Addition of IMD at a concentration

Addition of IMD at a concentration promotion info of 10 nM 15 min before the pressure increase prevented the rise in Kfc. The effect of IMD was comparable to that exerted by dibutyryl cAMP (DBcAMP) at a concentration of 130 nM (Fig. 8). Fig. 8. IMD dampens pressure-induced increase in pulmonary endothelial permeability. Repetitive hydrostatic challenges were performed in isolated, perfused, and ventilated mouse lungs to quantify the capillary filtration coefficient (Kfc). Left atrial pressure … DISCUSSION The present study identifies IMD as a hypoxia-regulated pulmonary endothelial peptide stabilizing endothelial barrier function. IMD reduced basal and thrombin-induced endothelial hyperpermeability in vitro, and in isolated, perfused mouse lungs it decreased pressure-induced hyperpermeability and edema formation, pointing to a pivotal role of IMD in the regulation of endothelial cell function in the pulmonary microvasculature.

Our quantitative RT-PCR data demonstrate that hypoxia increases the level of IMD mRNA. Fully in line with these observations are our results from transfection studies in HEK293T cells, in which we show the existence of four potential HRE sequences. Coexpression of HIF-1�� in HEK293T cells dose-dependently increases luciferase reporter activity of an IMD-promoter luciferase reporter, reminiscent of that of many HIF-1��-inducible genes (13, 34, 35). IMD shares ~30% sequence homology to AM (33, 37), and for both peptides similar effects on blood pressure and heart rate and cardioprotective functions are described (15, 18, 39, 43).

Even though there are structural and functional homologies between IMD and AM, the extent of hypoxia-induced mRNA expression differed for both peptides. In PMEC, hypoxia-induced IMD expression exceeded that of AM by about threefold, whereas in the other investigated cell types hypoxia-induced increase in AM surpassed that of IMD. This finding suggests that in the pulmonary microvasculature IMD rather than AM might be part of a protective regulatory mechanism involved in the adaptive response to hypoxia. As shown by Western blot analysis IMD induced phosphorylation of VASP at Ser157, which is a well-established endogenous substrate of PKA in endothelial cells (6). PKA site-specific VASP phosphorylation was abolished by two chemically nonrelated inhibitors of PKA, indicating that IMD can activate PKA signaling pathway in HMVEC-L.

Phosphorylation of VASP by PKA has been shown to play a role in the regulation of endothelial barrier function (11). However, analyzing the precise role of VASP phosphorylation Dacomitinib in the barrier-protective effects of IMD was beyond the scope of this study. The in vitro macromolecule permeability assay clearly shows that IMD reduced the permeability in a concentration- and time-dependent manner. Accordingly, at a concentration of 10 nM, IMD was as effective as forskolin (10 ��M) (21).

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