Tissue engineering a human phalanx.

A principal purpose of tissue engineering is the augmentation, repair or replacement of diseased or injured human tissue. This study was undertaken to determine whether human biopsies as a cell source could be utilized for successful engineering of human phalanges consisting of both bone and cartilage. This paper reports the use of cadaveric human chondrocytes and periosteum as a model for the development of phalanx constructs. Two factors, osteogenic protein-1 [OP-1/bone morphogenetic protein-7 (BMP7)], alone or combined with insulin-like growth factor (IGF-1), were examined for their potential enhancement of chondrocytes and their secreted extracellular matrices. Design of the study included culture of chondrocytes and periosteum on biodegradable polyglycolic acid (PGA) and poly-l-lactic acid (PLLA)-poly-ε-caprolactone (PCL) scaffolds and subsequent implantation in athymic nu/nu (nude) mice for 5, 20, 40 and 60 weeks. Engineered constructs retrieved from mice were characterized with regard to genotype and phenotype as a function of developmental (implantation) time. Assessments included gross observation, X-ray radiography or microcomputed tomography, histology and gene expression. The resulting data showed that human cell-scaffold constructs could be successfully developed over 60 weeks, despite variability in donor age. Cartilage formation of the distal phalanx models enhanced with both OP-1 and IGF-1 yielded more cells and extracellular matrix (collagen and proteoglycans) than control chondrocytes without added factors. Summary data demonstrated that human distal phalanx models utilizing cadaveric chondrocytes and periosteum were successfully fabricated and OP-1 and OP-1/IGF-1 accelerated construct development and mineralization. The results suggest that similar engineering and transplantation of human autologous tissues in patients are clinically feasible. Copyright © 2016 John Wiley & Sons, Ltd.

[Resection of a metastatic left ventricular tumor with an aid of thoracoscopy].

A 61-year-old woman who complained of chest pain and cough was admitted to our hospital. She was diagnosed with multiple metastasis of breast or lung cancer, and a cardiac tumor was detected by echocardiography during chemotherapy. The tumor was located on the papillary muscle near the apex, had a smooth surface, and was well mobile. Emergency operation was performed because the tumor was considered to be a cause of cerebral infarction. Under cardiopulmonary bypass, resection of the tumor was performed by trans-mitral-valve approach. By using a thoracoscope, we could share information and obtain the details of the tumor during the operation. Resection using a trans-mitral-valve approach with an aid of thoracoscopy is considered useful.

Effect of CYP2D6 polymorphism on pharmacokinetics of a novel ACAT inhibitor, pactimibe and its unique metabolite, R-125528.

PURPOSE: Pactimibe is a novel ACAT inhibitor. The pharmacokinetics of pactimibe and its pharmacologically inactive plasma metabolite, R-125528, of which the main clearance pathway is CYP2D6, was affected by coadministration of quinidine. The aim of this study was to investigate the influence of CYP2D6 polymorphism on pharmacokinetics of pactimibe and R-125528. In addition, exposure was examined after multiple doses of pactimibe sulfate in CYP2D6 poor metabolizer (PMs). METHODS: 24 healthy male Caucasian volunteers, genotyped as extensive, intermediate, and poor metabolizers, were received single dose of 25 mg pactimibe. In a multiple-dose study, six CYP2D6 PMs received 100 mg pactimibe for 21 days and exposure of pactimibe and R-125528 was examined. RESULTS: In contrast to the mild 1.7-fold increase in AUC0-inf of pactimibe, a marked 3.1-fold increase in AUC0-tz of R-125528 was observed in CYP2D6 PMs. After multiple doses of 100 mg pactimibe to CYP2D6 PMs, the accumulation ratio of R-125528 reached 8.8-fold, however, the exposure of R-125528 in CYP2D6 PMs was covered by the exposure in additional metabolite safety testing. CONCLUSIONS: Although CYP2D6 polymorphism greatly affected the pharmacokinetics of R-125528 rather than pactimibe, the exposure in CYP2D6 PMs after a multiple dose of 100 mg pactimibe sulfate was covered by additional non-clinical metabolite safety testing. The finding is clinically informative with respect to the safety testing of drug metabolite present at disproportionately high levels in a special population with specific genetic back ground.

Gemfibrozil and its glucuronide inhibit the hepatic uptake of pravastatin mediated by OATP1B1.

When pravastatin (40 mg/day) was co-administered with gemfibrozil (600 mg, b.i.d., 3 days) to man, the AUC of pravastatin increased approximately 2-fold. We have clarified that OATP1B1 is a key determinant of the hepatic uptake of pravastatin in humans. Thus, we hypothesized that gemfibrozil and the main plasma metabolites, a glucuronide (gem-glu) and a carboxylic acid metabolite (gem-M3), might inhibit the hepatic uptake of pravastatin and lead to the elevation of the plasma concentration of pravastatin. Gemfibrozil and gem-glu inhibited the uptake of (14)C-pravastatin by human hepatocytes with K(i) values of 31.7 microM and 15.7 microM, respectively and also inhibited pravastatin uptake by OATP1B1-expressing Xenopus laevis oocytes with K(i) values of 15.1 microM and 7.6 microM. Additionally, we examined the biliary transport of pravastatin and demonstrated that pravastatin was transported by MRP2 using both human canalicular membrane vesicles (hCMVs) and human MRP2-expressing vesicles. However, gemfibrozil, gem-glu and gem-M3 did not affect the biliary transport of pravastatin by MRP2. Considering the plasma concentrations of gemfibrozil and gem-glu in humans, the inhibition of OATP1B1-mediated hepatic uptake of pravastatin by gem-glu would contribute, at least in part, to the elevation of plasma concentration of pravastatin by the concomitant use of gemfibrozil.

Inhibition of human organic anion transporter 3 mediated pravastatin transport by gemfibrozil and the metabolites in humans.

Coadministration of gemfibrozil (600 mg, b.i.d., 3 days) with pravastatin (40 mg/day) decreased the renal clearance of pravastatin by approximately 40% in healthy volunteers. To investigate the mechanism of this drug-drug interaction in the renal excretion process, we undertook an uptake study of pravastatin using human organic anion transporters (hOATs)-expressing S2 cells. hOAT3 and hOAT4 transported pravastatin in a saturatable manner with Michaelis--Menten constants of 27.7 microM and 257 microM respectively. On the other hand, hOAT1 and hOAT2 did not transport pravastatin. Gemfibrozil and its glucuronide and carboxylic metabolite forms inhibited the uptake of pravastatin by hOAT3 with IC(50) values of 6.8 microM, 19.7 microM and 5.4 microM, respectively. Considering the plasma concentrations of gemfibrozil and its metabolites in humans, the inhibition of hOAT3-mediated pravastatin transport by gemfibrozil and its metabolites would lead to a decrease in the renal clearance of pravastatin in clinical settings.

Renal handling of CS-023 (RO4908463), a novel parenteral carbapenem antibiotic, in rabbits in comparison with meropenem.

The plasma half-life of CS-023 (RO4908463), a novel parenteral carbapenem antibiotic, is longer than that of meropenem in animals and humans. To address this issue, renal clearance studies were conducted in rabbits. A constant rate infusion of CS-023 and meropenem was conducted in male Japanese White rabbits. Concentrations in the plasma, urine and renal cortex were measured to evaluate renal clearance and renal tissue uptake. CS-023 showed a clearance ratio (renal clearance/glomerular filtration rate) of around 1, which was not affected by co-administration of probenecid or p-aminohippurate. On the other hand, meropenem exhibited a clearance ratio of around 3, which was significantly decreased to 1 by co-administration of probenecid. p-Aminohippurate, in contrast, had no effect. The renal cortex/plasma concentration ratio of CS-023 was around 0.6 with or without probenecid co-administration. This ratio of meropenem was around 3, which was decreased significantly by co-administration of probenecid to around 0.6. These data suggest that meropenem is secreted in the renal tubules via organic anion transporters, but CS-023 is not. The present findings in rabbits would indicate that a lack of renal tubular secretion of CS-023 is a reason for the long plasma half-life compared with meropenem.

Transatrial repair of ventricular septal rupture under preoperative localization by transesophageal echocardiography.

We report about a 71-year-old woman with postinfarction ventricular septal rupture who was successfully treated by the transatrial closure under preoperative localization by transesophageal echocardiography. In an attempt at transatrial repair of the ventricular septal rupture, the most important thing is preoperative localization of the defect in the septum, which is located high and posterior, where it is smooth with relatively few trabeculations and can be readily exposed by retraction of the tricuspid valve.

Mr flow measurement in the internal mammary artery-to-coronary artery bypass graft: comparison with graft stenosis at radiographic angiography.

PURPOSE: To evaluate the sensitivity and specificity of breath-hold magnetic resonance (MR) flow measurement for detection of significant stenosis in internal mammary artery bypass grafts. MATERIALS AND METHODS: Twenty-six consecutive patients who had undergone coronary artery bypass surgery were examined. Breath-hold velocity-encoded cine MR images were obtained at the midpoint of the internal mammary artery between its origin from the subclavian artery and the distal anastomosis to the left anterior descending artery. RESULTS: MR images were obtained successfully in 24 patients. At conventional angiography, no significant stenosis was observed in 17 patients (group A), and significant stenosis (diameter > 70%) was observed in seven patients (group B). The mean diastolic-to-systolic peak velocity ratio in group B (0.61 +/- 0.44 [SD]) was significantly lower than that in group A (1.88 +/- 0.96; P <.01). Evaluation of graft stenosis with the diastolic-to-systolic peak velocity ratio revealed a sensitivity of 86% and a specificity of 88%. The mean blood flow rate at baseline in group B (16.9 mL/min +/- 5.5) was significantly lower than that in group A (79.8 mL/min +/- 38.2; P <.01). The sensitivity and specificity of MR blood flow measurement in predicting significant stenosis were 86% and 94%, respectively. The mean pharmacologic flow reserve ratios were 2.00 +/- 1.43 in group A and 1.39 +/- 1.46 in group B (P >.05). CONCLUSION: Fast MR blood flow measurement at baseline is highly useful for predicting significant stenosis in internal mammary arterial grafts.

Human liver-specific organic anion transporter, LST-1, mediates uptake of pravastatin by human hepatocytes.

Involvement of LST-1 (a human liver-specific transporter, also called OATP2) as the major transporter in the uptake of pravastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, by human liver was demonstrated. The hepatic uptake of pravastatin evaluated using human hepatocytes was Na(+)-independent and reached saturation with a Michaelis constant (K(m)) of 11.5 +/- 2.2 microM. The uptake of pravastatin was temperature-dependent and was inhibited by estradiol-17beta-D-glucuronide, taurocholic acid, bromosulfophthalein, and simvastatin acid, but not by p-aminohippurate. Estradiol-17beta-D-glucuronide competitively inhibited pravastatin uptake with an inhibition constant comparable to the K(m) value for estradiol-17beta-D-glucuronide transport, indicating that a common transporter mediates the transport of pravastatin and estradiol-17beta-D-glucuronide in human hepatocytes. The results obtained with human hepatocytes agreed with those obtained with LST-1 expressing Xenopus oocytes. Oocytes microinjected with human liver polyadenylated mRNA showed Na(+)-independent uptake of pravastatin and estradiol-17beta-D-glucuronide. A simultaneous injection of LST-1 antisense oligonucleotides completely abolished this uptake. Expression of LST-1 was immunohistochemically demonstrated in the human hepatocytes, but not in Hep G2 cells, which showed very low uptake of pravastatin. Therefore, LST-1 was regarded as a key molecule for pravastatin in liver-specific inhibition of cholesterol synthesis, making pravastatin accessible to the target enzyme, which would otherwise not be inhibited by this hydrophilic drug.

LST-2, a human liver-specific organic anion transporter, determines methotrexate sensitivity in gastrointestinal cancers.

BACKGROUND & AIMS: One approach to the development of targeted cancer chemotherapy exploits increased uptake of the agent into neoplastic cells. In this scenario, higher concentrations of the agent in cancer cells are responsible for differential killing, whereas the low concentration in normal human cells decreases side effects. The aim of this study was to isolate an organic anion transporter that is weak in normal cells, but abundantly expressed in cancer cells, to deliver the anticancer drugs to the cells. METHODS: A human liver complementary DNA (cDNA) library was screened with liver-specific transporter (LST)-1 cDNA as a probe. Northern blot analyses were performed using the isolated cDNA (termed LST-2). An LST-2-specific antibody was raised, and immunohistochemical analyses including immunoelectron microscopy were performed. Xenopus oocyte expression system was used for functional analysis. We also established a permanent cell line that consistently expresses LST-2 to examine the relationship between methotrexate uptake and sensitivity. RESULTS: The isolated cDNA, LST-2, has 79.7% of overall homology with human LST-1. LST-2 exclusively expressed in the liver under normal conditions and its immunoreactivity was highest at the basolateral membrane of the hepatocytes around the central vein. Although its weak expression in the liver, LST-2 is abundantly expressed in the gastric, colon, and pancreatic cancers. On the other hand, the LST-1 was only detected in a hepatic cell line. LST-2 transports methotrexate in a saturable and dose-dependent manner. Furthermore, introduction of the LST-2 gene into mammalian cells potentiates sensitivity to methotrexate. CONCLUSIONS: LST-2 is one of the prime candidate molecules for determining methotrexate sensitivity and may be a good target to deliver anticancer drugs to the gastrointestinal cancers.

Identification of thyroid hormone transporters in humans: different molecules are involved in a tissue-specific manner.

We have recently identified that rat organic anion transporters, polypeptide2 (oatp2) and oatp3, both of which transport thyroid hormones. However, in humans the molecular organization of the organic anion transporters has diverged, and the responsible molecule for thyroid hormone transport has not been clarified, except for human liver-specific transporter (LST-1) identified by us. In this study we isolated and characterized a novel human organic anion transporter, OATP-E from human brain. The isolated complementary DNA encodes a polypeptide of 722 amino acids with 12 transmembrane domains. A rat counterpart, oatp-E, was also identified. Homology analysis and the phylogenetic tree analysis revealed that OATP-E/oatp-E is a subfamily of the organic anion transporter. Human OATP-E transported 3,3',5-triiodo-L-thyronine (K(m), 0.9 microM), thyronine, and rT(3) in a Na(+)-independent manner. Although the clone was isolated from the brain, OATP-E messenger RNA was abundantly expressed in various peripheral tissues. The rat counterpart, oatp-E, also transported 3,3',5-triiodo-L-thyronine. In addition, in this study we revealed that human OATP, which is exclusively expressed in the brain, transported 3,3',5-triiodo-L-thyronine (K(m), 6.5 microM), T(4) (K(m), 8.0 microM), and rT(3). These data suggest that in humans, several different molecules are involved in transporting thyroid hormone: OATP in the brain, LST-1 in the liver, and OATP-E in peripheral tissues.

Less invasive therapy using endovascular stent graft repair and video-assisted thoracoscopic surgery for ruptured acute aortic dissection.

We report a 75-year-old man with a ruptured acute thoracic aortic dissecting hematoma treated using endovascular stent grafting and video-assisted thoracoscopic surgery. This less invasive therapy is a good therapeutic option even in ruptured acute aortic dissections, particularly given the difficulty of surgery.

Molecular identification of a rat novel organic anion transporter moat1, which transports prostaglandin D(2), leukotriene C(4), and taurocholate.

We have isolated a rat novel multispecific organic anion transporter, moat1. The isolated clones were originated by alternative splicing of the moat1 mRNA. The nucleotide sequences predict a protein of 682 amino acids with moderate sequence similarity to LST-1, the oatp family, and the prostaglandin transporter. Northern blot analysis of rat moat1 identified a predominant transcript of 4.4 kilonucleotides in all tissues. Northern blot and in situ hybridization analyses of rat brain further indicated that moat1 mRNA is widely distributed in neuronal cells of the central nervous system, especially in the hippocampus and cerebellum. moat1 transports prostaglandin D(2) (K(m); 35.5 nM), leukotriene C(4) (K(m); 3.2 microM) and taurocholate (K(m); 17.6 microM) in a sodium-independent manner. moat1 also transports prostaglandin E(1), E(2), thromboxane B(2), and iloprost but not dehydroepiandrosterone sulfate and digoxin, of which the substrate specificity is similar, but definitively different from those of any other organic anion transporters.

[The effects of intravenous milrinone for the patient undergoing CABG].

The hemodynamic effects of intravenous infusion of milrinone were evaluated in 25 patients undergoing CABG using an internal mammary artery graft. Milrinone was administered to 9 patients at the time of weaning from cardiopulmonary bypass, at a dosage of 3 to 5 micrograms/kg/min. Postoperative cardiac function was compared in this group versus the other 17 patients who were treated without milrinone. We determined such parameters as cardiac index, wedge pressure and mean pulmonary pressure. Our findings did not show any significant difference between the 2 groups. We also studied a subject of low-output patients (EF < 0.5). In the patients with low-cardiac output, the use of milrinone in addition to standard postoperative administration of low-dose dopamine reduced mean pulmonary pressure and wedge pressure. Thus, milrinone not only improved the left ventricular function, but also expanded the pulmonary vascular bed.

Molecular characterization and functional regulation of a novel rat liver-specific organic anion transporter rlst-1.

BACKGROUND & AIMS: Recently, we isolated a new complementary DNA (cDNA) encoding human liver-specific organic anion transporter (LST-1), representing the multispecificity of human liver. The aim of this study was to isolate a rat counterpart of human LST-1 and examine the expression regulation of its messenger RNA (mRNA) to clarify the molecular basis of cholestasis. METHODS: A rat liver cDNA library was screened with human LST-1 cDNA as a probe. Xenopus oocyte expression system was used for functional analysis. Northern blot analyses were performed using the isolated cDNA (termed rlst-1). The bile duct ligation model and the cecum ligation and puncture model were used for expression analyses. RESULTS: rlst-1 encodes 652 amino acids, predicting at least 11 transmembrane regions. The overall homology with human LST-1 was 60.2%, which is the highest among all known organic anion transporters. rlst-1 also belongs to the same new gene family as human LST-1, located between the organic anion transporter family and the prostaglandin transporter. rlst-1 preferably transports taurocholate (K(m), 9.45 micromol/L) in an Na(+)-independent manner. The rlst-1 mRNA is exclusively expressed in the liver. In both the bile duct ligation model and the cecum ligation and puncture model, mRNA expression levels of rlst-1 were down-regulated. CONCLUSIONS: rlst-1 is a counterpart of human LST-1 and is one of the important transporters in rat liver for the clearance of bile acid. The expression of rlst-1 may be under feedback regulation of cholestasis by biliary obstruction and/or sepsis.

Pravastatin, an HMG-CoA reductase inhibitor, is transported by rat organic anion transporting polypeptide, oatp2.

PURPOSE: We previously demonstrated the HMG-CoA reductase inhibitor, pravastatin, is actively taken up into isolated rat hepatocytes through multispecific organic anion transporters. The present study examined whether a newly cloned organic anion transporting polypeptide (oatp2) transports pravastatin. METHODS: We investigated functional expression of oatp2 in Xenopus laevis oocytes, to examine [14C] pravastatin uptake. RESULTS: [14C] Pravastatin (30 microM) uptake into oatp2 cRNA-injected oocytes was 40 times higher than that of water-injected control oocytes. The oatp2-mediated pravastatin uptake was Na+-independent and saturable. The Michaelis-Menten constant was 37.5+/-9.9 microM, a level comparable to that obtained in isolated rat hepatocytes in our previous study. As is the case with rat hepatocytes, the uptake of pravastatin (30 microM) was inhibited by 300 microM concentrations of taurocholate, cholate, bromosulfophthalein, estradiol-17beta-glucuronide, and simvastatin acid, but not by para-aminohippurate. On the other hand, [14C] simvastatin acid (30 microM) uptake of oatp2 cRNA-injected oocytes was not significantly different from that of water-injected oocytes. CONCLUSIONS: The cloned oatp2 was identified as the transporter responsible for the active hepatocellular pravastatin uptake.

Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1.

We have isolated a novel liver-specific organic anion transporter, LST-1, that is expressed exclusively in the human, rat, and mouse liver. LST-1 is a new gene family located between the organic anion transporter family and prostaglandin transporter. LST-1 transports taurocholate (Km = 13.6 microM) in a sodium-independent manner. LST-1 also shows broad substrate specificity. It transports conjugated steroids (dehydroepiandrosterone sulfate, estradiol-17beta-glucuronide, and estrone-3-sulfate), eicosanoids (prostaglandin E2, thromboxane B2, leukotriene C4, leukotriene E4), and thyroid hormones (thyroxine, Km = 3.0 microM and triiodothyronine, Km = 2.7 microM), reflecting hepatic multispecificity. LST-1 is probably the most important transporter in human liver for clearance of bile acids and organic anions because hepatic levels of another organic anion transporter, OATP, is very low. This is also the first report of the human molecule that transports thyroid hormones.

A family of mammalian Na+-dependent L-ascorbic acid transporters.

Vitamin C (L-ascorbic acid) is essential for many enzymatic reactions, in which it serves to maintain prosthetic metal ions in their reduced forms (for example, Fe2+, Cu+), and for scavenging free radicals in order to protect tissues from oxidative damage. The facilitative sugar transporters of the GLUT type can transport the oxidized form of the vitamin, dehydroascorbic acid, but these transporters are unlikely to allow significant physiological amounts of vitamin C to be taken up in the presence of normal glucose concentrations, because the vitamin is present in plasma essentially only in its reduced form. Here we describe the isolation of two L-ascorbic acid transporters, SVCT1 and SVCT2, from rat complementary DNA libraries, as the first step in investigating the importance of L-ascorbic acid transport in regulating the supply and metabolism of vitamin C. We find that SVCT1 and SVCT2 each mediate concentrative, high-affinity L-ascorbic acid transport that is stereospecific and is driven by the Na+ electrochemical gradient. Despite their close sequence homology and similar functions, the two isoforms of the transporter are discretely distributed: SVCT1 is mainly confined to epithelial systems (intestine, kidney, liver), whereas SVCT2 serves a host of metabolically active cells and specialized tissues in the brain, eye and other organs.

Immunohistochemical distribution and functional characterization of an organic anion transporting polypeptide 2 (oatp2).

The rabbit polyclonal antibody against rat organic anion transporting polypeptide 2 (oatp2) was raised and immunoaffinity-purified. Western blot analysis for oatp2 detected two bands ( 74 and 76 kDa) in rat brain and a single band (76 kDa) in the liver. By immunohistochemical analysis, the oatp2 immunoreactivity was specifically high at the basolateral membrane of rat hepatocytes. Functionally, the oatp2-expressing oocytes were found to transport dehydroepiandrosterone sulfate, delta1 opioid receptor agonist [D-Pen2,D-Pen5]enkephalin, Leuenkephalin, and biotin significantly, as well as the substrates previously reported. These data reveal the exact distribution of the rat oatp2 at the protein level in the liver, and that oatp2 appears to be involved in the multispecificity of the uptaking substrates in the liver and brain.