No abstract

Abstract-Vascular endothelial growth factor (VEGF) promotes angiogenesis and endothelial cell (EC) migration and proliferation by affecting intracellular mediators, only some of which are known, distal to its receptors. Protein kinase C (PKC) participates in the function of VEGF, but the role of individual PKC isoenzymes is unknown. In this study, we tested the importance of the activity of specific PKC isoenzymes in human EC migration and proliferation in response to VEGF. PKC␦ specific activity was depressed by the addition of VEGF (by 41Ϯ8% [PϽ0.05] at 24 hours) in human umbilical vein ECs (HUVECs) and in a HUVEC-derived EC line, ECV, without changing the total amount of either protein or mRNA encoding PKC␦. Neither basic fibroblast growth factor (FGF-2) nor serum altered PKC␦ specific activity. The VEGF-induced decrease of PKC␦ activity, which began at 8 hours after stimulation, was strongly blocked by pretreatment with the nitric oxide (NO) synthase inhibitor N G -monomethyl-L-arginine in HUVECs; NO release peaked within 2 hours after stimulation. An exogenous NO donor, sodium nitroprusside, also decreased PKC␦ activity. The inhibition by N G -monomethyl-L-arginine of VEGF-induced HUVEC migration and proliferation, but not that induced by FGF-2 or serum, suggested that the decrease in PKC␦ via NO pathway is required for VEGF-induced EC migration and proliferation. Overexpression of PKC␦ in ECV cells specifically prevented EC response to VEGF but not to FGF-2 or serum. Thus, we conclude that suppression of PKC␦ activity via a NO synthase mechanism is required for VEGF-induced EC migration and proliferation, but not for that induced by FGF-2 or serum. (Circ Res. 1999;85:247-256.)

Abstract-Thromboxane A 2 (TxA 2 ) causes platelet aggregation, vasoconstriction, and inhibition of endothelial cell (EC) migration and prevents vascular tube formation via its specific receptors (TP), of which there are two isoforms (TP␣ and TP␤), both expressed in human ECs. In this study, we demonstrate that the TxA 2 mimetic IBOP increases apoptosis of human ECs and inhibits the phosphorylation of Akt kinase, an intracellular mediator required for cell survival. TxA 2 -induced functions are mediated by TPs that are members of the G protein-coupled receptor family. Thus far, two TP isoforms have been cloned, one from placenta (TP␣) 10 and one from ECs (TP␤). 11 These isoforms differ in their alternately spliced cytoplasmic C-terminal tails. These tails have an important role in regulating TP signaling and can be phosphorylated (eg, by the cGMP-dependent kinase). 12 The differences in the cytoplasmic domains have been shown to confer association with different G proteins. Although both receptors are linked to G q /G 11 , 13 TP␣ also interacts with G s to stimulate a concomitant increase in cAMP after ligand stimulation, whereas TP␤ causes a ligandinduced decrease in the elevated cAMP level produced by an adenylyl cyclase activator. 14 Recent reports show that the mechanisms and kinetics for desensitization and internalization differ between these two TP isoforms, 15,16 suggesting that each receptor is linked to a different signal transduction pathway. Whether each receptor triggers a discrete pathway in ECs or mediates distinct EC events is unknown. Recently, we reported that activation of the TP by the TxA 2 mimetics IBOP and U46619 inhibits EC migration in a denudationinjury model and prevents the formation of vascular networks in human umbilical vein ECs (HECs) in vitro, 17 both of which are fundamental events in angiogenesis and in remodeling. Because the balance between positive and negative regulators of EC survival determines vascular formation, we hypothesized that TP stimulation might be involved in the regulation of EC growth or survival. It has been established that activation of the serine/threonine protein kinase Akt, also known as protein kinase B or Rac kinase, is involved in embryonic vascular development and neoangiogenesis, and the phosphatidylinositol 3Ј-kinase (PI3-kinase)/Akt pathways