[Development of a novel aptamer blocking the interaction between the VWF A1 domain and platelet GP Ib for the treatment of arterial thrombosis].

A DNA aptamer to the von Willebrand factor (VWF) A1 domain, TAGX-0004, inhibits the binding of VWF to platelets. Nucleic acid aptamers are single-stranded DNA or RNA molecule forming three-dimensional structures that are capable of specifically binding to proteins and are expected to contribute to alternative medicine. ARC1779, an aptamer targeting the VWF A1 domain, had been evaluated in a phase II clinical trial of patients with acquired thrombotic thrombocytopenic purpura (aTTP); however, its development was terminated. Caplacizumab, an anti-VWF A1 domain nanobody, is now increasingly employed as first-line therapy for the treatment of aTTP in Western countries. However, there have been reports regarding adverse bleeding events and the high cost of the treatment. In this study, the inhibitory effects of TAGX-0004 were compared with those of ARC1779 and caplacizumab on in vitro platelet aggregation and thrombus formation. TAGX-0004 had an excellent high affinity to the VWF A1 domain and superior efficacy, such as its potent inhibitory activity in in vitro platelet aggregation and thrombus formation. Therefore, it can potentially overcome the problems associated with caplacizumab and can be developed as a promising drug not only for aTTP treatment but also for the treatment of the various VWF-mediated thrombotic disorders.

Novel aptamer to von Willebrand factor A1 domain (TAGX-0004) shows total inhibition of thrombus formation superior to ARC1779 and comparable to caplacizumab.

von Willebrand factor (VWF) is a blood glycoprotein that plays an important role in platelet thrombus formation through interaction between its A1 domain and platelet glycoprotein Ib. ARC1779, an aptamer to the VWF A1 domain, was evaluated in a clinical trial for acquired thrombotic thrombocytopenic purpura (aTTP). Subsequently, caplacizumab, an anti-VWF A1 domain nanobody, was approved for aTTP in Europe and the United States. We recently developed a novel DNA aptamer, TAGX-0004, to the VWF A1 domain; it contains an artificial base and demonstrates high affinity for VWF. To compare the effects of these three agents on VWF A1, their ability to inhibit ristocetin- or botrocetin-induced platelet aggregation under static conditions was analyzed, and the inhibition of thrombus formation under high shear stress was investigated in a microchip flow chamber system. In both assays, TAGX-0004 showed stronger inhibition than ARC1779, and had comparable inhibitory effects to caplacizumab. The binding sites of TAGX-0004 and ARC1779 were analyzed with surface plasmon resonance performed using alanine scanning mutagenesis of the VWF A1 domain. An electrophoretic mobility shift assay showed that R1395 and R1399 in the A1 domain bound to both aptamers. R1287, K1362, and R1392 contributed to ARC1779 binding, and F1366 was essential for TAGX-0004 binding. Surface plasmon resonance analysis of the binding sites of caplacizumab identified five amino acids in the VWF A1 domain (K1362, R1392, R1395, R1399, and K1406). These results suggested that TAGX-0004 possessed better pharmacological properties than caplacizumab in vitro and might be similarly promising for aTTP treatment.

Ets family members induce lymphangiogenesis through physical and functional interaction with Prox1.

Prox1 plays pivotal roles during embryonic lymphatic development and maintenance of adult lymphatic systems by modulating the expression of various lymphatic endothelial cell (LEC) markers, such as vascular endothelial growth factor receptor 3 (VEGFR3). However, the molecular mechanisms by which Prox1 transactivates its target genes remain largely unknown. Here, we identified Ets-2 as a candidate molecule that regulates the functions of Prox1. Whereas Ets-2 has been implicated in angiogenesis, its roles during lymphangiogenesis have not yet been elucidated. We found that endogenous Ets-2 interacts with Prox1 in LECs. Using an in vivo model of chronic aseptic peritonitis, we found that Ets-2 enhanced inflammatory lymphangiogenesis, whereas a dominant-negative mutant of Ets-1 suppressed it. Ets-2 also enhanced endothelial migration towards VEGF-C through induction of expression of VEGFR3 in collaboration with Prox1. Furthermore, we found that both Prox1 and Ets-2 bind to the VEGFR3 promoter in intact chromatin. These findings suggest that Ets family members function as transcriptional cofactors that enhance Prox1-induced lymphangiogenesis.

Th1/17 polarization and potential treatment by an anti-interferon-γ DNA aptamer in Hunner-type interstitial cystitis.

Hunner-type interstitial cystitis (HIC) is a rare, enigmatic inflammatory disease of the urinary bladder with no curative treatments. In this study, we aimed to characterize the unique cellular and immunological factors specifically involved in HIC by comparing with cystitis induced by Mycobacterium bovis bacillus Calmette-Guérin, which presents similar clinicopathological features to HIC. Here, we show that T helper 1/17 +polarized immune responses accompanied by prominent overexpression of interferon (IFN)-γ, enhanced cGAS-STING cytosolic DNA sensing pathway, and increased plasma cell infiltration are the characteristic inflammatory features in HIC bladder. Further, we developed a mouse anti-IFN-γ DNA aptamer and observed that the intravesical instillation of the aptamer significantly ameliorated bladder inflammation, pelvic pain and voiding dysfunction in a recently developed murine HIC model with little migration into the blood. Our study provides the plausible basis for the clinical translation of the anti-IFN-γ DNA aptamer in the treatment of human HIC.

Regulation of intracellular pH by p90Rsk-dependent activation of an Na(+)/H(+) exchanger in starfish oocytes.

Starfish oocytes arrest at metaphase of the first meiotic division (MI arrest) in the ovary and resume meiosis after spawning into seawater. MI arrest is maintained by lower intracellular pH (pH(i)) and release from arrest by cellular alkalization. To elucidate pH(i) regulation in oocytes, we cloned the starfish (Asterina pectinifera) Na(+)/H(+) exchanger 3 (ApNHE3) expressed in the plasma membrane of oocytes. The cytoplasmic domain of ApNHE3 contains p90 ribosomal S6 kinase (p90Rsk) phosphorylation sites, and injection of a constitutively active p90Rsk and the upstream regulator Mos to immature oocytes, stimulated an increase in pH(i). This increase was blocked by 5-(N-ethyl-N-isopropyl)-amiloride, a NHE inhibitor, and SL0101, a specific Rsk inhibitor. The MAPK kinase (MEK) inhibitor U0126 blocked the Mos-induced, but not the p90Rsk-induced, pH(i) increase, suggesting that the Mos-MEK-MAPK-p90Rsk pathway promotes ApNHE3 activation. In a cell-free extract, the Mos-MEK-MAPK-p90Rsk pathway phosphorylates ApNHE3 at Ser-590, -606, and -673. When p90Rsk-dependent ApNHE3 phosphorylation was blocked by a dominant-negative C-terminal fragment, or neutralizing antibody, the p90Rsk-induced pH(i) increase was suppressed in immature oocytes. However, ApNHE3 is up-regulated via the upstream phosphatidylinositol 3-kinase pathway before MAPK activation and the active state is maintained until spawning, suggesting that the p90Rsk-dependent ApNHE3 phosphorylation is unlikely to be the primary regulatory mechanism involved in MI arrest exit. After meiosis is completed, unfertilized eggs maintain their elevated pH(i) ( approximately 7.4) until the onset of apoptosis. We suggest that the p90Rsk/ApNHE3-dependent elevation of pH(i) increases fertilization success by delaying apoptosis initiation.

Identification of targets of Prox1 during in vitro vascular differentiation from embryonic stem cells: functional roles of HoxD8 in lymphangiogenesis.

During lymphatic development, Prox1 plays central roles in the differentiation of blood vascular endothelial cells (BECs) into lymphatic endothelial cells (LECs), and subsequently in the maturation and maintenance of lymphatic vessels. However, the molecular mechanisms by which Prox1 elicits these functions remain to be elucidated. Here, we identified FoxC2 and angiopoietin-2 (Ang2), which play important roles in the maturation of lymphatic vessels, as novel targets of Prox1 in mouse embryonic-stem-cell-derived endothelial cells (MESECs). Furthermore, we found that expression of HoxD8 was significantly induced by Prox1 in MESECs, a finding confirmed in human umbilical vein endothelial cells (HUVECs) and human dermal LECs (HDLECs). In mouse embryos, HoxD8 expression was significantly higher in LECs than in BECs. In a model of inflammatory lymphangiogenesis, diameters of lymphatic vessels of the diaphragm were increased by adenovirally transduced HoxD8. We also found that HoxD8 induces Ang2 expression in HDLECs and HUVECs. Moreover, we found that HoxD8 induces Prox1 expression in HUVECs and that knockdown of HoxD8 reduces this expression in HDLECs, suggesting that Prox1 expression in LECs is maintained by HoxD8. These findings indicate that transcriptional networks of Prox1 and HoxD8 play important roles in the maturation and maintenance of lymphatic vessels.

Aldosterone-induced kidney injury is mediated by NFκB activation.

BACKGROUND: Aldosterone induces inflammation and fibrosis in the kidney, while nuclear factor κB (NFκB) plays key roles in inflammation mediated by various cytokines. Here, we determined the roles of NFκB activation in aldosterone-induced kidney injury. METHODS: We used unilaterally nephrectomized rats with or without continuous aldosterone infusion and 0.9% saline as drinking water for 3 weeks. IMD-1041, an IKKß inhibitor, and spironolactone were orally administered to inhibit NFκB and mineralocorticoid receptor, respectively. RESULTS: The aldosterone-infused rats exhibited severe kidney injury, hypertension, and increased expression of pro-inflammatory and fibrotic proteins, osteopontin, fibrinogen, collagen type I, and PAI-1. Western blotting confirmed NFκB activation by aldosterone by the increased amount of p65 in the nuclear fraction of the kidney, and oral IMD-1041 prevented the kidney injury and lessened the increase in pro-inflammatory and fibrotic proteins without significant changes in blood pressures. In addition, changes in angiotensin-converting enzyme 2 (ACE2), which has been found to act as a protective factor in various kidney injury models, were examined. Immunofluorescence studies revealed the presence of ACE2 in the brush-border membrane of the proximal convoluted tubules and markedly blunted ACE2 staining in aldosterone-infused rats. The decrease in amount of ACE2 protein was confirmed by Western blotting, and IMD-1041 also prevented the decrease in ACE2. The administration of spironolactone also abolished the effects of aldosterone. CONCLUSION: Our results suggest that aldosterone induces kidney injury via activation of NFκB and mineralocorticoid receptor, and that decreased ACE2 expression may play an important role in aldosterone-induced kidney injury.

Anesthetic management of a patient with narcolepsy.

We report the anesthetic management of a narcoleptic patient performed using sevoflurane-remifentanil with bispectral index (BIS) monitoring. A 22-year-old man, who was diagnosed with narcolepsy at the age of 17, requested endoscopic sinus surgery, under general anesthesia, for chronic allergic rhinitis. On the morning of the day of operation, he took his daily dose of modafinil, used to control narcolepsy. Anesthesia was induced by 5% sevoflurane and maintained with sevoflurane and continuous infusion of remifentanil and 60% oxygen in conjunction with BIS monitoring. BIS values were between 47 and 58. Duration of surgery was 150 min. After surgery, the patient emerged from anesthesia within 10 min and was extubated. His recovery was uneventful. We found the use of BIS monitoring for titrating sevoflurane concentration in a narcoleptic patient is useful for preventing not only oversedation but also intraoperative awareness caused by the preoperative medication.

Awareness during anesthesia: the results of a questionnaire survey in Japan.

PURPOSE: We planned a survey to evaluate the current incidence and risk factors of intraoperative awareness. METHODS: A questionnaire survey was conducted via the Internet. The survey was designed to obtain information regarding cases involving intraoperative awareness in 2008. RESULTS: A total of 172 anesthesiologists answered the survey. The total number of reported anesthetic cases was 85,156. Twenty-four cases of definite or possible awareness were reported by 21 anesthesiologists, of which 14 were cases of definite awareness and ten of possible awareness. The incidence of awareness, including possible awareness, was 0.028%. Propofol was used in 21 cases, sevoflurane in two, and a high dose of fentanyl in one. Bispectral index (BIS) monitoring was used in seven cases (29%). Sixteen patients (67%) were <50 years old, six (26%) were men, and 17 (74%) were women. As the type of surgery, three cases (13%) involved gynecological surgeries and seven (30%) involved cervicofacial surgeries. During surgery, the memory at postural change was preserved in two cases. CONCLUSION: The most surprising finding of this study is that total intravenous anesthesia (TIVA) was used in 21 of the 24 (88%) cases of definite and possible awareness. Although the incidence of intraoperative awareness was compatible with the previous studies, meticulous care should be taken when anesthesia is performed by TIVA for high-risk patients. The results of this survey should be verified, as well as further continuous survey and prospective study, because this study was performed by an anonymous questionnaire survey conducted over only 1-year period.

[Coronary artery spasm during neurosurgical anesthesia].

We experienced a case of coronary artery spasm during neurosurgical anesthesia. A 69-year-old man was scheduled for craniotomy for cerebello-pontine angle meningioma. He had a history of cigarette smoking, but no history or evidence of ischemic heart disease. After the dura mater was opened, marked ST elevation on the ECG monitor followed by ventricular fibrillation was noticed. After successful resuscitation, the surgery was cancelled. Because the coronary angiography, immediately after surgery, demonstrated normal coronary arteries, coronary artery spasm was considered to be the cause of the ECG change. Possible triggering factor in this case was vagal stimulation due to surgical manipulation. Careful anesthetic management is required to prevent intraoperative coronary artery spasm even in patients without a history of ischemic heart disease during neurosurgery.

Osteochondral regeneration using scaffold-free constructs of adipose tissue-derived mesenchymal stem cells made by a bio three-dimensional printer with a needle-array in rabbits.

Osteoarthritis is a major joint disease for which medical interventions have been extensively investigated in humans and animals. In this study, we examined the regeneration of articular cartilage and subchondral bone using a scaffold-free construct consisting of adipose tissue-derived mesenchymal stem cells (AT-MSCs) fabricated using a bio three-dimensional (3D) printer. AT-MSCs were isolated from three rabbits and cultured to a number of sufficient for creation of 3D-printed constructs. One construct consisted of 960 spheroids obtained from 3.5 × 104 autologous AT-MSCs. The construct was then implanted into an osteochondral defect (diameter 4 mm and depth 4 mm) surgically bored into the left femoral trochlear groove of each rabbit. Three months after implantation, healing was assessed by computed tomography, magnetic resonance (MR) imaging, and pathology. MR images were evaluated based on a modified two-dimensional (2D)-magnetic resonance observation of cartilage repair tissue (MOCART) grading system, and gross and microscopic histology were scored according to the International Cartilage Repair Society scale. At the time of imaging, treated defects had become radiopaque, while control defects remained radiolucent. Total 2D-MOCART scores were higher in the implanted defects than in the controls, but not to a statistically significant extent. Similarly, average histological scores were comparable among all groups, although average gross scores were significantly higher in implanted defects than in controls. This is the first demonstration of a scaffold-free 3D-printed construct consisting of autologous AT-MSCs regenerating cartilage and subchondral bone within three months.

Hormone-induced cortical maturation ensures the slow block to polyspermy and does not couple with meiotic maturation in starfish.

Meiotic progression in starfish oocytes is reinitiated by a maturation-inducing hormone called 1-methyladenine (1-MeAde). In addition to meiotic maturation, 1-MeAde induces cortical maturation in which cortical granules become competent to discharge in response to fusion of a single sperm, which results in the formation of the fertilization envelope. We found that subthreshold concentrations of 1-MeAde induce cortical maturation without germinal vesicle breakdown (GVBD). During cortical maturation, the IP3 sensitivity of calcium stores was increased as well as during meiotic maturation. When oocytes were exposed with 1-MeAde only on a hemisphere of oocytes, the IP3 sensitivity of the cortical region was increased only in the exposed hemisphere, suggesting that signals and components involved in cortical maturation do not readily spread in the cytoplasm. Although a specific inhibitor of phosphatidylinositol-3 kinase, LY294002 blocked both GVBD and cortical maturation, a Cdc2 kinase inhibitor, roscovitine did not block cortical maturation. Inhibition of Akt activation by injecting the competitors for Akt phosphorylation and membrane recruitment also blocked cortical maturation. These results suggest that the signaling pathway leading to Akt activation is common in cortical maturation and meiotic maturation, and Cdc2 activation was not required for cortical maturation.

[Hypertension and tachycardia during endoscopic radial artery harvesting in two patients undergoing coronary artery bypass grafting].

Hypertension and tachycardia occurred during endoscopic radial artery harvesting in two patients undergoing coronary artery bypass grafting. Despite anesthesia maintained with moderate doses of fentanyl and isoflurane, hypertension and tachycardia occurred 10-15 min after tourniquet application and graft harvesting procedure. Tourniquet pain or direct stimulation to radial nerve by CO2 insufflation might be the causes. Although endoscopic radial artery harvesting is reported excellent for cosmetics with low incidence of complications such as infection and hematoma, hemodynamic change can occur during harvesting. Careful evaluation and management of hemodynamic changes should be exercised during this procedure in the patient with unstable angina and low cardiac function.

Modelling glioma invasion using 3D bioprinting and scaffold-free 3D culture.

Glioma is a highly aggressive form of brain cancer, with some subtypes having 5-year survival rates of less than 5%. Tumour cell invasion into the surrounding parenchyma seems to be the primary driver of these poor outcomes, as most gliomas recur within 2 cm of the original surgically-resected tumour. Many current approaches to the development of anticancer therapy attempt to target genetic weaknesses in a particular cancer, but may not take into account the microenvironment experienced by a tumour and the patient-specific genetic differences in susceptibility to treatment. Here we demonstrate the use of complementary approaches, 3D bioprinting and scaffold-free 3D tissue culture, to examine the invasion of glioma cells into neural-like tissue with 3D confocal microscopy. We found that, while both approaches were successful, the use of 3D tissue culture for organoid development offers the advantage of broad accessibility. As a proof-of-concept of our approach, we developed a system in which we could model the invasion of human glioma cells into mouse neural progenitor cell-derived spheroids. We show that we can follow invasion of human tumour cells using cell-tracking dyes and 3D laser scanning confocal microscopy, both in real time and in fixed samples. We validated these results using conventional cryosectioning. Our scaffold-free 3D approach has broad applicability, as we were easily able to examine invasion using different neural progenitor cell lines, thus mimicking differences that might be observed in patient brain tissue. These results, once applied to iPSC-derived cerebral organoids that incorporate the somatic genetic variability of patients, offer the promise of truly personalized treatments for brain cancer.

Degradation of polyubiquitinated cyclin B is blocked by the MAPK pathway at the metaphase I arrest in starfish oocytes.

In the starfish ovary, maturing oocytes stimulated by 1-methyladenine undergo synchronous germinal vesicle breakdown and then arrest in metaphase of the first meiotic division (metaphase I). Immediately after spawning, an increase of intracellular pH (pH(i)) from approximately 7.0 to approximately 7.3 is induced by Na(+)/H(+) antiporter in oocytes, and meiosis reinitiation occurs. Here we show that an endogenous substrate of the proteasome, polyubiquitinated cyclin B, was stable at pH 7.0, whereas it was degraded at pH 7.3. When the MAPK pathway was blocked by MEK inhibitor U0126, degradation of polyubiquitinated cyclin B occurred even at pH 7.0 without an increase of the peptidase activity of the proteasome. These results indicate that the proteasome activity at pH 7.0 is sufficient for degradation of polyubiquitinated cyclin B and that the MAPK pathway blocks the degradation of polyubiquitinated cyclin B in the maturing oocytes in the ovary. Immediately after spawning, the increase in pH(i) mediated by Na(+)/H(+) antiporter cancels the inhibitory effects of the MAPK pathway, resulting in the degradation of polyubiquitinated cyclin B and the release of the arrest. Thus, the key step of metaphase I arrest in starfish oocytes occurs after the polyubiqutination of cyclin B but before cyclin B proteolysis by the proteasome.

Metaphase I arrest of starfish oocytes induced via the MAP kinase pathway is released by an increase of intracellular pH.

Reinitiation of meiosis in oocytes usually occurs as a two-step process during which release from the prophase block is followed by an arrest in metaphase of the first or second meiotic division [metaphase I (MI) or metaphase II (MII)]. The mechanism of MI arrest in meiosis is poorly understood, although it is a widely observed phenomenon in invertebrates. The blockage of fully grown starfish oocytes in prophase of meiosis I is released by the hormone 1-methyladenine. It has been believed that meiosis of starfish oocytes proceeds completely without MI or MII arrest, even when fertilization does not occur. Here we show that MI arrest of starfish oocytes occurs in the ovary after germinal vesicle breakdown. This arrest is maintained both by the Mos/MEK/MAP kinase pathway and the blockage of an increase of intracellular pH in the ovary before spawning. Immediately after spawning into seawater, activation of Na+/H+ antiporters via a heterotrimeric G protein coupling to a 1-methyladenine receptor in the oocyte leads to an intracellular pH increase that can overcome the MI arrest even in the presence of active MAP kinase.