Cobalt chloride-induced signaling in endothelium leading to the augmented adherence of sickle red blood cells and transendothelial migration of monocyte-like HL-60 cells is blocked by PAF-Receptor antagonist

In response to hypoxia, sickle red blood cells (SS RBC) and leukocytes exhi bit increased adherence to the vascular endothelium, while diapedesis of le ukocytes through the blood vessel increases. However, the cellular signalin g pathway(s) caused by hypoxia is poorly understood. We utilized CoCl2 as a mimetic molecule for hypoxia to study cellular signaling pathways. We foun d that in human umbilical vein endothelial cells (HUVEC), CoCl2 at 2 mM con centration induced the surface expression of a subset of CAMs (VCAM-1) and activation of transcription factor NF-kB in the nuclear extracts of HUVEC. Furthermore, CoCl2 also caused time-dependent tyrosine phosphorylation of m itogen-activated protein (MAP) kinase isoform ERK2 without significantly af fecting ERK1, indicating ERK2 is the preferred substrate for upstream kinas e of the MAPK pathway. Inhibitors of MAP kinase (PD98059) or platelet-activ ating factor(PAF)- receptor antagonist (CV3988) inhibited the CoCl2-induced NF-kB activation and VCAM-1 expression. Augmented expression of VCAM-1 led to increased SS RBC adhesion, inhibitable by a VCAM-1 antibody. Additional ly, CoCl2 caused a two- to threefold increase in the rate of transendotheli al migration of monocyte-like HL-60 cells and a twentyfold increase in phos phorylation of platelet endothelial cell adhesion molecules (PECAM-1). The transendothelial migration of monocytes was inhibited by an antibody to PEC AM-1. Both phosphorylation of PECAM-1 and transendothelial migration of mon ocytes in response to CoCl2 were inhibited by protein kinase inhibitor (GF1 09203X) and augmented by protein phosphatase inhibitor (Calyculin A). Our d ata suggests that CoCl2-induced cellular signals directing increased expres sion of VCAM-1 in HUVEC involve downstream activation of MAP kinase and NF- kB, while the phosphorylation of PECAM-1 occurs as a result of activation o f PKC. We conclude that PAF-receptor antagonist inhibits the CoCl2- or hypo xia-induced increase in the adhesion of SS RBC, PECAM-1 phosphorylation, an d the concomitant transendothelial migration of monocytes. J. Cell. Physiol . 179:67-78, 1999. (C) 1999 Wiley-Liss, Inc.