The CYCLOCALYX study: Ovulatory cycle affects circulating compartments of the endothelial glycocalyx in blood.

PROBLEM: The endothelial glycocalyx (EGX) plays an important role in vascular integrity. Recently, increased levels of EGX components were detected in the circulating blood of healthy pregnant women and were related to the increased tendency to edema formation during gestation. However, the EGX has not yet been systematically studied in non-pregnant women during ovulatory cycles. METHOD OF STUDY: Serum levels of EGX components syndecan-1, heparan sulfate, and hyaluronan in healthy women (n = 16) at 3 phases of the ovulatory cycle (early follicular phase, at ovulation, and mid-luteal phase) were compared with a control group of healthy men (n = 10). Using immunofluorescence microscopy in cultured human umbilical vein endothelial cells, the effects of progesterone and estrogen on the EGX were measured. RESULTS: Syndecan-1 increased from 11.1 ± 2.4 ng/mL at ovulation to 12.6 ± 2.3 ng/mL in mid-luteal phase (P = .031) and of heparan sulfate from 663 ± 35 ng/mL to 782 ± 55 ng/mL (P = .011). In contrast to estrogen, there was a detrimental effect of progesterone on the EGX in HUVECs. CONCLUSION: The relationship between the natural menstrual cycle and the EGX as an indicator of vascular permeability may provide a new explanation for premenstrual edema in healthy women. This may be an attendant phenomenon of a regular physiological process, the hormonal downregulation of the vascular barrier during pregnancy.

Extracellular RNA promotes leukocyte recruitment in the vascular system by mobilising proinflammatory cytokines.

Extracellular RNA (eRNA), released from cells under conditions of injury or vascular disease, acts as potent prothrombotic factor and promotes vascular hyperpermeability related to oedema formation in vivo. In this study, we aimed to investigate the mechanism by which eRNA triggers inflammatory processes, particularly associated with different steps of leukocyte recruitment. Using intravital microscopy of murine cremaster muscle venules, eRNA (but not DNA) significantly induced leukocyte adhesion and transmigration in vivo, which was comparable in its effects to the function of tumour-necrosis-factor-α (TNF-α). In vitro, eRNA promoted adhesion and transmigration of monocytic cells on and across endothelial cell monolayers. eRNA-induced monocyte adhesion in vitro was mediated by activation of the vascular endothelial growth factor (VEGF)/VEGF-receptor-2 system and was abolished by neutralising antibodies against intercellular adhesion molecule-1 or the ß2-integrin Mac-1. Additionally, eRNA induced the release of TNF-α from monocytic cells in a time- and concentration-dependent manner, which involved activation of TNF-α-converting enzyme (TACE) as well as the nuclear factor κB signalling machinery. In vivo, inhibiton of TACE significantly reduced eRNA-induced leukocyte adhesion. Our findings present evidence that eRNA in connection with tissue/vascular damage provokes a potent inflammatory response by inducing leukocyte recruitment and by mobilising proinflammatory cytokines from monocytes.

Live in vivo imaging of Egr-1 promoter activity during neonatal development, liver regeneration and wound healing.

BACKGROUND: The zinc finger transcription factor Egr-1 (Early growth response 1) is central to several growth factors and represents an important activator of target genes not only involved in physiological processes like embryogenesis and neonatal development, but also in a variety of pathophysiological processes, for example atherosclerosis or cancer. Current options to investigate its transcription and activation in vivo are end-point measurements that do not provide insights into dynamic changes in the living organism. RESULTS: We developed a transgenic mouse (Egr-1-luc) in which the luciferase reporter gene is under the control of the murine Egr-1 promoter providing a versatile tool to study the time course of Egr-1 activation in vivo. In neonatal mice, bioluminescence imaging revealed a high Egr-1 promoter activity reaching basal levels three weeks after birth with activity at snout, ears and paws. Using a model of partial hepatectomy we could show that Egr-1 promoter activity and Egr-1 mRNA levels were increased in the regenerating liver. In a model of wound healing, we demonstrated that Egr-1 promoter activity was upregulated at the site of injury. CONCLUSION: Taken together, we have developed a transgenic mouse model that allows real time in vivo imaging of the Egr-1 promoter activity. The ability to monitor and quantify Egr-1 activity in the living organism may facilitate a better understanding of Egr-1 function in vivo.