Novel Screening System for Biliary Excretion of Drugs Using Human Cholangiocyte Organoid Monolayers with Directional Drug Transport.

It has recently been reported that cholangiocyte organoids can be established from primary human hepatocytes. The purpose of this study was to culture the organoids in monolayers on inserts to investigate the biliary excretory capacity of drugs. Cholangiocyte organoids prepared from hepatocytes had significantly higher mRNA expression of CK19, a bile duct epithelial marker, compared to hepatocytes. The organoids also expressed mRNA for efflux transporters involved in biliary excretion of drugs, P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP). The subcellular localization of each protein was observed. These results suggest that the membrane-cultured cholangiocyte organoids are oriented with the upper side being the apical membrane side (A side, bile duct lumen side) and the lower side being the basolateral membrane side (B side, hepatocyte side), and that each efflux transporter is localized to the apical membrane side. Transport studies showed that the permeation rate from the B side to the A side was faster than from the A side to the B side for the substrates of each efflux transporter, but this directionality disappeared in the presence of inhibitor of each transporter. In conclusion, the cholangiocyte organoid monolayer system has the potential to quantitatively evaluate the biliary excretion of drugs. The results of the present study represent an unprecedented system using human cholangiocyte organoids, which may be useful as a screening model to directly quantify the contribution of biliary excretion to the clearance of drugs.

The comparison of the clinical outcomes and costs between surgical aortic valve replacement and transcatheter aortic valve implantation based on the Japanese Diagnosis Procedure Combination database.

PURPOSE: We evaluated the clinical outcomes and costs of surgical aortic valve replacement (SAVR) and transfemoral transcatheter aortic valve implantation (TAVI) for aortic stenosis using the Japanese Diagnosis Procedure Combination (DPC) database. METHODS: Using our extraction protocol, we retrospectively analyzed summary tables in the DPC database from 2016 to 2019, which were provided by the Ministry of Health, Labor and Welfare. A total of 27,278 patients were available (SAVR, n = 12,534; TAVI, n = 14,744). RESULTS: The TAVI group was older than the SAVR group (SAVR vs. TAVI: 74.6 vs. 84.5 years; P < 0.01), with a lower in-hospital mortality (1.0 vs. 0.6%; P < 0.01) and a shorter hospital stay (26.9 vs. 20.3 days; P < 0.01). TAVI conferred more total medical service reimbursement points than SAVR (493,944 vs. 605,241 points; P < 0.01), especially materials points (147,830 vs. 434,609 points; P < 0.01). Total insurance claims for TAVI were approximately 1 million yen higher than those for SAVR. Regarding the details of costs, only the operation cost was higher, while other costs were lower with TAVI than with SAVR. CONCLUSION: Our analysis revealed that both SAVR and TAVI showed acceptable clinical outcomes. TAVI was associated with higher total insurance claims than SAVR. If the material cost of TAVI operations can be reduced, greater cost-effectiveness can be expected.

[Surgical Left Atrial Ablation Through the Left Atrial Appendage During Aortic Valve Replacement].

This is a 2-case report of concomitant left atrial( LA) surgical ablation through the left atrial appendage( LAA) for atrial fibrillation( AF) during aortic valve replacement( AVR). Case 1, a 74-year-old man, and Case 2, an 85-years-old woman, were presented for AVR and AF. Under the cardio-pulmonary bypass, right and left pulmonary vein isolations( PVIs) were performed with radiofrequency( RF) ablation devices. Through the opened LAA, an RF lesion was placed to connect the bilateral PVIs and the LAA, and another RF line was placed toward the mitral annulus. A right atrial( RA) isthmus ablation was also performed through a RA incision. Postoperatively, sinus rhythm and both RA and LA contraction were obtained, and have been maintained for 7.5 and 5 years without classⅠ/Ⅲ antiarrhythmic drugs in Case 1 and 2, respectively. This procedure is considered to be effective and safe, although further study is needed.

Directional Drug Transport through Membrane-Supported Monolayers of Human Liver-Derived Cell Lines.

The aim of this work is to develop a new assay system for screening biliary excretion drugs. When monolayers of human liver-derived cell lines HepG2 and Huh-7 were grown on an insert membrane, the efflux ratio (ER: ratio of the apparent permeability coefficient in the basal-to-apical direction (Papp,B-to-A) to that in the apical to basal direction (Papp,A-to-B)) of sulfobromophthalein (BSP), a model substrate of multidrug resistance-associated protein 2 (MRP2), was greater than 1.0, indicating transport of BSP in the efflux direction. The efflux transport was significantly suppressed by MK-571, an inhibitor of MRPs, in both cell lines. Expression of MRP2 mRNA in HepG2 and Huh-7 was 3.5- and 1.4-fold higher, respectively, than in primary human hepatocytes, while expression of P-glycoprotein and breast cancer resistance protein mRNAs was markedly lower, supporting the idea that MRP2 is the main mediator of directional BSP transport in this assay system. The advantage of our system is the potential to quantitatively evaluate biliary excretion of MRP2 substrates in vitro.

[Tricuspid Valve Ring Annuloplasty with Posterior Annular Plication for Functional Tricuspid Regurgitation].

This report presents a modified procedure of tricuspid valve ring annuloplasty (R-TAP) with posterior annular plication for functional tricuspid regurgitation (TR). Sutures on the native annulus were placed by a standard fashion in R-TAP, and those on the posterior annulus and its bilateral commissures were passed through in a narrow range between the 3 and 4 o'clock positions of the 26-mm ring. The other sutures were done with an usual manner and the ring was fixed to the annulus, resulting in the posterior annular plication( bicuspidization). Follow-up was performed for more than 5 years( mean: 7.9 years, range:5.5~11.5 years) by echocardiography in 13 cases. Postoperative TR reduced significantly to less than moderate, which was maintained during the entire follow-up period, even in the case with atrial fibrillation. There was no sign of tricuspid stenosis. R-TAP with posterior annular plication was feasible, reproducible, and effective, although further investigation is needed.

Early results of expanding the anatomical indications for using a Gore Iliac branch endoprosthesis to treat aortoiliac and iliac aneurysms.

PURPOSE: To assess the safety and anatomical suitability of using a Gore Iliac Branch Endoprosthesis (IBE) in aortoiliac and iliac aneurysm repair. METHODS: Between 2017 and 2020, 20 patients underwent endovascular aneurysm repair (EVAR) with a Gore IBE device (bilateral IBE, n = 1) after expanding the instructions for use (IFU) criteria. We evaluated the early clinical outcomes and suitability of the IFU criteria, retrospectively. RESULTS: Six patients (30%) met all the IFU criteria. Anatomical suitability according to the IFU criteria for the collective total of 21 IBE limbs was confirmed for 10 (47.6%) proximal common iliac arteries, 21 (100%) external iliac arteries, 18 (85.7%) internal iliac arteries, and in the length from the lowest renal artery to the iliac bifurcation in 15 (71.8%) patients. Assisted primary technical success was achieved in all patients with various bail-out techniques. One patient (5%) required a bare-stent insertion 7 days after EVAR for severe stenosis in the ipsilateral limb caused by a small terminal aorta. There was no case of occlusion of an iliac branch component device. CONCLUSIONS: Gore IBEs were implanted safely and effectively with various bail-out techniques to repair aortoiliac and iliac aneurysms in our Japanese patients with a low rate of inclusion IFU criteria.

[Repeated Heart Failure after Transcatheter Aortic Valve Implantation Using Self-expandable Bioprosthetic Valve:Report of a Case].

The patient was an 81-year-old man. Transcatheter aortic valve implantation( TAVI) was performed for severe aortic stenosis using Evolut R. The patient moved to intensive care unit without an adverse event after the operation. But repeated acute heart failure occurred several times during hospital stay. Mitral regurgitation (MR) was worsened from mild at baseline to moderate or more by transthoracic echocardiography. Various factors that worsened MR after TAVI have been reported, and treatment strategy for severe aortic stenosis patients with MR should be carefully developed.

[Preservation of the Spinal Perfusion Artery during Descending Thoracic and Thoracoabdominal Aortic Surgery].

This is a 3-case report of successful descending thoracic and thoracoabdominal aortic surgery by preserving the spinal perfusion artery (SPA) identified preoperatively. In Case 1, an 85-year-old woman, computed tomography (CT) showed Crawford type Ⅴ thoracoabdominal aortic aneurysm (TAAA:60 mm) and a SPA originated from L2. In Case 2, a 76-year-old man, CT revealed type Ⅳ TAAA( 58 mm) and a SPA originated from Th11. In Case 3, a 74-year-old man, CT detected an infectious pseudoaneurysm(44 mm) in the descending thoracic aorta with 2 SPAs originating from Th10 and L2. The ranges of graft replacement were Th7-Th12, Th12-L4, and Th8-Th10, respectively, while preserving all SPAs. All patients recovered well without postoperative neurological deficits. Although the protective effect of the SPA preservation against the spinal cord ischemia is still controversial, preoperative identification of the SPA was useful for planning a surgical strategy for descending thoracic and thoracoabdominal aortic repair surgery.

[Retrosternal Giant Aortic Aneurysm;Report of Two Cases].

This is a 2-case report of successful aortic repair surgery for the retrosternal giant aortic aneurysm. Our surgical strategy is "deep hypothermia and left ventricular( LV) unloading under cardiopulmonary bypass before approaching to the aortic aneurysm" in case of possible catastrophic bleeding. Case 1, a 64-year-old woman, had a retrosternal pseudoaneurysm (80 mm) at the distal anastomosis of a Dacron graft used to replace the ascending aorta 7 years before. An LV vent tube was cannulated via the right upper pulmonary vein through an inferior T-shaped ministernotomy. Case 2, an 86-year-old woman, had a retrosternal chronic aortic dissecting aneurysm (66 mm). An LV vent cannula was inserted via the LV apex through a left minithoracotomy. Arch replacement and ascending aorta replacement were performed in Case 1 and 2, respectively, without cardiac, neurological, or any other complications. This strategy is safe and useful in a case with complex aortic disease.

[Mitral Valve Replacement with the On-X Valve].

This is a 3-case report of mitral valve replacement (MVR) with an On-X mechanical valve followed up to 10 years. Case 1(64-year-old man) and case 2 (66-year-old woman) experienced traffic accident and traumatic event, respectively, in their chronic phase after MVR. Case 1 had multiple bone fractures of the bilateral lower limbs, which was followed by systemic infection and pyogenic spondylitis. He needed long-term antibiotics therapy for more than 4 years. Case 2 fell down at home and severely hit her head, which resulted in a traumatic subarachnoid hemorrhage. She was in a deep coma, and needed discontinuation of anticoagulation therapy for 4 weeks. Case1, 2, and 3(54-year-old man) are doing well in New York Heart Association functional class I without any valve-related thromboembolic or hemorrhagic events at 10, 9 and 8 years after MVR, respectively. On-X valve performance has also been found well maintained in all cases by echocardiography, even after traumatic accident or discontinuation of anticoagulation in Case 1 and 2. In this report, the On-X mechanical valve demonstrated good midterm result of its valve performance in the mitral position and its potential advantages in antithrombogenicity.

Evaluation of the metabolic capability of primary human hepatocytes in three-dimensional cultures on microstructural plates.

The NanoCulture Plate (NCP) is a novel microstructural plate designed as a base for the three-dimensional culture of cells/tissues. This study examined whether or not the metabolic capability of human primary hepatocytes is well maintained during culture on NCPs. The hepatocytes formed aggregates after seeding and their ATP content was well maintained during culture for 21 days. Expression of CYP1A2, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 mRNAs was detected throughout the 21-day culture period. Addition of CYP substrate drugs (midazolam, diclofenac, lamotrigine and acetaminophen) resulted in the formation of multiple metabolites with a corresponding decrease in the amounts of the unchanged compounds. The inducers omeprazole, phenobarbital and rifampicin increased the levels of CYP1A2, 2B6 and 3A4 mRNAs by 110-fold, 12.5-fold and 5.4-fold, respectively, at day 2, compared with control human hepatocytes. CYP activities were also increased at 2 days after inducer treatment (CYP1A2, 2.2-fold; CYP2B6, 20.6-fold; CYP3A4, 3.3-fold). The results indicate that the hepatocyte spheroids on NCP have detectable and inducible metabolic abilities during the 7-day culture period.

Time-lapse imaging assay using the BioStation CT: a sensitive drug-screening method for three-dimensional cell culture.

Three-dimensional (3D) cell culture is beneficial for physiological studies of tumor cells, due to its potential to deliver a high quantity of cell culture information that is representative of the cancer microenvironment and predictive of drug responses in vivo. Currently, gel-associated or matrix-associated 3D cell culture is comprised of intricate procedures that often result in experimental complexity. Therefore, we developed an innovative anti-cancer drug sensitivity screening technique for 3D cell culture on NanoCulture Plates (NCP) by employing the imaging device BioStation CT. Here, we showed that the human breast cancer cell lines BT474 and T47D form multicellular spheroids on NCP plates and compared their sensitivity to the anti-cancer drugs trastuzumab and paclitaxel using the BioStation CT. The anticancer drugs reduced spheroid migration velocity and suppressed spheroid fusion. In addition, primary cells derived from the human breast cancer tissues B58 and B61 grown on NCP plates also exhibited similar drug sensitivity. These results were in good agreement with the conventional assay method using ATP quantification. We confirmed the antitumor effects of the drugs on cells seeded in 96-well plates using the BioStation CT imaging technique. We expect this method to be useful in research for new antitumor agents and for drug sensitivity tests in individually-tailored cancer treatments.

[Intraoperative Endoscopic Evaluation of the Aortic Valve in Aortic Valve Repair].

This report discusses intraoperative endoscopic evaluation of the aortic valve performed in 2 cases of aortic valve repair. The "direct" or real image by the endoscopy helped to confirm the preoperatively-known lesion and even to detect a new legion which was not detected preoperatively. The endoscopy also enabled the evaluation of the aortic valve under the pressure-loaded condition without releasing the aortic clamp. Postoperative aortic regurgitation was grade I or less in both cases, although it progressed to grade II at 1 year in case 2. Echocardiographic parameters demonstrated no change in the size of the aortic root configuration for 8 and 5 years in case 1 and case 2, respectively. Intraoperative aortic endoscopy was useful to define the pathogenesis of aortic regurgitation and to evaluate the cusp repair procedures, which may contribute to a good mid-term result of aortic valve repair.

Operative technique for tracheo-innominate artery fistula repair.

Tracheo-innominate artery fistula is fatal unless treated surgically. We describe our surgical approach and results in seven patients. The average patient age was 15.7 years; all patients had prior severe neurological deficits. Three of seven patients were in hemorrhagic shock; control of preoperative bleeding was achieved with tracheostomy tube cuff overinflation. The innominate artery and the trachea were exposed through a collar incision and partial upper sternotomy. The innominate artery was divided at the aortic arch and at the bifurcation, with one exception. Cerebral blood flow was monitored by the blood pressure difference in the bilateral upper extremities and by near-infrared spectroscopy. The tracheal fistula was left adherent to the innominate artery in all but one patient. All patients were discharged without new neurologic deficits or severe morbidity. Overall survival was 84% at 37 months, without any vascular, tracheal, or neurological events.

Three-Dimensional Bio-Printed Tubular Tissue Using Dermal Fibroblast Cells as a New Tissue-Engineered Vascular Graft for Venous Replacement.

The current study was a preliminary evaluation of the feasibility and biologic features of three-dimensionally bio-printed tissue-engineered (3D bio-printed) vascular grafts comprising dermal fibroblast spheroids for venous replacement in rats and swine. The scaffold-free tubular tissue was made by the 3D bio-printer with normal human dermal fibroblasts. The tubular tissues were implanted into the infrarenal inferior vena cava of 4 male F344-rnu/rnu athymic nude rats and the short-term patency and histologic features were analyzed. A larger 3D bio-printed swine dermal fibroblast-derived prototype of tubular tissue was implanted into the right jugular vein of a swine and patency was evaluated at 4 weeks. The short-term patency rate was 100%. Immunohistochemistry analysis showed von Willebrand factor positivity on day 2, with more limited positivity observed on the luminal surface on day 5. Although the cross-sectional area of the wall differed significantly between preimplantation and days 2 and 5, suggesting swelling of the tubular tissue wall (both p < 0.01), the luminal diameter of the tubular tissues was not significantly altered during this period. The 3D bio-printed scaffold-free tubular tissues using human dermal or swine fibroblast spheroids may produce better tissue-engineered vascular grafts for venous replacement in rats or swine.

Detection of peripartum myocardial burden by vector-projected 187 channel electrocardiography and serum NT-proBNP.

There is no reliable method of screening for pregnant women at high risk of developing severe myocardial disorders. In this study, we used vector-projected 187 channel electrocardiography (DREAM-ECG) and serum biochemical markers to evaluate peripartum myocardial burden in pregnant women. Forty-one pregnant women were examined at 36-37 weeks gestation (GW36), 7 days postpartum (PPD7), and 1 month postpartum (PPM1). Ten non-pregnant control women were assessed at a single time point. Heart rate, sympathetic index, and repolarization index (RTc dispersion) were quantified using the DREAM-ECG system, and serum levels of NT-proBNP, cardiac troponin T, estrogen, and progesterone were determined. Heart rate and the sympathetic index decreased from GW36 to PPM1 (P = 0.0031). The repolarization index decreased over time and was greater than in non-pregnant controls (31 ± 13 ms). Estrogen and progesterone at PPD7 and PPM1 were significantly lower than those at GW36 (P < 0.0001, P < 0.001). NT-proBNP at PPD7 was greater than at GW36 (median 29 pg/mL at GW36, 86 pg/mL at PPD7), and decreased at PPM1 in comparison to PPD7 (median 18.5 pg/mL). Troponin T was in the normal range during the whole period (< 0.003 ng/mL). In conclusion, these results indicate that the peripartum myocardial burden in pregnant women does not return to normal nonpregnant levels by PPM1. We propose that both repolarization indexes such as RTc dispersion by DREAM-ECG and serum biochemical markers may identify pregnant women at high risk of developing severe myocardial damage in the peripartum period.

Scaffold-free human vascular calcification model using a bio-three-dimensional printer.

Morbidity and mortality rates associated with atherosclerosis-related diseases are increasing. Therefore, developing new research models is important in furthering our understanding of atherosclerosis and investigate novel treatments. Here, we designed novel vascular-like tubular tissues from multicellular spheroids composed of human aortic smooth muscle cells, endothelial cells, and fibroblasts using a bio-3D printer. We also evaluated their potential as a research model for Mönckeberg's medial calcific sclerosis. The tubular tissues were sufficiently strong to be handled 1 week after printing and could still be cultured for 3 weeks. Histological assessment showed that calcified areas appeared in the tubular tissues within 1 week after culture in a medium containing inorganic phosphate (Pi) or calcium chloride as the calcification-stimulating factors. Calcium deposition was confirmed using micro-computed tomography imaging. Real-time quantitative reverse transcription polymerase chain reaction analysis revealed that the expression of osteogenic transcription factors increased in calcified tubular tissues. Furthermore, the administration of Pi and rosuvastatin enhanced tissue calcification. The bio-3D printed vascular-like tubular structures, which are composed of human-derived cells, can serve as a novel research model for Mönckeberg's medial calcific sclerosis.

How to select the optimal size of frozen elephant trunk in total arch replacement for type A acute aortic dissection.

BACKGROUND: Total arch replacement (TAR) with a frozen elephant trunk (FET) is a common technique for acute aortic dissection, but there is no consensus on the optimal size of the FET. METHODS: Forty-four patients who underwent TAR with FET for acute aortic dissection at our hospital since 2014 were included. The aortic diameter obtained from FET was measured on postoperative computed tomography (CT) and the estimated oversizing ratio was calculated. We investigated the relationship between the estimated oversizing ratio and postoperative outcomes. We also measured the maximum true lumen diameter, circumference of the true lumen, and total aortic diameter at the same level as the FET end on preoperative CT and examined the correlation with the aortic diameter obtained from FET. RESULTS: The average estimated oversizing ratio was 109%. Early postoperative CT showed complete thrombosis of the false lumen in 41 (93.2%) patients. No distal stent graft-induced new entry occurred during follow-up. The correlation coefficients between the three measurements and aortic diameter obtained from FET were 0.64 (maximum true lumen diameter), 0.76 (true lumen diameter calculated from circumference), and 0.72 (total aortic diameter), respectively. CONCLUSIONS: The aortic diameter obtained from FET on postoperative CT was strongly correlated with the true lumen diameter calculated from the circumference and total aortic diameter on preoperative CT. It is reasonable to select a size of approximately 130% of the true lumen diameter calculated from the circumference or 80% to 85% of the total aortic diameter.

Fluid resuscitation of hemorrhagic shock with hemoglobin vesicles in Beagle dogs: pilot study.

Resuscitation of hemorrhagic shock requires volume replacement and restoration of oxygen metabolism. Artificial oxygen carriers that can both expand blood volume and deliver oxygen have been developed as resuscitation fluids. We employed hemoglobin vesicles (HbV), a cellular-type artificial oxygen carrier, in a Beagle dog hemorrhagic shock model to prove the efficacy of HbV. Hemorrhagic shock was introduced in splenectomized Beagle dogs by withdrawing 50% of circulating blood from the femoral artery. Shock was maintained for 60 minutes before isovolemic resuscitation with HbV dispersed in 5% albumin in saline (HbV), lactated Ringer's solution (LR), 5% human serum albumin in saline (HSA), or autologous shed blood (ASB). One animal in the LR group died 150 min after resuscitation. All other animals survived 4 h of the experiment. The mean arterial pressure remained significantly lower in the LR group than in the HbV group but did not differ significantly among the HbV, Alb, and ASB groups. Immediately after resuscitation, the HbV group showed a significantly higher mean pulmonary arterial pressure, which decreased within 10 minutes to the baseline level. The cardiac output was significantly higher in the Alb group than in the others, indicating compensation for low oxygen delivery per unit blood. The post-resuscitation hematocrit was 36% in the ASB group and decreased in the other groups (20-22%). Serum chemistry data from the HbV group were unremarkable. HbV contributed 32% of the post-resuscitation oxygen delivery. Collectively, HbV is comparable to ASB and HSA as a resuscitation fluid and is an effective oxygen carrier.

Body surface two-dimensional spectral map of atrial fibrillation using vector-projected 187 channel electrocardiography.

Atrial cycle length during atrial fibrillation and flutter waves may be correlated with atrial refractoriness and organization. The nature of the frequency by spectral analysis may reflect a profile of atrial cycle length. In this study, we developed a novel body surface 2-dimensional spectral map during fibrillation using vector-projected 187 channel ECG (187ch VP-ECG).The study consisted of 28 patients (24 with atrial fibrillation (AFIB) and 4 atrial flutter (AFL) with valvular heart disease). We performed spectral analysis by maximum entropy modeling (MEM) in 4 second nonaveraged 187ch electrical current waves by 187ch VP-ECG. Body surface spectral features were displayed according to the frequency and power magnitude components. We verified the accuracy of the spectral features by a 64ch magnetocardiography (MCG). The average dominant frequency in AFL by 187ch VP-ECG was lower than those in AFIB (4.6 ± 0.9 Hz in AFL, 7.2 ± 0.9 Hz in AFIB, P < 0.01). Comparison of average dominant frequency between 187ch VP-ECG and 64 ch MCG demonstrated good agreement (y = 0.86x+0.84, r(2) = 0.89, P < 0.0001). Body surface 2-dimensional spectral features demonstrated homogenous spectrum patterns in AFL, and in-homogenous spectrum patterns in AFIB.In conclusion, novel body surface spectral mapping using 187ch VP-ECG may represent a 2-dimensional spectral feature that may be related to atrial refractoriness and organization.