Biomedical subjects

T H Bugge

Publications and source records attributed to T H Bugge.

At least 19 recordsLinked to original sources

PubMed2006

Matriptase inhibition by hepatocyte growth factor activator inhibitor-1 is essential for placental development.

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a Kunitz-type transmembrane serine protease inhibitor that forms inhibitor complexes with several trypsin-like serine proteases and is required for mouse placental development and embryo survival. Here we show that the essential function of HAI-1 in placentation and all other embryonic processes is to restrict the activity of the type II transmembrane serine protease, matriptase. Enzymatic gene trapping of matriptase combined with HAI-1 immunohistochemistry revealed that matriptase is co-expressed with HAI-1 in both extraembryonic and embryonic tissues. As early as embryonic day 8.5, matriptase and HAI-1 were expressed in a population of chorionic trophoblasts. Ablation of HAI-1 disrupted the epithelial integrity of this cell population, causing disorganized laminin deposition and altered expression of E-cadherin and beta-catenin. This led to a complete loss of undifferentiated chorionic trophoblasts after embryonic day 9.5 and prevented the formation of the placental labyrinth. Genetic ablation of matriptase activity in HAI-1-deficient embryos, however, restored the integrity of chorionic trophoblasts and enabled placental labyrinth formation and development to term. Furthermore, matriptase/HAI-1 double-deficient mice were phenotypically indistinguishable from matriptase single-deficient littermates.

Animals↗

PubMed2001

Targeting of tumor cells by cell surface urokinase plasminogen activator-dependent anthrax toxin.

Urokinase plasminogen activator receptor (uPAR) binds pro-urokinase plasminogen activator (pro-uPA) and thereby localizes it near plasminogen, causing the generation of active uPA and plasmin on the cell surface. uPAR and uPA are overexpressed in a variety of human tumors and tumor cell lines, and expression of uPAR and uPA is highly correlated to tumor invasion and metastasis. To exploit these characteristics in the design of tumor cell-selective cytotoxins, we constructed mutated anthrax toxin-protective antigen (PrAg) proteins in which the furin cleavage site is replaced by sequences cleaved specifically by uPA. These uPA-targeted PrAg proteins were activated selectively on the surface of uPAR-expressing tumor cells in the presence of pro-uPA and plasminogen. The activated PrAg proteins caused internalization of a recombinant cytotoxin, FP59, consisting of anthrax toxin lethal factor residues 1-254 fused to the ADP-ribosylation domain of Pseudomonas exotoxin A, thereby killing the uPAR-expressing tumor cells. The activation and cytotoxicity of these uPA-targeted PrAg proteins were strictly dependent on the integrity of the tumor cell surface-associated plasminogen activation system. We also constructed a mutated PrAg protein that selectively killed tissue plasminogen activator-expressing cells. These mutated PrAg proteins may be useful as new therapeutic agents for cancer treatment.

Animals↗

PubMed2001

Plasminogen activators direct reorganization of the liver lobule after acute injury.

Tissue repair requires an adequate cellular proliferation coordinated with the timely proteolysis of matrix elements. Based on the properties of plasminogen activators in liver cell proliferation and tissue proteolysis, we explored the regulatory role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) in liver repair. Using carbon tetrachloride (CCl(4)) intoxication as a model of acute liver injury, we found that tPA-deficient mice displayed a mild defect in hepatic repair, whereas livers of uPA-deficient mice had a more substantial delay in repair, with injury of centrilobular hepatocytes persisting up to 14 days after CCl(4). Notably, functional cooperativity between plasminogen activators was strongly inferred from the profound reparative defect in livers of mice lacking tPA and uPA simultaneously, with persistence of centrilobular injury as far out as 35 days. The defective repair was not because of increased susceptibility of experimental mice to the toxin or to inadequate cellular proliferation. Instead, lack of plasminogen activators led to the accumulation of fibrin and fibronectin within injured areas and poor removal of necrotic cells. These data demonstrate that tPA and uPA play a critical role in hepatic repair via proteolysis of matrix elements and clearance of cellular debris from the field of injury.

Acute Disease↗

PubMed2001

The urokinase plasminogen activator receptor-associated protein/endo180 is coexpressed with its interaction partners urokinase plasminogen activator receptor and matrix metalloprotease-13 during osteogenesis.

The urokinase plasminogen activator receptor-associated protein/Endo180 (uPARAP/Endo180) is a newly discovered member of the macrophage mannose receptor family that was reported to interact with ligand-bound urokinase plasminogen activator receptor (uPAR), matrix metalloprotease-13 (MMP-13), and collagen V on the cell surface. We have determined the sites of expression of this novel receptor during murine postimplantation development. uPARAP/Endo180 was expressed in all tissues undergoing primary ossification, including the developing bones of the viscerocranium and calvarium that ossify intramembranously, and developing long bones undergoing endochondral ossification. uPARAP/Endo180 mRNA was expressed by both immature osteoblasts and by mature osteocalcin-producing osteoblasts-osteocytes, and was coexpressed with MMP-13. Interestingly, osteoblasts also expressed uPAR. Besides bone-forming tissues, uPARAP/Endo180 expression was detected only in a mesenchymal condensation of the midbrain and in the developing lungs. The data suggest a function of this novel protease receptor in bone development, possibly mediated through its interactions with uPAR, MMP-13, or collagen V.

Animals↗

PubMed2000

Fibrinogen is an important determinant of the metastatic potential of circulating tumor cells.

Detailed studies of tumor cell-associated procoagulants and fibrinolytic factors have implied that local thrombin generation and fibrin deposition and dissolution may be important in tumor growth and dissemination. To directly determine whether fibrin(ogen) or plasmin(ogen) are determinants of the metastatic potential of circulating tumor cells, this study examined the impact of genetic deficits in each of these key hemostatic factors on the hematogenous pulmonary metastasis of 2 established murine tumors, Lewis lung carcinoma and the B16-BL6 melanoma. In both tumor models, fibrinogen deficiency strongly diminished, but did not prevent, the development of lung metastasis. The quantitative reduction in metastasis in fibrinogen-deficient mice was not due to any appreciable difference in tumor stroma formation or tumor growth. Rather, tumor cell fate studies indicated an important role for fibrin(ogen) in sustained adhesion and survival of tumor cells within the lung. The specific thrombin inhibitor, hirudin, further diminished the metastatic potential of circulating tumor cells in fibrinogen-deficient mice, although the inhibitor had no apparent effect on tumor cell proliferation in vitro. The absence of plasminogen and plasmin-mediated fibrinolysis had no significant impact on hematogenous metastasis. The authors concluded that fibrin(ogen) is a critical determinant of the metastatic potential of circulating tumor cells. Furthermore, thrombin appears to facilitate tumor dissemination through at least one fibrin(ogen)-independent mechanism. These findings suggest that therapeutic strategies focusing on multiple distinct hemostatic factors might be beneficial in the containment of tumor metastasis.

Animals↗

PubMed2000

Plasminogen is a critical determinant of vascular remodeling in mice.

Extracellular proteolysis is likely to be a feature of vascular remodeling associated with atherosclerotic and restenotic arteries. To investigate the role of plasminogen-mediated proteolysis in remodeling, polyethylene cuffs were placed around the femoral arteries of mice with single and combined deficiencies in plasminogen and fibrinogen. Neointimal development occurred in all mice and was unaffected by genotype. Significant compensatory medial remodeling occurred in the cuffed arteries of control mice but not in plasminogen-deficient mice. Furthermore, focal areas of medial atrophy were frequently observed in plasminogen-deficient mice but not in control animals. A simultaneous deficit of fibrinogen restored the potential of the arteries of plasminogen-deficient mice to enlarge in association with neointimal development but did not eliminate the focal medial atrophy. An intense inflammatory infiltrate occurred in the adventitia of cuffed arteries, which was associated with enhanced matrix deposition. Adventitial collagen deposition was apparent after 28 days in control and fibrinogen-deficient arteries but not in plasminogen-deficient arteries, which contained persistent fibrin. These studies demonstrate that plasmin(ogen) contributes to favorable arterial remodeling and adventitial collagen deposition via a mechanism that is related to fibrinogen, presumably fibrinolysis. In addition, these studies reveal a fibrin-independent role of plasminogen in preventing medial atrophy in challenged vessels.

Animals↗

PubMed2000

The tissue plasminogen activator (tPA)/plasmin extracellular proteolytic system regulates seizure-induced hippocampal mossy fiber outgrowth through a proteoglycan substrate.

Short seizure episodes are associated with remodeling of neuronal connections. One region where such reorganization occurs is the hippocampus, and in particular, the mossy fiber pathway. Using genetic and pharmacological approaches, we show here a critical role in vivo for tissue plasminogen activator (tPA), an extracellular protease that converts plasminogen to plasmin, to induce mossy fiber sprouting. We identify DSD-1-PG/phosphacan, an extracellular matrix component associated with neurite reorganization, as a physiological target of plasmin. Mice lacking tPA displayed decreased mossy fiber outgrowth and an aberrant band at the border of the supragranular region of the dentate gyrus that coincides with the deposition of unprocessed DSD-1-PG/phosphacan and excessive Timm-positive, mossy fiber termini. Plasminogen-deficient mice also exhibit the laminar band and DSD- 1-PG/phosphacan deposition, but mossy fiber outgrowth through the supragranular region is normal. These results demonstrate that tPA functions acutely, both through and independently of plasmin, to mediate mossy fiber reorganization.

Amygdala↗

PubMed2000

Plasminogen-independent initiation of the pro-urokinase activation cascade in vivo. Activation of pro-urokinase by glandular kallikrein (mGK-6) in plasminogen-deficient mice.

The plasminogen activation (PA) system is involved in the degradation of fibrin and various extracellular matrix proteins, taking part in a number of physiological and pathological tissue remodeling processes including cancer invasion. This system is organized as a classical proteolytic cascade, and as for other cascade systems, understanding the physiological initiation mechanism is of central importance. The attempts to identify initiation routes for activation of the proform of the key enzyme urokinase-type plasminogen activator (pro-uPA) in vivo have been hampered by the strong activator potency of the plasmin, that is generated during the progress of the cascade. Using gene-targeted mice deficient in plasminogen (Plg -/- mice) [Bugge, T. H., Flick, M. J., Daugherty, C. C., and Degen, J. L. (1995) Genes Dev. 9, 794-807], we have now demonstrated and identified a component capable of initiating the cascade by activating pro-uPA. The urine from Plg -/- mice contained active two-chain uPA as well as a proteinase capable of activating exogenously added pro-uPA. The active component was purified and identified by mass spectrometry-based peptide mapping as mouse glandular kallikrein mGK-6 (true tissue kallikrein). The pro-uPA converting activity of the mGK-6 enzyme, as well as its ability to cleave a synthetic substrate for glandular kallikrein, was inhibited by the serine proteinase inhibitor leupeptin but not by other serine proteinase inhibitors such as aprotinin, antithrombin III, or alpha(1)-antitrypsin. We suggest that mouse glandular kallikrein mGK-6 is an activator of pro-uPA in the mouse urinary tract in vivo. Since this kallikrein is expressed in a number of tissues and also occurs in plasma, it can also be considered a candidate for a physiological pro-uPA activator in other locations.

Amino Acid Sequence↗

PubMed2000

Lactational competence and involution of the mouse mammary gland require plasminogen.

Urokinase-type plasminogen activator expression is induced in the mouse mammary gland during development and post-lactational involution. We now show that primiparous plasminogen-deficient (Plg(-/-)) mice have seriously compromised mammary gland development and involution. All mammary glands were underdeveloped and one-quarter of the mice failed to lactate. Although the glands from lactating Plg(-/-) mice were initially smaller, they failed to involute after weaning, and in most cases they failed to support a second litter. Alveolar regression was markedly reduced and a fibrotic stroma accumulated in Plg(-/-) mice. Nevertheless, urokinase and matrix metalloproteinases (MMPs) were upregulated normally in involuting glands of Plg(-/-) mice, and fibrin did not accumulate in the glands. Heterozygous Plg(+/-) mice exhibited haploinsufficiency, with a definite, but less severe mammary phenotype. These data demonstrate a critical, dose-dependent requirement for Plg in lactational differentiation and mammary gland remodeling during involution.

Animals↗

PubMed2000

Persistent corneal haze after excimer laser photokeratectomy in plasminogen-deficient mice.

PURPOSE: Excimer laser photorefractive keratectomy creates a nonvascular wound of the cornea. Fibrin deposition and resolution after excimer laser photokeratectomy were investigated in relation to corneal repair and restoration of clarity in mice with a genetic deficiency of plasminogen. METHODS: A Summit Apex Laser (Summit, Waltham, MA) was used to perform 2-mm, 175-pulse, transepithelial photoablations that resulted in deep stromal keratectomies. Photokeratectomy was performed on the corneas of plasminogen-deficient (Plg-/-) mice and littermate control animals. Eyes were examined for re-epithelialization and clarity throughout the 21-day observational period. Histologic sections were taken during the observational period and fibrin(ogen) was detected immunohistochemically. RESULTS: Re-epithelialization was rapid and complete within 3 days in both control and Plg-/- animals. Exuberant corneal fibrin(ogen) deposition was noted in Plg-/- mice and sparse fibrin(ogen) deposition in control mice on days 1 and 3 after injury. Fibrin(ogen) deposits resolved in control mice but persisted in Plg-/- mice (74% of eyes at 21 days; P < 0.004). Corneal opacification, scarring, and the presence of anterior chamber fibrin(ogen) occurred in plasminogen-deficient mice but not in control mice. CONCLUSIONS: Fibrin(ogen) deposition occurs during corneal wound repair after photokeratectomy. Impaired fibrinolysis in Plg-/- mice caused persistent stromal fibrin deposits that correlated with the development of corneal opacity.

Animals↗

PubMed1999

Plasminogen deficiency leads to impaired remodeling after a toxic injury to the liver.

Cellular proliferation and tissue remodeling are central to the regenerative response after a toxic injury to the liver. To explore the role of plasminogen in hepatic tissue remodeling and regeneration, we used carbon tetrachloride to induce an acute liver injury in plasminogen-deficient (Plg(o)) mice and nontransgenic littermates (Plg(+)). On day 2 after CCl(4), livers of Plg(+) and Plg(o) mice had a similar diseased pale/lacy appearance, followed by restoration of normal appearance in Plg(+) livers by day 7. In contrast, Plg(o) livers remained diseased for as long as 2.5 months, with a diffuse pale/lacy appearance and persistent damage to centrilobular hepatocytes. The persistent centrilobular lesions were not a consequence of impaired proliferative response in Plg(o) mice. Notably, fibrin deposition was a prominent feature in diseased centrilobular areas in Plg(o) livers for at least 30 days after injury. Nonetheless, the genetically superimposed loss of the Aalpha fibrinogen chain (Plg(o)/Fib(o) mice) did not correct the abnormal phenotype. These data show that plasminogen deficiency impedes the clearance of necrotic tissue from a diseased hepatic microenvironment and the subsequent reconstitution of normal liver architecture in a fashion that is unrelated to circulating fibrinogen.

Afibrinogenemia↗

PubMed1999

Neuronal death and blood-brain barrier breakdown after excitotoxic injury are independent processes.

Neuronal damage in the CNS after excitotoxic injury is correlated with blood-brain barrier (BBB) breakdown. We have used a glutamate analog injection model and genetically altered mice to investigate the relationship between these two processes in the hippocampus. Our results show that BBB dysfunction occurs too late to initiate neurodegeneration. In addition, plasma infused directly into the hippocampus is not toxic and does not affect excitotoxin-induced neuronal death. To test plasma protein recruitment in neuronal degeneration, we used plasminogen-deficient (plg(-/-)) mice, which are resistant to excitotoxin-induced degeneration. Plasminogen is produced in the hippocampus and is also present at high levels in plasma, allowing us to determine the contribution of each source to cell death. Intrahippocampal delivery of plasminogen to plg(-/-) mice restored degeneration to wild-type levels, but intravenous delivery of plasminogen did not. Finally, although the neurons in plg(-/-) mice do not die after excitotoxin injection, BBB breakdown occurs to a similar extent as in wild-type mice, indicating that neuronal death is not necessary for BBB breakdown. These results indicate that excitotoxin-induced neuronal death and BBB breakdown are separable events in the hippocampus.

Animals↗

PubMed1999

Functional overlap between two classes of matrix-degrading proteases in wound healing.

Retarded wound healing was found in mice deficient in the serine protease precursor plasminogen, as well as in wild-type mice treated with the metalloprotease inhibitor galardin, but in both cases wound closure was ultimately completed in all mice within 60 days. The expression of several matrix metalloproteases in keratinocytes migrating to cover the wound was strongly enhanced by galardin treatment. However, when plasminogen-deficient mice were treated with galardin, healing was completely arrested and wound closure was not seen during an observation period of 100 days, demonstrating that protease activity is essential for skin wound healing. The requirement for both plasminogen deficiency and metalloprotease inhibition for complete inhibition of the healing process indicates that there is a functional overlap between the two classes of matrix-degrading proteases, probably in the dissection of the fibrin-rich provisional matrix by migrating keratinocytes. Each class alone is capable of maintaining sufficient keratinocyte migration to regenerate the epidermal surface, although this function would normally be performed by both classes acting in parallel. Since there are strong similarities between the proteolytic mechanisms in wound healing and cancer invasion, these results predict that complete arrest of this latter process in therapeutic settings will require the use of inhibitors of both classes of proteases.

Animals↗

PubMed1999

The plasminogen activation system enhances brain and heart invasion in murine relapsing fever borreliosis.

The role of the plasminogen activation system (PAS) was investigated during the course of infection of a relapsing fever Borrelia species in plasminogen-deficient (plg -/-) and control (plg +/+ and plg +/-) mice. Subcutaneous inoculation of 10(4) spirochetes resulted in a peak spirochetemia five days after infection with 20-23 x 10(6) organisms per milliliter of whole blood in all mice, indicating that the PAS had no effect on the development of this phase of the infection. Anemia, thrombocytopenia, hepatitis, carditis, and splenomegaly were noted in all mice during and immediately after peak spirochetemia. Fibrin deposition in organs was noted in plg -/- mice but not in controls during these stages. Significantly greater spirochetal DNA burdens were consistently observed in the hearts and brains of control mice 28-30 days after infection, as determined by PCR amplification of this organism's flagellin gene (flaB), followed by quantitative densitometry. Furthermore, the decreased spirochetal load in brains of plg -/- mice was associated with a significant decrease in the degree of inflammation of the leptomeninges in these mice. These findings indicate a role for the PAS in heart and brain invasion by relapsing fever Borrelia, resulting in organ injury.

Animals↗

PubMed1998

Prothrombin deficiency results in embryonic and neonatal lethality in mice.

The conversion of prothrombin (FII) to the serine protease, thrombin (FIIa), is a key step in the coagulation cascade because FIIa triggers platelet activation, converts fibrinogen to fibrin, and activates regulatory pathways that both promote and ultimately suppress coagulation. However, several observations suggest that FII may serve a broader physiological role than simply stemming blood loss, including the identification of multiple G protein-coupled, thrombin-activated receptors, and the well-documented mitogenic activity of FIIa in in vitro test systems. To explore in greater detail the physiological roles of FII in vivo, FII-deficient (FII-/-) mice were generated. Inactivation of the FII gene leads to partial embryonic lethality with more than one-half of the FII-/- embryos dying between embryonic days 9.5 and 11.5. Bleeding into the yolk sac cavity and varying degrees of tissue necrosis were observed in many FII-/- embryos within this gestational time frame. However, at least one-quarter of the FII-/- mice survived to term, but ultimately they, too, developed fatal hemorrhagic events and died within a few days of birth. This study directly demonstrates that FII is important in maintaining vascular integrity during development as well as postnatal life.

Alleles↗

PubMed1998

Reduced metastasis of Polyoma virus middle T antigen-induced mammary cancer in plasminogen-deficient mice.

To investigate the role of plasmin(ogen) in mammary tumor development and progression, plasminogen-deficient mice were crossed with transgenic mice expressing Polyoma middle T antigen under the control of the mouse mammary tumor virus long terminal repeat. Virgin females carrying the Polyoma middle T antigen uniformly developed multiple, bilateral mammary tumors, regardless of the presence or absence of circulating plasminogen. Both the age at which these tumors became palpable and subsequent tumor growth were indistinguishable between plasminogen-deficient mice and plasminogen-expressing littermates. However, plasminogen was found to greatly modify the metastatic potential in this model system; lung metastasis in plasminogen-deficient mice was significantly reduced as compared to littermate controls with respect to frequency of occurrence, total number of metastases, and total metastatic tumor burden. Plasminogen activators, as well as other key factors that govern the conversion of plasminogen to plasmin, were expressed within the mammary tumors, suggesting that the plasminogen/plasmin system may promote metastasis by contributing to tumor-associated extracellular proteolysis. The data provide direct evidence that plasmin(ogen) is a tumor progression factor in PymT-induced mammary cancer, and support the hypothesis that hemostatic factors play an important role in tumor biology.

Animals↗

PubMed1998

Ligneous conjunctivitis in plasminogen-deficient mice.

Ligneous conjunctivitis is a rare form of chronic pseudomembranous conjunctivitis that is associated with systemic membranous pathological changes. A probable link between plasminogen and ligneous conjunctivitis has been indicated by the recent diagnoses of plasminogen deficiency in five patients suffering from ligneous conjunctivitis. The current study reports that plasminogen-deficient mice develop conjunctival lesions indistinguishable from human ligneous conjunctivitis in both appearance and histology. Both human and mouse lesions contain acellular material rich in fibrin, and aberrant or disrupted epithelium. The incidence of lesion development in mice increases with age and is strongly influenced by genetic background. Interestingly, ligneous conjunctivitis was not observed in plasminogen-deficient mice simultaneously lacking fibrinogen. This study provides direct evidence that plasminogen deficiency is one cause of ligneous conjunctivitis and suggests that plasminogen-deficient mice may be an excellent model for the development of therapeutic strategies for the treatment of this debilitating disease.

Aging↗

PubMed1998

Healing of corneal epithelial defects in plasminogen- and fibrinogen-deficient mice.

PURPOSE: The local deposition of fibrinogen and other plasma products from tears within corneal wounds and the expression of plasminogen activator by corneal epithelial cells suggest that the coagulation and fibrinolytic systems play an important role in corneal wound healing. The authors used mouse lines deficient in plasminogen (Plg), fibrinogen (Fib), or both to elucidate the roles of these key fibrinolytic and coagulation factors in the healing of corneal epithelial defects. METHODS: Mice were anesthetized, and corneal epithelial defects (3 mm) were created with a blade. The authors conducted histologic examination and immunohistochemical analysis on the healing of injured corneas. RESULTS: The corneal epithelial defects of wild-type mice with transparent corneas healed quickly in 7 days, whereas the healing of plasminogen-deficient mice was impaired and complicated by severe and persistent inflammatory responses, the formation of retrocorneal fibrin deposits, corneal cloudiness caused by scar-tissue formation, and often stromal neovascularization. To determine whether these defects in corneal wound repair were specifically related to an impediment in fibrinolysis, corneal wound healing was compared in mice with a combined deficiency in plasminogen and fibrinogen. The loss of fibrinogen in mice lacking plasminogen resulted in the restoration of normal healing with transparent corneas in 7 days, similar to that of wild-type mice. CONCLUSIONS: These results provide direct evidence that hemostatic factors play a crucial role in corneal wound repair despite the lack of local hemorrhage. Furthermore, they demonstrate that the essential role of plasmin in corneal would healing is fibrinolysis. It prevents the adverse inflammatory responses caused by prolonged fibrin and fibrinogen deposition in injured corneas.

Afibrinogenemia↗