Role of fibrinogen in acute ischemic kidney injury.

Renal ischemia-reperfusion (I/R) is associated with activation of the coagulation system and accumulation of blood clotting factors in the kidney. The aim of the present study was to examine the functional impact of fibrinogen on renal inflammation, damage, and repair in the context of I/R injury. In this study, we found that I/R was associated with a significant increase in the renal deposition of circulating fibrinogen. In parallel, I/R stress induced the de novo expression of fibrinogen in tubular epithelial cells, as reflected by RT-PCR, immunofluorescence, and in situ hybridization. In vitro, fibrinogen expression was induced by oncostatin M and hyper-IL-6 in primary tubular epithelial cells, and fibrinogen-containing medium had an inhibitory effect on tubular epithelial cell adhesion and migration. Fibrinogen(+/-) mice showed similar survival as wild-type mice but better preservation in early postischemic renal function. In fibrinogen(-/-) mice, renal function and survival were significantly worse than in fibrinogen(+/-) mice. Renal transplant experiments revealed reduced expression of tubular damage markers and attenuated proinflammatory cytokine expression but increased inflammatory cell infiltrates and transforming growth factor-ß expression in fibrinogen(-/-) isografts. These data point to heterogeneous effects of fibrinogen in renal I/R injury. While a complete lack of fibrinogen may be detrimental, partial reduction of fibrinogen in heterozygous mice can improve renal function and overall outcome.

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.

Impaired wound healing in mice with a disrupted plasminogen gene.

Activation of plasminogen (Plg) has been proposed to play a role in proteolytic degradation of extracellular matrices in tissue remodeling events, including wound healing. However, there has been no definitive proof of involvement of Plg in such processes. We now report that healing of skin wounds is severely impaired in mice made deficient in Plg by targeted gene disruption. The results demonstrate that Plg is required for normal repair of skin wounds in mice and support the assumption that it also plays a central role in other disease processes involving extracellular matrix degradation, such as cancer invasion.

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.

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.

Replacement of diseased mouse liver by hepatic cell transplantation.

Adult liver has the unusual ability to fully regenerate after injury. Although regeneration is accomplished by the division of mature hepatocytes, the replicative potential of these cells is unknown. Here, the replicative capacity of adult liver cells and their medical usefulness as donor cells for transplantation were investigated by transfer of adult mouse liver cells into transgenic mice that display an endogenous defect in hepatic growth potential and function. The transplanted liver cell populations replaced up to 80 percent of the diseased recipient liver. These findings demonstrate the enormous growth potential of adult hepatocytes, indicating the feasibility of liver cell transplantation as a method to replace lost or diseased hepatic parenchyma.

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.

Biological effects of targeted inactivation of hepatocyte growth factor-like protein in mice.

Hepatocyte growth factor-like protein (HGFL) is a liver-derived serum glycoprotein involved in cell proliferation and differentiation, and is proposed to have a fundamental role in embryogenesis, fertility, hematopoiesis, macrophage activation, and tissue repair. To assess the in vivo effects of total loss of HGFL, we generated mice with targeted disruption of the gene resulting in loss of the protein. Disruption of the HGFL gene allowed for normal embryogenesis, and followed a Mendelian pattern of genetic transmission. Mice homozygous for the targeted allele (HGFL-/- mice) are fertile, and grow to adulthood without obvious phenotypic abnormalities in unchallenged animals, except for development of lipid-containing cytoplasmic vacuoles in hepatocytes throughout the liver lobules. These histologic changes are not accompanied by discernible changes in synthetic or excretory hepatic functions. Hematopoiesis appears unaltered, and although macrophage activation is delayed in the absence of HGFL, migration to the peritoneal cavity upon challenge with thioglycollate was similar in HGFL-/- and wild-type mice. Challenged with incision to skin, HGFL-/- mice display normal wound healing. These data demonstrate that HGFL is not essential for embryogenesis, fertility, or wound healing. HGFL-deficient mice will provide a valuable means to assess the role of HGFL in hepatic and systemic responses to inflammatory and infectious stimuli in vivo.

Fibrinogen deficiency is compatible with the development of atherosclerosis in mice.

A critical role of the coagulation system in the development of atherosclerosis has been frequently postulated based on a variety of indirect observations, including the expression of procoagulants and fibrinolytic factors within atherosclerotic vessels, the presence of substantial amounts of fibrin(ogen) and fibrin degradation products within intimal lesions, the cellular infiltration and assimilation of mural thrombi into developing plaques, and the identification of high plasma fibrinogen (Fib) levels as an independent risk factor for the development of ischemic heart disease. To directly examine the role of fibrin(ogen) in atherogenesis, Fib-deficient mice were crossed to atherosclerosis-prone apolipoprotein E (apo E)-deficient mice. Both apo E-/- and apo E-/-/Fib-/- mice developed lesions throughout the entire aortic tree, ranging in appearance from simple fatty streaks to complex fibrous plaques. Furthermore, remarkably little difference in lesion size and complexity was observed within the aortae of age- and gender-matched apo E-/- and apo E-/-/Fib-/- mice. These results indicate that the contribution of fibrin(ogen) to intimal mass and local cell adhesion, migration, and proliferation is not strictly required for the development of advanced atherosclerotic disease in mice with a severe defect in lipid metabolism.

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.

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.

The Ron/STK receptor tyrosine kinase is essential for peri-implantation development in the mouse.

The Ron/STK receptor tyrosine kinase is a member of the c-Met family of receptors and is activated by hepatocyte growth factor-like protein (HGFL). Ron activation results in a variety of cellular responses in vitro, such as activation of macrophages, proliferation, migration, and invasion, suggesting a broad biologic role in vivo. Nevertheless, HGFL-deficient mice grow to adulthood with few appreciable phenotypic abnormalities. We report here that in striking contrast to the loss of its only known ligand, complete loss of Ron leads to early embryonic death. Embryos that are devoid of Ron (Ron-/-) are viable through the blastocyst stage of development but fail to survive past the peri-implantation period. In situ hybridization analysis demonstrates that Ron is expressed in the trophectoderm at embryonic day (E) 3.5 and is maintained in extraembryonic tissue through E7.5, compatible with an essential function at this stage of development. Hemizygous mice (Ron+/-) grow to adulthood; however, these mice are highly susceptible to endotoxic shock and appear to be compromised in their ability to downregulate nitric oxide production. These results demonstrate a novel role for Ron in early mouse development and suggest that Ron plays a limiting role in the inflammatory response.

Fibrin and fibrinolysis in infection and host defense.

Bacterial pathogens have frequently evolved and maintained the capacity to engage and/or activate hemostatic system components of their vertebrate hosts. Recent studies of mice with selected alterations in host plasminogen and other hemostatic factors have begun to reveal a seminal role of bacterial plasminogen activators and fibrin clearance in microbial pathogenesis. Bacterial pathogens appear to exploit host plasmin-mediated proteolysis to both support microbial dissemination and evade innate immune surveillance systems. The contribution of bacterial plasminogen activation to the evasion of the inflammatory response is particularly conspicuous with the plague agent, Yersinia pestis. Infection of control mice with wild-type Y. pestis leads to the formation of widespread foci containing massive numbers of free bacteria with little inflammatory cell infiltrate, whereas the loss of either the bacterial plasminogen activator, Pla, or the elimination of host plasminogen results in the accumulation of robust inflammatory cell infiltrates at sites of infection and greatly improved survival. Interestingly, fibrin(ogen) deficiency undermines the local inflammatory response observed with Pla-deficient Y. pestis and effectively eliminates the survival benefits posed by the elimination of either host plasminogen or bacterial Pla. These studies, and complementary studies with other human pathogens, illustrate that plasminogen and fibrinogen are extremely effective modifiers of the inflammatory response in vivo and critical determinants of bacterial virulence and host defense. Detailed studies of the inflammatory response in mice with genetically-imposed modifications in coagulation and fibrinolytic factors underscore the regulatory crosstalk between the hemostatic and immune systems.

Differential modulation of plasminogen activator gene expression by oncogene-encoded protein tyrosine kinases.

Urokinase-type plasminogen activator (uPA) gene transcription is increased > or = 50-fold in chicken embryo fibroblasts (CEF) following transformation by the protein tyrosine kinase pp60v-src. Protein phosphorylation appears to play a critical role in uPA gene expression in these cells; protein kinase C-activating phorbol esters cooperate with pp60v-src to synergistically increase uPA mRNA, whereas cyclic AMP (cAMP)-dependent protein kinase-activating agents (e.g., 8-bromo cAMP) repress uPA mRNA levels. To explore the relationship between transforming oncogenes and uPA gene expression, uPA mRNA levels were measured in CEF infected with selected avian retroviruses. We report that v-ras and the transforming protein tyrosine kinases v-src, v-yes, and v-ros all increase cellular uPA mRNAs. However, transformation with the protein tyrosine kinase encoded by v-erbB, or the nuclear proteins encoded by v-jun, v-ski, or v-myc, did not increase uPA mRNA detectably. Ras and all of the protein tyrosine kinases analyzed, including the v-erbB product, but none of the nuclear oncoproteins sensitized cells to phorbol ester induction of uPA gene expression. Thus, increased uPA gene expression is not simply a secondary consequence of cell transformation but, rather, is regulated or comodulated by only a subset of oncogene products. Analysis of cells expressing site-directed mutants of pp60v-src showed that the induction of the uPA gene is dependent on protein tyrosine kinase catalytic activity, myristylation, and plasma membrane localization. However, these properties together are not sufficient; an additional feature in the src homology 2 domain is also required. The major sites of serine phosphorylation, serines 12 and 17, and the autophosphorylation site, tyrosine 416, are not essential for uPA gene induction. However, the reduction of uPA mRNA in pp60v-src-transformed cells by 8-bromo cAMP is dependent on tyrosine 416.

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.

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.

Purification, properties and regulation of the level of bovine S-adenosylmethionine decarboxylase during lymphocyte mitogenesis.

S-adenosylmethionine decarboxylase was purified from the livers of calves treated with methylglyoxal bis(guanylhydrazone) to elevate the level of the enzyme. Purified bovine S-adenosylmethionine decarboxylase was similar in specific activity and subunit molecular weight (32,000) to the enzymes previously isolated from rat and mouse. The bovine liver enzyme immunologically crossreacted with S-adenosylmethionine decarboxylase from resting and mitogenically activated bovine lymphocytes. The rate of enzyme synthesis in activated lymphocytes was determined by labeling the cells with [3H]leucine and isolating the radioactive decarboxylase by affinity chromatography and sodium dodecyl sulfate gel electrophoresis. The rate of enzyme synthesis was increased 10-fold by 9 h after mitogen treatment, which accounts for the initial increase in cellular enzymatic activity. There was no further increase in the rate of S-adenosylmethionine decarboxylase synthesis that correlated with a second elevation of activity occurring at approx. 24 h after mitogenic activation. It was concluded that the second increase in enzyme activity was due to lengthening the intracellular half-life of the enzyme by 2-fold.

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.

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.