B. Van Hoef

Recombinant human microplasmin: production and potential therapeutic properties

Summary.  The effect of recombinant human microplasmin was studied in ischemic stroke models in mice and in an extracorporeal loop thrombosis model in rabbits. Human microplasminogen (µPlg), which lacks the five ‘kringle’ domains of plasminogen was expressed with high yield in Pichia pastoris. It was purified, converted to microplasmin (µPli) and equilibrated with 5 mmol L−1 citrate, pH 3.1, yielding a stable preparation. In mice with middle cerebral artery (MCA) ligation, an intravenous (i.v.) bolus of 5.0 mg kg−1µPli reduced infarct size at 24 h from 27 (26–30) to 25 (21–28) mm3 (median and range, n= 16 each, P= 0.0001), whereas 4.0 mg kg−1 rt‐PA and 40 mg kg−1µPlg had no effect. Infarct reduction was observed with administration at 4 h after occlusion. In mice with MCA, infarct size at 24 h was reduced from 20 (14–30) to 9.1 (3.1–25) mm3 with 5.0 mg kg−1µPli (n = 15 each, P < 0.002) and to 11 (5.2–27) mm3 with 4.0 mg kg−1 rt‐PA (n = 6; P= 0.02). Infarct reduction was still observed at 10 h after occlusion with µPli but not with t‐PA. In rabbits with radiolabeled clots in an extracorporeal arteriovenous loop, local infusion of 2.5 mg kg−1µPli over 2 h, induced 51 ± 15% lysis (mean ± SD, n= 11) vs. a control value of 23 ± 5.5%. µPli did not prolong template bleeding times, whereas equipotent doses of rt‐PA were associated with extensive rebleeding. The potency of µPli in both models was similar to that of intact plasmin. These findings indicate that recombinant µPli may be useful for treatment of ischemic stroke and arterial thrombosis.

On the role of plasminogen activator inhibitor‐1 in adipose tissue development and insulin resistance in mice

Summary.  Objectives: To investigate the role of plasminogen activator inhibitor‐1 (PAI‐1) in adipose tissue development and insulin metabolism. Methods: Aged male wild‐type (WT) or transgenic mice with adipose tissue overexpression of PAI‐1 (45–55 weeks) in 50% C57Bl/6: 50% Friend Virus B‐strain (FVB) genetic background, kept on normal chow, were used without or with administration of a synthetic low molecular weight PAI‐1 inhibitor (PAI‐039) to the food (1 mg g−1) for 4 weeks. Results: The PAI‐1 transgenic mice showed somewhat lower body weight and adipose tissue mass than WT mice, whereas fasting insulin levels were higher. Glucose and insulin tolerance tests did not reveal significant differences between both genotypes. Addition of PAI‐039 to the food did not significantly affect total body fat, weight of the isolated s.c. and gonadal fat territories or their adipocyte size and blood vessel composition in either genotype. Fasting glucose levels and glucose tolerance tests were, for both genotypes, comparable with those without inhibitor treatment. Insulin levels and insulin tolerance tests in WT, but not in PAI‐1 transgenic mice, suggested a higher insulin sensitivity after inhibitor treatment (insulin level 30 min after glucose injection of 2.0 ± 0.17 ng mL−1 vs. 3.2 ± 0.48 ng mL−1 without inhibitor treatment; P = 0.028). Conclusions: In this model, overexpression of PAI‐1 moderately impaired adipose tissue formation without affecting glucose or insulin tolerance. Administration of a synthetic PAI‐1 inhibitor for 4 weeks did not affect adipose tissue development in WT or PAI‐1 transgenic mice, but induced a higher insulin sensitivity in WT mice.

Effects of plasminogen activator inhibitor‐1 on ischemic brain injury in permanent and thrombotic middle cerebral artery occlusion models in mice

Summary.  Background and Objectives: Tissue plasminogen activator (t‐PA) improves the outcome of ischemic stroke by recanalization of occluded vessels, but has neurotoxic side effects in experimental stroke models. Here, the effect of plasminogen activator inhibitor‐1 (PAI‐1), an endogenous inhibitor of t‐PA, on ischemic infarct volume was studied. Methods: After either permanent ligation or thrombotic occlusion of the middle cerebral artery (MCA), infarct volume, spontaneous reperfusion of thrombosed MCA, t‐PA/PAI‐1 complex level, and blood–brain barrier (BBB) permeability in the ischemic region was studied in transgenic mice with overexpression of PAI‐1 and wild‐type littermate controls and in mice with intracerebroventricular injection of human PAI‐1. Results: Infarct volume was smaller in PAI‐1 transgenic mice (2.9 ± 3.7 mm3, mean ± SD) than in controls (8.9 ± 5.0 mm3, P < 0.05) after permanent MCA ligation (plasma PAI‐1 level 39 ± 23 ng mL−1 in transgenic mice vs. 1.5 ± 0.6 ng mL−1 in controls), whereas after MCA thrombosis it was larger in transgenics (13.1 ± 3.1 mm3) than in controls (8.0 ± 3.2 mm3, P < 0.05). Spontaneous reperfusion of the thrombosed MCA was significantly delayed in transgenic vs. control mice. In the ligation model, t‐PA/PAI‐1 complex levels were higher and BBB disruption was more pronounced in the ischemic region. Human PAI‐1 injection reduced infarct volume by about 50% in wild‐type mice but not in t‐PA gene deficient mice. Conclusions: High PAI‐1 levels reduced infarct volume in the permanent MCA ligation model, but enhanced it in the MCA thrombosis model.

Plasminogen activator inhibitor-1 contributes to the deleterious effect of obesity on the outcome of thrombotic ischemic stroke in mice

Summary.  Background: It is widely accepted that obesity is a risk factor for ischemic heart disease, but the association with stroke is less clear. Adipose tissue is an important source of plasminogen activator inhibitor‐1 (PAI‐1), the main inhibitor of plasminogen activation. Objective: To test the hypothesis that elevated PAI‐1 levels associated with obesity negatively affect the outcome of thrombotic ischemic stroke. Methods: Middle cerebral artery (MCA) occlusion was induced photochemically in mice with nutritionally induced or genetically determined obesity and their lean counterparts. Results: The MCA occlusion time (to obtain complete occlusion) was significantly shorter in obese (nutritionally induced) than in lean wild‐type (WT) C57Bl/6 mice, whereas the infarct size was significantly larger and intracranial hemorrhage (ICH) was enhanced (all P < 0.05). Similar observations were made in genetically obese ob/ob mice, as compared to lean WT littermates. In both strains, obesity was associated with markedly elevated circulating PAI‐1 levels, probably originating from the fat tissue. In contrast, PAI‐1‐deficient lean and obese mice did not display significant differences in MCA occlusion time, infarct volume or ICH. Conclusions: Plasminogen activator inhibitor‐1 may play a functional role in the deleterious effect of obesity on the outcome of thrombotic ischemic stroke in mice.

Biochemical Properties of Natural and Recombinant Staphylokinase

Abstract The biochemical properties of natural staphylokinase (STAN), purified from the culture broth of a selected Staphylococcus aureus strain, were compared with those of different molecular species of recombinant staphylokinase (STAR), produced by expression of a genomic DNA fragment of the Staphylococcus aureus strain in E. coli. STAR was obtained from the supernatant (STAR-S) or periplasmic fraction (STAR-P) of the transformed E. coli either as a low molecular weight form lacking the 10 NHZ-terminal amino acids (STA-Δ10) (peak I following chromatography of STAR-S or STAR-P on CM-Sephadex, STA-CM-I) or as a mixture of mature STA (STA-M) and a proteolytic derivative lacking the 6 NH2-terminal amino acids (STA-Δ6) (peak II following chromatography of STAR-S or STAR-P on CM-Sephadex, STA-CM-II), or it was obtained from the cytosol fraction (STAR-C) as STA-Δ10. STAN, STA-CM-I, STA-CM-II and STAR-C were found to be similar with respect to following properties: rate and extent of complex formation with plasminogen, plasminogen activating potential in the absence or the presence of a fibrin-like stimulator and inhibition rate of the plasminogen-STA complex by a2-antiplasmin, and fibrinolytic and fibrinogenolytic potential in a human plasma milieu in vitro. In a human plasma milieu in vitro, STAN had a somewhat lower fibrinolytic activity versus platelet-rich plasma ((PRP); 4 × 105 platelets/μl) clots than versus platelet-poor plasma ((PPP); Thus, STAN, STA-CM-I, STA-CM-II and STAR-C are functionally indistinguishable, and in a human plasma milieu STA is relatively more potent towards retracted or compressed plasma clots than streptokinase.

Neointima formation and thrombosis after vascular injury in transgenic mice overexpressing plasminogen activator inhibitor‐1 (PAI‐1)

Summary.  The controversial role of plasminogen activator inhibitor‐1 (PAI‐1) in neointima formation and restenosis was studied with the use of a vascular injury model in transgenic mice overexpressing murine PAI‐1 (PAI‐1 Tg) and in wild‐type (WT) controls. Despite the high circulating PAI‐1 levels in the PAI‐1 Tg mice (52 ± 9.8 ng mL−1 vs. 0.76 ± 0.17 ng mL−1 in WT mice), no significant fibrin deposition was observed in non‐injured femoral arteries of 8‐ to 12‐week‐old mice. Two weeks after severe electric injury, extensive and comparable fibrin deposition was observed in both genotypes, despite a significantly reduced in situ fibrinolytic activity in arterial sections of the PAI‐1 Tg mice. The neointimal and medial areas were similar in WT and PAI‐1 Tg mice, resulting in comparable intima/media ratios (e.g. 0.94 ± 0.25 and 1.04 ± 0.17 at the center of the injury). Nuclear cell counts in cross‐sectional areas of the neointima of the injured region were also comparable in arteries from WT and PAI‐1 Tg mice (224 ± 63, 233 ± 20), and the distribution pattern of α‐actin‐positive smooth muscle cells was similar. These findings indicate that in a vascular injury model that induces extensive and persistent fibrin deposition in femoral arteries of mice, overexpression of PAI‐1 does not affect neointima formation.

α2-Antiplasmin Gene Deficiency in Mice Does Not Affect Neointima Formation After Vascular Injury

Abstract —The hypothesis that α2-antiplasmin (α2-AP), the main physiological plasmin inhibitor, plays a role in neointima formation was tested with use of a vascular injury model in wild-type (α2-AP+/+) and α2-AP–deficient (α2-AP−/−) mice. The neointimal and medial areas were similar 1 to 3 weeks after electric injury of the femoral artery in α2-AP+/+ and α2-AP−/− mice, resulting in comparable intima/media ratios (eg, 0.43±0.12 and 0.42±0.11 2 weeks after injury). Nuclear cell counts in cross-sectional areas of the intima of the injured region were also comparable in arteries from α2-AP+/+ and α2-AP−/− mice (78±19 and 69±8). Fibrin deposition was not significantly different in arteries of both genotypes 1 day after injury, and no mural thrombosis was detected 1 week after injury. Fibrinolytic activity in femoral arterial sections, as monitored by fibrin zymography, was higher in α2-AP−/− mice 1 week after injury ( P <0.001) but was comparable in both genotypes 2 and 3 weeks after injury. Staining for elastin did not reveal significant degradation of the internal elastica lamina in either genotype. Immunocytochemical analysis revealed a comparable distribution pattern of α-actin–positive smooth muscle cells in both genotypes. These findings indicate that the endogenous fibrinolytic system of α2-AP+/+ mice is capable of preventing fibrin deposition after vascular injury and suggest that α2-AP does not play a major role in smooth muscle cell migration and neointima formation in vivo.

Stromelysin-2 (MMP-10) deficiency does not affect adipose tissue formation in a mouse model of nutritionally induced obesity

Adipose tissue development is associated with angiogenesis, adipogenesis and extracellular matrix degradation. The class of matrix metalloproteinases contributes to these processes, but little information is available on the role of individual proteinases. We report that stromelysin-2 (MMP-10) deficiency has no significant effect on total body weight or on subcutaneous (SC) or gonadal (GON) adipose tissue mass of mice kept on a high fat diet for 15 weeks. The adipocyte size and density in SC and GON adipose tissues were also comparable in MMP-10 deficient and wild-type control mice. Similarly, blood vessel size and density in obese SC and GON adipose tissues was not affected by MMP-10 deficiency. Metabolic parameters and blood cell composition were similar for both genotypes. Stromelysin-1 (MMP-3) expression was significantly reduced in adipose tissues of the deficient mice as compared to the wild-type controls. These data indicate that MMP-10 does not significantly contribute to adipose tissue development and associated angiogenesis in a mouse model of nutritionally induced obesity.

Plasminogen/plasmin system function in mice deficient in stromelysin-1 (MMP-3) or in tissue inhibitor of metalloproteinases type-1 (TIMP-1)

Summary Specific interactions between the plasminogen/plasmin (fibrinolytic) and matrix metalloproteinase (MMP) systems suggest that both systems may cooperate in extracellular matrix degradation. Therefore, the plasminogen/plasmin system function was evaluated in mice with stromelysin-1 (MMP-3) or tissue inhibitor of MMP type-1 (TIMP-1) gene inactivation. Urinary urokinase-type plasminogen activator (u-PA) antigen (120–190 ng/ml) and activity (13–18 IU/ml) levels were similar in both wild-type and gene-deficient mice. Vascular plasminogen activator activity, measured in aorta extracts, was similar for both tissue-type plasminogen activator (t-PA)-mediated activity (8–14 arbitrary units (AU) per mg protein) and for u-PA-mediated activity (25–34 AU per mg protein). The spontaneous thrombolytic potential (lysis of a 125 I-fibrin labelled pulmonary embolus) was comparable in wild-type (MMP-3 +/+ ) and MMP-3 −/− mice (36 ± 6% versus 49 ± 5% after 8 h; P = 0.13), and in wild-type (TIMP-1 +/+ ) and TIMP-1 −/− mice (48 ± 4% versus 48 ± 2% after 8 h). Organ sections did not reveal significant fibrin deposition in the liver of any of the genotypes. Furthermore, in vivo thioglycollate-stimulated macrophages of all 4 genotypes expressed comparable u-PA-mediated plasminogen activating potential and 125 I-fibrin matrix degrading potential. Thus, these data suggest that MMP-3 and TIMP-1 do not play a major role in the generation of fibrinolytic activity mediated via the plasminogen/plasmin system.

Deficiency of vascular endothelial growth factor-D does not affect murine adipose tissue development

Vascular endothelial growth factor (VEGF)-D deficiency had no significant effect on total body weight or on subcutaneous (SC) or gonadal (GON) adipose tissue mass of mice kept on a standard fat (SFD) or a high fat diet (HFD) for 15 weeks. The composition of SC and GON adipose tissues of VEGF-D deficient mice in terms of size and density of adipocytes or blood vessels was also comparable to that of wild-type control mice. Staining of lymphatic vessels in adipose tissue sections did not reveal marked differences between both genotypes. The absence of an effect of VEGF-D deficiency could not be explained by compensatory increases of VEGF-C expression in adipose tissues of the deficient mice. Thus, our data do not support an important role of VEGF-D in (lymph) angiogenesis or in adipose tissue development.