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.

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.

Inhibition of vascular endothelial growth factor receptor tyrosine kinases impairs adipose tissue development in mouse models of obesity.

We have studied the effect of PTK787 (Vatalanib), an inhibitor of vascular endothelial growth factor receptor (VEGFR) tyrosine kinases, on adipose tissue development. Oral administration of PTK787 for 4 weeks (2 mg/g high fat diet, HFD) to C57Bl/6 mice resulted in a significant reduction in total body weight and of subcutaneous (SC) and gonadal (GON) adipose tissue mass, as compared to control animals fed HFD only (all p<0.0005). In the GON adipose tissue adipocytes were hypertrophic after PTK787 treatment. Blood vessel size and density were not significantly affected by PTK787 treatment. Expression of Flk-1 (VEGFR-2) mRNA was significantly reduced in SC and GON adipose tissues of PTK787 treated mice. De novo fat pad formation following injection of preadipocytes in NUDE mice was significantly (p<0.005) impaired by PTK787 administration (2 mg/g HFD for 4 weeks), without associated effect on blood vessel size or density. Thus, in nutritionally induced murine obesity models, oral administration of the VEGFR tyrosine kinases inhibitor PTK787 resulted in reduced adipose tissue development.

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

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.

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

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.

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.

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

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 alpha-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.

Recombinant human microplasmin: production and potential therapeutic properties.

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 ( micro Plg), which lacks the five 'kringle' domains of plasminogen was expressed with high yield in Pichia pastoris. It was purified, converted to microplasmin ( micro Pli) 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(-1) micro Pli 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(-1) micro Plg 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(-1) micro Pli (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 micro Pli but not with t-PA. In rabbits with radiolabeled clots in an extracorporeal arteriovenous loop, local infusion of 2.5 mg kg(-1) micro Pli over 2 h, induced 51 +/- 15% lysis (mean +/- SD, n= 11) vs. a control value of 23 +/- 5.5%. micro Pli did not prolong template bleeding times, whereas equipotent doses of rt-PA were associated with extensive rebleeding. The potency of micro Pli in both models was similar to that of intact plasmin. These findings indicate that recombinant micro Pli may be useful for treatment of ischemic stroke and arterial thrombosis.

Matrix metalloproteinase inhibition impairs adipose tissue development in mice.

The effect of galardin, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, was studied in mice kept on a high fat diet (HFD). Five-week-old male wild-type mice were fed the HFD (42% fat) for up to 12 weeks and were daily injected intraperitoneally with the inhibitor (100 mg/kg) or with vehicle. After 12 weeks of the HFD, the body weights of both groups were comparable, but the weight of the isolated subcutaneous (SC) or gonadal (GON) fat deposits was significantly lower in the inhibitor-treated group than in the control group (88 +/- 11 versus 251 +/- 66 mg, respectively, for SC fat [P<0.05]; 90 +/- 24 versus 217 +/- 30 mg, respectively, for GON fat [P<0.02]). The number of adipocytes was somewhat higher and the diameter was somewhat smaller (but not significantly) in adipose tissues of the inhibitor-treated group. Adipose tissue of the inhibitor-treated mice contained more collagen than did that of the vehicle-treated mice (Sirius red-stained area of 42 +/- 2.6% versus 22 +/- 4.4%, respectively, for SC fat [P<0.05]; 21 +/- 5.1% versus 4.7 +/- 0.92%, respectively, for GON fat [P<0.01]); a distinct collagen-rich cap was formed around the inhibitor-treated tissue. In situ zymography with casein- or gelatin-containing gels confirmed a reduced MMP activity in SC and GON adipose tissues of inhibitor-treated mice. Thus, in this model, growth and development of adipose tissue appears to be limited by the formation of a collagen-rich matrix cap around the inhibitor-treated tissue. These data suggest a functional role for MMPs in the development of adipose tissue.

Adipocyte hypertrophy in stromelysin-3 deficient mice with nutritionally induced obesity.

Several matrix metalloproteinases (MMPs), including the stromelysins MMP-3 and MMP-11, are expressed in adipose tissue. To investigate a potential role of MMP-11 (stromelysin-3) in adipose tissue development, five-week-old male wild-type mice (MMP-11+/+) or mice with deficiency of MMP-11 (MMP-11-/-) were fed a high fat diet (HFD, 42% fat) for 15 weeks. Haematologic parameters, including white and red blood cells, platelets, haemoglobin and haematocrit, and metabolic parameters including glucose, triglycerides and total cholesterol were not different for both genotypes. At the time of sacrifice, the body weight of the MMP-11-/- mice was higher than that of the MMP-11+/+ mice (36+/-1.4 g versus 29+/-0.9 g, p = 0.0002). The weight of the isolated subcutaneous (SC) and gonadal (GON) fat deposits was also higher in MMP-11-/- mice (620+/-150 mg versus 280+/-28 mg for SC fat, and 970+/-180 mg versus 430+/-62 mg, p < 0.05, for GON fat). Adipocytes in MMP-11-/- adipose tissue were hypertrophic as compared to MMP-11+/+ adipocytes (volume of 57+/-12 x 10(3) microm3 versus 31+/-2.4 x 10(3) microm3 for SC fat, and 100+/-18 x 10(3) microm3 versus 57+/-7.6 x 10(3) microm3 for GON fat; both p < 0.06). In nutritionally induced obesity models in mice a potential role of the fibrinolytic system was suggested in adipocyte hypertrophy. The hypertrophy observed in this model is, however, not related to changes in fibrinolytic parameters, as suggested by our finding that levels of t-PA, u-PA and PAI-1 antigen as well as t-PA and u-PA activity were not different in SC or GON adipose tissue extracts of both genotypes. As the main biological function of MMP-11 remains unknown, it is not clear whether the adipocyte hypertrophy in MMP-11-/- adipose tissue is directly related to the deficiency or to other pathways affected by MMP-11.

Influence of t-pA and u-PA on adipose tissue development in a murine model of diet-induced obesity.

To investigate the potential role of tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA) in development of adipose tissue, we have used a nutritionally induced obesity model in t-PA (t-PA-/-) and u-PA (u-PA-/-) deficient mice. Five week old male wild-type (WT), t-PA-/- or u-PA-/- mice (n = 9 to 16) were fed a high fat diet (HFD, 42% fat). After 16 weeks of HFD, the body weight of t-PA-/- mice was significantly higher than that of WT mice (48 +/- 1.1 g vs. 39 +/- 2.2 g, p = 0.004). The total weight of the isolated subcutaneous (sc) fat deposit was higher in t-PA-/- than in WT mice (2.4 +/- 0.22 g vs. 1.2 +/- 0.29 g, p = 0.002). accompanied with higher adipocyte diameters (80 +/- 1.7 microm vs. 61 +/- 4.7 microm, p < 0.01). These differences were not observed in the intra-abdominal fat deposit. The number of stroma cells in both adipose tissue territories was increased in t-PA-/- as compared to WT mice (2.0 +/- 0.13 vs. 1.5 +/- 0.10, p = 0.2 and 3.0 +/- 0.17 vs 1.6 +/- 0.17, p = 0.0001, stroma cells/adipocytes in sc and intra-abdominal tissue, respectively), partly as a result of an increased number of endothelial cells (192 +/- 9 vs. 154 +/-18, p = 0.06 and 108 +/- 13 vs. 69 +/- 8, p = 0.04 CD31 stained/adipocyte area). In contrast the weight gain and adipose tissue development in u-PA-/- mice was not different from that in WT mice. These data suggest that t-PA but not u-PA plays a role in adipose tissue development.

Inactivation of the serpin alpha(2)-antiplasmin by stromelysin-1.

Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) hydrolyzes the Met(374)-Ser(375) (P3-P2), Glu(416)-Leu(417) and Ser(432)-Leu(433) peptide bonds in human alpha(2)-antiplasmin (alpha(2)-AP), the main physiological plasmin inhibitor. Cleavage is completely abolished in the presence of the MMP inhibitors EDTA or 1,10-phenanthroline. At enzyme/substrate ratio of 1:10 at 37 degrees C, alpha(2)-AP protein cleavage occurs with a half-life of 8 min, and is associated with rapid loss of inhibitory activity towards plasmin with a half-life of 5 min. alpha(2)-AP cleaved by MMP-3 does no longer form a stable complex with plasmin, as shown by SDS-PAGE, and does no longer interact with plasminogen, as shown by crossed immunoelectrophoresis with plasminogen added to the gel. These data are compatible with the removal of a COOH-terminal fragment containing the reactive site peptide bond and the plasmin(ogen)-binding site. In addition, MMP-3 cleaves the Pro(19)-Leu(20) peptide bond in alpha(2)-AP, thereby removing the fibrin-binding site from the inhibitor. A dysfunctional alpha(2)-AP variant (Ala-alpha(2)-AP or alpha(2)-AP Enschede), with an alanine insertion in the reactive site sequence converting it from a plasmin inhibitor into a substrate, was also efficiently cleaved by MMP-3 (half-life of 13 min at 37 degrees C and enzyme/substrate ratio of 1:10). Cleavage and inactivation of alpha(2)-AP by MMP-3 may constitute a mechanism favoring local plasmin-mediated proteolysis.

Depletion of circulating alpha(2)-antiplasmin by intravenous plasmin or immunoneutralization reduces focal cerebral ischemic injury in the absence of arterial recanalization.

In the absence of arterial recanalization, thrombolytic agents induce a dose-related extension of focal cerebral ischemic injury (FII) in experimental animals. However, FII is smaller in mice lacking alpha(2)-antiplasmin (alpha(2)-AP), the physiologic inhibitor of plasmin, suggesting its depletion might reduce FII in the absence of reperfusion. Therefore, the effect of human plasmin (Pli), human miniplasmin (mPli), and an Fab fragment neutralizing murine alpha(2)-AP (Fab-4H9) on FII after middle cerebral artery (MCA) ligation was studied in mice and in hamsters. In BALB/c mice, the median FII after 24 hours was 28 microL (range, 20-34) (n = 10) with saline and 23 microL (range, 17-26) (n = 9) with a single bolus of 0.07 mg Pli, given after MCA ligation (P =.010), which reduced alpha(2)-AP to 44% and fibrinogen from 0.75 to 0.44 g/L. FII was 20 microL (range, 13-26) (n = 6, P =.025) with 0.2 mg mPli and was 24 microL (range, 20-27) (n = 6, P =.020) with 1.7 mg Fab-4H9. Neuronal atrophy and reduction of laminin immunoreactivity were comparably observed in the infarct area after saline and Pli. In hamsters, a single bolus injection of 1 mg Pli, after MCA ligation, depleted alpha(2)-AP and fibrinogen and reduced FII at 24 hours from 20 microL (range, 9.9-38) (n = 6) to 7.0 microL (range, 0.44-31) (n = 7, P =.032). Thus, reduction of circulating alpha(2)-AP, with a single bolus of plasmin or of a neutralizing antibody fragment, significantly reduced FII after MCA ligation in mouse and hamster models, suggesting that, provided these observations can be extrapolated to human beings, transient depletion of circulating alpha(2)-AP might reduce ischemic stroke in the absence of reperfusion.

Inactivation of plasminogen activator inhibitor-1 by specific proteolysis with stromelysin-1 (MMP-3).

Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) specifically hydrolyzes the Ser(337)-Ser(338) (P10-P9) and Val(341)-Ile(342) (P6-P5) peptide bonds in human plasminogen activator inhibitor-1 (PAI-1). Cleavage is completely abolished in the presence of the metal chelators EDTA or 1,10-phenanthroline. A stabilized active PAI-1 variant was also cleaved by MMP-3. At an enzyme/substrate ratio of 1/10 at 37 degrees C, PAI-1 protein cleavage occurred with half-lives of 27 or 14 min for active or stable PAI-1 and was associated with rapid loss of inhibitory activity toward tissue-type plasminogen activator with half-lives of 15 or 13 min, respectively. A substrate-like variant of PAI-1, lacking inhibitory activity but with exposed reactive site loop, was cleaved with a half-life of 23 min, whereas latent PAI-1 in which a major part of the reactive site loop is inserted into the molecule, was resistant to cleavage. Biospecific interaction analysis indicated comparable binding of active, stable, and substrate PAI-1 to both proMMP-3 and MMP-3 (K(A) of 12-22 x 10(6) m(-1)), whereas binding of latent PAI-1 occurred with lower affinity (1.7-2.3 x 10(6) m(-1)). Stable PAI-1 bound to vitronectin was cleaved and inactivated by MMP-3 in a manner comparable with that of free PAI-1; however, the cleaved protein did not bind to vitronectin. Cleavage and inactivation of PAI-1 by MMP-3 may thus constitute a mechanism decreasing the antiproteolytic activity of PAI-1 and impairing the potential inhibitory effect of vitronectin-bound PAI-1 on cell adhesion and/or migration.

Alpha(2)-antiplasmin gene deficiency in mice does not affect neointima formation after vascular injury.

The hypothesis that alpha(2)-antiplasmin (alpha(2)-AP), the main physiological plasmin inhibitor, plays a role in neointima formation was tested with use of a vascular injury model in wild-type (alpha(2)-AP(+/+)) and alpha(2)-AP-deficient (alpha(2)-AP(-/-)) mice. The neointimal and medial areas were similar 1 to 3 weeks after electric injury of the femoral artery in alpha(2)-AP(+/+) and alpha(2)-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 alpha(2)-AP(+/+) and alpha(2)-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 alpha(2)-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 alpha-actin-positive smooth muscle cells in both genotypes. These findings indicate that the endogenous fibrinolytic system of alpha(2)-AP(+/+) mice is capable of preventing fibrin deposition after vascular injury and suggest that alpha(2)-AP does not play a major role in smooth muscle cell migration and neointima formation in vivo.

Specific proteolysis of human plasminogen by a 24 kDa endopeptidase from a novel Chryseobacterium Sp.

A novel single polypeptide endopeptidase of 24 kDa (24k-endopeptidase) was purified with a yield of 300-400 microg/L from conditioned medium of a bacterial strain which was identified as a new species in the genus Chryseobacterium Sp. on the basis of its 16S rDNA sequence and DNA:DNA hybridizations. The NH(2)-terminal amino acid sequence (Val-Ala-Thr-Pro-Asn-Leu-Glu-.) was not found in the availabe databases. The 24k-endopeptidase specifically hydrolyzed the Ser(441)-Val(442) peptide bond in human plasmin(ogen), with additional cleavage of the Lys(78)-Val(79) and Pro(447)-Val(448) peptide bonds, and a secondary cleavage at Lys(615)-Val(616). Thereby, plasminogen is converted into an angiostatin-like fragment containing kringles 1-4 (K1-4) and miniplasminogen (kringle 5 and the serine proteinase domain). The purified K1-4 fragment showed a comparable cytotoxicity toward endothelial cells as the elastase-derived K1-3 fragment (12.7% versus 10.6% at a concentration of 10 microg/mL). Plasminogen, bound to monocytoid THP-1 cells, was also cleaved by the 24k-endopeptidase, resulting in generation of an angiostatin-like fragment and in a decreased capacity to generate cell-associated plasmin following activation by urokinase. The 24k-endopeptidase was not efficiently neutralized by specific inhibitors against the serine, cysteine, aspartic, or matrix metalloproteinase classes of enzymes. In human plasma or serum, however, it induced only very limited plasminogen degradation, apparently due to neutralization of its activity by alpha(2)-macroglobulin. Interaction of this novel 24k-endopeptidase with plasminogen thus yields an angiostatin-like fragment and affects plasmin-mediated cellular proteolytic activity.

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.

Guiding a docking mode by phage display: selection of correlated mutations at the staphylokinase-plasmin interface.

During co-evolution of interacting proteins, functionally disruptive mutations on one side of the interface may be compensated by local amino acid changes on the other to restore binding affinity. This information can be useful for geometry-based docking approaches by reducing the translational and rotational space available to the proteins. Here, we demonstrate that correlated mutations at a protein-protein interface can be rapidly identified by selecting a phage-displayed library of a randomly mutated component of the complex for complementation of mutations that decreased binding in the interacting partner. This approach was used to deduce the binding mode of staphylokinase (Sak), a 15.5 kDa "indirect" plasminogen activator on microplasmin (microPli), the 28 kDa serine protease domain of plasmin. Biopanning indicated that residues Arg94 and Gly174 in microPli are located in close proximity to Glu75 and the Glu88:Ile128 pair in Sak, respectively. The coupled mutations Glu94<-->Lys75 reversed and Gly174<-->Lys88:Val128 introduced a salt bridge, whereby the binding affinities (with coupling energies of 1.8 to 2.3 kcal mol-1, respectively) and the plasminogen activation ability of the mutated complexes were partially restored. These findings suggested a unique docking mode of Sak at the western rim of the active-site cleft of microPli, that is in agreement with the structure of the Sak-microPli complex as recently derived by other methods.

Structural and functional basis of plasminogen activation by staphylokinase.

Staphylokinase (Sak), a 15.5-kDa bacterial protein, forms a complex with human plasmin, which in turn activates other plasminogen molecules to plasmin. Three recombinant DNA-based approaches, (i) site directed substitution with alanine, (ii) search for proximity relationships at the complex interface, and (iii) active-site accessibility to protease inhibitors have been used to deduce a coherent docking model of the crystal structure of Sak on the homology-based model of microplasmin (microPli), the serine protease domain of plasmin. Sak binding on microPli is primarily mediated by two surface-exposed loops, loops 174 and 215, at the rim of the active-site cleft, while the binding epitope of Sak on microPli involves several residues located in the flexible NH2-terminal arm and in the five-stranded mixed beta-sheet. Several Sak residues located within the unique alpha-helix and the beta2 strand do not contribute to the binding epitope but are essential to induce plasminogen activating potential in the Sak:microPli complex. These residues form a topologically distinct activation epitope, which, upon binding of Sak to the catalytic domain of microPli, protrudes into a broad groove near the catalytic triad of microPli, thereby generating a competent binding pocket for micro-plasminogen (microPlg), which buries approximately 2500 A of the Sak:microPli complex upon binding. This structural and functional model may serve as a template for the design of improved Sak-derived thrombolytic agents. Following the completion and presentation of the present study, the deduced Sak:microPli:microPlg complex was fully confirmed by X-ray crystallography, which further illustrates the power and potential of the present approach.

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.