Jun Mimuro

Essential roles of sphingosine 1-phosphate/S1P1 receptor axis in the migration of neural stem cells toward a site of spinal cord injury

Neural stem/progenitor cells (NSPCs) migrate toward a damaged area of the central nervous system (CNS) for the purpose of limiting and/or repairing the damage. Although this migratory property of NSPCs could theoretically be exploited for cell‐based therapeutics of CNS diseases, little is known of the mechanisms responsible for migratory responses of NSPCs. Here, we found that sphingosine 1‐phosphate (Sph‐1‐P), a physiological lysophospholipid mediator, had a potent chemoattractant activity for NSPCs, in which, of Sph‐1‐P receptors, S1P1 was abundantly expressed. Sph‐1‐P‐induced NSPC migration was inhibited by the pretreatment with pertussis toxin, Y‐27632 (a Rho kinase inhibitor), and VPC23019 (a competitive inhibitor of S1P1 and S1P3). Sph‐1‐P does not act as intracellular mediator or in an autocrine manner, because [3H]sphingosine, incorporated into NSPCs, was mainly converted to ceramide and sphingomyeline intracellularly, and the stimulation‐dependent formation and extracellular release of Sph‐1‐P were not observed. Further, Sph‐1‐P concentration in the spinal cord was significantly increased at 7 days after a contusion injury, due to accumulation of microglia and reactive astrocytes in the injured area. This locally increased Sph‐1‐P concentration contributed to the migration of in vivo transplanted NSPCs through its receptor S1P1, given that lentiviral transduction of NSPCs with a short hairpin RNA interference for S1P1 abolished in vivo NSPC migration toward the injured area. This is the first report to identify a physiological role for a lipid mediator in NSPC migration toward a pathological area of the CNS and further indicates that the Sph‐1‐P/S1P1 pathway may have therapeutic potential for CNS injuries.

Aspirin resistance detected with aggregometry cannot be explained by cyclooxygenase activity : involvement of other signaling pathway(s) in cardiovascular events of aspirin-treated patients

Summary.  Objectives: Although the concept of aspirin resistance is extensively reported in medical literature, its precise mechanisms and clinical outcomes are largely unknown. In this study, we examined individual thromboxane biosynthesis and platelet aggregation in aspirin‐treated patients, and whether the results of a platelet aggregation test influenced clinical outcomes. Results: Subjects taking 81 mg of aspirin (n = 50) and controls (n = 38) were evaluated for platelet aggregation and platelet cyclooxygenase‐1 (COX‐1) activity by measuring collagen‐induced thromboxane B2 production. For aggregometry, both light transmission (LT) and laser‐light scattering methods were employed to quantitatively evaluate aggregate sizes and numbers. Aspirin treatment resulted in the inhibition of collagen‐induced platelet aggregation, particularly the transition from small to large platelet aggregates. Although platelet COX‐1 activity seemed to be uniformly inhibited in all patients, platelet aggregation studies showed great inter‐individual differences; variation in platelet COX‐1 activity only accounted for 6–20% of the individual aggregations. Factor analysis revealed the existence of a common factor (other than platelet COX‐1) that explained 48.4% of the variations in platelet aggregation induced by collagen, adenosine diphosphate (ADP), and collagen‐related peptide. We then prospectively enrolled 136 aspirin‐treated patients in our study, and we found that being in the upper quartile level of LT, or with large aggregate formation induced by collagen, was an independent risk factor for developing cardiovascular events within 12 months [hazard ratio (HR) = 7.98, P = 0.008 for LT; HR = 7.76, P = 0.007 for large aggregates]. On the other hand, the existence of diabetes mellitus was an independent risk factor for overall outcomes (HR 1.30–11.9, P = 0.015–0.033). Conclusions: Aspirin resistance expressed as unsuppressed platelet COX‐1 activity is a rare condition in an out‐patient population. Other factor(s) affecting collagen‐induced platelet aggregation may influence early outcomes in aspirin‐treated patients.

Plasminogen Activator Inhibitor 1 Promotes a Poor Prognosis in Sepsis-Induced Disseminated Intravascular Coagulation

Sepsis-induced disseminated intravascular coagulation (DIC) is a serious condition because it is closely linked to the development of multiple organ dysfunctions.We compared molecular fibrinolysis markers for 117 patients with sepsis-induced DIC and 1627 patients with nonseptic DIC. Levels of fibrinogen and fibrin degradation products and D-dimer were significantly lower in sepsis-induced DIC cases than in nonseptic DIC cases. In septic DIC cases, plasma plasminogen activator inhibitor 1 (PAI-1) levels were significantly higher than in nonseptic DIC cases. D-dimer levels were negatively correlated with plasma PAI-1 levels in septic DIC cases. Multiple Organ Dysfunction Scores were significantly higher in septic DIC patients with PAI-1 levels >90 ng/mL than in the group with PAI-1 levels <30 ng/mL. The Kaplan-Meier survival functions until 28 days after DIC diagnosis were significantly lower in the group with PAI-1 levels >90 ng/mL than in the other groups. In a multivariate analysis, plasma PAI-1 levels at DIC diagnosis were an independent risk factor for mortality in sepsis-induced DIC (hazard ratio, 1.012; P = .008). These data suggest that plasma PAI-1 plays an important role in sustaining DIC in septic DIC cases and contributes to multiple organ failure and decreased survival in such patients.

Interactions between type 1 plasminogen activator inhibitor, extracellular matrix and vitronectin

Regulation of plasminogen activation is a key process in controlling proteolytic events in the extracellular matrix (ECM) and this regulation is achieved through the action of specific plasminogen activator (PA) inhibitors (PAIs). Type I PAI (PAI-1) is the physiological inhibitor both of urinary-type PA (u-PA) and tissue-type PA (t-PA) (Loskutoff et al., 1989) and is a major component of the ECM of cultured cells. This inhibitor may protect ECM constituents against cellular proteases and thus influence the cell migration and tissue destruction that occurs during development, inflammation and tumor metastasis. In this review, we discuss the properties of PAI-1 and the evidence that the binding of PAI-1 to ECM is mediated by serum-derived vitronectin (Vn).

Induction of immune tolerance by neonatal intravenous injection of human factor VIII in murine hemophilia A

Inhibitory antibody formation is the most serious complication of factor (F)VIII replacement therapy in hemophilia A patients. FVIII‐deficient mice were used to study new approaches for induction of immune tolerance. Neither antiFVIII inhibitory antibodies nor antiFVIII IgGs were observed in 13 of 14 adult mice that received 0.05 U g−1 body weight of human FVIII intravenously within 24 h after birth and repeated injections as adults. In contrast, high FVIII antibody titers (>50 Bethesda Units mL−1) developed in seven of 13 mice injected on day 3 postpartum and in all adult mice not treated neonatally. One of nine mice and three of 17 mice developed high‐titer antiFVIII inhibitory antibody when they were treated initially with 2‐fold (0.1 U g−1 body weight) and 10‐fold higher doses (0.5 U g−1 body weight) FVIII on day 0, respectively. A human FVIII‐specific T‐cell proliferative response was absent in splenocytes from neonatally treated mice. The tolerance was FVIII specific because antitoxoid antibodies developed after immunization with tetanus toxoid. Splenocytes failed to proliferate or produce interferon (IFN)‐γ in response to FVIII stimulation, yet still secreted interleukin‐2. A proliferative response was restored with exogenous IFN‐γ or interleukin‐12, suggesting that lack of inhibitor to FVIII was due to IFN‐γ‐dependent anergy. Thus, exposure on day 0 to physiological levels of FVIII antigen might be important for induction of immune tolerance. This immune tolerance model may provide a basis for new approaches to prevention of FVIII inhibitors during replacement therapy.

Protection of Plasminogen Activator Inhibitor-1-Deficient Mice from Nasal Allergy

This study was performed to clarify the relationship between fibrinolytic components and the pathology of allergy, particularly that during the development of nasal allergy and nasal tissue changes. Intranasal OVA challenge after sensitization by i.p. administration of OVA induced a higher level of excess subepithelial collagen deposition in wild-type (WT) C57BL/6J mice than in plasminogen activator inhibitor (PAI)-1-deficient (PAI-1−/−) mice. The excess PAI-1 induction in the nasal mucosa and higher level of active PAI-1 in the nasal lavage fluid of WT-OVA mice compared with those in WT-control mice suggested that the decrease of proteolytic activity inhibits the removal of subepithelial collagen. The frequency of sneezing, nasal rubbing, nasal hyperresponsiveness, production of specific IgG1 and IgE in the serum, and production of IL-4 and IL-5 in splenocyte culture supernatant increased significantly in WT-OVA mice. In PAI-1−/− mice, these reactions were absent, and specific IgG2a in serum and IFN-γ in splenocyte culture medium increased significantly. Histopathologically, there were marked goblet cell hyperplasia and eosinophil infiltration into the nasal mucosa in WT-OVA mice, but these were absent in PAI-1−/− mice. These results indicate that the immune response in WT-OVA mice can be classified as a dominant Th2 response, which would promote collagen deposition. In contrast, the Th2 response in PAI-1−/− mice was down-regulated, and the immune response shifted from Th2-dominant reaction to a Th1-dominant one. Taken together, these findings suggest that PAI-1 plays an important role not only in thrombolysis but also in immune response.

Retinal pigment epithelial cells produce a latent fibrinolytic inhibitor that is antigenically and biochemically related to type 1 plasminogen activator inhibitor produced by vascular endothelial cells

Conditioned media from retinal pigment epithelial (RPE) cells in culture contain active and latent plasminogen activator inhibitors (PAIs). Latent activity is unmasked by denaturants and accounts for the vast majority of total inhibitor activity. Activation by denaturants is an unusual characteristic previously described for PAI-1, the inhibitor produced by vascular endothelial cells. This property is not shared by PAI-2 or protease nexin. Reverse fibrin autography demonstrates that the PAI activity in RPE-conditioned media (RPE-CM) comigrates with purified endothelial cell-derived PAI-1 and has an apparent Mr of 50,000. Immunoblotting with a monospecific antiserum directed against endothelial cell-derived PAI-1 demonstrates a cross-reacting protein in RPE-CM at 50 kDa, and this same antiserum is able to immunoprecipitate a 50 kDa protein from [35S]methionine-labeled RPE-CM. These data suggest that RPE cells produce a PAI that is biochemically and immunologically related to PAI-1.

Adenoassociated Virus–Mediated Prostacyclin Synthase Expression Prevents Pulmonary Arterial Hypertension in Rats

Prostacyclin synthase (PGIS) is the final committed enzyme in the metabolic pathway of prostacyclin production. The therapeutic option of intravenous prostacyclin infusion in patients with pulmonary arterial hypertension is limited by the short half-life of the drug and life-threatening catheter-related complications. To develop a better delivery system for prostacyclin, we examined the feasibility of intramuscular injection of an adenoassociated virus (AAV) vector expressing PGIS for preventing monocrotaline-induced pulmonary arterial hypertension in rats. We developed an AAV serotype 1–based vector carrying a human PGIS gene (AAV-PGIS). AAV-PGIS or the control AAV vector expressing enhanced green fluorescent protein was injected into the anterior tibial muscles of 3-week–old male Wistar rats; this was followed by the monocrotaline administration at 7 weeks. Eight weeks after injecting the vector, the plasma levels of 6-keto-prostaglandin F1α increased in a vector dose-dependent manner. At this time point, the PGIS transduction (1×1010 genome copies per body) significantly decreased mean pulmonary arterial pressure (33.9±2.4 versus 46.1±3.0 mm Hg; P<0.05), pulmonary vascular resistance (0.26±0.03 versus 0.41±0.03 mm Hg · mL−1 · min−1 · kg−1; P<0.05), and medial thickness of the peripheral pulmonary artery (14.6±1.5% versus 23.5±0.5%; P<0.01) as compared with the controls. Furthermore, the PGIS-transduced rats demonstrated significantly improved survival rates as compared with the controls (100% versus 50%; P<0.05) at 8 weeks postmonocrotaline administration. An intramuscular injection of AAV-PGIS prevents monocrotaline-pulmonary arterial hypertension in rats and provides a new therapeutic alternative for preventing pulmonary arterial hypertension in humans.

Induction of factor VIII‐specific unresponsiveness by intrathymic factor VIII injection in murine hemophilia A

Summary.  Background: Hemophilia A is a congenital bleeding disorder caused by a deficiency of coagulation factor VIII. Approximately 30% of hemophilia A patients develop inhibitors against FVIII following replacement therapy. We have reported that neonatal exposure of FVIII antigen can induce antigen‐specific immune tolerance by interferon‐γ (IFN‐γ)‐dependent T‐cell anergy in hemophilia A mice. Objective: The thymus plays crucial roles in self‐tolerance, with negative selection of self‐reactive effector T cells and positive selection of self‐reactive regulatory T cells. We investigated the possibility of the induction of antigen‐specific immune tolerance by intrathymic injection of FVIII in hemophilia A mice. Methods: Hemophilia A mice were injected with recombinant FVIII into the thymus under real‐time high‐resolution image guidance. Results: Anti‐FVIII inhibitory antibody titers in mice challenged with intravenous administration of FVIII were significantly lower in mice (n = 22) that had received thymic FVIII injection than in mice (n = 18) without thymic injection (9.4 ± 2.3 vs. 122.5 ± 27.6 BU mL−1, respectively, P = 0.00078). The CD4+ T cells from thymic‐injected mice could not proliferate or produce interleukin (IL)‐2, IL‐12 and IFN‐γ in response to FVIII. The CD4+CD25+ T cells generated from thymic‐treated mice but not from naïve mice efficiently suppressed the in vitro proliferative response of CD4+ T cells and blocked the in vivo development of anti‐FVIII antibodies in the adoptive transfer. Conclusion: These data suggest that intrathymic administration of FVIII could result in immune tolerance by induction of FVIII‐specific regulatory T cells.

Silencing of a Targeted Protein in In Vivo Platelets Using a Lentiviral Vector Delivering Short Hairpin RNA Sequence

Objective— Because platelets are anucleate cells having a limited life span, direct gene manipulation cannot in principle be used to investigate the involvement of a specific signal transduction pathway in platelet activation. In this study, we examined whether the expression of a short hairpin RNA (shRNA) sequence in hematopoietic stem cells is maintained during megakaryocyte differentiation, thus resulting in inhibition of targeted protein in platelets. Methods and Results— To identify platelets derived from transduced stem cells, we generated a lentiviral vector that simultaneously expresses the shRNA sequence driven by the U6 promoter and GFP under the control of the glycoprotein (GP) Ib&agr; promoter. Transplantation of mouse bone marrow cells transduced with the vector facilitated specifically mark platelets derived from the transduced cells. Transplantation of cells transduced with shRNA sequence targeting integrin &agr;IIb caused a significant reduction of integrin &agr;IIb&bgr;3 (&agr;IIb&bgr;3) expression in GFP-positive platelets. It also inhibited &agr;IIb&bgr;3 activation assessed by the binding of JON/A, an antibody that recognizes activated &agr;IIb&bgr;3. Talin-1 silencing by the same method resulted in normal &agr;IIb&bgr;3 expression but deficient inside-out &agr;IIb&bgr;3 signaling. Conclusions— shRNA expression driven by the U6 promoter is preserved during megakaryopoiesis. This method facilitates functional analysis of targeted protein in platelet activation.