Erik W. Holy

Sirt1 inhibition promotes in vivo arterial thrombosis and tissue factor expression in stimulated cells

AIMS The mammalian silent information regulator-two 1 (Sirt1) blunts the noxious effects of cardiovascular risk factors such as type 2 diabetes mellitus and obesity. Nevertheless, the role of Sirt1 in regulating the expression of tissue factor (TF), the key trigger of coagulation, and arterial thrombus formation remains unknown. METHODS AND RESULTS Human as well as mouse cell lines were used for in vitro experiments, and C57Bl/6 mice for in vivo procedures. Sirt1 inhibition by splitomicin or sirtinol enhanced cytokine-induced endothelial TF protein expression as well as surface activity, while TF pathway inhibitor protein expression did not change. Sirt1 inhibition further enhanced TF mRNA expression, TF promoter activity, and nuclear translocation as well as DNA binding of the p65 subunit of nuclear factor-kappa B (NFκB/p65). Sirt1 siRNA enhanced TF protein and mRNA expression, and this effect was reduced in NFκB/p65(-/-) mouse embryonic fibroblasts reconstituted with non-acetylatable Lys(310)-mutant NFκB/p65. Activation of the mitogen-activated protein kinases p38, c-Jun NH(2)-terminal kinase, and p44/42 (ERK) remained unaffected. In vivo, mice treated with the Sirt1 inhibitor splitomicin exhibited enhanced TF activity in the arterial vessel wall and accelerated carotid artery thrombus formation in a photochemical injury model. CONCLUSION We provide pharmacological and genetic evidence that Sirt1 inhibition enhances TF expression and activity by increasing NFκB/p65 activation in human endothelial cells. Furthermore, Sirt1 inhibition induces arterial thrombus formation in vivo. Hence, modulation of Sirt1 may offer novel therapeutic options for targeting thrombosis.

Carbamylated low-density lipoprotein induces endothelial dysfunction

AIMS Cardiovascular events remain the leading cause of death in Western world. Atherosclerosis is the most common underlying complication driven by low-density lipoproteins (LDL) disturbing vascular integrity. Carbamylation of lysine residues, occurring primarily in the presence of chronic kidney disease (CKD), may affect functional properties of lipoproteins; however, its effect on endothelial function is unknown. METHODS AND RESULTS Low-density lipoprotein from healthy donors was isolated and carbamylated. Vascular reactivity after treatment with native LDL (nLDL) or carbamylated LDL (cLDL) was examined in organ chambers for isometric tension recording using aortic rings of wild-type or lectin-like-oxidized LDL receptor-1 (LOX-1) transgenic mice. Reactive oxygen species (ROS) and nitric oxide (NO) production were determined using electron spin resonance spectroscopy. The effect of LDL-carbamyl-lysine levels on cardiovascular outcomes was determined in patients with CKD during a median follow-up of 4.7 years. Carbamylated LDL impaired endothelium-dependent relaxation to acetylcholine or calcium-ionophore A23187, but not endothelium-independent relaxation to sodium nitroprusside. In contrast, nLDL had no effect. Carbamylated LDL enhanced aortic ROS production by activating NADPH-oxidase. Carbamylated LDL stimulated endothelial NO synthase (eNOS) uncoupling at least partially by promoting S-glutathionylation of eNOS. Carbamylated LDL-induced endothelial dysfunction was enhanced in LOX-1 transgenic mice. In patients with CKD, LDL-carbamyl-lysine levels were significant predictors for cardiovascular events and all-cause mortality. CONCLUSIONS Carbamylation of LDL induces endothelial dysfunction via LOX-1 activation and increased ROS production leading to eNOS uncoupling. This indicates a novel mechanism in the pathogenesis of atherosclerotic disease which may be pathogenic and prognostic in patients with CKD and high plasma levels of cLDL.

Dietary α-Linolenic Acid Inhibits Arterial Thrombus Formation, Tissue Factor Expression, and Platelet Activation

Objective—Plant-derived &agr;-linolenic acid (ALA) may constitute an attractive cardioprotective alternative to fish-derived n-3 fatty acids. However, the effect of dietary ALA on arterial thrombus formation remains unknown. Methods and Results—Male C57Bl/6 mice were fed a high-ALA or low-ALA diet for 2 weeks. Arterial thrombus formation was delayed in mice fed a high-ALA diet compared with those on a low-ALA diet (n=7; P<0.005). Dietary ALA impaired platelet aggregation to collagen and thrombin (n=5; P<0.005) and decreased p38 mitogen-activated protein kinase activation in platelets. Dietary ALA impaired arterial tissue factor (TF) expression, TF activity, and nuclear factor-&kgr;B activity (n=7; P<0.05); plasma clotting times and plasma thrombin generation did not differ (n=5; P=not significant). In cultured human vascular smooth muscle and endothelial cells, ALA inhibited TF expression and activity (n=4; P<0.01). Inhibition of TF expression occurred at the transcriptional level via the mitogen-activated protein kinase p38 in smooth muscle cells and p38, extracellular signal-regulated kinases 1 and 2, and c-Jun N-terminal kinases 1 and 2 in endothelial cells. Conclusion—ALA impairs arterial thrombus formation, TF expression, and platelet activation and thereby represents an attractive nutritional intervention with direct dual antithrombotic effects.

Berberine, a natural lipid-lowering drug, exerts prothrombotic effects on vascular cells

Berberine (BBR) is a novel natural hypolipidemic agent. This study investigates whether BBR, similar to statins, exerts pleiotropic effects on endothelial tissue factor (TF) expression. BBR enhanced tumor necrosis factor-alpha (TNF-alpha) and thrombin induced TF expression in human endothelial cells by 3.5-fold. These effects were paralleled by an enhanced TF surface activity. In contrast, expression of TF pathway inhibitor was impaired. BBR enhanced TNF-alpha induced TF mRNA expression; however, TF promoter activity was inhibited. Activation of ERK and p38 remained unaffected, while c-Jun terminal NH(2) kinase was inhibited. BBR reduced TF mRNA degradation rates, prolonging its half-life from 1.1 to 4.3 h. The HMG-CoA reductase inhibitor simvastatin impaired thrombin induced TF expression, and BBR blunted this inhibition. Simvastatin did not affect TNF-alpha induced TF expression, and BBR enhanced TF under these conditions. Administration of BBR (100 mg/kg/d) increased TF activity and impaired TFPI expression in carotid artery of ApoE(-/-) mice. BBR enhances TF via mRNA stabilization at clinically relevant concentrations. Clinical application of BBR, either as an alternative to or in combination with statins, should be considered with caution.

Guggulsterone, an anti-inflammatory phytosterol, inhibits tissue factor and arterial thrombosis

BackgroundThe phytosterol guggulsterone is a potent anti-inflammatory mediator with less side effects than classic steroids. This study assesses the impact of guggulsterone on tissue factor (TF) expression and thrombus formation.Methods and resultsGuggulsterone inhibited TNF-α-induced endothelial TF protein expression and surface activity in a concentration-dependent manner; in contrast, dexamethasone did not affect TNF-α-induced TF expression. Guggulsterone enhanced endothelial tissue factor pathway inhibitor and impaired plasminogen activator inhibitor-1 as well as vascular cell adhesion molecule-1 protein. Real-time polymerase chain reaction revealed that guggulsterone inhibited TNF-α-induced TF mRNA expression; moreover, it impaired activation of the MAP kinases JNK and p38, while that of ERK remained unaffected. In vivo, guggulsterone inhibited TF activity and photochemical injury induced thrombotic occlusion of mouse carotid artery. Guggulsterone also inhibited TF expression, proliferation, and migration of vascular smooth muscle cells in a concentration-dependent manner.ConclusionsGuggulsterone inhibits TF expression in vascular cells as well as thrombus formation in vivo; moreover, it impairs vascular smooth muscle cell activation. Hence, this phytosterol offers novel therapeutic options, in particular in inflammatory diseases associated with an increased risk of thrombosis.

PI3K/p110α inhibition selectively interferes with arterial thrombosis and neointima formation, but not re-endothelialization: potential implications for drug-eluting stent design

BACKGROUND Impaired re-endothelialization and stent thrombosis are a safety concern associated with drug-eluting stents (DES). PI3K/p110α controls cellular wound healing pathways, thereby representing an emerging drug target to modulate vascular homoeostasis after injury. METHODS AND RESULTS PI3K/p110α was inhibited by treatment with the small molecule inhibitor PIK75 or a specific siRNA. Arterial thrombosis, neointima formation, and re-endothelialization were studied in a murine carotid artery injury model. Proliferation and migration of human vascular smooth muscle cell (VSMC) and endothelial cell (EC) were assessed by cell number and Boyden chamber, respectively. Endothelial senescence was evaluated by the β-galactosidase assay, endothelial dysfunction by organ chambers for isometric tension. Arterial thrombus formation was delayed in mice treated with PIK75 when compared with controls. PIK75 impaired arterial expression and activity of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1); in contrast, plasma clotting and platelet aggregation did not differ. In VSMC and EC, PIK75 inhibited expression and activity of TF and PAI-1. These effects occurred at the transcriptional level via the RhoA signalling cascade and the transcription factor NFkB. Furthermore, inhibition of PI3K/p110α with PIK75 or a specific siRNA selectively impaired proliferation and migration of VSMC while sparing EC completely. Treatment with PIK75 did not induce endothelial senescence nor inhibit endothelium-dependent relaxations. In line with this observation, treatment with PIK75 selectively inhibited neointima formation without affecting re-endothelialization following vascular injury. CONCLUSION Following vascular injury, PI3K/p110α inhibition selectively interferes with arterial thrombosis and neointima formation, but not re-endothelialization. Hence, PI3K/p110α represents an attractive new target in DES design.

Tissue Factor in Cardiovascular Disease: Pathophysiology and Pharmacological Intervention

Tissue factor (TF) is the major trigger of the coagulation cascade and thereby crucially involved in the maintenance of vascular hemostasis. By binding factor VIIa, the resulting TF:VIIa complex activates the coagulation factors IX and X ultimately leading to fibrin and clot formation. In the vessel wall, TF expression and activity is detectable in vascular smooth muscle cells and fibroblasts and, at a much lower level, in endothelial cells and can be induced by various stimuli including cytokines. In addition, TF is found in the bloodstream in circulating cells such as monocytes, in TF containing microparticles, and as a soluble splicing isoform. Besides its well-known extracellular role as a trigger of coagulation, TF also functions as a transmembrane receptor, and TF-dependent intracellular signaling events regulate the expression of genes involved in cellular responses such as proliferation and migration. TF indeed appears to be involved in the pathogenesis of neointima formation and tumor growth, and increased levels of TF have been detected in patients with cardiovascular risk factors or coronary artery disease as well as in those with cancer. Therefore, pharmacological or genetic inhibition of TF may be an attractive target for the treatment of cardiovascular disease and cancer. Different strategies for inhibition of TF have been developed such as inhibition of TF synthesis and blockade of TF action. Clinical applications of such strategies need to be tested in appropriate trials, in particular for evaluating the advantages of targeted versus systemic delivery of the inhibitors.

Laminin receptor activation inhibits endothelial tissue factor expression

Tissue factor (TF) is an important trigger of arterial thrombosis. The green tea catechin epigallocatechin-3-gallate (EGCG) is a ligand of the 67-kDa laminin receptor (67LR) and exhibits cardioprotective effects. This study investigates whether 67LR regulates TF expression in human endothelial cells. Immunofluorescence demonstrated that human aortic endothelial cells expressed 67LR. Cells grown on laminin expressed 35% less TF in response to TNF-alpha (TNF-alpha) than those grown on fibronectin (n=6; p<0.001). EGCG (1-30 microM) inhibited TNF-alpha and histamine induced endothelial TF expression and activity in a concentration dependent manner resulting in 87% reduction of TF expression (n=5; p<0.001); in contrast, expression of tissue factor pathway inhibitor was not affected (n=4; p=NS). In vivo administration of EGCG (30 mg/kg/day) inhibited TF activity in carotid arteries of C57BL6 mice. Real-time PCR and promoter studies revealed that EGCG decreased TF expression at the transcriptional level and impaired activation of the mitogen activated protein (MAP) kinase JNK 1/2, but not ERK or p38. Similarly, the JNK 1/2 inhibitor SP600125 (1 microM) impaired TF promoter activity (n=4; p<0.001) and protein expression (n=4; p<0.001). 67LR blocking antibodies blunted the inhibitory effect of EGCG on both TF protein expression and JNK activation. In contrast, vascular cell adhesion molecule 1 (VCAM-1) was not affected by laminin nor EGCG, and its expression was not regulated by JNK. EGCG did not affect TNF-alpha stimulated NFkB activation. Laminin receptor activation inhibits endothelial TF expression by impairing JNK phosphorylation. Thus, 67LR may be a potential target for the development of novel anti-thrombotic therapies.

Aging Induces Endothelial Dysfunction While Sparing Arterial Thrombosis

Objective—To assess the effects of aging on arterial thrombus formation by comparing 2-year-old with 11-week-old C57Bl6 mice. Methods and Results—Aging is a major risk factor for cardiovascular disease. In humans, assessing the direct effects of aging on vascular homeostasis is difficult because it occurs in the presence of other risk factors. Arterial thrombosis is the critical event in cardiovascular diseases; however, it is not known whether aging per se promotes its occurrence. Mice represent an interesting system to address this issue because they age without spontaneously developing other risk factors. Organ chamber experiments confirmed the advanced level of aging of old mice. As previously shown, old mice exhibited endothelial dysfunction; however, arterial thrombosis induced by photochemical injury was unchanged. Arterial tissue factor expression and activity; expressions of tissue factor pathway inhibitor, thrombomodulin, and plasminogen activator inhibitor 1; prothrombin time; partial thromboplastin time; thrombin-antithrombin complex; and platelet activation were comparable in both groups. Conclusion—Although these results cannot be directly extrapolated to humans, this study contributes novel important information on the direct effect of aging on arterial thrombosis and underscores the importance of controlling modifiable risk factors in aged individuals.

High-density lipoprotein from patients with coronary heart disease loses anti-thrombotic effects on endothelial cells: impact on arterial thrombus formation.

Thrombus formation is determined by the balance between pro- thrombotic mediators and anti-thrombotic factors.High-density lipoprotein (HDL) from healthy subjects exerts anti-thrombotic properties. Whether this is also the case for HDL from patients with stable coronary heart disease (CHD) or acute coronary syndrome (ACS) is unknown.In human aortic endothelial cells in culture,HDL (50 µg/ml) from healthy subjects (HS) inhibited thrombin-induced tissue factor (TF) expression and activity, while HDL (50 µg/ml) from CHD and ACS patients did not. Similarly, only healthy HDL increased endothelial tissue factor pathway inhibitor (TFPI) expression and tissue plasminogen activator (tPA) release, while HDL from CHD and ACS patients had no effect. Healthy HDL inhibited thrombin-induced plasminogen activator inhibitor type 1 (PAI-1) expression, while HDL from ACS patients enhanced endothelial PAI-1 expression. Inhibition of nitric oxide (NO) formation with L-NAME (100 µmol/l) abolished the anti-thrombotic effects of healthy HDL on TF, TFPI, and tPA expression. The exogenous nitric oxide donor, DETANO, mimicked the effects of healthy HDL and counterbalanced the loss of anti-thrombotic effects of HDL from CHD and ACS patients in endothelial cells. In line with this observation, healthy HDL, in contrast to HDL from CHD and ACS patients, increased endothelial NO production. In the laser-injured carotid artery of the mouse, thrombus formation was delayed in animals treated with healthy HDL compared with mice treated with vehicle or HDL from patients with CHD or ACS. In conclusion, HDL from CHD and ACS patients loses the ability of healthy HDL to suppress TF and to increase TFPI and t-PA and instead enhances PAI-1 and arterial thrombus formation.