Berthe Van Hoef

Function of the Plasminogen/Plasmin and Matrix Metalloproteinase Systems After Vascular Injury in Mice With Targeted Inactivation of Fibrinolytic System Genes

The matrix metalloproteinase (MMP) system, which may be activated via the plasminogen (Plg)/plasmin system, is claimed to play a role in matrix degradation and smooth muscle cell migration. To test the role of both systems, expression of fibrinolytic and gelatinolytic activity was quantified after vascular injury in mice with targeted inactivation of tissue-type Plg activator (tPA-/-), urokinase-type Plg activator (uPA-/-), or Plg (Plg-/-). Neointima formation 1 week after vascular injury was impaired in uPA-/- and Plg-/- mice compared with wild-type (WT) mice or tPA-/- mice (reduction of neointimal area to 30% and 10% of WT, respectively). Cell accumulation at the borders of the injury was significantly (P<0.01) impaired compared with that in WT mice. One week after injury of the femoral artery, tPA-mediated fibrinolytic activity in arterial sections or extracts of WT, uPA-/-, or Plg-/- mice was not altered, whereas uPA activity levels in tPA-/- and Plg-/- mice were 2- to 3-fold higher than in uninjured controls. Total levels (latent plus active) of MMP-2 (gelatinase A) were increased by 2- to 4-fold, whereas the contribution of active MMP-2 represented 38% to 63% of the total in the different genotypes. MMP-9 (gelatinase B) was not detectable in the majority of control arteries, whereas total MMP-9 levels after injury were dramatically increased (up to 50-fold above the detection limit). Active MMP-9 represented 20% to 46% of total MMP-9 in WT, tPA-/-, and uPA-/- mice but was not consistently detectable in Plg-/- mice. Similar results were obtained in carotid arteries. Thus, the unaltered ratios of active and latent MMP-2 suggest that proMMP-2 activation may occur in the absence of tPA, uPA, or Plg, whereas no active MMP-9 was detected in the absence of Plg. The data of this study confirm a role for uPA and Plg but not for tPA in smooth muscle cell migration and neointima formation after vascular injury and indicate that impairment of these phenomena may occur despite the observed increases in MMP-2 or MMP-9 levels after vascular injury.

Nutritionally Induced Obesity Is Attenuated in Transgenic Mice Overexpressing Plasminogen Activator Inhibitor-1

Objective—The objective of this study was to investigate the role of plasminogen activator inhibitor-1 (PAI-1) in adipose tissue development in vivo. Methods and Results—Transgenic (Tg) mice overexpressing murine PAI-1 under control of the adipocyte promoter aP2 and wild-type (WT) controls were kept on standard food (SFD) or on high-fat diet (HFD) for 15 weeks. The body weight and the weight of the isolated subcutaneous and gonadal fat deposits of the Tg mice kept on the HFD were significantly lower than those of the WT mice. The number of adipocytes in the adipose tissue was similar for Tg and WT mice on the HFD, but adipocyte hypotrophy and a significantly lower ratio of stroma cells/adipocytes were observed in the Tg mice. A significant negative correlation (P <0.01) was observed between expression of preadipocyte factor-1, which blocks adipocyte differentiation, and adipose tissue weight. Fasting insulin and total cholesterol levels on the HFD were lower in Tg than in WT mice. Conclusions—High circulating PAI-1 levels attenuate nutritionally induced obesity. This may be related to modifications in adipose tissue cellularity affecting weight and plasma metabolic parameters.

Comparative kinetic analysis of the activation of human plasminogen by natural and recombinant single-chain urokinase-type plasminogen activator

Single-chain urokinase-type plasminogen activator (scu-PA) may be obtained from conditioned cell culture media (natural scu-PA) or by expression of the cDNA encoding human scu-PA in Escherichia coli (recombinant scu-PA). The activation of Glu-plasminogen by natural and recombinant scu-PA can be described by a sequence of three reactions, each of which obeys Michaelis-Menten kinetics. Initial activation of plasminogen to plasmin by scu-PA (reaction I) occurs with a high affinity (Km below 0.8 microM) for both scu-PAs, while the catalytic rate constant (k2) is 0.017 s-1 for recombinant scu-PA but only 0.0009 s-1 for natural scu-PA. Subsequent conversion of scu-PA to urokinase (two-chain urokinase-type plasminogen activator, tcu-PA) by generated plasmin (reaction II) occurs with a comparable affinity (Km about 5 microM) for natural and recombinant scu-PA and with a k2 of 0.23 s-1 for natural and 1.2 s-1 for recombinant scu-PA. Finally, activation of plasminogen by tcu-PA (reaction III) occurs with low affinity (Km 30-50 microM) but with a high catalytic rate constant (k2 about 5 s-1) for both natural and recombinant tcu-PA. The differences in the kinetic parameters of the activation of plasminogen by natural or recombinant scu-PA are thus mainly due to differences in turnover rate in the first reaction. Indeed, the catalytic rate constant of the first reaction is about 20-times higher for recombinant scu-PA than for natural scu-PA. Thus, surprisingly, the artificial, unglycosylated recombinant scu-PA molecule has a better catalytic efficiency than its natural glycosylated counterpart.

Accelerated Neointima Formation After Vascular Injury in Mice With Stromelysin-3 (MMP-11) Gene Inactivation

The hypothesis that stromelysin-3 (MMP-11), a unique member of the matrix metalloproteinase (MMP) family, plays a role in neointima formation was tested with the use of a vascular injury model in wild-type (MMP-11(+/+)) and MMP-11-deficient (MMP-11(-/-)) mice. Neointima formation 2 to 3 weeks after electric injury of the femoral artery was significantly enhanced in MMP-11(-/-) as compared with MMP-11(+/+) mice, in both mice of a pure 129SV genetic background (0.014 versus 0.0010 mm(2) at 2 weeks, P<0.001) and those of a 50/50 mixed 129SV/BL6 background (0.030 versus 0.013 mm(2) at 3 weeks, P<0.05). The medial areas were comparable, resulting in intima/media ratios that were significantly increased in MMP-11(-/-) as compared with MMP-11(+/+) arteries, in mice of both the 129SV (1. 0 versus 0.18, P<0.001) and mixed (1.5 versus 0.70, P<0.05) backgrounds. Nuclear cell counts in cross-sectional areas of the intima of the injured region were higher in arteries from MMP-11(-/-) mice than in those from MMP-11(+/+) mice (210 versus 48, P<0.001, in pure 129SV mice and 290 versus 150, P<0.01, in mice of the mixed genetic background). Immunocytochemical analysis revealed that alpha-actin-positive and CD45-positive cells were more abundant in intimal sections of MMP-11(-/-) mice. Degradation of the internal elastic lamina was more extensive in arteries of MMP-11(-/-) mice than in those of MMP-11(+/+) mice (39% versus 6.8% at 3 weeks, P<0. 005). The mechanisms by which MMP-11 could impair elastin degradation and cellular migration in this model remain, however, unknown.

Fumagillin reduces adipose tissue formation in murine models of nutritionally induced obesity.

The effect of fumagillin (a methionine aminopeptidase‐type 2 (Met‐AP2) inhibitor, with antiangiogenic properties) was investigated in murine models of diet‐induced obesity. Eleven‐week‐old male C57Bl/6 mice (group 1) were given fumagillin by oral gavage at a dose of 1 mg/kg/day during 4 weeks while fed a high‐fat diet (HFD) (20.1 kJ/g), and control mice (group 2) received solvent and were pair‐fed. At the end of the experiment, body weights in group 1 were significantly lower as compared to group 2 (P < 0.0005). The subcutaneous (SC) and gonadal (GON) fat mass was also significantly lower in group 1 (P < 0.005 and P < 0.05, respectively). Adipocytes were smaller in adipose tissues of mice in group 1, associated with higher adipocyte density. Blood vessel density normalized to adipocyte density was lower in group 1 adipose tissues. However, in mice with established obesity monitored to maintain the same body weight and fat mass as controls, short‐term fumagillin administration was also associated with adipocyte hypotrophy (P = 0.01) without affecting blood vessel size or density. Thus, treatment with fumagillin impaired diet‐induced obesity in mice, associated with adipocyte hypotrophy but without marked effect on adipose tissue angiogenesis.

On the molecular interactions between plasminogen-staphylokinase, α2-antiplasmin-formula> and fibrin

The molecular interactions between the plasminogen-staphylokinase complex, α 2 -antiplasmin-formula> and fibrin were studied by measuring the effect of CNBr-digested fibrinogen on the inhibition rate of the plasminogen-staphylokinase complex by α 2 -antiplasmin-formula>. The second-order rate constant for the inhibition of plasminogen-staphylokinase by α 2 -antiplasmin-formula> was 2.7±0.3 · 10 6 M −1 s −1 -formula> (mean±S.D.-formula>; n = 7-formula>). Addition of CNBr-digested fibrinogen, but not of fibrinogen, resulted in a concentration-dependent reduction of the apparent inhibition rate constant, with a 50 percent reduction at a concentration of 5 nM CNBr-digested fibrinogen. The second-order rate constant for the inhibition of the low- M r -formula> plasminogen-staphylokinase complex (plasminogen lacking the kringle structures comprising the lysine-binding sites) by α 2 -antiplasmin-formula> was about 30-fold lower (9.3 ± 0.7 · 10 4 M −1 -formula>, mean±S.D.-formula>; n = 4-formula>) than that of plasminogen-staphylokinase and was not affected by addition of CNBr-digested fibrinogen. Inhibition of the plasminogen-staphylokinase complex by the chloromethylketone d -Val-Phe-Lys-CH 2 Cl is 9-fold less efficient than that of plasmin ( k 2 /K i -formula> of 700 M −1 s −1 versus 6300 M −1 s −1 ). Our results confirm and establish that rapid inhibition of plasminogen-staphylokinase by α 2 -antiplasmin-formula> requires the availability of the lysine-binding sites in the plasminogen moiety of the complex. Fibrin, but not fibrinogen, reduces the inhibition rate by α 2 -antiplasmin-formula> by competition for interaction with the lysine-binding site. Protection of the plasminogen-staphylokinase complex bound to fibrin from rapid inhibition by α 2 -antiplasmin-formula> thus appears to contribute to the fibrin-specificity of clot lysis with staphylokinase in a plasma milieu, by allowing preferential plasminogen activation at the fibrin surface, while the free complex is rapidly inhibited in plasma.

Adverse Adipose Phenotype and Hyperinsulinemia in Gravid Mice Deficient in Placental Growth Factor

Pregnancy-induced metabolic changes are regulated by signals from an expanded adipose organ. Placental growth factor (PlGF), acting through vascular endothelial growth factor receptor-1, may be among those signals. There is a steep rise in circulating PlGF during normal pregnancy, which is repressed in gravidas who develop preeclampsia. PlGF-deficiency in mice impairs adipose vascularization and development. Here we studied young-adult PlGF-deficient (PlGF(-/-)) and wild-type mice on a high-fat diet in the nongravid state and at embryonic day (E) 13.5 or E18.5 of gestation. Litter size and weight were normal, but E18.5 placentas were smaller in PlGF(-/-) pregnancies. PlGF(-/-) mice showed altered intraadipose dynamics, with the following: 1) less blood vessels and fewer brown, uncoupling protein (UCP)-1-positive, adipocytes in white sc and perigonadal fat compartments and 2) white adipocyte hypertrophy. The mRNA expression of beta(3)-adrenergic receptors, peroxisome proliferator-activated receptor-gamma coactivator-1alpha, and UCP-1 was decreased accordingly. Moreover, PlGF(-/-) mice showed hyperinsulinemia. Pregnancy-associated changes were largely comparable in PlGF(-/-) and wild-type dams. They included expanded sc fat compartments and adipocyte hypertrophy, whereas adipose expression of key angiogenesis/adipogenesis (vascular endothelial growth factor receptor-1, peroxisome proliferator-activated receptor-gamma(2)) and thermogenesis (beta(3)-adrenergic receptors, peroxisome proliferator-activated receptor-gamma coactivator-1alpha, and UCP-1) genes was down-regulated; circulating insulin levels gradually increased during pregnancy. In conclusion, reduced adipose vascularization in PlGF(-/-) mice impairs adaptive thermogenesis in favor of energy storage, thereby promoting insulin resistance and hyperinsulinemia. Pregnancy adds to these changes by PlGF-independent mechanisms. Disturbed intraadipose dynamics is a novel mechanism to explain metabolic changes in late pregnancy in general and preeclamptic pregnancy in particular.

Short-term ethinylestradiol treatment suppresses inferior caval vein thrombosis in obese mice

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.

Factor V Leiden mutation is associated with enhanced arterial thrombotic tendency in lean but not in obese mice

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.

Rofecoxib impairs adipose tissue development in a murine model of nutritionally induced obesity

The effect of rofecoxib (Vioxx), a cyclooxygenase (COX)-2 inhibitor, on adipose tissue development was studied in a murine model of diet-induced obesity. Oral administration of Vioxx for six weeks (34 mg/kg/day) to C57Bl/6 mice kept on high-fat diet (n = 19) resulted in a significant reduction in total body weight (p < 0.01) and of subcutaneous (p < 0.05) and gonadal (p < 0.01) adipose tissue mass, as compared to placebo-treated animals (n = 21). There was no significant difference in food intake between both groups (2.8 +/- 0.09 vs. 3.0 +/- 0.10 g/day; p = 0.20). Administration of Vioxx resulted in reduced total cholesterol and high-density lipoprotein (HDL) cholesterol levels (p < 0.0001) and in enhanced levels of liver enzymes, as compared to place-bo. In the gonadal but not in the subcutaneous adipose tissue, adipocytes were smaller after Vioxx treatment (p < 0.05). The macrophage content was significantly lower in gonadal adipose tissues of Vioxx-treated mice (p < 0.05), but not in the subcutaneous adipose tissues. This was, however, not associated with differences in adipose tissue levels of the pro-inflammatory tumor necrosis factor (TNF)-alpha. Blood vessel size or density in either fat pad were not affected by Vioxx treatment. Thus, in a nutritionally induced murine obesity model, oral administration of Vioxx, as compared to placebo, resulted in reduced adipose tissue development, associated with lower feeding efficiency and smaller adipocytes.