Shogo Kasuda

A matrix metalloprotease-PAR1 system regulates vascular integrity, systemic inflammation and death in sepsis.

Sepsis is a deadly disease characterized by the inability to regulate the inflammatory–coagulation response in which the endothelium plays a key role. The cause of this perturbation remains poorly understood and has hampered the development of effective therapeutics. Matrix metalloproteases (MMPs) are involved in the host response to pathogens, but can also cause uncontrolled tissue damage and contribute to mortality. We found that human sepsis patients had markedly elevated plasma proMMP‐1 and active MMP‐1 levels, which correlated with death at 7 and 28 days after diagnosis. Likewise, septic mice had increased plasma levels of the MMP‐1 ortholog, MMP‐1a. We identified mouse MMP‐1a as an agonist of protease‐activated receptor‐1 (PAR1) on endothelial cells. MMP‐1a was released from endothelial cells in septic mice. Blockade of MMP‐1 activity suppressed endothelial barrier disruption, disseminated intravascular coagulation (DIC), lung vascular permeability as well as the cytokine storm and improved survival, which was lost in PAR1‐deficient mice. Infusion of human MMP‐1 increased lung vascular permeability in normal wild‐type mice but not in PAR1‐deficient mice. These findings implicate MMP‐1 as an important activator of PAR1 in sepsis and suggest that therapeutics that target MMP1‐PAR1 may prove beneficial in the treatment of sepsis.

Suppression of Arterial Thrombosis without Affecting Hemostatic Parameters with A Cell-Penetrating PAR1 Pepducin

Background— Thrombin-dependent platelet activation is heightened in the setting of percutaneous coronary intervention and may cause arterial thrombosis with consequent myocardial necrosis. Given the high incidence of adverse effects in patients with acute coronary syndromes, there remains an unmet need for the development of new therapeutics that target platelet activation without unduly affecting hemostasis. The thrombin receptor, PAR1, has recently emerged as a promising new target for therapeutic intervention in patients with acute coronary syndromes. Methods and Results— We report the development of a first-in-class intracellular PAR1 inhibitor with optimized pharmacokinetic properties for use during percutaneous coronary intervention in patients with acute coronary syndromes. PZ-128 is a cell-penetrating pepducin inhibitor of PAR1 that targets the receptor–G-protein interface on the inside surface of platelets. The structure of PZ-128 closely resembles the predicted off-state of the corresponding juxtamembrane region of the third intracellular loop of PAR1. The onset of action of PZ-128 was rapid and suppressed PAR1 aggregation and arterial thrombosis in guinea pigs and baboons and strongly synergized with oral clopidogrel. There was full recovery of platelet function by 24 hours. Importantly, PZ-128 had no effect on bleeding or coagulation parameters in primates or in blood from patients undergoing percutaneous coronary intervention. Conclusions— Based on the efficacy data in nonhuman primates with no noted adverse effects on hemostasis, we anticipate that the rapid onset of platelet inhibition and reversible properties of PZ-128 are well suited to the acute interventional setting of percutaneous coronary intervention and may provide an alternative to long-acting small-molecule inhibitors of PAR1.

Protease-Activated Receptor-2 Modulates Protease-Activated Receptor-1–Driven Neointimal Hyperplasia

Objective—Emerging evidence suggests that protease-activated receptors-1 and -2 (PAR1 and PAR2) can signal together in response to proteases found in the rapidly changing microenvironment of damaged blood vessels. However, it is unknown whether PAR1 and PAR2 promote or mitigate the hyperplastic response to arterial injury. Using cell-penetrating PAR1 pepducins and mice deficient in PAR1 or PAR2, we set out to determine the respective contributions of the receptors to hyperplasia and phenotypic modulation of smooth muscle cells (SMCs) in response to arterial injury. Methods and Results—SMCs were strongly activated by PAR1 stimulation, as evidenced by increased mitogenesis, mitochondrial activity, and calcium mobilization. The effects of chronic PAR1 stimulation following vascular injury were studied by performing carotid artery ligations in mice treated with the PAR1 agonist pepducin, P1pal-13. Histological analysis revealed that PAR1 stimulation caused striking hyperplasia, which was ablated in PAR1−/− and, surprisingly, PAR2−/− mice. P1pal-13 treatment yielded an expression pattern consistent with a dedifferentiated phenotype in carotid artery SMCs. Detection of PAR1-PAR2 complexes provided an explanation for the hyperplastic effects of the PAR1 agonist requiring the presence of both receptors. Conclusion—We conclude that PAR2 regulates the PAR1 hyperplastic response to arterial injury leading to stenosis.

Anticoagulant activity of M-LAO, l-amino acid oxidase purified from Agkistrodon halys blomhoffii, through selective inhibition of factor IX

One of haemorrhagic toxins present in snake venoms is L-amino acid oxidase (LAO), which catalyzes the oxidative deamination of L-amino acids with the generation of hydrogen peroxide. Although it is widely accepted that LAO alters platelet function, the effects of LAO on human blood coagulation remain largely unknown. The present study demonstrated, for the first time, that M-LAO, LAO purified from the venom of Agkistrodon halys blomhoffii (Japanese mamushi), possesses an anticoagulant activity. Thrombelastography (TEG) showed that M-LAO significantly delayed the onset and the progress of the coagulation process. In addition, the enzyme prolonged the activated partial thromboplastin time (aPTT) dose-dependently, but had little effect on the prothrombin time (PT), suggesting that its principal activity was mediated in the intrinsic coagulation pathway. Furthermore, M-LAO reduced factor IX procoagulant activity in a dose-dependent manner and did not affect other coagulation factors. These results indicate that M-LAO has an anticoagulant activity that impairs the intrinsic clotting by inhibiting factor IX.

The homeobox gene Hex regulates hepatocyte differentiation from embryonic stem cell–derived endoderm

We investigated the role of the hematopoietically expressed homeobox (Hex) in the differentiation and development of hepatocytes within embryonic stem cell (ESC)–derived embryoid bodies (EBs). Analyses of hepatic endoderm derived from Hex−/− EBs revealed a dramatic reduction in the levels of albumin (Alb) and alpha‐fetoprotein (Afp) expression. In contrast, stage‐specific forced expression of Hex in EBs from wild‐type ESCs led to the up‐regulation of Alb and Afp expression and secretion of Alb and transferrin. These inductive effects were restricted to c‐kit+ endoderm‐enriched EB‐derived populations, suggesting that Hex functions at the level of hepatic specification of endoderm in this model. Microarray analysis revealed that Hex regulated the expression of a broad spectrum of hepatocyte‐related genes, including fibrinogens, apolipoproteins, and cytochromes. When added to the endoderm‐induced EBs, bone morphogenetic protein 4 acted synergistically with Hex in the induction of expression of Alb, Afp, carbamoyl phosphate synthetase, transcription factor 1, and CCAAT/enhancer binding protein α. These findings indicate that Hex plays a pivotal role during induction of liver development from endoderm in this in vitro model and suggest that this strategy may provide important insight into the generation of functional hepatocytes from ESCs. (HEPATOLOGY 2010.)

Establishment of embryonic stem cells secreting human factor VIII for cell-based treatment of hemophilia A

Summary.  Background: Hemophilia A is an X‐chromosome‐linked recessive bleeding disorder resulting from an F8 gene abnormality. Although various gene therapies have been attempted with the aim of eliminating the need for factor VIII replacement therapy, obstacles to their clinical application remain. Objectives: We evaluated whether embryonic stem (ES) cells with a tetracycline‐inducible system could secrete human FVIII. Methods and results: We found that embryoid bodies (EBs) developed under conditions promoting liver differentiation efficiently secreted human FVIII after doxycycline induction. Moreover, use of a B‐domain variant F8 cDNA (226aa/N6) dramatically enhanced FVIII secretion. Sorting based on green fluorescent protein (GFP)–brachyury (Bry) and c‐kit revealed that GFP–Bry+/c‐kit+ cells during EB differentiation with serum contain an endoderm progenitor population. When GFP–Bry+/c‐kit+ cells were cultured under the liver cell‐promoting conditions, these cells secreted FVIII more efficiently than other populations tested. Conclusion: Our findings suggest the potential for future development of an effective ES cell‐based approach to treating hemophilia A.

Effectiveness of factor VIII infusions in haemophilia A patients with high responding inhibitors

Summary.  We report here the efficacy of factor VIII (FVIII) infusions in two haemophiliacs with inhibitors using clot waveform analysis on the MDA® II system, which was possible to detect very low levels of FVIII activity < 1.0 U dL−1. In the presence of type 1 inhibitors at the level of 6.2 (patient 1) and 14.4 (patient 2) Bethesda Units mL−1, 3.2 and 6.5 U dL−1 of FVIII:C remained 30 min after the infusion of FVIII (100 U kg−1), respectively. Moreover, 0.9 U dL−1 of FVIII:C remained 24 h after infusion in patient 2. In both cases, these changes were reflected by qualitative improvement in the aPTT clot waveform and quantitative changes in the minimum value of the second derivative of the aPTT waveform (Min2) that reflects clot acceleration. These results suggest that FVIII infusion may be continued with clinical benefit in some haemophiliacs with high responding inhibitors. Furthermore, the haemostatic response may be monitored accurately and efficiently by clot waveform analysis.

Expression of coagulation factors from murine induced pluripotent stem cell-derived liver cells

A protocol to differentiate liver cells from induced pluripotent stem (iPS) cells is being established. However, the ability of these differentiated iPS cells to express liver-specific proteins, such as coagulation cascade and related factors, has yet to be assessed. This study evaluated whether liver-like populations differentiated from murine iPS cells gain the ability to produce coagulation-related factors. Following differentiation of murine iPS cells into hematopoietic-like and liver-like embryoid bodies, we assessed gene expression profiles for coagulation-related markers, including fibrinogen, factors II, V, VII, VIII, IX, X, XI, XII, and XIIIβ, protein C, protein S, antithrombin, plasminogen, von Willebrand factor, and ADAMTS13 by real-time reverse transcription PCR. Liver-like embryoid bodies demonstrated strong expression levels of nearly all the coagulation-related genes assessed, compared with undifferentiated iPS cells and hematopoietic-like embryoid bodies. We also confirmed efficient translation and secretion of fibrinogen and albumin (hepatocyte-specific marker proteins) into the conditioned medium by these differentiated cells, suggesting successful differentiation of iPS cells into the liver lineage. These findings suggest that iPS cells can be differentiated into liver-like populations that express coagulation-related factors. Liver-like embryoid bodies may provide a source for cell-based therapies directed toward liver diseases, including coagulation factor deficiencies in the future.

Respiratory complications of Ehlers-Danlos syndrome type IV.

We describe a case of Ehlers-Danlos syndrome (EDS) type IV in a male in early half in his twenties, who experienced recurrent and eventually fatal pulmonary hemorrhage. EDS type IV is a rare disorder of type III collagen synthesis that is characterized by unusual facies, thin translucent skin with a venous vascular pattern, easy bruising, and hypermobility of the small joints. Autopsy findings showed hypermobility of the joints and distensibility of the skin. Microscopically, the abdominal skin showed substantially decreased dermal thickness. Moreover, the reticular dermis showed fine collagen bundles and large interstitial spaces compared with the skin from a normal control that showed large collagen bundles. Individual elastic fibers were also thicker than those observed in the skin of a normal control. The thoracic aorta showed thin adventitia and a relative increase in elastic fibers. The parenchyma of both the lungs showed markedly diffuse hemorrhage with hemosiderin-laden alveolar macrophages or old thrombi and organized thrombi in the small bronchi. Furthermore, both sections of the lung showed multiple fibrous nodules containing benign metaplastic bone. Vascular wall disruption and tearing of the vessel walls in the lung parenchyma were also observed. We concluded that EDS type IV led to the patients death because of pulmonary hemorrhage. Because this syndrome resulted in the patients death from arterial and bowel rupture, it is important to consider EDS as a potential cause of sudden death.

Application of energy dispersive X-ray fluorescent spectrometry (EDXRF) in drug-related cases.

We applied here energy dispersive X-ray fluorescent spectrometry (EDXRF) to two medico-legal autopsy cases of bromvalerylurea ingestion. Rapid elemental analysis using EDXRF identified bromide in blood, urine and stomach contents of victims during autopsy. The present cases indicate that screening with EDXRF, an instrument suitable for non-destructive, rapid elemental analysis, provides useful information for identification of drugs.