Adrenomedullin is a peptide hormone with multifunctional biological properties. Its most characteristic effects are the regulation of circulation and the control of fluid and electrolyte homeostasis through peripheral and central nervous system actions. Although adrenomedullin is a vasodilator of cerebral vasculature, and it may be implicated in the pathomechanism of cerebrovascular diseases, the source of adrenomedullin in the cerebral circulation has not been investigated thus far. We measured the secretion of adrenomedullin by radioimmunoassay and detected adrenomedullin mRNA expression by Northern blot analysis in primary cultures of rat cerebral endothelial cells (RCECs), pericytes and astrocytes. We also investigated the expression of specific adrenomedullin receptor components by reverse transcriptase-polymerase chain reaction and intracellular cAMP concentrations in RCECs and pericytes. RCECs had approximately one magnitude higher adrenomedullin production (135 +/- 13 fmol/10(5) cells per 12 h; mean +/- SD, n = 10) compared to that previously reported for other cell types. RCECs secreted adrenomedullin mostly at their luminal cell membrane. Adrenomedullin production was not increased by thrombin, lipopolysaccharide or cytokines, which are known inducers of adrenomedullin release in peripheral endothelial cells, although it was stimulated by astrocyte-derived factors. Pericytes had moderate, while astrocytes had very low basal adrenomedullin secretion. In vivo experiments showed that adrenomedullin plasma concentration in the jugular vein of rats was approximately 50% higher than that in the carotid artery or in the vena cava. Both RCECs and pericytes, which are potential targets of adrenomedullin in cerebral microcirculation, expressed adrenomedullin receptor components, and exhibited a dose-dependent increase in intracellular cAMP concentrations after exogenous adrenomedullin administration. Antisense oligonucleotide treatment significantly reduced adrenomedullin production by RCECs and tended to decrease intraendothelial cAMP concentrations. These findings may suggest an important autocrine and paracrine role for adrenomedullin in the regulation of cerebral circulation and blood-brain barrier functions. Cerebral endothelial cells are a potential source of adrenomedullin in the central nervous system, where adrenomedullin can also be involved in the regulation of neuroendocrine functions.

Adrenomedullin (AM) is an important vasodilator in cerebral circulation, and cerebral endothelial cells are a major source of AM. This in vitro study aimed to determine the AM-induced changes in blood-brain barrier (BBB) functions. AM administration increased, whereas AM antisense oligonucleotide treatment decreased transendothelial electrical resistance. AM incubation decreased BBB permeability for sodium fluorescein (mol. wt 376 Da) but not for Evan's blue albumin (mol. wt 67 kDa), and it also attenuated fluid-phase endocytosis. AM treatment resulted in functional activation of P-glycoprotein efflux pump in vitro. Our results indicate that AM as an autocrine mediator plays an important role in the regulation of BBB properties of the cerebral endothelial cells.

SummaryTo investigate the effects of transforming growth factor-betas (TGF-βs) on endothelial anticoagulant activity, we assayed thrombomodulin (TM) activity and antigen levels of human umbilical vein endothelial cells (HUVECs) incubated with TGF-βs in vitro. TGF-β1 suppressed surface TM activity and surface TM antigen levels maximally 12 h after incubation in dose-dependent manners. TGF-β2 was almost equipotent with TGF-β1 for the suppression of them. Both TGF-βs suppressed total TM antigen level in HUVECs, and the time course of the suppression was similar to that of the cell surface TM antigen level. The maximal reductions of TM mRNA levels by TGF-βs were observed at several hours ahead of those observed in both surface and total TM antigen levels, suggesting that the TGF-β-mediated suppression of TM antigen of HUVECs is primarily regulated at the TM mRNA level. Our present work suggests that the down-modulation of TM level induced by TGF-βs in HUVECs contributes in vivo to promoting the thrombogenesis either at the sites of injury of vessel walls, such as atherosclerotic lesions where TGF-β1 is released from platelets, smooth muscle cells and monocytes, or at neovascular walls in tumors secreting TGF-β2.

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Both untreated and treated primary neuroblastomas from 52 patients were analyzed to determine the correlation between the amplification of N-myc oncogene and various prognostic factors. Amplification of N-myc was observed in eight of 28 untreated cases and in 12 of 24 treated cases. As a whole, 12 of 18 tumors (67%) in Stage IV had N-myc amplification, but there were fewer cases in the unadvanced disease stage, as reported previously by others. Furthermore, the authors detected N-myc amplification in three of nine tumors in Stage IV-S, although the amplification was less than 50 copies. Analysis of progression-free survival at 24 months revealed that amplification of N-myc was associated with the worst prognosis (P less than 0.001). In the untreated group, no amplification of N-myc was detected in any of two ganglioneuromas and four ganglioneuroblastomas, whereas amplification of N-myc was observed in all two round-cell and six of 20 rosette fibrillary neuroblastomas. On the other hand, the authors detected amplification of N-myc in three of eight less differentiated ganglioneuroblastomas in the treated group and observed the worst prognosis in these three patients. The total percentage of the cases from both untreated and treated groups suggest that amplification of N-myc may occur more frequently in undifferentiated types of neuroblastomas than in less malignant types. In conclusion, the amplification of N-myc in neuroblastomas was closely associated with the worst prognosis, which was suggested by both disease stage and histologic characteristics.