Biomedical subjects

J L Degen

Publications and source records attributed to J L Degen.

At least 19 recordsLinked to original sources

PubMed2004

Role of fibrinogen- and platelet-mediated hemostasis in mouse embryogenesis and reproduction.

Studies of mice with genetic deficits in specific coagulation factors have shown that many, but not all, components of the hemostatic system serve an essential role in mouse embryogenesis and pregnancy. Although the developmental failures observed in these mice are typically associated with severe hemorrhage, it is uncertain whether the role of coagulation factors in embryogenesis and reproduction is specifically tied to their function in thrombus formation and prevention of blood loss (i.e. hemostasis). Here, we show (i) that a complete loss of fibrinogen- and platelet-dependent hemostatic capacity does not reproduce the developmental defects occurring in mice with either deficits in thrombin generation or unfettered thrombin consumption; (ii) that the essential role of fibrinogen in the maintenance of pregnancy does not involve interaction with platelets; and (iii) that the previously described in utero growth retardation of gene-targeted mice lacking NF-E2, a transcription factor critical for megakaryopoieis, is not caused by a loss of platelet-dependent hemostatic function. In addition, we demonstrate (iv) that fibrinogen can confer physiologically relevant hemostatic function in the absence of platelets, but that a complete loss of hemostatic capacity results if a combined absence of these components is genetically imposed. These findings support the notion that the function of coagulation factors for in utero development and pregnancy is mechanistically distinct from their ability to mediate the formation of hemostatic platelet-fibrin(ogen) aggregates.

Adenosine Diphosphate↗

PubMed2001

Wound-healing defects in mice lacking fibrinogen.

In addition to its key role in the control of blood loss following injury, fibrin(ogen) has been proposed to play an important role in tissue repair by providing an initial matrix that can stabilize wound fields and support local cell proliferation and migration. To test directly these concepts, the effect of fibrinogen deficiency on cutaneous tissue repair in mice was investigated using incisional and excisional wounds. The time required to overtly heal wounds was similar in fibrinogen-deficient and control mice, but histologic evaluation revealed distinct differences in the repair process, including an altered pattern of epithelial cell migration and increased epithelial hyperplasia. Furthermore, granulation tissue in fibrinogen-deficient mice failed to adequately close the wound gap, resulting in persistent open wounds or partially covered sinus tracts. The tensile strength of these wounds was also reduced compared with control mice. The most profound defect in wound tissue organization was observed in fibrinogen-deficient mice following the subcutaneous implantation of a porous tubing chamber. Cells migrated into the wall of the implants at a similar rate as control mice, but cells from fibrinogen-deficient animals were unable to efficiently organize and migrate into wound fluid-filled dead space within the center of the implants. These studies show that re-epithelialization, granulation tissue formation, including the establishment of neovasculature, and the formation of fibrotic scar tissue can proceed in the absence of fibrin(ogen) and all of its proteolytic derivatives. However, fibrin (ogen) is important for appropriate cellular migration and organization within wound fields and in initially establishing wound strength and stability. (Blood. 2001;97:3691-3698)

Animals↗

PubMed2001

Plasminogen deficiency leads to impaired lobular reorganization and matrix accumulation after chronic liver injury.

To determine the regulatory role of plasminogen in hepatic repair following a chronic liver injury, we injected carbon tetrachloride (CCl(4)) biweekly into mice lacking plasminogen (Plg(0)) and plasminogen-sufficient littermates (Plg(+)). On gross examination, we found that Plg(0) livers became enlarged and pale with foci of red nodules as early as 4 weeks after CCl(4) injection, while Plg(+) livers appeared minimally affected by 6 weeks. Microscopically, Plg(0) livers had a pronounced pericentral linking, with accumulation of centrilobular eosinophilic material in injured areas, which resulted in a significant increase in liver mass and total protein. Immunohistochemistry revealed that fibrin accumulated progressively in injured regions. However, the combined genetic loss of plasminogen and fibrinogen did not correct the abnormal phenotype. Mason's trichrome staining revealed intense signal in centrilobular regions and electron microscopy showed a marked increase in fibrillary material demonstrating an excessive accumulation of extracellular matrix in Plg(0) mice. The zone-specific increase in matrix components was associated with an increase in the number of activated hepatic stellate cells within injured sites of Plg(0) livers. Taken together, these data suggest that the progressive accumulation of fibrin-unrelated matrix substrates in Plg(0) livers after a chronic injury results from the combined effects of impaired proteolysis and increased matrix production.

Afibrinogenemia↗

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

Plasminogen deficiency results in poor clearance of non-fibrin matrix and persistent activation of hepatic stellate cells after an acute injury.

BACKGROUND/AIMS: Plasminogen directs matrix proteolysis during liver repair. Based on the role of hepatic stellate cells (HSCs) on matrix production, we investigated whether plasminogen-driven matrix proteolysis modulates the phenotype of HSCs. METHODS: Carbon tetrachloride was injected intraperitoneally into mice deficient in plasminogen, fibrinogen, or both, and to normal littermates, followed by determination of the phenotype of HSCs, matrix deposition, and apoptosis. RESULTS: Activation of HSCs was restricted to the zone of injury and increased >ten-fold above baseline regardless of the plasminogen status 2 days after toxin. Thereafter, the number of activated HSCs decreased to baseline levels between 7 and 14 days in normal mice, but remained elevated in plasminogen-deficient livers approximately ten-fold above non-targeted littermates. Despite the zonal increase in activated HSCs, the total number of desmin-stained HSCs was similar along the lobule in both genotypes. No appreciable difference in apoptosis of perisinusoidal cells was found between genotypes; however, fibrillary material was present in the subsinusoidal space of Plg(0) livers. This fibrillary material was not fibrin, as demonstrated by similar findings in Plg(0)/Fib(0) mice, which accumulated fibronectin in injured areas. CONCLUSIONS: Proteolytic clearance of non-fibrin matrix components by plasminogen must occur for HSCs to restore the quiescent phenotype during liver repair.

Animals↗

PubMed2001

Genetic manipulation of fibrinogen and fibrinolysis in mice.

Vascular integrity is maintained by a sophisticated system of circulating and cell associated hemostatic factors that control local platelet deposition, the conversion of soluble fibrinogen to an insoluble fibrin polymer, and the dissolution of fibrin matrices. However, hemostatic factors are likely to be biologically more important than merely maintaining vascular patency and controlling blood loss. Specific hemostatic factors have been associated with a wide spectrum of physiological processes, including development, reproduction, tissue remodeling, wound repair, angiogenesis, and the inflammatory response. Similarly, it has been proposed that hemostatic factors are important determinants of a variety of pathological processes, including vessel wall disease, tumor dissemination, infectious disease, and inflammatory diseases of the joint, lung, and kidney. The development of gene targeted mice either lacking or expressing modified forms of selected hemostatic factors has provided a valuable opportunity to test prevailing hypotheses regarding the biological roles of key coagulation and fibrinolytic system components in vivo. Genetic analyses of fibrin(ogen) and its interacting factors in transgenic mice have proven to be particularly illuminating, often challenging long standing concepts. This review summarizes the key findings made in recent studies of gene targeted mice with single and combined deficits in fibrinogen and fibrinolytic factors. Studies illustrating the role and interplay of these factors in disease progression are highlighted.

Animals↗

PubMed2001

Crescentic glomerulonephritis is diminished in fibrinogen-deficient mice.

Crescentic forms of glomerulonephritis are characterized by the accumulation of fibrin and cells in Bowman's space and are associated with a rapid loss of renal function. Accumulation of fibrin in the glomerular tufts is thought to promote macrophage infiltration and glomerular injury. To directly explore the role of fibrin(ogen) in the development of crescentic glomerulonephritis, antiglomerular basement membrane nephritis was induced in fibrinogen-deficient and control mice. Glomeruli from control mice developed severe disease including fibrin deposits, inflammatory cell accumulation, and crescent formation (46.3 +/- 7.3% of glomeruli). Fibrinogen-deficient mice developed significantly milder disease with fewer glomerular crescents (24.0 +/- 4.7% of glomeruli; P < 0.03). Glomerular macrophage accumulation was diminished in fibrinogen-deficient mice (0.9 +/- 0.4 macrophages/glomerular cross section) relative to control mice (3.9 +/- 1.4 macrophages/glomerular cross section; P < 0.03). Finally, renal function as assessed by serum creatinine was better maintained in fibrinogen-deficient mice. These results indicate that although fibrin(ogen) is not essential for the development of glomerular crescents, it contributes significantly to the pathogenesis of crescentic glomerulonephritis by promoting glomerular macrophage accumulation and impairing filtration.

Animals↗

PubMed2001

Genetic interactions between the coagulation and fibrinolytic systems.

Nearly all of the genes encoding the established coagulation and fibrinolytic factors have been successfully altered or disrupted in transgenic mice. Although comprehensive studies of each of these gene-targeted mouse lines are still ongoing, the initial findings have significantly refined our understanding of the roles of selected hemostatic factors in vivo, and occasionally altered long-standing concepts. This review summarizes some of the progress that has been made in the generation and phenotypic characterization of mice lacking key hemostatic factors, including coagulation, fibrinolytic, platelet and endothelial cell-associated factors. New insights regarding the role(s) and interplay of hemostatic factors that have emerged from detailed studies of mice carrying multiple deficits in coagulation and fibrinolytic system components are highlighted.

Afibrinogenemia↗

PubMed2001

Fibrinogen and tumor cell metastasis.

Detailed studies tumor cell-associated procoagulants and fibrinolytic factors have strongly suggested that local thrombin and plasmin generation may be important in tumor progression. Given that one target for both these serine proteases is fibrinogen, a logical extension of this hypothesis is that local fibrin deposition and dissolution may be key determinants of tumor growth and/or dissemination. To directly test this concept, we initiated studies of tumor growth, experimental metastasis, and spontaneous metastasis in C57Bl/6-inbred mice with and without fibrinogen. Using two established C57Bl/6-derived tumor cell lines, Lewis lung carcinoma and B16-BL6 melanoma, fibrinogen deficiency was found to strongly diminish, but not prevent, the development of lung metastases in both experimental and spontaneous metastasis assays. This difference was not a consequence of any obvious difference in tumor stroma formation or the growth of primary or secondary tumors. Rather, tumor cell fate studies argued that there is an important role of fibrin(ogen) in the sustained adhesion and/or survival of tumor cell emboli within the lung. The specific thrombin inhibitor, hirudin, was also shown to strongly diminish metastatic potential, consistent with earlier reports. More importantly, hirudin was found to further diminish the already low metastatic potential of tumor cells in fibrinogen-deficient mice. We conclude that fibrin(ogen) is a critical determinant of metastatic potential, but thrombin appears to contribute to tumor cell dissemination through at least one fibrinogen-independent mechanism. Further, these findings suggest that therapeutic strategies directed at several hemostatic factors might be useful in the suppression of metastatic disease.

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

Tissue plasminogen activator-mediated fibrinolysis protects against axonal degeneration and demyelination after sciatic nerve injury.

Tissue plasminogen activator (tPA) is a serine protease that converts plasminogen to plasmin and can trigger the degradation of extracellular matrix proteins. In the nervous system, under noninflammatory conditions, tPA contributes to excitotoxic neuronal death, probably through degradation of laminin. To evaluate the contribution of extracellular proteolysis in inflammatory neuronal degeneration, we performed sciatic nerve injury in mice. Proteolytic activity was increased in the nerve after injury, and this activity was primarily because of Schwann cell-produced tPA. To identify whether tPA release after nerve damage played a beneficial or deleterious role, we crushed the sciatic nerve of mice deficient for tPA. Axonal demyelination was exacerbated in the absence of tPA or plasminogen, indicating that tPA has a protective role in nerve injury, and that this protective effect is due to its proteolytic action on plasminogen. Axonal damage was correlated with increased fibrin(ogen) deposition, suggesting that this protein might play a role in neuronal injury. Consistent with this idea, the increased axonal degeneration phenotype in tPA- or plasminogen-deficient mice was ameliorated by genetic or pharmacological depletion of fibrinogen, identifying fibrin as the plasmin substrate in the nervous system under inflammatory axonal damage. This study shows that fibrin deposition exacerbates axonal injury, and that induction of an extracellular proteolytic cascade is a beneficial response of the tissue to remove fibrin. tPA/plasmin-mediated fibrinolysis may be a widespread protective mechanism in neuroinflammatory pathologies.

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

Role of the pleiotropic effects of plasminogen deficiency in infection experiments with plasminogen-deficient mice.

Plasminogen-deficient mice hold great promise as tools for analyzing the contribution of plasminogen activators produced by infectious agents to pathogenesis. However, the pathology caused by congenital plasminogen deficiency complicates the interpretation of infection experiments conducted with these animals. This pathology, the most prominent features of which are poor weight gain, wasting after about 60 days of age, and shortened lifespan, results from the inability of the mice to clear small fibrin thrombi. This article describes strategies for distinguishing the contribution of this pathology from the direct effects of depriving infectious agents of plasminogen. These strategies depend on the use of mouse genotypes in which the correlation of plasminogen deficiency with fibrin-dependent pathology is broken. Mice with plasminogen activator deficiencies are unable to generate plasmin and develop pathologies identical to those seen in plasminogen-deficient mice. However, unlike plasminogen-deficient mice, they do make plasminogen available to the infectious agent. Fibrinogen-deficient mice also deficient for plasminogen do not develop the pathology typical of plasminogen deficiency. These mice allow examination of plasminogen deficiency in the absence of fibrin-dependent pathology. Use of fibrinogen-deficient mice is complicated by the possibility that fibrin may be the key substrate of plasmin generated by the infectious agent.

Animals↗

PubMed2000

Hepatocyte transplantation into diseased mouse liver. Kinetics of parenchymal repopulation and identification of the proliferative capacity of tetraploid and octaploid hepatocytes.

To examine the process of liver repopulation by transplanted hepatocytes, we developed transgenic mice carrying a mouse major urinary protein-urokinase-type plasminogen activator fusion transgene. Expression of this transgene induced diffuse hepatocellular damage beginning at 3 weeks of age, and homozygous mice supported up to 97% parenchymal repopulation by healthy donor hepatocytes transplanted into the spleen. Using this transplantation model, we determined that 1) a mean of 21% of splenically injected hepatocytes engraft in liver parenchyma; 2) a mean of 6.6% of splenically injected hepatocytes (or one-third of engrafted cells) can give rise to proliferating hepatocyte foci; 3) transplanted cells in proliferating foci display an initial cell-doubling time of 28 hours, and focus growth continues through a mean of 12 cell doublings; 4) hepatocytes isolated from young and aged adult mice display similar focus repopulation kinetics; 5) the extent of repopulated parenchyma remains stable throughout the life of the recipient mouse; and 6) tetraploid and octaploid hepatocytes can support clonal proliferation.

Aging↗

PubMed2000

Hepatocyte transplantation in a model of toxin-induced liver disease: variable therapeutic effect during replacement of damaged parenchyma by donor cells.

To provide long-term therapy in patients with severe toxin-induced hepatic parenchymal damage, donor hepatocytes would need to replicate and replace a large portion of the damaged parenchyma. Using a mouse model developed to reproduce this type of hepatic injury, we found that hepatocyte transplantation only slightly improved survival after transplantation despite the fact that many non-survivors showed moderate liver repopulation by donor cells. Perhaps accounting for this outcome, donor parenchyma in non-survivors did not have typical lobular organization. These results indicate that the re-creation of functional parenchyma by transplanted hepatocytes requires time, during which donor cells proliferate and then establish normal parenchymal architecture.

Alkaline Phosphatase↗

PubMed2000

Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice.

Mice deleted for the plasminogen activator inhibitor-1 (PAI-1) gene are relatively protected from developing pulmonary fibrosis induced by bleomycin. We hypothesized that PAI-1 deficiency reduces fibrosis by promoting plasminogen activation and accelerating the clearance of fibrin matrices that accumulate within the damaged lung. In support of this hypothesis, we found that the lungs of PAI-1(-/-) mice accumulated less fibrin after injury than wild-type mice, due in part to enhanced fibrinolytic activity. To further substantiate the importance of fibrin removal as the mechanism by which PAI-1 deficiency limited bleomycin-induced fibrosis, bleomycin was administered to mice deficient in the gene for the Aalpha-chain of fibrinogen (fib). Contrary to our expectation, fib(-/-) mice developed pulmonary fibrosis to a degree similar to fib(+/-) littermate controls, which have a plasma fibrinogen level that is 70% of that of wild-type mice. Although elimination of fibrin from the lung was not in itself protective, the beneficial effect of PAI-1 deficiency was still associated with proteolytic activity of the plasminogen activation system. In particular, inhibition of plasmin activation and/or activity by tranexamic acid reversed both the accelerated fibrin clearance and the protective effect of PAI-1 deficiency. We conclude that protection from fibrosis by PAI-1 deficiency is dependent upon increased proteolytic activity of the plasminogen activation system; however, complete removal of fibrin is not sufficient to protect the lung.

Animals↗

PubMed2000

Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen.

We used intravital microscopy to observe the formation of platelet plugs in ferric chloride-injured arterioles of live mice. With this model, we evaluated thrombus growth in mice lacking von Willebrand factor (vWF) and fibrinogen (Fg), the two key ligands known to mediate platelet adhesion and aggregation. In vWF(-/-) mice, despite the presence of arterial shear, delayed platelet adhesion occurred and stable thrombi formed. In many mice, a persisting high-shear channel never occluded. Abundant thrombi formed in Fg(-/-) mice, but they detached from the subendothelium, which ultimately caused downstream occlusion in all cases. Surprisingly, mice deficient in both vWF and Fg successfully formed thrombi with properties characteristic of both mutations, leading to vessel occlusion in the majority of vessels. Platelets of these doubly deficient mice specifically accumulated fibronectin in their alpha-granules, suggesting that fibronectin could be the ligand supporting the platelet aggregation.

Afibrinogenemia↗