Activated Protein C Induces Endothelial Cell Proliferation by Mitogen-Activated Protein Kinase Activation In Vitro and Angiogenesis In Vivo

Abstract

Abstract-Activated protein C (APC), a natural anticoagulant, has recently been demonstrated to activate the mitogenactivated protein kinase (MAPK) pathway in endothelial cells in vitro. Because the MAPK pathway is implicated in endothelial cell proliferation, it is possible that APC induces endothelial cell proliferation, thereby causing angiogenesis. We examined this possibility in the present study. APC activated the MAPK pathway, increased DNA synthesis, and induced proliferation in cultured human umbilical vein endothelial cells dependent on its serine protease activity. Antibody against the endothelial protein C receptor (EPCR) inhibited these events. Early activation of the MAPK pathway was inhibited by an antibody against protease-activated receptor-1, whereas neither late and complete activation of the MAPK pathway nor endothelial cell proliferation were inhibited by this antibody. APC activated endothelial nitric oxide synthase (eNOS) via phosphatidylinositol 3-kinase-dependent phosphorylation, followed by activation of protein kinase G, suggesting that APC bound to EPCR might activate the endothelial MAPK pathway by a mechanism similar to that of VEGF. APC induced morphogenetic changes resembling tube-like structures of endothelial cells, whereas DIP-APC did not. When applied topically to the mouse cornea, APC clearly induced angiogenesis in wild-type mice, but not in eNOS knockout mice. These in vitro events induced by APC might at least partly explain the angiogenic activity in vivo. This angiogenic activity of APC might contribute to maintain proper microcirculation in addition to its antithrombotic activity. Key Words: angiogenesis Ⅲ activated protein C Ⅲ endothelial protein C receptor Ⅲ protease-activated receptor-1 Ⅲ mitogen-activated protein kinase A ctivated protein C (APC) is a serine protease that plays a central role in physiological anticoagulation. Activation of protein C occurs on the endothelial cell surface by thrombin bound to thrombomodulin. 1 The endothelial protein C receptor (EPCR), which is a glycoprotein on the vascular endothelium, binds to protein C, thereby augmenting protein C activation by the thrombin-thrombomodulin complex. 2 APC regulates the coagulation cascade by inactivating activated forms of factors V and VIII in the presence of protein S, 3 and it exerts a profibrinolytic effect by inactivating plasminogen activator inhibitor-1. 4 The importance of APC as a natural anticoagulant is illustrated by the development of severe thrombosis in patients with congenital protein C deficiency. 5 In addition to the anticoagulant effect, APC has been shown to be capable of activating protease activated receptor (PAR)-1 on endothelial cells, 6 thereby activating the mitogen activated protein kinase (MAPK) pathway. The MAPK pathway is activated during mitosis, meiosis, and G0-G1 transition 7 and is implicated in regulation of the cell cycle, mitogen-induced cell growth, and proliferation. 8 The MAPK pathway plays an important role in signal transduction from growth factor receptor...

Activated protein C promotes breast cancer cell migration through interactions with EPCR and PAR-1

“…In the PROWESS trial, sepsis patients that were treated with APC have a steady-state level of 45 ng/ ml [45]. However, our overall goal was to characterize the mechanism of APC to promote cell migration and we used similar APC concentrations as in previous in vitro studies (0.5-50 μg/ml) [20,26,29]. Interestingly, levels of APC used in HUVEC experiments are closer to physiological concentrations of APC (0.1-10 μg/ml) (data not shown).…”

“…Using zymogen PC, chemically inactivated DEGR-APC, and an active site mutant of APC (S195A) with the MDA-MB-231 cancer cells, one finding of our study showed that the active protease was needed to increase cell migration. Therefore, unlike the inhibitory role of APC with lymphocytes, the pro-migratory role of APC in the MDA-MB-231 cells requires the active site of the protease, most likely to bind and activate receptors, such as PAR-1 [20,21,26,30,31], and to activate extracellular matrix proteases, such as MMP-2 and MMP-9 [27,46,47]. It is possible that when bound to EPCR, APC may undergo modifications to its macromolecular substrate recognition.…”

“…PAR-1 has been shown to be involved in mediating the nonhemostatic effects of APC [20,21,26,31]. With Western blots of cell lysates (Fig.…”

“…APC/PC reduces the migration of immune cells towards chemoattractants through its binding to EPCR [17] and to epidermal growth factor receptor (EGFR) [25]. In a concentrationdependent manner, APC increases human umbilical vein endothelial cells (HUVEC) proliferation through the activation of the MAPK, PI3K, and eNOS pathways primarily via binding to EPCR rather than to PAR-1 [26]. In a mouse cornea angiogenesis assay, APC was shown to increase angiogenesis via the eNOS pathway [26].…”

“…In a concentrationdependent manner, APC increases human umbilical vein endothelial cells (HUVEC) proliferation through the activation of the MAPK, PI3K, and eNOS pathways primarily via binding to EPCR rather than to PAR-1 [26]. In a mouse cornea angiogenesis assay, APC was shown to increase angiogenesis via the eNOS pathway [26]. APC increases proliferation and migration in keratinocytes by increasing the expression and activation of matrix metalloprotease-2 (MMP-2) [27].…”