Pericellular proteolytic cascade by plasmin/plasminogen activator system

Pericellular proteolytic cascade by plasmin/plasminogen activator system     

Regulation of the plasminogen activator/plasmin system by epidermal growth factor in cultured human endometrial cells

Recent studies have suggested that epidermal growth factor (EGF)and plasminogen activators (PA) are involved in the implantationof concepti. Therefore we attempted to evaluate the effect ofEGF on the release of PA in primary cultures of human endometrialcells. The addition of EGF to culture medium increased the releaseof tissue-type PA (t-PA). The effect of EGF was significantat 5 ng/ml, which produced a 45% increase in t-PA release. EGFalso stimulated the release of urokinase-type PA (u-PA), aswell as the release of PA inhibitor type 1 (PAI-1). These stimulatoryeffects on the release of t-PA and PAI-1, but not of u-PA, wereenhanced by the concomitant addition of progesterone. The modulatoryeffect of EGF on PA release seemed to be selective in that othergrowth factors, such as basic fibroblast growth factor, transforminggrowth factor and interleukin-1, did not affect the releaseof PA. From these data, we propose that EGF in concert withprogesterone participates in embryo implantation, the processentailing tissue remodelling and cell migration in part by modulatingthe PA/plasmin system.     

Amelioration of collagen II-induced arthritis in rats by the type IV phosphodiesterase inhibitor Rolipram

The effect of Rolipram, a selective inhibitor of the cyclic AMP specific phosphodiesterase (PDE IV) was evaluated in the rat collagen type II (RCII)-induced arthritis model in the DA rat. Rolipram was given either shortly before expected onset of disease (days 10–14) or shortly after the onset of clinically evident arthritis (days 15–19 after immunization). Administration at days 10–14 delayed the onset of arthritis for approximately 5 days, but the severity of arthritis was thereafter comparable to that seen in a non-treated control group. Rolipram treatment of animals with manifest arthritis inhibited further arthritis development and also tended to diminish its severity at a phase of disease where non-treated control animals showed a rapidly progressing disease development. Serum levels of antibodies to RCII were in all experiments similar between Rolipram-treated and control animals. An in situ hybridization method for determining cytokine mRNA synthesis in regional lymph nodes, after administration of Rolipram (at days 2–7), demonstrated a strong inhibitory effect on tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) mRNA expression, whereas no effects were seen on IL-2 mRNA synthesis after in vivo challenge with native RCII emulsified in Freund's incomplete adjuvant. The results thus demonstrate strong preventive as well as therapeutic effects of Rolipram in a model for arthritis that is very similar to human rheumatoid arthritis with respect to cytokine regulation, and suggest that Rolipram has its major effects in the effector stage of the arthritogenic immune response.     

Suppression of collagen type II-induced arthritis by transfer of lymphoid cells from rats immunized with collagen.

Rats were immunized with type II collagen to induce polyarthritis. Spleen and lymph node cells were taken at various times and transferred to normal syngeneic animals. Disease was not observed in recipients of cells taken from donors either during the pre-clinical phase or the acute clinical phase of the disease. However, arthritis could not be induced in the recipient animals that had received cells taken from donors during the preclinical phase. Animals receiving cells from donors with clinical disease appeared to have a normal susceptibility to disease induced subsequently. In contrast with the differences in their susceptibility to induced disease, all recipient animals made unmodified antibody responses to a challenging injection of type II collagen. The results showed that before the appearance of clinical disease in CII immunized rats, there were cells in the spleen and lymph nodes that inhibit the development of disease but not antibody responses.     

An essential role for CCL3 in the development of collagen antibody-induced arthritis

CCL3 is a C–C family chemokine detected at high levels in the synovial tissue and fluids of rheumatoid arthritis (RA) patients. CCL3 binds to the chemokine receptors CCR1 and CCR5, which are expressed by inflammatory leukocytes such as macrophages and T cells present in the affected joints of RA patients. The present study was undertaken to investigate whether absence of CCL3 prevented development of inflammation and joint destruction in anti-type II collagen monoclonal antibody (anti-CII mAb)-induced arthritis. “CCL3 null mice were different from wild-type control mice in terms of body weight loss”. In addition, CCL3 null mice exhibited milder clinical and histopathological scores following administration of anti-CII mAb and endotoxin. Moreover, the release of TNF in response to systemic administration of endotoxin was not affected in CCL3 null mice compared to wild-type mice, indicating that the phenotype was not attributable to a defect in endotoxin response. These results indicate that CCL3 plays an essential role in the development of inflammation and joint destruction induced by anti-CII mAb.     

Role of tissue plasminogen activator/plasmin cascade in delayed neuronal death after transient forebrain ischemia

We studied the possible involvement of the tissue plasminogen activator (t-PA)/plasmin system on both delayed neuronal death in the hippocampus and the associated enhancement of locomotor activity in rats, after transient forebrain ischemia induced by a four-vessel occlusion (FVO). Seven days after FVO, locomotor activity was abnormally increased and, after 10 days, pyramidal cells were degraded in the CA1 region of the hippocampus. FVO increased the t-PA antigen level and its activity in the hippocampus, which peaked at 4 h. Both the enhanced locomotor activity and the degradation of pyramidal cells were significantly suppressed by intracerebroventricular injection of aprotinin, a plasmin inhibitor, at 4 h but not during FVO. These results suggest the importance of the t-PA/plasmin cascade during the early pathological stages of delayed neuronal death in the hippocampus following transient forebrain ischemia.     

Low levels of plasminogen activator inhibitor type 1 in patients with inflammatory bowel disease

Recent investigations suggest that microthrombi formation in bowel capillaries could be a determinant factor in inflammatory bowel disease (IBD) pathogenesis. To evaluate the implication of fibrinolysis in these thrombotic events, we analysed plasmatic values of physiologic activators, effectors and inhibitors of fibrinolysis. In a sample of 112 IBD patients we found decreased plasminogen activator inhibitor type 1 (PAI-1:Ag) levels, a finding which has not been reported to date: 8.8±1.1 ng/mL (mean±SEM) versus 17.8±1.1 ng/mL in controls (p<0.0001). Urokinase-type plasminogen activator (u-PA:Ag) from patients with inflammatory activity was decreased: 0.41±0.03 ng/mL in active disease versus 0.52±0.02 ng/mL in inactive disease (p=0.01) and the same applied to patients with Crohn's disease (CD) (0.38±0.03 ng/mL) with respect to ulcerative colitis (UC) patients (0.52±0.025 ng/mL), p=0.001. Levels of plasminogen, alpha 2 antiplasmin and tissue-type plasminogen activator (t-PA:Ag) showed no differences with respect to the controls. With the exception of u-PA:Ag, there were no differences between UC and CD. These results demonstrate plasmatic disturbances in the flbrinolytic system of IBD patients.     

The myofibroblast is the predominant plasminogen activator inhibitor-1-expressing cell type in human breast carcinomas

The tumor level of plasminogen activator inhibitor-1 (PAI-1) is an informative biochemical marker of a poor prognosis in several cancer types. However, the tumor biological functions of PAI-1 and the identity of PAI-1-expressing cells are controversial. With the aim of immunohistochemically localizing PAI-1 in formalin-fixed, paraffin-embedded invasive ductal breast carcinoma samples, we raised new polyclonal antibodies against PAI-1 from different expression systems. The antibodies were affinity purified by absorption on immobilized preparations of PAI-1 different from those used for immunization. The specificity of the antibodies was ensured by immunoblotting analysis. In immunohistochemistry, the staining pattern obtained with the antibodies showed a good correlation with the PAI-1 mRNA expression pattern. In all 25 cases analyzed, PAI-1 immunoreactivity was predominantly localized in fibroblast-like cells. Double-immunofluorescence analyses showed co-expression of PAI-1 and alpha-smooth muscle actin in these cells, suggesting that they are myofibroblasts. PAI-1 was also seen in some myoepithelial cells surrounding occasional foci of ductal carcinoma in situ (9 of 25), some endothelial cells (8 of 25), some cancer cells (3 of 25), and some mast cells (6 of 25). In conclusion, we have provided a robust immunohistochemical procedure for detection of PAI-1 and shown that the majority of the PAI-1-expressing cells in invasive ductal breast carcinomas are myofibroblasts.     

Cell invasion is affected by differential expression of the urokinase plasminogen activator/urokinase plasminogen activator receptor system in muscle satellite cells from normal and dystrophic patients

The aim of this study was to evaluate the differential expression and the function in cell movement and proliferation of the urokinase plasminogen activator (u-PA) system in muscle satellite cells (MSC) of normal individuals and patients with Duchenne muscular dystrophy (DMD). By immunoenzymatic, zymographic, and radioligand binding methods and by quantitative polymerase chain reaction of the specific mRNA we have shown that both normal and DMD MSC produce u-PA and the plasminogen activator inhibitor-1 and express u-PA receptors (u-PAR). During the proliferation phase of their growth-differentiation program, MSC from DMD patients show more u-PAR than their normal counterpart, produce more plasminogen activator inhibitor-1, and release low amounts of u-PA into the culture medium. By Boyden chamber Matrigel invasion assays we have shown that normal MSC are more prone than DMD cells to spontaneous invasion but, when subjected to a chemotactic gradient of u-PA, DMD MSC sense the ligand much better and to a greater extent than normal MSC. u-PA also stimulates proliferation of MSC, but no difference is observable between normal and DMD patients. Antagonization of u-PA/u-PAR interaction with specific anti-u-PA and anti-u-PAR monoclonal antibodies and with antisense oligonucleotides inhibiting u-PAR expression indicates that u-PA/u-PAR interaction is required in spontaneous and u-PA-induced invasion, as well as in u-PA-induced proliferation.     

The influence of recombinant tissue-type plasminogen activator and plasmin on platelet aggregation: a quenched-flow study

Our study was designed to investigate the influence of human recombinant tissue-type plasminogen activator (rt-PA) and plasmin on platelet aggregation kinetics. Quenched-flow aggregometry coupled to single-particle counting was used with washed human platelets, which were pre-incubated for 5 min at 37°C with physiological and pharmacological doses of rt-PA (0.14–70 nM). Aggregation induced by adenosine diphosphate (ADP) under physiological flow conditions was followed over 15s. Depending on donor and reaction time, potentiation (one third of subjects), inhibition (one third of subjects), and no effect on ADP-induced (10 μM) aggregation were noted within the first 5s, while from 5.0 to 15.O s no influence was seen. On the other hand, using 2.5 μM ADP, aggregation was inhibited in all sensitive donors. Pre-incubation of platelets with plasmin (10nM, 100 nM) for 5 min at 37°C elicited a biphasic response to ADP. Depending on concentration and reaction time, plasmin potentiated or inhibited ADP-induced (10 μM) aggregation. Our data reveal that some individuals are susceptible for potentiation of platelet aggregation by both rt-PA and plasmin. Such susceptibility may contribute to the phenomenon of early reocclusion observed during thrombolytic therapy with rt-PA.     

The possible role of tissue-type plasminogen activator and the plasminogen system in the pathogenesis of major depression

Major depressive disorder (MDD) is one of the most common psychiatric illnesses with an unknown etiology. Evidence from animal and human studies has suggested that brain-derived neurotrophic factor (BDNF) function may be implicated in the pathogenesis of MDD. Tissue-type plasminogen activator (tPA) is a highly specific serine proteinase that catalyses the generation of zymogen plasminogen from the proteinase plasmin. Recent studies have found that the proteolytic cleavage of proBDNF, a BDNF precursor, to BDNF by the plasmin represents a mechanism by which the direction of BDNF action is controlled. Furthermore, studies using mice deficient in tPA has demonstrated that tPA is important for the stress reaction, a common precipitating factor for MDD. A study of the serum levels of the plasminogen activator inhibitor-1 (PAI-1), the major inhibitor of tPA, found that women with MDD had a higher PAI-1 concentration than normal controls. From these findings, it is proposed that the tPA/plasminogen system may play a role in the pathogenesis of MDD. Attempts to confirm the tPA/plasminogen hypothesis may lead to new directions in the study of the pathogenesis of MDD and the development of a novel intervention of this disorder.     

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.     

Role of heparin and tissue type plasminogen activator in high altitude adaptation of the hemostasis system

All-Union Hematology Research Center, Ministry of Health of the USSR. Department of Human and Animal Physiology, Faculty of Biology, M. V. Lomonosov State University, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. I. Vorob'ev.) Translated from Byulletin Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 11, pp. 494–497, November, 1991.     

Tissue plasminogen activator in the amygdala is critical for stress-induced anxiety-like behavior

Although neuronal stress circuits have been identified, little is known about the mechanisms that underlie the stress-induced neuronal plasticity leading to fear and anxiety. Here we found that the serine protease tissue-plasminogen activator (tPA) was upregulated in the central and medial amygdala by acute restraint stress, where it promoted stress-related neuronal remodeling and was subsequently inhibited by plasminogen activator inhibitor-1 (PAI-1). These events preceded stress-induced increases in anxiety-like behavior of mice. Mice in which the tPA gene has been disrupted did not show anxiety after up to three weeks of daily restraint and showed attenuated neuronal remodeling as well as a maladaptive hormonal response. These studies support the idea that tPA is critical for the development of anxiety-like behavior after stress.     

Immunization of rats with homologous type XI collagen leads to chronic and relapsing arthritis with different genetics and joint pathology than arthritis induced with homologous type II collagen

The most commonly used animal model for rheumatoid arthritis (RA) is collagen-induced arthritis (CIA), induced by immunization with type II collagen (CII), a cartilage restricted protein. In this work we show that type XI collagen (CXI), which is a minor component in cartilage, induces a different form of erosive and chronic relapsing polyarthritis in rats. Using a series of inbred rat strains involving various genetic backgrounds (DA, LEW, E3), and congenic MHC regions (a, u, f, n, c, d), we found that CXI induced arthritis (C(XI)IA) is associated with the RT1f haplotype in contrast to CII induced arthritis (C(II)IA), which is associated with the RT1a and RT1u haplotypes. The C(XI)IA follows a chronic disease course affecting peripheral joints with both progression and relapses, which appear not to cease (occurring >800 days). Susceptible strains showed a sustained antibody response to CXI with time indicating that the autoimmune response was self-perpetuated. Microscopic analysis of the joints at different stages demonstrated the severe destruction of bone and cartilage by pannus tissue consisting of activated macrophages and T cells. The main difference to joints from rats with C(II)IA was larger numbers of infiltrating lymphocytes and these tended to form follicle-like aggregates. Surprisingly, males were more susceptible to C(XI)IA than females whereas the opposite has been observed in other rat arthritis models, including C(II)IA. Taken together, C(XI)IA is a chronic relapsing and erosive polyarthritis that is MHC associated, which in fact fulfills the criteria for diagnosis of RA. Thus the C(XI)IA model will be useful as a novel and relevant animal model for RA.     

Effects of the Magenstrasse and Mill operation for obesity on plasma plasminogen activator inhibitor type 1, tissue plasminogen activator,fibrinogen and insulin

Plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator (t-PA), fibrinogen and insulin were measured in 43 patients 3 years after they had undergone the Magenstrasse and Mill (MM) procedure and in 43 morbidly obese (MO) patients. Mean plasma PAI-1 was 61 ng/ml in the MO group compared to 30 ng/ml in the MM group (p < 0.0001); mean plasma t-PA was 10 ng/ml in the MO group compared to 7 ng/ml in the MM group (p < 0.001). Mean fibrinogen was 3.6 g/l in the MO group compared to 3.2 g/l in the MM group (p < 0.05). Mean plasma insulin levels were 32 U/ml in the MO group compared to 15 U/ml in the MM group. These changes suggest that use of the MM procedure may reduce mortality and morbidity from coronary heart disease in these high-risk obese patients.