Data providing direct evidence for a causative link between endothelial dysfunction, microvascular disease and diabetic end-organ damage are scarce. Here we show that activated protein C (APC) formation, which is regulated by endothelial thrombomodulin, is reduced in diabetic mice and causally linked to nephropathy. Thrombomodulin-dependent APC formation mediates cytoprotection in diabetic nephropathy by inhibiting glomerular apoptosis. APC prevents glucose-induced apoptosis in endothelial cells and podocytes, the cellular components of the glomerular filtration barrier. APC modulates the mitochondrial apoptosis pathway via the protease-activated receptor PAR-1 and the endothelial protein C receptor EPCR in glucose-stressed cells. These experiments establish a new pathway, in which hyperglycemia impairs endothelial thrombomodulin-dependent APC formation. Loss of thrombomodulin-dependent APC formation interrupts cross-talk between the vascular compartment and podocytes, causing glomerular apoptosis and diabetic nephropathy. Conversely, maintaining high APC levels during long-term diabetes protects against diabetic nephropathy.
The coagulation protease activated protein C (aPC) confers cytoprotective effects in various in vitro and in vivo disease models, including diabetic nephropathy. The nephroprotective effect may be related to antioxidant effects of aPC. However, the mechanism through which aPC may convey these antioxidant effects and the functional relevance of these properties remain unknown. Here, we show that endogenous and exogenous aPC prevents glomerular accumulation of oxidative stress markers and of the redox-regulating protein p66 Shc in experimental diabetic nephropathy. These effects were predominately observed in podocytes. In vitro, aPC inhibited glucose-induced expression of p66 Shc mRNA and protein in podocytes (via PAR-1 and PAR-3) and various endothelial cell lines, but not in glomerular endothelial cells. Treatment with aPC reversed glucose-induced hypomethylation and hyperacetylation of the p66 Shc promoter in podocytes. The hyperacetylating agent sodium butyrate abolished the suppressive effect of aPC on p66 Shc expression both in vitro and in vivo. Moreover, sodium butyrate abolished the beneficial effects of aPC in experimental diabetic nephropathy. Inhibition of p66 Shc expression and mitochondrial translocation by aPC normalized mitochondrial ROS production and the mitochondrial membrane potential in glucose-treated podocytes. Genetic ablation of p66 Shc compensated for the loss of protein C activation in vivo, normalizing markers of diabetic nephropathy and oxidative stress. These studies identify a unique mechanism underlying the cytoprotective effect of aPC. Activated PC epigenetically controls expression of the redox-regulating protein p66 Shc , thus linking the extracellular protease aPC to mitochondrial function in diabetic nephropathy.
Preeclampsia (PE) is a placenta-induced inflammatory disease associated with maternal and fetal morbidity and mortality. The mechanisms underlying PE remain enigmatic and delivery of the placenta is the only known remedy. PE is associated with coagulation and platelet activation and increased extracellular vesicle (EV) formation. However, thrombotic occlusion of the placental vascular bed is rarely observed and the mechanistic relevance of EV and platelet activation remains unknown. Here we show that EVs induce a thromboinflammatory response specifically in the placenta. Following EV injection, activated platelets accumulate particularly within the placental vascular bed. EVs cause adenosine triphosphate (ATP) release from platelets and inflammasome activation within trophoblast cells through purinergic signaling. Inflammasome activation in trophoblast cells triggers a PE-like phenotype, characterized by pregnancy failure, elevated blood pressure, increased plasma soluble fms-like tyrosine kinase 1, and renal dysfunction. Intriguingly, genetic inhibition of inflammasome activation specifically in the placenta, pharmacological inhibition of inflammasome or purinergic signaling, or genetic inhibition of maternal platelet activation abolishes the PE-like phenotype. Inflammasome activation in trophoblast cells of women with preeclampsia corroborates the translational relevance of these findings. These results strongly suggest that EVs cause placental sterile inflammation and PE through activation of maternal platelets and purinergic inflammasome activation in trophoblast cells, uncovering a novel thromboinflammatory mechanism at the maternal-embryonic interface.
Background-Clinical studies failed to provide clear evidence for a proatherogenic role of hypercoagulability. This is in contrast to the well-established detrimental role of hypercoagulability and thrombin during acute atherosclerotic complications. These seemingly opposing data suggest that hypercoagulability might exert both proatherogenic and antiatherogenic effects. We therefore investigated whether hypercoagulability mediates a beneficial effect during de novo atherogenesis. Methods and Results-De novo atherogenesis was evaluated in 2 mouse models with hyperlipidemia and genetically imposed hypercoagulability (TM Pro/Pro ApoE Ϫ/Ϫ and FVL Q/Q ApoE Ϫ/Ϫ mice). In both mouse models, hypercoagulability resulted in larger plaques, but vascular stenosis was not enhanced secondary to positive vascular remodeling. Importantly, plaque stability was increased in hypercoagulable mice with less necrotic cores, more extracellular matrix, more smooth muscle cells, and fewer macrophages. Long-term anticoagulation reversed these changes. The reduced frequency of intraplaque macrophages in hypercoagulable mice is explained by an inhibitory role of thrombin and protease-activated receptor-1 on monocyte transendothelial migration in vitro. This is dependent on phospholipase-C, phosphoinositide 3-kinase, and nitric oxide signaling in monocytes but not in endothelial cells. Conclusions-Here, we show a new function of the coagulation system, averting stenosis and plaque destabilization during de novo atherogenesis. The in vivo and in vitro data establish that thrombin-induced signaling via protease-activated receptor-1, phospholipase-C, phosphoinositide 3-kinase, and nitric oxide in monocytes impairs monocyte transendothelial migration. This likely accounts for the reduced macrophage accumulation in plaques of hypercoagulable mice. Thus, in contrast to their role in unstable plaques or after vascular injury, hypercoagulability and thrombin convey a protective effect during de novo atherogenesis. (Circulation. 2009;120:774-784.)Key Words: atherosclerosis Ⅲ blood coagulation Ⅲ endothelium Ⅲ plaque Ⅲ monocytes A therosclerosis is a slowly progressive disease characterized by vascular remodeling and intraplaque accumulation of monocyte-derived macrophages. The role of thrombin and the protease-activated receptor-1 (PAR-1) in atherosclerosis is generally perceived as being detrimental on the basis of their pathogenic role in acute atherosclerotic complications or after vascular injury. [1][2][3] This perception is fostered by a study evaluating de novo atherogenesis in experimental murine atherosclerosis, which showed larger plaques in hypercoagulable factor V Leiden mice (FVL Q/Q ). 4 However, several large studies evaluating the role of genetic risk factors of hypercoagulability (eg, factor V Leiden [FV G1691A] or prothrombin variant [FII G20210A]) failed to show an association between hypercoagulability and the prevalence of Received January 8, 2009; accepted June 29, 2009. From the Department of Medicine I and Clinical Chemistr...
Whereas it is generally perceived to be harmful, enhanced coagulation activation can also convey salutary effects. The high prevalence of the prothrombotic factor V Leiden (FVL) mutation in whites has been attributed to a positive selection pressure (eg, resulting from reduced blood loss or improved survival in sepsis). The consequences of enhanced coagulation activation, as observed in FVL carriers, on microvascular diabetic complications remain unknown. We therefore investigated the role of FVL in diabetic nephropathy. In heterozygous or homozygous diabetic FVL mice, albuminuria and indices of diabetic nephropathy were reduced compared with diabetic wild-type mice. This was associated with reduced glomerular apoptosis and preservation of podocytes in diabetic FVL-positive mice. In vitro, low-dose thrombin (50pM) prevented, whereas high-dose thrombin (20nM) aggravated, glucose-induced apoptosis in podocytes. In diabetic patients, the FVL mutation, but not the plasminogen activator inhibitor-1 4G/5G polymorphism, is associated with reduced albuminuria, which is consistent with a nephroprotective role of low but sustained thrombin generation. Consistently, anticoagulation of diabetic FVL-positive mice with hirudin abolished the nephroprotective effect. These results identify a nephroprotective function of low but sustained thrombin levels in FVL carriers, supporting a dual, context-dependent function of thrombin in chronic diseases. (Blood. 2011;117(19):5231-5242) IntroductionThe coagulation system provides an "on-demand" vascular repair system. After vascular injury, the coagulation system is activated, inducing fibrin formation and platelet activation, thus sealing the vascular leak and initiating a healing process leading in most cases to a restitutio ad integrum. As is evident in patients with severe hemophilia, this beneficial function of the hemostatic system is required for normal survival. Enhanced coagulation activation, however, is generally assumed to be disadvantageous for an individual's health, because some genetic polymorphisms associated with increased thrombin generation mediate an increased risk of venous thrombosis. 1 Considering the impending health risks associated with prothrombotic genetic polymorphisms, the high prevalence of some of them, in particular the factor V Leiden (FVL) mutation, led to the assumption that these genetic polymorphisms must be coupled with a positive selection pressure during evolution. 2 The FVL mutation is a missense mutation in the factor V gene (R506Q), resulting in the resistance of activated factor V to inactivation by activated protein C (aPC). The prevalence of the FVL mutation in whites is 4%-6%. 1 The potential benefits associated with FVL include a higher embryonic implantation rate, reduced puerperal maternal blood loss, and improved survival from severe infections. [3][4][5] The latter has been a matter of debate, because not all studies were able to reproduce the improved survival of FVL carriers in sepsis. [6][7][8][9][10][11] We have previously shown...
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