ADAMTS13 deficiency promotes microthrombosis in a murine model of diet-induced liver steatosis.

ADAMTS13 cleaves ultralarge multimeric von Willebrand Factor (VWF), thereby preventing formation of platelet-rich microthrombi. ADAMTS13 is mainly produced by hepatic stellate cells, and numerous studies have suggested a functional role of ADAMTS13 in the pathogenesis of liver diseases. The aim of our study was to investigate a potential role of ADAMTS13 in formation of hepatic microthrombi and development of non-alcoholic steatohepatitis (NASH), and furthermore to evaluate whether plasmin can compensate for the absence of ADAMTS13 in removal of thrombi. Therefore, we used a model of high-fat diet-induced steatosis in Adamts13 deficient (Adamts13-/-) and wild-type (WT) control mice. Microthrombi were more abundant in the liver of obese Adamts13-/- as compared to obese WT or to lean Adamts13-/- mice. Obese Adamts13-/- mice displayed lower platelet counts and higher prevalence of ultra-large VWF multimers. Hepatic plasmin-α2-antiplasmin complex levels were comparable for obese WT and Adamts13-/- mice and were lower for lean Adamts13-/- than WT mice, not supporting marked activation of the fibrinolytic system. High fat diet feeding, as compared to normal chow, resulted in enhanced liver triglyceride levels for both genotypes (p < 0.0001) and steatosis (p < 0.0001 for WT mice, p = 0.002 for Adamts13-/- mice) without differences between the genotypes. Expression of markers of inflammation, oxidative stress, steatosis and fibrosis was affected by diet, but not by genotype. Thus, our data confirm that obesity promotes NASH, but do not support a detrimental role of ADAMTS13 in its development. However, Adamts13 deficiency in obese mice promotes hepatic microthrombosis, whereas a compensatory role of plasmin in removal of microthrombi in the absence of ADAMTS13 could not be demonstrated.

Novel Role for Matrix Metalloproteinase 9 in Modulation of Cholesterol Metabolism.

BACKGROUND: The development of atherosclerosis is strongly linked to disorders of cholesterol metabolism. Matrix metalloproteinases (MMPs) are dysregulated in patients and animal models with atherosclerosis. Whether systemic MMP activity influences cholesterol metabolism is unknown. METHODS AND RESULTS: We examined MMP-9-deficient (Mmp9-/-) mice and found them to have abnormal lipid gene transcriptional responses to dietary cholesterol supplementation. As opposed to Mmp9+/+ (wild-type) mice, Mmp9-/- mice failed to decrease the hepatic expression of sterol regulatory element binding protein 2 pathway genes, which control hepatic cholesterol biosynthesis and uptake. Furthermore, Mmp9-/- mice failed to increase the expression of genes encoding the rate-limiting enzymes in biliary cholesterol excretion (eg, Cyp7a and Cyp27a). In contrast, MMP-9 deficiency did not impair intestinal cholesterol absorption, as shown by the 14C-cholesterol and 3H-sitostanol absorption assay. Similar to our earlier study on Mmp2-/- mice, we observed that Mmp9-/- mice had elevated plasma secreted phospholipase A2 activity. Pharmacological inhibition of systemic circulating secreted phospholipase A2 activity (with varespladib) partially normalized the hepatic transcriptional responses to dietary cholesterol in Mmp9-/- mice. Functional studies with mice deficient in other MMPs suggested an important role for the MMP system, as a whole, in modulation of cholesterol metabolism. CONCLUSIONS: Our results show that MMP-9 modulates cholesterol metabolism, at least in part, through a novel MMP-9-plasma secreted phospholipase A2 axis that affects the hepatic transcriptional responses to dietary cholesterol. Furthermore, the data suggest that dysregulation of the MMP system can result in metabolic disorder, which could lead to atherosclerosis and coronary heart disease.

Bacterial pathogens activate plasminogen to breach tissue barriers and escape from innate immunity.

Both coagulation and fibrinolysis are tightly connected with the innate immune system. Infection and inflammation cause profound alterations in the otherwise well-controlled balance between coagulation and fibrinolysis. Many pathogenic bacteria directly exploit the host's hemostatic system to increase their virulence. Here, we review the capacity of bacteria to activate plasminogen. The resulting proteolytic activity allows them to breach tissue barriers and evade innate immune defense, thus promoting bacterial spreading. Yersinia pestis, streptococci of group A, C and G and Staphylococcus aureus produce a specific bacterial plasminogen activator. Moreover, surface plasminogen receptors play an established role in pneumococcal, borrelial and group B streptococcal infections. This review summarizes the mechanisms of bacterial activation of host plasminogen and the role of the fibrinolytic system in infections caused by these pathogens.

Plasminogen activation by staphylokinase enhances local spreading of S. aureus in skin infections.

BACKGROUND: Staphylococcus aureus (S. aureus) is a frequent cause of skin and soft tissue infections. A unique feature of S. aureus is the combined presence of coagulases that trigger fibrin formation and of the plasminogen activator staphylokinase (SAK). Whereas the importance of fibrin generation for S. aureus virulence has been established, the role of SAK remains unclear. We studied the role of plasminogen activation by SAK in a skin infection model in mice and evaluated the impact of alpha-2-antiplasmin (α2AP) deficiency on the spreading and proteolytic activity of S. aureus skin infections. The species-selectivity of SAK was overcome by adenoviral expression of human plasminogen. Bacterial spread and density was assessed non-invasively by imaging the bioluminescence of S. aureus Xen36. RESULTS: SAK-mediated plasmin activity increased the local invasiveness of S. aureus, leading to larger lesions with skin disruption as well as decreased bacterial clearance by the host. Even though fibrin and bacterial surfaces protected SAK-mediated plasmin activity from inhibition by α2AP, the deficiency of α2AP resulted in increased bacterial spreading. SAK-mediated plasmin also induced secondary activation of gelatinases, shown both in vitro and in lesions from the in vivo model. CONCLUSION: SAK contributes to the phenotype of S. aureus skin infections by enhancing bacterial spreading as a result of fibrinolytic and proteolytic activation.

Tissue-type plasminogen activator is an extracellular mediator of Purkinje cell damage and altered gait.

Purkinje neurons are a sensitive and specialised cell type important for fine motor movement and coordination. Purkinje cell damage manifests as motor incoordination and ataxia - a prominent feature of many human disorders including spinocerebellar ataxia and Huntington's disease. A correlation between Purkinje degeneration and excess cerebellar levels of tissue-type plasminogen activator (tPA) has been observed in multiple genetically-distinct models of ataxia. Here we show that Purkinje loss in a mouse model of Huntington's disease also correlates with a 200% increase in cerebellar tPA activity. That elevated tPA levels arise in a variety of ataxia models suggests that tPA is a common mediator of Purkinje damage. To address the specific contribution of tPA to cerebellar dysfunction we studied the T4 mice line that overexpresses murine tPA in postnatal neurons through the Thy1.2 gene promoter, which directs preferential expression to Purkinje cells within the cerebellum. Here we show that T4 mice develop signs of cerebellar damage within 10 weeks of birth including atrophy of Purkinje cell soma and dendrites, astrogliosis, reduced molecular layer volume and altered gait. In contrast, T4 mice displayed no evidence of microgliosis, nor any changes in interneuron density, nor alteration in the cerebellar granular neuron layer. Thus, excess tPA levels may be sufficient to cause targeted Purkinje cell degeneration and ataxia. We propose that elevated cerebellar tPA levels exert a common pathway of Purkinje cell damage. Therapeutically lowering cerebellar tPA levels may represent a novel means of preserving Purkinje cell integrity and motor coordination across a wide range of neurodegenerative diseases.

Differential effects of a gelatinase inhibitor on adipocyte differentiation and adipose tissue development.

(1) A potential role for the gelatinases in adipocyte differentiation in vitro and adipose tissue development in vivo was investigated using the gelatinase inhibitor tolylsam ((R)-3-methyl-2-[4-(3-p-tolyl-[1,2,4]oxadiazol-5-yl)-benzenesulphonylamino]-butyric acid). (2) Differentiation of murine 3T3-F442A preadipocytes (12 days after reaching confluence) into mature adipocytes in vitro was promoted in the presence of tolylsam (10-100 µmol/L). (3) De novo development of fat tissue in nude mice injected with preadipocytes and kept on a high-fat diet was significantly impaired following treatment with tolylsam (100 mg/kg per day for 4 weeks). (4) Adipose tissue development in matrix metalloproteinase (MMP)-2 deficient mice, kept on a high-fat diet, was significantly impaired following administration of tolylsam (100 mg/kg per day for 15 weeks). This was associated with markedly enhanced metabolic rate. (5) Treatment of MMP-2-deficient mice with tolylsam (100 mg/kg per day, 15 weeks) was associated with the preservation of collagen and a reduction in blood vessel size in adipose tissues in vivo. (6) Furthermore, plasma levels of triglycerides and free fatty acids were reduced by tolylsam treatment of MMP-2-deficient mice (100 mg/kg per day, 15 weeks), whereas nutrient adsorption in the intestine was not affected. (7) The results of the present study indicate that tolylsam promotes preadipocyte differentiation in vitro, but impairs adipose tissue development in vivo.

Host-derived plasminogen activator inhibitor-1 (PAI-1) concentration is critical for in vivo tumoral angiogenesis and growth.

Plasminogen activator inhibitor type 1 (PAI-1) plays a key role in tumor progression and is believed to control proteolytic activity and cell migration during angiogenesis. We report here that host PAI-1, at physiological concentration, promotes in vivo tumor invasion and angiogenesis. In sharp contrast, inhibition of tumor vascularization was observed when PAI-1 was produced at supraphysiologic levels, either by host cells (transgenic mice overexpressing PAI-1) or by tumor cells (after transfection with murine PAI-1 cDNA). This study provides for the first time in vivo evidence for a dose-dependent effect of PAI-1 on tumor angiogenesis. Of great interest is the finding that PAI-1 produced by tumor cells, even at high concentration, did not overcome the absence of PAI-1 in the host, emphasizing the importance of the cellular source of PAI-1.