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.

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.

Cyclosporine treatment reduces early atherosclerosis in the cholesterol-fed rabbit.

While T helper cell infiltration is an early event in the development of atherosclerosis in cholesterol-fed rabbits, their functional contribution to atherogenesis is not clear. To investigate their role, T cell activation was blocked with cyclosporine A (CsA) in New Zealand White (NZW) rabbits fed a 1% cholesterol diet. CsA was administered at a dose of 16 mg/kg body weight, intramuscularly every second day, resulting in circulating whole blood levels of 460 +/- 39 micrograms/l. After 4 weeks on the cholesterol diet, untreated rabbits developed atherosclerotic plaques covering 74.4% +/- 3.5% of their aortic arch, 19.8% +/- 7.8% of their thoracic aorta and 19.8% +/- 6.2% of their abdominal aorta. T cells were observed in plaques of their aortic arches (CD5 positive, 11.1 +/- 7.3 cells/mm2; CD4 positive, 9.9 +/- 4.9 cells/mm2) by immunofluorescence using monoclonal anti-rabbit CD5 and CD4 antibodies. Rabbits treated with CsA developed significantly less extensive plaques after 4 weeks (aortic arch 33.0% +/- 6.2%, P < 0.001; thoracic aorta 6.3% +/- 1.5%, P < 0.05; abdominal aorta 2.7% +/- 0.5%, P < 0.005) than untreated rabbits. No CD4 or CD5 positive cells were observed in their plaques. Treatment with CsA did not affect the weight gain of rabbits or reduce their serum cholesterol levels. Circulating T cell numbers and subsets were unaffected. These studies suggest that inhibition of T cell activation prevents their localisation in plaques and reduces the extent of early lesions, suggesting a role for T cells in the initiation of atherosclerosis.

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.

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.

Animal models of diet-induced atherosclerosis.

Animals models of atherosclerosis develop lesions either spontaneously or by interventions such as dietary, mechanical, chemical, or immunological induction. Animal models provide a means for studying the underlying mechanisms behind the atherosclerotic disease process, as well as a means for studying the effect of interventions, dietary or otherwise, on the development or regression of disease, while under controlled conditions. The effect of risk factors for atherosclerotic disease development has been evaluated in animal models, with the advantage of excluding other influences. Animal models have provided valuable information regarding diagnostic and therapeutic strategies, with extensive investigation of events occurring in the artery wall throughout these procedures. Animal models have provided information about factors contributing to disease progression and regression that apply to human situations.

Collection and processing of arterial specimens for histological analysis.

After atherosclerotic development has proceeded for the allotted time, arteries can be harvested by several techniques to optimize both quantitative and qualitative histological analysis. A study design must include consideration of the parameters to be analyzed, to determine how tissue should be harvested. Often, studies increase the numbers of samples in each group to allow specimen collection by several techniques, and hence several different analyses can be performed.

T helper cell infiltration and foam cell proliferation are early events in the development of atherosclerosis in cholesterol-fed rabbits.

The involvement of T cells in the early cellular events in atherosclerosis was studied in rabbits fed a 1% cholesterol diet by use of specific monoclonal anti-rabbit CD5 and CD4 antibodies. T cells were not seen in the aortic intimas of rabbits not fed cholesterol but were seen in intimal lesions in cholesterol-fed rabbits. Accumulation of T cells in plaques occurred between 2 and 4 weeks after commencement of cholesterol feeding, and the greatest density of CD5-positive T cells were observed after 4 weeks (11.2 +/- 6.0 cells/mm2 [mean +/- SEM]; P < .02 compared with normal control rabbits, P < .03 compared with 2-week plaques). Staining for CD4 indicated that the majority of these T cells were T helper cells (9.9 +/- 4.9 cells/mm2). At this time, plaques showed a dense cellular infiltrate of macrophages (3623 +/- 467 cells/mm2) and macrophage proliferation was evident (2.1 +/- 1.1% of total plaque cells). As the cross-sectional area of intimal lesions increased progressively in subsequent weeks, their cellularity declined (8 weeks, 2239 +/- 271 cells/mm2; 12 weeks, 1535 +/- 55 cells/mm2; 16 weeks, 1747 +/- 242 cells/mm2, P < .05 for all groups compared with the 4-week group). The density of the T cell infiltrate (8 weeks, 6.7 +/- 3.0 cells/mm2; 12 weeks, 0.6 +/- 0.2 cells/mm2; 16 weeks, 1.0 +/- 0.4 cells/mm2) and the proliferative index of cells within plaques (8 weeks, 0.6 +/- 0.2%; 12 weeks, 0.8 +/- 0.3%; 16 weeks, 0.2 +/- 0.2%) also declined.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Tissue factor pathway inhibitor expression in atherosclerosis.

Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of tissue factor (TF) -initiated coagulation and may play a role in regulating coagulation in atherosclerotic plaques. The expression of TFPI protein and mRNA was examined by immunohistology and in situ hybridization in normal human and rabbit arteries, in human carotid arteries with advanced atherosclerosis, and in atherosclerotic aortas from cholesterol-fed rabbits. In normal human and rabbit arteries, TFPI protein and mRNA were detected in the adventitial layer but were undetectable in the luminal endothelium. In the medial smooth muscle layer of rabbits, weak expression of TFPI mRNA, but not protein, was detected; in that of humans, neither TFPI mRNA nor protein was detectable. In atherosclerotic arteries, TFPI protein and mRNA were detected in three of six internal carotid plaques from patients undergoing endarterectomy, and mRNA alone was detected in one further specimen. TFPI protein was found in areas of the plaque where TF was abundant and colocalized with macrophages, suggesting that these cells are responsible for TFPI synthesis. TFPI protein and mRNA were also detected in fatty-streak lesions in 18 of 19 rabbits fed a high-cholesterol diet for periods between 4 and 16 weeks. In these macrophage-rich lesions, expression of TFPI protein and mRNA was most intense at the base of the plaques. These studies suggest that TFPI is expressed in the adventitial layer of large arteries and that in atherosclerotic vessels, TFPI is expressed by macrophages in focal areas throughout the plaque. Local production of TFPI may regulate procoagulant activity and thrombotic events within atherosclerotic plaques.

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.

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.

Correlation of tumor- and stromal-derived MT1-MMP expression with progression of human ovarian tumors in SCID mice.

OBJECTIVE: Human ovarian carcinoma samples were orthotopically implanted into SCID mice to investigate the contribution of matrix metalloproteases (MMPs) to the spread of ovarian tumors. METHODS: Mice were inoculated with patient tumor samples, and developed ovarian tumors over a 16-week period with metastasis occurring in some mice. Species-specific quantitative RT-PCR was used to identify the source of tumor-associated MMPs. RESULTS: Membrane-type (MT)1-MMP mRNA was significantly increased in high-grade tumors, tumors with evidence of serosal involvement, and tumors in which distant metastases were detected. The increase in MT1-MMP expression was predominantly from the human tumor cells, with a minor contribution from the mouse ovarian stroma. Neither human nor mouse MT2-MMP were correlated with tumor progression and MT3-MMP levels were negligible. While tumor cells did not produce significant amounts of MMP-2 or MMP-9, the presence of tumor was associated with increased levels of MMP-2 expression by mouse ovarian stroma. Stromal-derived MT1-MMP was greater in large tumors and was associated with stromal MMP-2 expression but neither was significantly linked with metastasis. CONCLUSIONS: These studies indicate that tumor-derived MT1-MMP, more so than other gelatinolytic MMPs, is strongly linked to aggressive tumor behavior. This orthotopic model of human ovarian carcinoma is appropriate for studying ovarian tumor progression, and will be valuable in the further investigation of the metastatic process.

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.

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.