Bart J M van Vlijmen

Leiden University Medical Centre, Leyden, South Holland, Netherlands

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Publications (78)409.28 Total impact

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    ABSTRACT: To clarify the role of ABCB1, ABCG2, and CYP3A in blood and brain exposure of everolimus using knockout mouse models. We used wild-type, Abcb1a/1b(-/-), Abcg2(-/-), Abcb1a/1b;Abcg2(-/-) and Cyp3a(-/-) mice to study everolimus oral bioavailability and brain accumulation. Following everolimus administration, brain concentrations and brain-to-liver ratios were substantially increased in Abcb1a/1b(-/-) and Abcb1a/1b;Abcg2(-/-), but not Abcg2(-/-) mice. The fraction of everolimus located in the plasma compartment was highly increased in all knockout strains. In vitro, everolimus was rapidly degraded in wild-type but not knockout plasma. Carboxylesterase 1c (Ces1c), a plasma carboxylesterase gene, was highly upregulated (~80-fold) in the liver of knockout mice relative to wild-type mice, and plasma Ces1c likely protected everolimus from degradation by binding and stabilizing it. This binding was prevented by preincubation with the carboxylesterase inhibitor BNPP. In vivo knockdown experiments confirmed the involvement of Ces1c in everolimus stabilization. Everolimus also markedly inhibited the hydrolysis of irinotecan and p-nitrophenyl acetate by mouse plasma carboxylesterase and recombinant human CES2, respectively. After correcting for carboxylesterase binding, Cyp3a(-/-), but not Abcb1a/1b(-/-), Abcg2(-/-), or Abcb1a/1b;Abcg2(-/-) mice, displayed highly (>5-fold) increased oral availability of everolimus. Brain accumulation of everolimus was restricted by Abcb1, but not Abcg2, suggesting the use of coadministered ABCB1 inhibitors to improve brain tumor treatment. Cyp3a, but not Abcb1a/1b, restricted everolimus oral availability, underscoring drug-drug interaction risks via CYP3A. Upregulated Ces1c likely mediated the tight binding and stabilization of everolimus, causing higher plasma retention in knockout strains. This Ces upregulation might confound other pharmacological studies.
    Clinical Cancer Research 04/2014; · 7.84 Impact Factor
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    ABSTRACT: Key points RNA interference of antithrombin and/or protein C allows studying the function of these genes, alone or in combination, in normal adult miceRNA interference of antithrombin and protein C provides a novel, controlled mouse model for spontaneous venous thrombosis.
    Blood 04/2013; · 9.78 Impact Factor
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    ABSTRACT: Single nucleotide polymorphisms (SNPs) in a 4q35.2 locus that harbors the coagulation factor XI (F11), prekallikrein (KLKB1), and a cytochrome P450 family member (CYP4V2) genes are associated with deep venous thrombosis (DVT). These SNPs exert their effect on DVT by modifying the circulating levels of FXI. However, SNPs associated with DVT were not necessarily all in F11, but also in KLKB1 and CYP4V2. Here, we searched for evidence for common regulatory elements within the 4q35.2 locus, outside the F11 gene, that might control FXI plasma levels and/or DVT risk. To this end, we investigated the regulation of the orthologous mouse gene cluster under several metabolic conditions that impact mouse hepatic F11 transcription. In livers of mice in which HNF4α, a key transcription factor controlling F11, was ablated, or reduced by siRNA, a strong decrease in hepatic F11 transcript levels was observed that correlated with Cyp4v3 (mouse orthologue of CYP4V2), but not by Klkb1 levels. Estrogens induced hepatic F11 and Cyp4v3, but not Klkb1 transcript levels, whereas thyroid hormone strongly induced hepatic F11 transcript levels, and reduced Cyp4v3, leaving Klkb1 levels unaffected. Mice fed a high-fat diet also had elevated F11 transcription, markedly paralleled by an induction of Klkb1 and Cyp4v3 expression. We conclude that within the mouse F11, Klkb1, Cyp4v3 gene cluster, F11 and Cyp4v3 frequently display striking parallel transcriptional responses suggesting the presence of shared regulatory elements.
    PLoS ONE 01/2013; 8(9):e74637. · 3.53 Impact Factor
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    ABSTRACT: Venous thrombosis represents a serious complication of oral contraceptive use and hormone replacement therapy. The estrogen component, often 17α-ethinylestradiol, is considered to be the predominant thrombotic constituent and we have previously shown that oral ethinylestradiol (EE) in mice also has profound effects on the plasma coagulation profile, at least at the level of individual pro- and anticoagulant factors.(1) The overall effect of alterations in the hemostatic balance can be determined by assessing thrombin generation and subsequent calculation of the endogenous thrombin potential (ETP). © 2012 International Society on Thrombosis and Haemostasis.
    Journal of Thrombosis and Haemostasis 09/2012; · 6.08 Impact Factor
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    ABSTRACT: Hepatocyte nuclear factor 4α (HNF4α) and CCAAT/enhancer-binding protein α (C/EBPα) are important for the transcriptional control of coagulation factors. To determine in vivo the direct role of HNF4α and C/EBPα in control of genes encoding coagulation factors, a synthetic small interfering (si)RNA approach was used that enabled strong reduction of mouse hepatic HNF4α and C/EBPα under conditions that minimized target-related secondary effects. For both HNF4α and C/EBPα, intravenous injection of specific synthetic siRNAs (siHNF4α and siC/EBPα) resulted in more than 75% reduction in their liver transcript and protein levels 2 days post-injection. For siHNF4α, this coincided with marked and significantly reduced transcript levels of the coagulation genes Hrg, Proz, Serpina5, F11, F12, F13b, Serpinf2, F5, and F9 (in order of magnitude of effect) as compared to levels in control siRNA injected animals. Significant decreases in HNF4α target gene mRNA levels were also observed at 5 days post-siRNA injection, despite a limited level of HNF4α knockdown at this time point. Compared to HNF4α, C/EBPα knockdown had a modest impact on genes encoding coagulation factors. A strong reduction in C/EBPα transcript and protein levels resulted in significantly affected transcript levels of the control genes Pck1 and Fasn and a modest downregulation for coagulation genes Fba, Fbg and F5. F5 and F11 were the sole coagulation genes that were significantly affected upon prolonged (5 day) C/EBPα knockdown. We conclude that in the mouse, HNF4α has a direct and essential regulatory role for multiple hepatic coagulation genes, while a role for C/EBPα is more restricted. In addition, this study demonstrates that synthetic siRNA provides a simple and fast means for determining liver transcription factor involvement in vivo.
    PLoS ONE 01/2012; 7(6):e38104. · 3.53 Impact Factor
  • Thrombosis Research 05/2011; 128(2):200-1. · 3.13 Impact Factor
  • Journal of Thrombosis and Haemostasis 10/2010; 8(12):2839-41. · 6.08 Impact Factor
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    ABSTRACT: Oral estrogen use is associated with changes in plasma levels of many coagulation proteins. To gain more insight into the underlying mechanism of estrogen-induced changes in coagulation. Ovariectomized female mice were used to study the impact of oral 17α-ethinylestradiol (EE) on plasma coagulation, hepatic coagulation gene transcript levels, and dependence on estrogen receptor (ER) α and ERβ. Ten days of oral EE treatment resulted in significantly reduced plasma activity levels of factor (F)VIII, FXII, combined FII/FVII/FX and antithrombin, whereas FIX activity significantly increased. Regarding hepatic transcript levels, oral EE caused significant decreases in fibrinogen-γ, FII, FV, FVII, FX, FXII, antithrombin, protein C, protein Z, protein Z inhibitor and heparin cofactor II mRNA levels, whereas FXI levels significantly increased and transcript levels of FVIII, FIX, protein S and α(2) -antiplasmin remained unaffected. All EE-induced coagulation-related changes were neutralized by coadministration of the non-specific ER antagonist ICI182780. In addition, ERα-deficient mice lacked the EE-induced changes in plasma coagulation and hepatic transcript profile, whereas ERβ-deficient mice responded similarly to non-deficient littermate controls. A crucial role for the ER was further demonstrated by its rapid effects on transcription, within 2.5-5 h after EE administration, suggesting a short chain of events leading to its final effects. Oral EE administration has a broad impact on the mouse coagulation profile at the level of both plasma and hepatic mRNA levels. The effects on transcription are rapidly induced, mostly downregulatory, and principally mediated by ERα.
    Journal of Thrombosis and Haemostasis 01/2010; 8(8):1838-1846. · 6.08 Impact Factor
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    ABSTRACT: Obesity and oral estrogens are independent risk factors for venous thrombosis, and their combined effect is stronger than the sum of the isolated factors. It was the objective of this study to investigate the interaction between obesity and estrogens at the level of venous thrombotic tendency, coagulation and inflammation in a mouse model. Female C57Bl/6J mice were fed a standard fat diet (SFD) or a high fat diet (HFD) to induce nutritional obesity. After 14 weeks, while maintaining their diet, mice were orally treated eight days with 1 microg ethinylestradiol or vehicle (n=25 per group), and subsequently subjected to an inferior caval vein (ICV) thrombosis model. The ICV thrombosis model resulted in an increased thrombus weight in vehicle-treated HFD mice (3.0 +/- 0.7 mg) compared to vehicle-treated SFD mice (1.4 +/- 0.4 mg; p=0.064). Surprisingly, estrogens reduced thrombus weight, which was significant for the HFD group (0.8 +/- 0.5 mg; p=0.013). As compared to SFD feeding, HFD feeding significantly increased plasma levels of coagulation factor VIII, combined factor II/VII/X (p < 0.001), and plasminogen activator inhibitor-1 (p=0.009), causing a prothrombotic shift of the coagulation profile. Estrogens had no significant effects on this profile with either diet, whereas serum amyloid A and hepatic inflammatory cytokines were minimally affected. The synergistic effect of obesity and estrogens on the venous thrombotic risk in women could not be translated into the mouse context. Short-term ethinylestradiol administration in a mouse ICV thrombosis model counteracts the prothrombotic phenotype associated with nutritionally induced obesity, despite a comparable activated plasma coagulation profile in estrogen-treated and untreated obese mice.
    Thrombosis and Haemostasis 11/2009; 102(5):993-1000. · 5.76 Impact Factor
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    ABSTRACT: The cellular composition of atherosclerotic lesions is determined by many factors including cell infiltration, proliferation and cell death. Tumor suppressor gene p53 has been shown to regulate both cell proliferation and cell death in many cell types. In the present study, we investigated the role of macrophage p53 in the pathogenesis of early and advanced atherosclerosis. Using the Cre-loxP system we found that absence of macrophage p53 (p53(del)) strongly reduces apoptosis of macrophages both in early and advanced atherosclerotic lesions (-59% and -37%, respectively). Consequently, in advanced atherosclerosis, reduced apoptosis upon absence of macrophage p53, coincided with increased acellular necrotic core formation (+96%), increased macrophage content (+24%), and reduced cholesterol cleft accumulation (-41%). Proliferation was not affected by the absence of macrophage p53 in both early and advanced atherosclerosis. However, these significant changes in lesional cell death did not affect total lesion area in both early and advanced atherosclerosis, neither in the aortic root nor in the aortic arch and thoracic aorta in ApoE-deficient mice. Our data demonstrate that macrophage p53 is an important regulator of macrophage apoptosis, thereby preventing necrotic death of lesional macrophages. The regulation of this cell death balance directly affects lesion composition.
    Atherosclerosis 07/2009; 207(2):399-404. · 3.71 Impact Factor
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    ABSTRACT: Pregnancy, oral contraceptive (OC)use and hormone replacement therapy (HRT) are established risk factors for venous thrombosis. Acquired resistance to activated protein C (APC) has been proposed to contribute to the increased thrombosis risk. Mouse models are often used for preclinical testing of newly developed hormone preparations. However, it is not known whether hormone-induced APC resistance is also observed in laboratory animals. To investigate whether hormonal changes modulate APC resistance in mice, we used pregnant mice as a model of hormone-induced APC resistance. The effect of pregnancy on APC resistance was studied in wild-type and factor (F)V Leiden mice. APC resistance was determined in mouse plasma using a thrombin generation-based APC resistance test. APC resistance determinants,i.e. prothrombin, FV, FX, antithrombin and protein S levels,and of tissue factor pathway inhibitor (TFPI) activity were evaluated in plasma from non-pregnant and pregnant mice. In contrast to humans, pregnancy induced a decrease in APC resistance in wild-type and in FV Leiden mice.Pregnant mice had higher levels of prothrombin, FV, FX,protein S and TFPI activity as compared with non-pregnant mice. Pregnancy causes a decrease in APC resistance in mice, which can be explained by the elevation of protein S levels and increased TFPI activity in plasma. Our findings show species specificity in the effects of pregnancy on the major determinants of the protein C system and suggest that protein S and TFPI play an important role in the development of pregnancy-induced APC resistance in humans.
    Journal of Thrombosis and Haemostasis 12/2008; 7(2):312-8. · 6.08 Impact Factor
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    ABSTRACT: LPL activity plays an important role in preceding the VLDL remnant clearance via the three major apolipoprotein E (apoE)-recognizing receptors: the LDL receptor (LDLr), LDL receptor-related protein (LRP), and VLDL receptor (VLDLr). The aim of this study was to determine whether LPL activity is also important for VLDL remnant clearance irrespective of these receptors and to determine the mechanisms involved in the hepatic remnant uptake. Administration of an adenovirus expressing LPL (AdLPL) into lrp(-)ldlr(-/-)vldlr(-/-) mice reduced both VLDL-triglyceride (TG) and VLDL-total cholesterol (TC) levels. Conversely, inhibition of LPL by AdAPOC1 increased plasma VLDL-TG and VLDL-TC levels. Metabolic studies with radiolabeled VLDL-like emulsion particles showed that the clearance and hepatic association of their remnants positively correlated with LPL activity. This hepatic association was independent of the bridging function of LPL and HL, since heparin did not reduce the liver association. In vitro studies demonstrated that VLDL-like emulsion particles avidly bound to the cell surface of primary hepatocytes from lrp(-)ldlr(-/-)vldlr(-/-) mice, followed by slow internalization, and involved heparin-releaseable cell surface proteins as well as scavenger receptor class B type I (SR-BI). Collectively, we conclude that hepatic VLDL remnant uptake in the absence of the three classical apoE-recognizing receptors is regulated by LPL activity and involves heparan sulfate proteoglycans and SR-BI.
    The Journal of Lipid Research 08/2008; 49(7):1553-61. · 4.39 Impact Factor
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    ABSTRACT: Blood coagulation and platelet activation are mutually dependent processes, but contribute differently to venous and arterial thrombosis. We investigated the interplay of these processes in vivo in a mouse model of arteriolar and venular thrombus formation. Thrombus formation was studied by intravital (fluorescence) microscopy after topical application of FeCl3 on mouse mesenteric microvessels. Both in arterioles and venules, the thrombus-forming process relied on tissue factor-factor VII(a) interaction, collagen exposure, and glycoprotein VI-mediated platelet activation. Arterial thrombus formation was impaired by mild thrombin inhibition or platelet inhibition, while venous thrombosis was only suppressed by strong thrombin inhibition or by mild thrombin inhibition together with platelet inhibition. Phosphatidylserine-exposing platelets were present in thrombi of both vessel types, as detected with fluorescently labeled annexin A5. Injection of annexin A5 to shield exposed phosphatidylserine abolished thrombus formation in arterioles and venules, while mutant M1234-annexin A5 was ineffective. Arterial and venous thrombus formations were only slightly affected in mice carrying the factor V Leiden mutation, suggesting insensitivity to factor Va inactivation. In this microvascular model, the formation of both arterial and venous thrombi relies on collagen-induced platelet activation and tissue factor-induced thrombin generation. Activated, phosphatidylserine-exposing platelets play a key role in thrombus growth in arterioles and venules.
    Microcirculation 06/2008; 15(4):269-82. · 2.76 Impact Factor
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    ABSTRACT: An FeCl(3) induced femoral arterial thrombosis model was applied to lean (47+/-1.4 g) and obese (64+/-1.7 g) mice (Swiss genetic background) in order to study the relation between obesity and thrombotic risk. As compared to lean mice, obese mice showed a significantly shorter occlusion time (9.9+/-1.0 min versus 13+/-0.5 min; p=0.04) and lower total blood flow (37+/-7.3% versus 69+/-6.7%; p=0.008). A significant negative correlation was observed between body weight and both occlusion time (r=-0.57; p=0.014) and blood flow (r=-0.57; p=0.028). Analysis of the coagulation profile revealed significantly higher levels of plasminogen activator inhibitor-1 (PAI-1), thrombin-antithrombin complex, Factor V activity and combined Factors II/VII/X activity, and moderately elevated Factor VIII activity in obese mice. The degree of arterial damage and the thrombus extension were, however, not significantly different. A significant positive correlation was observed between body weight and either PAI-1 (r=0.63; p=0.003), Factors II/VII/X levels (r=0.80; p<0.0001) or Factor V levels (r=0.65; p=0.003). Thus, this injury induced femoral artery thrombosis model in mice establishes experimentally a correlation between obesity and prothrombotic tendency.
    Thrombosis Research 02/2008; 122(4):549-55. · 3.13 Impact Factor
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    ABSTRACT: Since activation of the haemostatic system is an important feature of the wound healing response triggered by arterial injury, variations in genes involved in thrombus formation may play a role in restenosis after percutaneous coronary interventions (PCI). Therefore, our aim was to examine the relationship between polymorphisms that are known to play a role in the haemostatic system and the risk of clinical restenosis in the GENetic DEterminants of Restenosis (GENDER) study, a multicenter prospective study design that enrolled 3,104 consecutive patients after successful PCI. Target vessel revascularization (TVR) was the primary endpoint. All patients were genotyped for six polymorphisms in the Factor II, Factor V, Factor VII and PAI-1 genes. The PAI-1 4G variant was associated with an increased risk of TVR. When compared to 5G/5G homozygotes, heterozygous patients were at higher risk for TVR (HR: 1.46, 95% CI: 1.05-2.03), whereas patients with the 4G/4G genotype had an even further increased risk (HR: 1.69, 95% CI: 1.19-2.41). In contrast, the factor V 506Gln (factor V Leiden) amino acid substitution was associated with a decreased risk of TVR (HR: 0.41, 95% CI: 0.19-0.86). Our findings indicate that polymorphisms in the factorV and PAI-1 genes may play a role in the process of restenosis.
    Thrombosis and Haemostasis 01/2008; 98(6):1323-8. · 5.76 Impact Factor
  • L Hu, N Bovenschen, L M Havekes, B J M van Vlijmen, J T Tamsma
    Journal of Thrombosis and Haemostasis 12/2007; 5(11):2301-4. · 6.08 Impact Factor
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    ABSTRACT: The homozygous factor V Leiden mutation is associated with enhanced venous thrombotic risk. Obesity is a major risk factor for development of thrombotic cardiovascular disease. It was the objective of this study to investigate whether obesity affects the thrombotic risk associated with the mutation. Male mice with homozygous factor V Leiden mutation (Arg 504 to Gln) (FVQ/Q) and corresponding wild-type (WT) mice were kept on a standard fat diet (SFD) or high fat diet (HFD) for 14 weeks, and femoral artery thrombosis was induced by FeCl3 treatment. As compared to SFD, HFD feeding for 14 weeks resulted in significantly higher body weight and fat mass associated with adipocyte hypertrophy, which were, however, similar for both genotypes. In the FeCl3-induced arterial thrombosis model, FVQ/Q mice kept on SFD had a 40% shorter occlusion time (p = 0.015) and 40% lower blood flow (p = 0.03), as compared to WT mice. However, on HFD the occlusion time and blood flow were not significantly different for both genotypes. This finding could not be explained by differential changes of coagulation factors in either genotype fed on SFD or HFD. In conclusion, on SFD, but not on HFD, the factor V Leiden mutation is associated with enhanced thrombotic tendency after FeCl3 injury of the femoral artery, suggesting that in this model obesity rescues the increased thrombotic risk associated with the factor V Leiden mutation.
    Thrombosis and Haemostasis 11/2007; 98(4):858-63. · 5.76 Impact Factor
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    S N Tchaikovski, B J M VAN Vlijmen, J Rosing, G Tans
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    ABSTRACT: Mouse models have become increasingly important in thrombosis research. However, only a limited number of assays are available for assessment of the coagulation system in mouse plasma. To quantify tissue factor-initiated thrombin generation in murine platelet-rich and platelet-free plasma and to develop a test for measurement of resistance to activated protein C (APC) in mouse plasma. Thrombin generation was monitored with calibrated automated thrombography (CAT) using a low-affinity fluorogenic substrate for thrombin. To overcome the higher activity of coagulation inhibitors in mouse plasma as compared with human plasma, the reaction temperature was lowered to 33 degrees C and the assay was carried out at a 2-fold higher final plasma dilution (1:3) than commonly used for CAT in human plasma. This increased the endogenous thrombin potential (ETP) 4- to 5-fold and enabled reliable measurement of thrombin generation in both platelet-free and platelet-rich mouse plasma. For the APC resistance measurement, the reaction conditions were further optimized with respect to tissue factor, phospholipid, APC and CaCl(2) concentrations. The test was validated using plasma of mice with different genetic background with respect to the factor V Leiden mutation (FV Leiden). Mice homozygous for FV Leiden had higher APC sensitivity ratios (mean 5.46; 95% CI 4.88-6.03) than heterozygous FV Leiden mice (mean 4.21; 95% CI 3.53-4.89) and than wild-type mice (mean 2.71; 95%CI 2.15-3.27). We have established reaction conditions for measurement of thrombin generation and APC resistance in mouse plasma. This assay enables evaluation of the coagulation system and the function of the protein C system in mouse models.
    Journal of Thrombosis and Haemostasis 11/2007; 5(10):2079-86. · 6.08 Impact Factor
  • Audrey C A Cleuren, Bart J M van Vlijmen, Pieter H Reitsma
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    ABSTRACT: During the last 15 years, transgenic mice have been generated that carry defective and/or mutant alleles of the natural anticoagulant pathways and display a spontaneous thrombotic phenotype. With the generation of these mouse lines, better opportunities became available for investigating both existing and novel risk factors for venous thrombosis. In addition, these models could serve as a tool for evaluating novel antithrombotic strategies. This review summarizes these mouse models and evaluates whether they have fulfilled the expectations.
    Seminars in Thrombosis and Hemostasis 10/2007; 33(6):610-6. · 4.22 Impact Factor
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    ABSTRACT: p53 is a potent inhibitor of cell growth and an inducer of apoptosis. During embryonic development, Mdm2 and Mdm4 inhibit the growth suppressive activities of p53. However, whether tight surveillance of p53 activity is required in quiescent cells is unknown. To test this, conditional inactivation of mdm2 and mdm4 was carried out in smooth muscle cells (SMCs). Upon SMC-specific inactivation of mdm2, and not of mdm4, mice rapidly became ill and died. Necropsy showed small intestinal dilation, and histological analyses indicated a severe reduction in the number of intestinal SMCs. Increased p53 levels and activity were detected in the remaining SMCs, and the phenotype was completely rescued on a p53-null background. Interestingly, intestinal SMCs are caspase-3-negative and therefore did not undergo caspase-3-dependent apoptotic cell death. Together, Mdm2, but not Mdm4, prevents accumulation of active p53 in quiescent SMCs and thereby the induction of p53-mediated caspase-3-independent cell death.
    Cell Death and Differentiation 01/2007; 13(12):2089-98. · 8.37 Impact Factor

Publication Stats

2k Citations
409.28 Total Impact Points

Institutions

  • 1999–2013
    • Leiden University Medical Centre
      • • Department of Thrombosis and Hemostasis
      • • Department of Cardiology
      • • Department of Human Genetics
      Leyden, South Holland, Netherlands
  • 1998
    • Maastricht University
      Maestricht, Limburg, Netherlands
  • 1996–1997
    • TNO
      Delft, South Holland, Netherlands