endothelial growth factor (VEGF)-A and placental growth factor (PlGF) are involved in the placental vascular development. We investigated whether dysregulation in the VEGF family may explain the defective uteroplacental vascularization characterizing preeclampsia. We compared pregnancies complicated by early onset severe preeclampsia or intrauterine growth retardation to normal pregnancies. Maternal plasma, placentas, and placental bed biopsies were collected. The mRNA levels of VEGF-A, PlGF, and their receptors were quantified in placentas and placental beds. Levels of VEGF-A, PlGF, and soluble VEGF receptor (VEGFR) were assessed in maternal plasma. In compromised pregnancies, elevated levels of VEGF-A and VEGFR-1 mRNAs may reflect the hypoxic status of the placenta. On contrast, the membrane-bound VEGFR-1 was decreased in the placental bed of preeclamptic patients. Preeclampsia was associated with low levels of circulating PlGF and increased levels of total VEGF-A and soluble VEGFR-1. Free VEGF-A was undetectable in maternal blood. Immunohistochemical studies revealed that VEGF-A and PlGF were localized in trophoblastic cells. Altogether, our results suggest two different pathophysiological mechanisms associated with preeclampsia. The first one is related to an overproduction of competitive soluble VEGFR-1 that may lead to suppression of VEGF-A and PlGF effects. The second one is the down-regulation of its membrane bound form (VEGFR-1) in the placental bed, which may result in the defective uteroplacental development.