Takayuki Iwaki

PAI-1, progress in understanding the clinical problem and its aetiology

Plasminogen activator inhibitor‐1 (PAI‐1, also known as SERPINE1) is a member of the serine protease inhibitor (SERPIN) superfamily and is the primary physiological regulator of urokinase‐type plasminogen activator (uPA) and tissue‐type plasminogen activator (tPA) activity. Although the principal function of PAI‐1 is the inhibition of fibrinolysis, PAI‐1 possesses pleiotropic functions besides haemostasis. In the quarter century since its discovery, a number of studies have focused on improving our understanding of PAI‐1 functions in vivo and in vitro. The use of Serpine1‐deficient mice has particularly enhanced our understanding of the functions of PAI‐1 in various physiological and pathophysiological conditions. In this review, the results of recent studies on PAI‐1 and its role in clinical conditions are discussed.

Fibrinogen Stabilizes Placental-Maternal Attachment During Embryonic Development in the Mouse

In humans, maternal fibrinogen (Fg) is required to support pregnancies by maintaining hemostatic balance and stabilizing uteroplacental attachment at the fibrinoid layer found at the fetal-maternal junction. To examine relationships between low Fg levels and early fetal loss, a genetic model of afibrinogenemia was developed. Pregnant mice homozygous for a deletion of the Fg-gamma chain, which results in a total Fg deficiency state (FG(-/-)), aborted the fetuses at the equivalent gestational stage seen in humans. Results obtained from timed matings of FG(-/-) mice showed that vaginal bleeding was initiated as early as embryonic day (E)6 to 7, a critical stage for maternal-fetal vascular development. The condition of afibrinogenemia retarded embryo-placental development, and consistently led to abortion and maternal death at E9.75. Lack of Fg did not alter the extent or distribution pattern of other putative factors of embryo-placental attachment, including laminin, fibronectin, and Factor XIII, indicating that the presence of fibrin(ogen) is required to confer sufficient stability at the placental-decidual interface. The results of these studies demonstrate that maternal Fg plays a critical role in maintenance of pregnancy in mice, both by supporting proper development of fetal-maternal vascular communication and stabilization of embryo implantation.

Plasma levels of bradykinin are suppressed in factor XII-deficient mice

A genetically-transmissible factor (F) XII-inactivated allele has been produced in mice by targeted replacement of exons 3-8 of the FXII gene with the neomycin resistance gene. Interbreeding of these mice provided offspring homozygous for two inactivated FXII alleles (FXII(-/-)). Male and female FXII-deficient mice bred normally in all genotypic combinations of the heterozygous and homozygous states, and the offspring survived to adulthood, suggesting that a total FXII deficiency does not affect embryonic development and survival. Neither FXII transcripts nor FXII antigen was found in various tissues of adult FXII(-/-) mice. No obvious unchallenged coagulopathies were present in FXII(-/-) adult mice, despite greatly prolonged activated partial thromboplastin times in this mouse cohort. FXII(-/-) mice were then used to assess the in vivo importance of the plasma FXII/prekallikrein/kininogen pathway in provision of resting plasma bradykinin (BK) levels and in generation of plasma BK stimulated by contact with an artificial surface, using a new and greatly improved plasma BK assay developed during these studies. It was found that approximately 50% of resting BK, and all of the contact-stimulated plasma BK, was provided by this FXII-dependent pathway, without a requirement for FXI. These results provide clear evidence that surface-stimulated BK production, in mice, is dependent on the activation of FXII.

Life-threatening hemorrhage and prolonged wound healing are remarkable phenotypes manifested by complete plasminogen activator inhibitor-1 deficiency in humans

Summary.  Background: Plasminogen activator inhibitor‐1 (PAI‐1) is the primary physiological regulator of urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) activity. A number of studies have shown that elevated levels of PAI‐1 are related to pathological states such as an increased risk of arterial thrombotic events and a poor prognosis for cancer patients; however, there are few reports about PAI‐1 deficiency in humans because the disorder is very rare. Objective: To understand the in vivo impact of a complete PAI‐1 deficiency, Serpine1−/− mice were generated; a number of in vivo studies have been conducted to elucidate the function of PAI‐1 using Serpine1−/− mice. The phenotypes demonstrated in Serpine1−/− mice, however, were quite different from those in humans. Therefore, it is necessary to find out and analyze SERPINE1 deficiency in humans. Patient and methods: The patient is a 47‐year‐old woman who has had multiple episodes of major bleeding. Although most of the patient’s blood coagulation factors were functionally normal, her PAI‐1 antigen levels were undetectable. Therefore, DNA sequencing of the SERPINE1 gene were analyzed. Results: The proband had a homozygous 1‐bp duplication (C) at exon 3 (c.356dupC; p.Ile120AspfsX42). Both wild‐type PAI‐1 (42.7 kDa) and mutated (Mut) PAI‐1 (14.7 kDa) were expressed in COS‐1 cells, although the level of Mut PAI‐1 expressed in the cell lysates was much lower. Wild‐type PAI‐1 was observed in the culture supernatant, whereas no Mut PAI‐1 was detected in the supernatant. Conclusions: Considering the results of the present study, the translation of mouse studies to humans must be performed with great care.

A single plasmid transfection that offers a significant advantage associated with puromycin selection in Drosophila Schneider S2 cells expressing heterologous proteins

The Drosophila Schneider S2 (S2) Expression System enables expression of recombinant proteins constitutively, as well as inductively. This system can establish both transient and stable transformants with various selection markers. The generation of stable cell lines for increased expression or large scale expression of the desired protein is currently accomplished by cotransfection of both the expression and selection vectors. The selection vectors, pCoHYGRO and pCoBLAST, are commercially available using hygromycin-B and blasticidin S, respectively. Recently, we generated a plasmid, pCoPURO, for selection of transfected S2 cells using puromycin, which allows significant acceleration of the selection time. Although co-transfection of the selection marker with the plasmid for heterologous protein expression is functional in stable expression at short culture periods, the expression levels of stable transformants are continuously decreased during long culture times. To overcome this limitation, we generated pMT-PURO, a new plasmid that contains both the expression cassette and puromycin selection marker in a single plasmid. This system allows rapid selection and maintenance of the transformed S2 lines for extended culture periods.

A Missense Mutation in the Sodium Phosphate Co-transporter Slc34a1 Impairs Phosphate Homeostasis

The sodium phosphate co-transporters Npt2a and Npt2c play important roles in the regulation of phosphate homeostasis. Slc34a1, the gene encoding Npt2a, resides downstream of the gene encoding coagulation factor XII (f12) and was inadvertently modified while generating f12(-/-) mice. In this report, the renal consequences of this modification are described. The combined single allelic mutant Slc34a1m contains two point mutations in exon 13: A499V is located in intracellular loop 5, and V528M is located in transmembrane domain 11. In addition to the expected coagulopathy of the f12(-/-) phenotype, mice homozygous for the double allelic modification (f12(-/-)/slc34a1(m/m)) displayed hypophosphatemia, hypercalcemia, elevated levels of alkaline phosphatase, urolithiasis, and hydronephrosis. Strategic cross-breedings demonstrated that the kidney-related pathology was associated only with autosomal recessive transmission of the slc34a1(m) gene and was not influenced by the simultaneous inactivation of f12. Npt2a[V528M] could be properly expressed in opossum kidney cells, but Npt2a[A499V] could not. These results suggest that a single amino acid substitution in Npt2a can lead to improper translocation of the protein to the cell membrane, disturbance of phosphate homeostasis, and renal calcification. Whether point mutations in the SLC34A1 gene can lead to hypophosphatemia and nephrolithiasis in humans remains unknown.

Delayed inflammatory responses to endotoxin in fibrinogen-deficient mice

Severe inflammation leads to haemostatic abnormalities, such as the development of microvascular thrombi. As a result, ischaemia‐related downstream organ damage can occur. The present study demonstrates that mice with a total deficiency of fibrinogen (Fg−/−) present with altered responses to challenge with Gram‐negative lipopolysaccharide (LPS). Early survival in response to continuous LPS challenge was increased in Fg−/− mice and histological findings indicated that this improvement correlated with a lack of fibrin deposition in organs. Neutrophils appeared early in the lungs of challenged wild‐type (WT) mice, but occurred in Fg−/− mice at later times. This delayed response in Fg−/− mice was confirmed by studies that showed a strong dependence on Fg of binding of neutrophils to endothelial cells in the presence of LPS. While cytokines were also elevated in both WT and Fg−/− mice, their levels were generally lower at early times in this latter group. The time course of MIP‐2 expression correlated with the occurrence of pulmonary leakage after LPS challenge, which was delayed in Fg−/− mice. These results suggest that fibrin(ogen) plays a role as an early mediator in the cross‐talk between coagulation and inflammation. Copyright

Anti-Macrophage Inhibitory Factor Antibody Inhibits PMSG-hCG-Induced Follicular Growth and Ovulation in Mice

AbstractPurpose: To investigate the effect of an anti-MIF antibody on PMSG-hCG-induced murine follicular growth and ovulation and to determine whether MIF plays an essential role in this process. Methods: Mice were primed with an intraperitoneal injection of pregnant mare serum gonadotropin (PMSG) and were treated with an anti-rat MIF antibody and human chorionic gonadotropin (hCG) to induce ovulation. After that, the ovulated ova were counted. The ovaries were studied using standard histological procedures. Results: Ovaries treated with the anti-MIF antibody showed reduced numbers of growing follicles surrounded by granulosa cells and theca cells with a little proliferation compared with the control. The average numbers of ova collected from mice treated with the anti-MIF antibody were reduced compared with those collected from control mice. Conclusions: Anti-MIF antibody inhibits the follicular growth and ovulation in mice, and MIF may play an important role in the inflammatory reactions during follicle growth and ovulation.

Recombinant adenovirus vector bearing antisense macrophage migration inhibitory factor cDNA prevents acute lipopolysaccharide-induced liver failure in mice.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine involved in delayed hypersensitivity and cellular immunity. MIF also acts as a proinflammatory cytokine and counterregulates the anti-inflammatory effects of glucocorticoids. Exogenous gene transfer mediated by adenovirus is useful to study a particular molecular function as well as to develop gene therapy strategies. A recombinant adenovirus containing sense and antisense murine MIF (mMIF) cDNA inserts was constructed using a cosmid-terminal protein complex method. The sense mMIF adenovirus (AxCA-mMIFS) efficiently induced mMIF in COS-7 cells that endogenously lack mMIF in a dose-dependent manner. In contrast, the antisense mMIF adenovirus (AxCA-mMIFAS) inhibited the expression of mMIF in NIH3T3 cells in a dose-dependent manner. To assess the pathophysiologic role of MIF in acute liver failure, we induced acute onset of liver damage in mice (male Jcl:ICR) by a combined treatment of Bacille Calmette-Guerin (BCG) and lipopolysaccharide (LPS). mMIF level in the liver of mice infected with AxCA-mMIFAS showed a significant reduction in MIF production in response to BCG-LPS compared with mice treated without viral infection and with AxCA-mMIFS. In addition, the immunohistochemical staining demonstrated that F4/80 antigen on macrophage was enhanced in liver infected with AxCA-mMIFS but reduced in liver infected with AxCA-mMIFAS. The staining intensity is correlated with the mMIF antigen level in liver tissue. The survival rate of mice infected with AxCA-mMIFAS was significantly higher than that of mice treated with PBS and infected with AxCA-LacZ in BCG-LPS. These results suggest that inhibition of MIF production, using recombinant adenovirus bearing the antisense MIF gene, reduced the mortality rate in BCG-LPS–induced liver failure in mice. This finding might aid in the further development of gene therapy targeting MIF.

A complete factor XII deficiency does not affect coagulopathy, inflammatory responses, and lethality, but attenuates early hypotension in endotoxemic mice.

The contact coagulation system is activated in lethal endotoxemia, and levels of coagulation Factor XII (FXII) decline in the consumptive phases of this disease. Thus, we examined the effects of a total inactivation of the endogenous F12 gene in the major phases of endotoxemia using a continuous infusion lethal dose model of lipopolysaccharide (LPS) administration in mice. Blood levels of coagulation and inflammatory parameters were measured, along with BK levels and BP parameters. For this study, we employed 7–8 wk of age male wild-type (WT) mice and mice with a total targeted inactivation of the FXII gene (F12−/−) that were fully backcrossed into the C57BL/6 strain [1]. These mice were subjected to continuous challenge with LPS (Escherichia coli; Serotype O111; B4, Sigma, St. Louis, MO) in saline (5 μg/ml) that was placed in osmotic pumps and the latter implanted into the peritoneal cavities [2]. The pumps release their contents at the rate of 1 μl/hr. Under these conditions, WT mice showed approximately 50% lethality at 96 hr. No survival differences were observed between WT and F12−/− mice under these conditions (Figure 1A). Figure 1 responses of WT and F12−/− mice to LPS challenge. (A) Survival rates of WT and F12−/− mice after implantation of LPS containing osmotic pumps. The solid line depicts the survival curve of WT mice (N=19). The survival at ... Coagulation parameters and inflammation markers were then determined on 6–12 mice at 0 time, and at 3, 6, 9, 12, 18, and 24 hr post-LPS challenge. ELISA-based measurements of thrombin-antithrombin (TAT) levels in WT and F12−/− mice were evaluated with a TAT ELISA Kit (ERL, South Bend, IN, USA) during continuous LPS challenge. The plasma levels of TAT in F12−/− mice (76 ± 16 pM) were significantly (p < 0.05, pairwise comparison) lower than those same values in WT mice (125 ± 15 pM) under resting conditions (time = 0). The TAT levels rapidly increased to 276 ± 67 pM for WT and 308 ± 44 pM for F12−/− mice at 3 hr post-LPS, and progressively decreased at later time points. TAT levels reached baseline at approximately 12 hr, and declined further to approximately one-half of the pre-LPS challenge values at 24 hr, likely because of a consumptive coagulopathy. Plasma fibrinogen levels, determined with the Fibri-Prest Automate Kit (Diagnostica Stago, Asnieres-Sur-Seine, France) continuously increased during the challenge, from 197 ± 12 at time = 0 to 440 ± 21 at 24 hr subsequent to LPS challenge for WT mice and 197 ± 5 at rest to 536 ± 55 at 24 hr post-LPS challenge, for F12−/− mice. This likely reflects an acute phase response. Platelet counts, analyzed using an automated CBC analyzer continually decreased from 687 × 103/μl to 303 ± 40 × 103/μl for WT mice and 683 ± 28 × 103/μl to 284 ± 66 × 103/μl during this same 24 hr time period. Thus, a significant coagulopathy was found in each genotypic group under this LPS dosage regimen, but no significant differences were found between the WT and F12−/− groups in these markers at any post-LPS timepoint examined. Select inflammation markers, viz., tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and macrophage inflammatory protein-2 (MIP-2), were measured in plasmas of WT and F12−/− mice at various times during continuous LPS challenge using Quantikine-M Immunoassay kits (R&D Systems, Minneapolis, MN, USA). Plasma levels of TNF-α in resting WT (12.7 ± 1.5 pg/ml) and F12−/− (9.6 ± 0.3) mice significantly increased to 408 ± 51 pg/ml and 357 ± 66 pg/ml, respectively, at 3 hr post-LPS, and gradually declined through the next 24 hr, but did not reach resting levels at that later timepoint. Similarly, while IL-6 was undetectable in plasmas of both resting WT and F12−/− mice, the peak values of 37.6 ± 1.6 ng/ml and 36.1 ± 2.5 ng/ml, respectively, at 3 hr, also gradually declined through 24 hr, but remained above resting levels in each case. Lastly, the MIP-2 levels paralleled the IL-6 data. MIP-2 was undetectable in plasma of WT and F12−/− mice, reached peak levels at 3 hr post-LPS (51.6 ± 6.3 ng/ml for WT and 41.8 ± 6.1 ng/ml for F12−/− mice), and gradually declined to just above detection levels at 24 hr after LPS administration. No statistical differences were found between the WT and F12−/− groups in these particular inflammatory markers at any timepoint. Plasma bradykinin (BK) levels were evaluated at various times post-LPS by RIA [1]. The plasma levels of BK in WT mice were significantly higher those in F12−/− mice under resting conditions (Figure 1B), the latter of which were below levels of detection at all challenge times. This further illustrates the importance of the FXII-dependent pathway in BK generation. The BK levels in WT mice were increased at 3 hr and 6 hr after challenge, and then diminished after 9 hr (Figure 1B). On the other hand, the BK levels in F12−/− mice were below detection in all time points, and did not change over time (Figure 1B). Because of the lack of response of BK to LPS-administration in F12−/− mice, we examined heart rates (HR) and central arterial BPs of the mice during endotoxemia [3]. HRs of both WT and F12−/− mice were slightly elevated from 0 to 2 hr after LPS-containing pump implantation, and then decreased from 2–6 hr, reaching a nadir of approximately 500 bpm (Figure 1C). Although the drop in heart rates of F12−/− mice was less severe than WT mice from 2–6 hr, statistical significance between the groups was not achieved. The mean arterial BPs (MAP) of WT mice showed patterns similar to their heart rates (Figure 1D). On the other hand, such drops of the MAP of F12−/− mice were not observed from 2–6 hr, and the differences between WT and F12−/− mice were statistically significant. After these initial periods, the parameters gradually decreased throughout the time period measured (Figure 1D). Recent studies have indicated that activation of the FVII/TF pathway [4, 5], and downregulation of the anticoagulant Protein C pathway [2, 6–8], most likely play key roles in spontaneous bleeding and induction of coagulopathy in inflammatory diseases, such as endotoxemia, while the contact activation pathway does not appear to be as important in provoking or further establishing DIC in sepsis [9, 10] despite the pronounced activation of this system in human meningococcal disease [11] and in experimental models of bacteremia in baboons [12]. These conclusions correlate with the findings that patients with diminished levels of contact factors do not display spontaneous hemostasis defects in the absence of challenge. However, protection from fibrin and thrombus formation is afforded in various challenge models by deficiencies of components of the contact system, e.g., FXII [13, 14] and kininogen-1 [15]. In summary, the absence of FXII did not contribute to the coagulopathy, inflammatory changes, and mortalities of endotoxemic mice, but the absence of FXII attenuated early hypotensive changes that correlated with BK stimulation. Therefore, we conclude that the initiation of coagulopathy in endotoxemia is primarily triggered by the extrinsic pathway. Activation of the contact pathway in endotoxemia did not substantially contribute to the coagulopathy and inflammatory changes observed, but played a major role in hemodynamic changes via BK production.