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Angiogenesis is involved in various physiologic and pathological conditions, including tumor growth, and is tightly regulated by the orchestration of proangiogenic and antiangiogenic factors. Inhibition of vascular endothelial growth factor (VEGF), the best-established antiangiogenic treatment in cancer, has shown some effectiveness; however, the identification of novel regulators, whose function is independent of VEGF, is required to achieve better outcomes. Here, we show that transcription factor 8 (TCF8) is up-regulated in endothelial cells during angiogenesis, acting as a negative regulator. Furthermore, TCF8 is specifically expressed in the endothelium of tumor vessels. Tcf8-heterozygous knockout mice are more permissive than wild-type mice to the formation of tumor blood vessels in s.c. implanted melanoma, which seems to contribute to the more aggressive growth and the lung metastases of the tumor in mutant mice. Suppression of TCF8 facilitates angiogenesis in both in vitro and ex vivo models, and displays comprehensive cellular phenotypes, including enhanced cell invasion, impaired cell adhesion, and increased cell monolayer permeability due to, at least partly, MMP1 overexpression, attenuation of focal adhesion formation, and insufficient VE-cadherin recruitment, respectively. Taken together, our findings define a novel, integral role for TCF8 in the regulation of pathologic angiogenesis, and propose TCF8 as a target for therapeutic intervention in cancer. [Cancer Res 2009;69(4):1678–84]

Supplementary Figures 1-3 from Integral Role of Transcription Factor 8 in the Negative Regulation of Tumor Angiogenesis

BACKGROUND Falls may cause elderly people to be bedridden, requiring professional intervention; thus, prevention is crucial. The use of electronic health records (EHRs) is expected to provide highly accurate risk assessment and length-of-stay data related to falls, which may be used to estimate the costs and benefits of prevention. However, no studies exist that investigate the extent to which hospital stays could be shortened through fall avoidance resulting from the use of prediction tools. OBJECTIVE First, the extended length of hospital stays caused by falls among elderly inpatients was estimated. Next, we developed a model that predicts falls by using clinical text as input and evaluated its accuracy. Finally, we estimated the potentially shortened hospital stay that would be made possible by appropriate interventions based on the prediction model. METHODS Patients 65 years or older were selected as subjects, and the EHRs of 1,728 falls and 70,586 non-falls were subjected to analysis. The extended stay lengths were estimated using the propensity score-matching of 49 associated variables. Bidirectional encoder representations from transformers and bidirectional Long Short-Term Memory (LSTM) methods were used to predict falls from clinical text. The estimated length of stay and the outputs of the prediction model were used to determine stay reductions. RESULTS The extended length of hospital stay due to falls was estimated to be 17.8 days (95% CI: 16.6–19.0), and it fell to 8.6 days when there were unobserved covariants at an odds ratio of 2.0. The accuracy of the prediction model was as follows: area under the receiver operating characteristic curve, 0.851; F-value, 0.165; recall, 0.737; precision, 0.093; and specificity, 0.839. When assuming interventions with 25 or 100% effectiveness against cases where the model predicted a fall, the stay reduction was estimated at 0.038 and 0.154 days/day, respectively. CONCLUSIONS The accuracy of the prediction model using clinical text is thought to be higher than the prediction accuracy of conventional assessments. However, our model’s precision remained low at 9.3%. This may be due, in part, to the inclusion of cases in which falls did not occur because of preventative interventions during hospitalization. Nonetheless, it is estimated that interventions for cases when falls were predicted will reduce medical costs by 1,520 yen/day of intervention, even if the preventative effect is 25%. Limitations include the fact that these results cannot be extrapolated to short- or long-term hospitalization cases, and that this was a single-center study.

BACKGROUND The rate of adoption of electronic medical record (EMR) systems has increased internationally, and new EMR adoption is currently a major topic in Japan. However, no study has performed a detailed analysis of longitudinal data to evaluate the changes in the EMR adoption status over time. OBJECTIVE This study aimed to evaluate the changes in the EMR adoption status over time in hospitals and clinics in Japan and to examine the facility and regional factors associated with these changes. METHODS Secondary longitudinal data were created by matching data in fiscal year (FY) 2011 and FY 2014 using reference numbers. EMR adoption status was defined as “EMR adoption,” “specified adoption schedule,” or “no adoption schedule.” Data were obtained for hospitals (n=4410) and clinics (n=67,329) that had no adoption schedule in FY 2011 and for hospitals (n=1068) and clinics (n=3132) with a specified adoption schedule in FY 2011. The EMR adoption statuses of medical institutions in FY 2014 were also examined. A multinomial logistic model was used to investigate the associations between EMR adoption status in FY 2014 and facility and regional factors in FY 2011. Considering the regional variations of these models, multilevel analyses with second levels were conducted. These models were constructed separately for hospitals and clinics, resulting in four multinomial logistic models. The odds ratio (OR) and 95% Bayesian credible interval (CI) were estimated for each variable. RESULTS A total of 6.9% of hospitals and 14.82% of clinics with no EMR adoption schedules in FY 2011 had adopted EMR by FY 2014, while 10.49% of hospitals and 33.65% of clinics with specified adoption schedules in FY 2011 had cancelled the scheduled adoption by FY 2014. For hospitals with no adoption schedules in FY 2011, EMR adoption/scheduled adoption was associated with practice size characteristics, such as number of outpatients (from quantile 4 to quantile 1: OR 1.67, 95% CI 1.005-2.84 and OR 2.40, 95% CI 1.80-3.21, respectively), and number of doctors (from quantile 4 to quantile 1: OR 4.20, 95% CI 2.39-7.31 and OR 2.02, 95% CI 1.52-2.64, respectively). For clinics with specified EMR adoption schedules in FY 2011, the factors negatively associated with EMR adoption/cancellation of scheduled EMR adoption were the presence of beds (quantile 4 to quantile 1: OR 0.57, 95% CI 0.45-0.72 and OR 0.74, 95% CI 0.58-0.96, respectively) and having a private establisher (quantile 4 to quantile 1: OR 0.27, 95% CI 0.13-0.55 and OR 0.43, 95% CI 0.19-0.91, respectively). No regional factors were significantly associated with the EMR adoption status of hospitals with no EMR adoption schedules; population density was positively associated with EMR adoption in clinics with no EMR adoption schedule (quantile 4 to quantile 1: OR 1.49, 95% CI 1.32-1.69). CONCLUSIONS Different approaches are needed to promote new adoption of EMR systems in hospitals as compared to clinics. It is important to induce decision making in small- and medium-sized hospitals, and regional postdecision technical support is important to avoid cancellation of scheduled EMR adoption in clinics.

Background To secure human resources for occupational medicine, it is important to analyse occupational physician retention trends and the factors associated with retention. However, little is currently known about this topic. Aims To identify occupational physician retention trends, to identify factors associated with this retention and to discuss the policy implications of the findings. Methods We analysed data from the biannual national physician census surveys conducted by the government of Japan from 2002 to 2014. In this study, those who chose 'working as an occupational physician' as their workplace/type of work from a pre-determined list in the survey questionnaire were considered full-time occupational physicians. We presented retention trends by calculating the annual retention rate for each set of two consecutive surveys. We then used logistic regression to identify factors associated with retention among occupational physicians. Results The annual retention rate of full-time occupational physicians from 2012 to 2014 was estimated as 76%, which represents a 6% improvement in retention over the study period. The odds of continuing to practise as an occupational physician were higher for occupational physicians working in cities compared with those working in towns or villages. Conclusions Improving and facilitating smooth transitions between clinical practice and occupational medicine would help to secure human resources in occupational medicine, even if the current trend of low retention continues.

Background The board certification system serves as a quality assurance system for physicians, and its design and operation are important health policy issues. In Japan, board certification was established and operated independently by academic societies and has not been directly linked to reimbursement systems. The phenomenon of younger physicians seeking specialist careers has raised concerns about acceleration of the tendency of fewer physicians working in rural areas and the maldistribution of physicians. Little is known about the associations between physicians’ geographical migration patterns and board certification status changes or between the continuation of urban/rural practice and the maintenance of board certification. This study aimed to identify these associations and to discuss their policy implications. Methods We analyzed 2012 and 2014 data from the Survey of Physicians, Dentists, and Pharmacists, a national census survey. To analyze geographical migration patterns, transitions in practice location (rural, intermediate, and urban) were analyzed by board certification status change (new, lost, consistently certified, and consistently uncertified). Logistic regression analysis was conducted to assess whether the odds of migrating to more urban/rural municipalities were associated with board certification status changes, adjusting for covariates, and whether practicing in a rural area was associated with maintaining board certification. Results Among 18,726 newly board-certified physicians, 94.9% (13,435/14,150) of those working in urban areas before certification remained in urban areas, whereas 64.6% (393/608) of those working in rural areas stayed in rural areas. Those who were newly certified had higher odds of moving to more urban areas, adjusting for covariates. Those who stayed in rural areas showed lower odds of maintaining board certification, adjusting for covariates. Conclusions Newly board-certified physicians are more likely to migrate to other types of areas, particularly more urban areas, than other physicians. Allocating more training quotas to rural areas could be one option for leveling the distribution of specialists. It also appeared that those practicing in rural areas have difficulty maintaining their certification, so the need to establish a support system for already-certified physicians in rural areas should be emphasized. Electronic supplementary material The online version of this article (10.1186/s12913-018-3441-y) contains supplementary material, which is available to authorized users.

We comprehensively evaluated cognitive and social functioning in patients requiring long-term inpatient psychiatric care using the International Classification of Functioning, Disability, and Health.We surveyed 1967 patients receiving long-term inpatient psychiatric care. Patients were further categorized into an old long-stay group (n = 892,5 years in hospitals) and a new long-stay group (n = 1075, 1-5 years in hospitals). We obtained responses for all the International Classification of Functioning, Disability, and Health items in domain b (Body Functions) and domain d (Activities and Participation). We estimated weighted means for each item using the propensity score to adjust for confounding factors.Responses were received from 307 hospitals (response rate of hospitals: 25.5%). Cognitive and social functioning in the old long-stay group was more severely impaired than in the new long-stay group. No statistically significant differences were observed regarding the International Classification of Functioning, Disability, and Health items associated with basic activities of daily living between the two groups.Combined therapy consisting of cognitive remediation and rehabilitation on social functioning for this patient population should be started from the early stage of hospitalization. Non-restrictive, independent environments may also be optimal for this patient population. Implications for rehabilitation Rehabilitation of cognitive and social functioning for patients requiring long-term inpatient psychiatric care should be started in the early stages of hospitalization. In psychiatric fields, the International Classification of Functioning, Disability, and Health checklist could facilitate individualized rehabilitation planning by allowing healthcare professionals to visually assess the comprehensive functioning of each patient using graphics such as radar charts.

Since an X-ray Free Electron Laser (XFEL) facility is a linac-based single-user machine, a multi-beamline mode of operation, which improves the efficiency of user experiments, is critical for accommodating users' rapidly increasing demand for beamtime. A key supporting technology is a highly stable pulsed power supply (PS), which enables stable XFEL operations by precisely switching the beam route. We developed a high-power pulsed PS to drive a kicker magnet installed in a SACLA's beam switching system. SiC MOSFETs were adapted as switching elements to reduce the required size and to increase the electric power efficiency. The PS we developed provides two key capabilities: (i) a high current stability of 20 ppm (peak-to-peak) at a peak power of 0.24 MW and (ii) generation of controllable, bipolar, and trapezoidal current waveforms at 60 Hz. This paper describes the overall concept, the detailed design, the performance achieved, and the initial beam test results.

Recent studies indicate the importance of spatiotemporal regulation in the diversity and specificity of intracellular signaling. Here, we show that Ras-PI3K signaling plays an important role in the local regulation of phosphatidylinositol metabolism in the endosome through live-cell imaging by using a bimolecular fluorescence complementation technique, in which molecular interaction is indicated by fluorescence emission. Using several possible combinations of Ras and the Ras-binding domain, we identified an optimal set of probe molecules that yielded the most significant increase in fluorescence intensity between the active and inactive forms of Ras. This combination revealed that, among the Ras effectors tested, phosphatidylinositol 3-kinase (PI3K) was specifically implicated in signaling in the endosome. We also found that full length PI3K was recruited to the endosome in EGF- and Ras-dependent manners, which appears to be essential for the activation of PI3K in this compartment. Taken together, these findings demonstrate that the spatiotemporal regulation of Ras-PI3K signaling may dictate the activation of PI3K and subsequent downstream signaling in the endosome.

Radiotherapy is an important noninvasive treatment for many types of cancer. However, it has been reported that the proliferative, invasive, and metastatic capacities of tumor cells can be increased in the repopulated tumors that survive radiotherapy. We have previously established a radiation-surviving cell model for the human non-small cell lung cancer cell line H1299 by harvesting relic cells 14 days after irradiation (IR cells). Here, we report that cell invasion, cell migration, and cell adhesion are enhanced in these surviving cancer cells. The mRNA expression levels of matrix metalloproteinases (MMPs), including mmp1, mmp2, and mmp9, were upregulated in IR cells compared with parental cells. A gelatin zymogram, wound healing assay, and invasion assay showed increased MMP activity, cell motility, and invasiveness in IR cells, respectively. Moreover, IR cells adhered more tightly to collagen-coated dishes than parental cells. Consistently, paxillin, phosphorylated FAK, integrin beta1, and vinculin were strongly localized at focal adhesions in IR cells, as visualized by immunofluorescence. In this report, we identify molecules responsible for the malignant properties of tumor cells that survive irradiation. These molecules may be important therapeutic targets for the control of repopulated tumors after radiotherapy.

Parathyroid hormone-related protein (PTHrP) is detected in many aggressive tumors and involved in malignant conversion; however, the underlying mechanism remains obscure. Here, we identified PTHrP as a mediator of epidermal growth factor receptor (EGFR) signaling to promote the malignancies of oral cancers. PTHrP mRNA was abundantly expressed in most of the quiescent oral cancer cells, and was significantly upregulated by EGF stimulation via ERK and p38 MAPK. PTHrP silencing by RNA interference, as well as EGFR inhibitor AG1478 treatment, significantly suppressed cell proliferation, migration, and invasiveness. Furthermore, combined treatment of AG1478 and PTHrP knockdown achieved synergistic inhibition of malignant phenotypes. Recombinant PTHrP substantially promoted cell motility, and rescued the inhibition by PTHrP knockdown, suggesting the paracrine/autocrine function of PTHrP. These data indicate that PTHrP contributes to the malignancy of oral cancers downstream of EGFR signaling, and may thus provide a therapeutic target for oral cancer.

The parallel operation of plural undulator beam lines is an important means of improving the efficiency and usability of x-ray free-electron laser facilities. After the installation of a second undulator beam line (BL2) at SPring-8 Angstrom compact free-electron laser (SACLA), pulse-by-pulse switching between two beam lines was tested using kicker and dc twin-septum magnets. To maintain a compact size, all undulator beam lines at SACLA are designed to be placed within the same undulator hall located downstream of the accelerator. In order to ensure broad tunability of the laser wavelength, the electron bunches are accelerated to different beam energies optimized for the wavelengths of each beam line. In the demonstration, the 30 Hz electron beam was alternately deflected to two beam lines and simultaneous lasing was achieved with 15 Hz at each beam line. Since the electron beam was deflected twice by 3° in a dogleg to BL2, the coherent synchrotron radiation (CSR) effects became non-negligible. Currently in a wavelength range of 4–10 keV, a laser pulse energy of 100–150 μJ can be obtained with a reduced peak current of around 1 kA by alleviating the CSR effects. This paper reports the results and operational issues related to the multi-beam-line operation of SACLA.

Objective— C-reactive protein (CRP), an obesity-related inflammatory marker, is a promising predictor for cardiovascular disease and may be a mediator for atherogenesis. It has been reported that diet-induced weight loss lowered CRP levels. However, the effect of exercise training, another therapy that can reduce weight, on CRP is still unclear. We examined effects of exercise training with weight loss on CRP levels and conventional cardiovascular risks. Methods and Results— A total of 227 apparently healthy women were recruited, and 199 subjects (average age 52 years) completed a 2-month weight reduction program consisting of supervised aerobic exercises. After the program, weight was reduced from 65.8 to 62.8 kg ( P P Conclusions— Exercise training with weight reduction disproportionately lowered CRP levels. Considering inflammatory status, there might be an optimal pace of exercise with weight loss.

✓ Letter-perception centers are not held in as high regard as motor- and language-related cortices during planning of neurosurgical procedures, and there have been no reports suggesting cortical reorganization of reading ability. The authors describe a patient with a left mesial temporal glioma in whom two letter-perception centers (the anterior portion of the left superior temporal gyrus and the left fusiform gyrus) were successfully localized before surgery by performing magnetoencephalography (MEG) during reading tasks. Control MEG examinations of 15 healthy volunteers were also performed to assist in a careful interpretation of patient results. Although a radical resection of the mesial temporal glioma, which involved the left fusiform gyrus, caused severe dyslexia, the patient's impaired reading skills improved gradually during a 1-year postoperative period. In the meantime, the spared left superior temporal gyrus displayed an overshot recovery of MEG responses. During the postoperative period there was no obvious recovery in MEG signals and no compensatory activity in the contralateral fusiform gyrus. This case demonstrates that lexicosemantic centers involved in the reading process can be noninvasively localized using MEG and that the results obtained are highly reliable for surgical planning. The results of the repeated MEG reflected sequentially the patient's recovery from dyslexia. This is the first report in which MEG studies have been shown to predict preoperatively the risk of dyslexia and demonstrate its serial physiological recovery.

In the hearts of spontaneously hypertensive rats, mRNA of the renin-angiotensin system (renin, angiotensinogen, and angiotensin converting enzyme) was expressed. Level of angiotensinogen and renin mRNA expressed in ventricles, and angiotensinogen mRNA expressed in fibroblasts from SHR were higher than those from WKY. ACE mRNA was also more strongly expressed in the ventricles and fibroblasts from SHR compared with those of WKY. We investigated the effects of angiotensin II on cardiac collagen synthesis in cardiac fibroblasts of 10-week-old spontaneously hypertensive rats and age-matched Wistar-Kyoto rats. Basal collagen synthesis in cardiac fibroblasts from spontaneously hypertensive rats was 1.6-fold greater than that in the cell of Wistar-Kyoto rats. Angiotensin II stimulated collagen synthesis in cardiac fibroblasts in a dose-dependent manner. The responsiveness of collagen production to angiotensin II was significantly enhanced in cardiac fibroblasts from spontaneously hypertensive rats (100 nM angiotensin II resulted in 185 ± 18% increase above basal levels, 185 ± 18 versus 128 ± 19% in Wistar-Kyoto rats, p < 0.01). This effect was receptor-specific, because the competitive inhibitor saralasin and MK 954 blocked it. These results indicate that collagen production was enhanced in cardiac fibroblasts from spontaneously hypertensive rats, that angiotensin II had a stimulatory effect on collagen synthesis in cardiac fibroblasts, and that cardiac fibroblasts from spontaneously hypertensive rats were hyperresponsive to stimulation by angiotensin II. These findings suggest that the cardiac renin-angiotensin system may play an important role in collagen accumulation in hypertensive cardiac hypertrophy.

The effects of L-arginine on spontaneous contraction of endothelium-denuded longitudinal preparations of the rat portal vein were studied. L-arginine increased the frequency of spontaneous contraction concentration-dependently between 10 microM and 1 mM. Changes in contraction amplitude and duration were not remarkable. D-arginine had a negligible effect on spontaneous contraction. N(omega)-nitro-L-arginine (1 mM) did not affect spontaneous contraction or the response to L-arginine. Addition of N(G)-monomethyl-L-arginine (1 mM), l-lysine (1 mM) or N-ethymaleimide (0.1 mM) increased the frequency of spontaneous contractions and inhibited the effect of L-arginine. Glibenclamide (10 microM) did not affect spontaneous contraction or the response to L-arginine. Spontaneous increase in concentration of intracellular Ca2+, estimated as the ratio of Fura-PE3 fluorescence occurred synchronously with spontaneous contraction. Spontaneous increase in concentration of intracellular Ca2+ occurred more frequently in the presence of L-arginine (1 mM). L-arginine (1 mM) also increased the number of action potential bursts/min in the longitudinal smooth muscle layer. L-arginine (1 mM) also depolarized cell membranes. This study indicates that L-arginine increases the frequency of spontaneous contraction of longitudinal muscle in the rat portal vein by membrane depolarization through mechanisms that do not involve nitric oxide or the inhibition of ATP-sensitive K+ channels.

A family of specific carrier protein designated as monocarboxylate transporter (MCT) has been known to transport the lactate and other moncarboxylates in mammalian cells. We hypothesized the presence of serum protein in human circulation that may works as a lactate carrier and that biochemical structure would possesses common structure with MCT on the plasma membrane.

Although the types of pathophysiological stimulation that initiate an overexpression of OPN have yet to be determined, we hypothesized that mechanical stress is one of the candidates which initiates OPN expression in vascular smooth muscle cells. Cell proliferation assay indicated that a pure atmospheric pressure of 160 mmHg activated cell proliferation by 11% in human aortic smooth muscle cells (HASMC) compared to nonpressurized controls. Immunoblot analysis probed with an anti-OPN antibody demonstrated a 50% increase in OPN. Dual-luciferase reporter assay demonstrated that OPN promoter, corresponding to the −771 through −1 region of OPN gene, was highly responsive to pure atmospheric pressure by ten times that of the control. From these observations, we concluded that pure atmospheric pressure directly promotes an expression of OPN in HASMC, with these results also suggesting that high blood pressure-mediated mechanical compression is involved in the process of atherosclerosis and remodeling via OPN expression in HASMC.

Mechanical forces related to pressure and flow are important for the etiology of atherosclerosis and hypertension. We hypothesized the presence of mechanosensors that were solely sensitive to pure atmospheric pressure in the absence of shear and tensile stresses. A pressure-loading apparatus was set up to examine the effects of atmospheric pressure on human aortic smooth muscle cells (HASMC). Pressure application of 140 to 180 mmHg produced DNA synthesis in a pressure-dependent manner. In contrast, pressure of 120 mmHg or less produced no significant change. Both extracellular signal-regulated kinase and c-Jun N-terminal kinase activities, but not p38 activity, were stimulated by pressures of more than 160 mmHg. Pertussis toxin (PTx) completely inhibited the pressure-induced increase of DNA synthesis under the high pressure of 200 mmHg. These data suggest that HASMC have a mechanosensing cellular switch for DNA synthesis which is sensitive to pure atmospheric pressure, and that the molecular switch is activated by pressure of more than 140 mmHg. The activation mechanism consists of PTx-sensitive and -insensitive pathways, and the former is activated by high pure pressure.

This study examined the effects of long-term treatments with the angiotensin-converting enzyme inhibitor, enalapril, and the calcium antagonist, amlodipine, on the morphologic changes, progressive left ventricular dysfunction, and gene expression of the ryanodine receptor (RyR) and phospholamban (PLN) in dilated cardiomyopathy. From the ages of 5 through 20 weeks, dilated cardiomyopathic hamsters, BIO53.58 (BIO), and control hamsters, F1b, orally received either enalapril or amlodipine. Cardiac function was assessed by echocardiography. At the age of 20 weeks, the collagen volume fractions were analyzed by the stereologic method. RyR and PLN messenger RNAs (mRNAs) were examined by Northern blot in the amlodipine group. In BIO, the reduction of left ventricular percentage of fractional shortening was attenuated in the enalapril group (p < 0.05) and amlodipine group (p < 0.001), and the increase in the collagen volume fraction and the loss of myocytes were suppressed in the amlodipine group compared with the untreated group. RyR mRNA level decreased in BIO (p < 0.01) compared with F1b, but PLN mRNA level was unchanged. RyR and PLN mRNA levels were unaffected by the treatment with amlodipine. Enalapril and amlodipine prevent progressive remodeling and reduce cardiac dysfunction in BIO. Amlodipine prevents fibrosis and cell death without modifying RyR and PLN mRNA levels in BIO.

Hypertensive cardiac hypertrophy is associated with the accumulation of collagen in the myocardial interstitium. Previous studies have demonstrated that this myocardial fibrosis accounts for impaired myocardial stiffness and ventricular dysfunction. Although cardiac fibroblasts are responsible for the synthesis of fibrillar collagen, the factors that regulate collagen synthesis in cardiac fibroblasts are not fully understood. We investigated the effects of angiotensin II on cardiac collagen synthesis in cardiac fibroblasts. Cardiac fibroblasts of 10 week old spontaneously hypertensive rats and age-matched Wistar-Kyoto rats were prepared and maintained in culture medium supplemented with 10% fetal calf serum. The expression of mRNA of the renin-angiotensin system (renin, angiotensinogen, angiotensin converting enzyme) was determined by using a ribonuclease protection assay. Basal collagen synthesis in cardiac fibroblasts from spontaneously hypertensive rats was 1.6 fold greater than that in the cell of Wistar-Kyoto rats. Angiotensin II stimulated collagen synthesis in cardiac fibroblasts in a dose-dependent manner. The responsiveness of collagen production to angiotensin II was significantly enhanced in cardiac fibroblasts from spontaneously hypertensive rats (100 nM angiotensin II resulted in 185 ± 18% increase above basal levels, 185 ± 18 versus 128 ± 19% in Wistar-Kyoto rats p < 0.01). This effect was receptor-specific, because it was blocked by the competitive inhibitor saralasin and MK 954. These results indicate that collagen production was enhanced in cardiac fibroblasts from spontaneously hypertensive rats, that angiotensin II had a stimulatory effect on collagen synthesis in cardiac fibroblasts, and that cardiac fibroblasts from spontaneously hypertensive rats were hyper-responsive to stimulation by angiotensin II. Level of angiotensin and renin mRNA expressed in ventricles, and angiotensinogen mRNA expressed in fibroblasts from SHR were higher than those from WKY. These findings suggest that the cardiac renin-angiotensin system may play an important role in collagen accumulation in hypertensive cardiac hypertrophy.

近年の生化学,遺伝子工学の研究の発展によって循環器領域でも,日常外来検査で簡単に行う事が出来,しかも正確な診断が出来る検査が幾つか出現してきた.たとえば心房性ナトリウム利尿ペプチド(ANP, BNP),は心不全時に増加し,その予後との関連が検討されている.また心筋梗塞時にも増加し,心筋梗塞巣の大きさや心室リモデリングとの関連も検討されてきた.トロポニンT,ミオシン軽鎖,ミオグロビンなどは心筋梗塞時にそれぞれ特異な遊出パターンを示すので,これらを組み合わせて測定することによって,より正確な病態把握が可能になる.

To examine the contribution of the renin-angiotensin system to hypertrophic cardiomyopathy (HCM), we studied 96 patients with HCM (mean age 50 years, 55% male), 105 of their unaffected siblings and offspring, and 160 healthy subjects without known hypertension and left ventricular hypertrophy (LVH) who were frequency matched to cases by age and sex. Patients were divided into familial or sporadic HCM (FHCM or SHCM) groups with or without affected members of their family. The region of interest in the angiotensinogen (AGT) gene, the missense mutation with methione-to-threonine amino acid substitution at codon 235 in angiotensinogen (M235T), was amplified by polymerase chain reaction with the use of allele-specific oligonucleotide primers flanking the polymorphic region of the AGT gene to amplify template deoxyribonucleic acid prepared from peripheral leukocytes. The T allele frequency was higher in the SHCM group than in unaffected siblings and offspring (88% vs 78%, X2 = 4.6, p < 0.05). The M allele frequency was higher in unaffected siblings and offspring than in patients with SHCM (23% vs 12%, X2 = 4.6, p < 0.05). The T allele frequency among unaffected siblings and offspring was similar to that observed in healthy subjects (78% vs 78%). We conclude that HCM, especially in sporadic cases, is partially determined by genetic disposition. The molecular variant of angiotensinogen T235 seems to be a predisposing factor for cardiac hypertrophy in HCM and carries an approximately twofold increased risk.

Congestive heart failure (CHF) patients share several similar features, such as reduced cardiac contractility and neurohumoral activation to compensate the impaired cardiac function. In CHF patients, the cardiac renin-angitensin (RA) system, receptors, GTP-binding proteins, and their effector molecules are inevitably exposed to chronically elevated neurohumoral stimulation. A widely recognized concept is that a chronic increase in such stimulation can desensitize target cell receptors and the post-receptor signal transducing pathway. Recently, reports of several studies have indicated that the inhibitory GTP-binding protein (Gi) can be increased in CHF patients and animal models. Although direct evidence for a change in catalytic protein of adenylyl cyclase has not been found, limited information has suggested a reduced catalytic activity in terminally failing hearts. In this paper, we have assessed the changes in beta AR, GTP-binding protein, catalytic protein and beta ARK. We also examined angiotensinogen mRNA expression in failing heart. It was detected not only in the liver, but also in both the atrial and ventricular heart tissues, suggesting that angiotensinogen is synthesized in the human heart. Immunohistochemical studies revealed a stronger reaction in the endocardial layer of the human left ventricle than in the epicardial layer, and intense immunoreactivity in the conduction system and right atrium. Our experiments revealed a widespread immunopositive reaction for angiotensinogen in the left ventricle of diseased hearts. In the non-diseased heart, ACE and AT1 receptor RNA are present in ventricular muscles. Renin and Ao mRNA could not be detected in the subendocardium of non-diseased left ventricle, but both were present in the left ventricle of diseased hearts. These data indicate that the cardiac RA system plays an important role in the deterioration of cardiac function.

The expression of three different subtypes of GTP binding protein beta-subunits (beta 1, beta 2 and beta 3) was examined in rat hearts at both mRNA and protein levels. Among three subtypes, beta 3 mRNA was more abundant in ventricles while beta 1 and beta 2 mRNAs were expressed ubiquitously among ventricles, atria and aortas. The immunodetectable beta 1- and beta 3-proteins were predominantly found in the cytosolic fraction of rat ventricles. In this study, we demonstrate ventricle-specific expression of beta 3-subunit and localization of G protein beta 3-subunit in the cytosol of rat hearts. This unusual cytosolic localization of G protein beta 3-subunit might indicate subtype-specific function of G protein beta (gamma)-subunits in rat hearts.

The effects of transforming growth factor beta-1 on concentration of intracellular Ca2+ changes in the presence of isoproterenol were studied in cardiac fibroblasts. Transforming growth factor beta-1 effect on cyclic-AMP production and ligand assay of beta-adrenergic receptors were also examined. Production of cyclic-AMP in fibroblasts preincubated with transforming growth factor beta-1 decreased compared with non-transforming growth factor beta-1-treated fibroblasts in the presence of isoproterenol. An increase of intracellular Ca2+ concentration in the presence of isoproterenol was also inhibited in transforming growth factor beta-1-treated fibroblasts. Islet-activating protein did not restore these reactions of transforming growth factor beta-1. Concentration change of intracellular calcium under the treatment of transforming growth factor beta-1 was unaffected by forskolin addition. Binding of [3H]CGP-12177 was decreased to 47% of control preincubated for 24 h with transforming growth factor beta-1 in fibroblasts. Scatchard plots suggested a decrease in beta-adrenergic receptor number without specific change in receptor affinity. These results suggested that transforming growth factor beta-1 modulates the signal transduction through beta-adrenergic receptor and intracellular Ca2+ concentration by regulating the number of receptor in fibroblasts.

Extrahepatic synthesis and localization of angiotensinogen (ATN) have been described in animals, thus establishing the tissue renin-angiotensin (RA) system. However, there had been no reports of tissue RA systems in human organs, including the heart. In earlier, we have reported the possibility of ATN synthesis in the human heart using ribonuclease protection assay system. ATN mRNA was detected not only in the liver, but also in both the atrial and ventricular heart tissues, suggesting that ATN is synthesized in the human heart. In this report, we looked for the distribution of ATN in diseased human heart. Northern blot hybridization of cDNA with total RNA extracted from human liver, brain, kidney, atrial and ventricular tissues revealed that ATN mRNA exists in cardiac ventricule. Immunohistochemical studies using a specific antibody to ATN revealed a stronger reaction in the endocardial layer of the human left ventricle, than in the epicardial layer, and intense immunoreactivity in the conduction system and right atrium. This distribution pattern was similar to that of human atrial natriuretic peptide (hANP), which functions a smooth muscle relaxant. Double immunostaining of ATN and hANP demonstrated that all myocytes in the right atrium had immunopositive reactions to ATN, hANP or both of ATN and hANP. Double immunoelectron staining enabled us to show more detailed localization of ATN and hANP; hANP only existed in the specific granules and ATN existed in the myofibril, but not in the granule. Furthermore, our experiments provide evidence of ATN in healthy human hearts and also reveal a widespread immunopositive reaction for ATN in the left ventricle of diseased hearts.

The accumulation of inositol 1,4,5-trisphosphate (IP3) after hormonal stimulation has a physiological role, possibly by alteration of Ca2+ levels in cardiac myocyte. However, this accumulation has not been studied under pathophysiological conditions. In this report, we examine phosphatidylinositol metabolism during cellular response to norepinephrine in pressure-overloaded hypertrophic rat heart. After stimulation with norepinephrine, the accumulations of IP3 and diacylglyceride significantly increased in isolated myocytes from stroke-prone spontaneously hypertensive rat (SHRSP) heart, indicating phosphatidylinositol-specific phospholipase C activity increased in SHRSP heart cells. Protein kinase C activity was also enhanced in SHRSP, with a marked increase in particulate activity. We determined the intracellular calcium concentration and found it to be higher in SHRSP than in Wistar-Kyoto (WKY) rats at 30-40 weeks of age. Ca2+ influx was also elevated in SHRSP stimulated by norepinephrine. In SHRSP heart, cytosolic Ca2+ concentration may rise quickly in response to some stimuli, such as alpha 1-adrenergic stimulation, which is shown to be one of the pathways that increases cytosolic Ca2+ levels in hypertrophied rat heart. These data suggest that a part of the phosphatidylinositol-turnover pathway, such as the phosphatidylinositol 4,5-bisphosphate-IP3-Ca2+ pathway or the diacylglyceride-protein kinase C pathway, may play an important role in the development of hypertrophy in SHRSP heart.

It is known that inositol (1, 4, 5)-trisphosphate (IP3) stimulates Ca2+ release from sarcoplasmic reticulum (SR) in several tissues, but in cardiac myocytes this phenomenon has not been confirmed. The purpose of the present study was to confirm the effect of (1, 4, 5)-IP3 on Ca2+ release from SR in cardiac myocytes. The effect of IP3 on Ca2+ release from SR in hypertrophic cardiac cells was also determined. We examined the effects of IP3 on Ca2+ release from cardiac myocyte SR by the digital-image method in a single cell. We also determined the effect of IP3 on calcium release from isolated SR. SR was prepared from spontaneous hypertensive rat hearts and Wistar kyoto rat hearts. The SR was prelabeled with 45Ca2+, and then incubated with the indicated concentrations of IP3 for 1 min at 37 degrees C. In cardiac myocytes treated with saponin, Ca2+ release stimulated by 10 microM (1, 4, 5)-IP3 was detected by fura-2. In 45Ca2+ prelabeled SR, the maximal Ca2+ release was achieved at 10 microM IP3 incubated for 1 min. The release of Ca2+ was higher in SR of SHR than in the SR of WKY. IP3 stimulates Ca2-release from cardiac SR, and this release is greater in SHR than in WKY. However, it is uncertain whether this phenomenon plays a role in cardiac hypertrophy.

著者等は有害金属を含むスラッジを無害固形化する方法に関する一連の実験を行っている.こまれでにCr2O3をスラッジのモデルとし, Na2O-CaO-SiO2三成分系ケイ酸塩の組成が化学的固定化剤として有効かどうかを調べた.そのため, Cr2O3にNa2CO3, CaCO3およびSiO2を調合して焼成し, クロムの溶出試験を行った.本研究では焼成の際に少量の活性炭を加えることによる還元効果が, クロムの溶出濃度に及ぼす影響について調べた.

The accumulations of inositol-1,4,5-trisphosphate (IP3) and inositol-1,3,4,5-tetrakisphosphate (IP4) after hormonal stimulation may have a physiological role, possibly by alteration of Ca2+ levels in cardiac tissue. But the accumulation of inositol polyphosphate in a pathophysiological condition has not been studied. We investigated phosphatidylinositol-4,5-bisphosphate (PIP2) metabolism in hypertrophic cardiac myocytes, and clarified that the accumulations of IP3, IP4 and diacylglyceride after stimulation with norepinephrine were significantly enhanced in isolated myocytes from spontaneously hypertensive rat heart. Phospholipase C activity increased with age in SHRSP heart cells. These data suggest that PI turnover pathways, which can be mediated by both phosphatidylinositol-4,5-bisphosphate and diacylglyceride, may play an important role in development of hypertrophy in the hearts of rats with spontaneous hypertension.

BACKGROUND There have been no reports on the presence of the tissue renin-angiotensin system in the human heart, although the presence of angiotensinogen has been described in the animal heart. METHODS AND RESULTS To determine whether angiotensinogen is synthesized in the human heart, we examined angiotensinogen messenger RNA (mRNA) synthesis in autopsy hearts by using ribonuclease protection assay. As a result, angiotensinogen mRNA was detected in the atrial muscle, muscles of the conduction system, and the left ventricular wall. In the left ventricular wall, mRNA expression was more prominent in the subendocardial muscles than in the midcardial or epicardial muscles. Using a monoclonal antibody to human angiotensinogen in immunoblotting experiments, we detected two closely spaced bands at approximately 70 kd in the heart, which was quite consistent with the human angiotensinogen molecule. Immunohistochemical studies with this monoclonal antibody demonstrated intense immunoreactivity in the atrial muscles, the muscles of the conduction system, and those of the subendocardial layers. CONCLUSIONS We conclude that angiotensinogen was synthesized in the human heart. It was evident that the localization of angiotensinogen was not ubiquitous in the cardiac muscles, showing its predilection for the atrial muscles, muscles of the conduction system, and subendocardial layer of the left ventricle.

The aim of this study was to investigate the correlation between hypoxic myocardial cell injury and intracellular protease activity. Cardiac myocytes were isolated from neonatal rat hearts and cultured in Eagle's modified minimum essential medium. Myocytes were incubated in hypoxic conditions for 6 hours. The cell death rate during hypoxia rose to 80% after 6 hours. Extracellular protease activity was elevated to 4 units during hypoxia, much higher than the 0.7 units in aerobic states at 6 hours. This extracellular protease activity in hypoxic conditions was markedly inhibited by leupeptin and EDTA, and weakly inhibited by the cysteine protease inhibitor, NCO-700, but phenylmethyl sulfonyl fluoride did not inhibit the protease activity. To identify the protease activated during hypoxia, calpain-specific inhibitors were added to the incubation mixture. Calpain inhibitor 1 and calpastatin, an endogenous selective calpain inhibitor, markedly inhibited extracellular protease activity during hypoxia. NCO-700 also inhibited intracellular protease activity. NCO-700 reduced hypoxic cell death to 30% after 6 hours of hypoxy-genation. These observations indicate that calpain is activated during hypoxia and leads to irreversible cell membrane degradation after 6 hours of hypoxygenation.

The adaptor protein Crk mediates intracellular signaling related to cell motility and proliferation and is implicated in human tumorigenesis. The role of Crk in the growth of human sarcoma has remained unclear, however. The present study shows that Crk-induced activation of Src and subsequent signaling by p38 mitogen-activated protein kinase (MAPK) contribute to the enhanced proliferation of human synovial sarcoma cells. Depletion of Crk by RNA interference markedly inhibited proliferation of the synovial sarcoma cell lines HS-SYII, SYO-1, and Fuji as well as prevented anchorage-independent growth. Conversely, reconstitution with CrkII by authentic small interfering RNA–resistant Crk gene restored proliferation in Crk-silenced SYO-1 cells. Crk-depleted synovial sarcoma cells manifested enhanced transcriptional activity and expression of the p16INK4A gene, resulting in their accumulation in G1 phase of the cell cycle. In response to hepatocyte growth factor stimulation, Crk prominently induced the tyrosine phosphorylation of Grb2-associated binder 1 through activation of Src and focal adhesion kinase, and the Src family kinase inhibitor PP2 almost completely inhibited the proliferation of SYO-1 cells. Crk also induced the phosphorylation of p38 MAPK, and SB203580, a p38 MAPK–specific inhibitor, increased expression of p16INK4A gene in SYO-1 cells. Furthermore, SB203580 or depletion of p38 MAPK by small interfering RNA suppressed both the phosphorylation of Akt triggered by hepatocyte growth factor and the proliferation of SYO-1 cells. These results suggest that Crk promotes proliferation of human synovial sarcoma cells through activation of Src and its downstream signaling by a novel p38 MAPK-Akt pathway, with these signaling molecules providing potent new targets for molecular therapeutics. (Mol Cancer Res 2009;7(9):1582–92)

Angiogenesis is involved in various physiologic and pathological conditions, including tumor growth, and is tightly regulated by the orchestration of proangiogenic and antiangiogenic factors. Inhibition of vascular endothelial growth factor (VEGF), the best-established antiangiogenic treatment in cancer, has shown some effectiveness; however, the identification of novel regulators, whose function is independent of VEGF, is required to achieve better outcomes. Here, we show that transcription factor 8 (TCF8) is up-regulated in endothelial cells during angiogenesis, acting as a negative regulator. Furthermore, TCF8 is specifically expressed in the endothelium of tumor vessels. Tcf8-heterozygous knockout mice are more permissive than wild-type mice to the formation of tumor blood vessels in s.c. implanted melanoma, which seems to contribute to the more aggressive growth and the lung metastases of the tumor in mutant mice. Suppression of TCF8 facilitates angiogenesis in both in vitro and ex vivo models, and displays comprehensive cellular phenotypes, including enhanced cell invasion, impaired cell adhesion, and increased cell monolayer permeability due to, at least partly, MMP1 overexpression, attenuation of focal adhesion formation, and insufficient VE-cadherin recruitment, respectively. Taken together, our findings define a novel, integral role for TCF8 in the regulation of pathologic angiogenesis, and propose TCF8 as a target for therapeutic intervention in cancer. [Cancer Res 2009;69(4):1678–84]

Cell death during hypoxia rose to 80% after 6 h. Calpain activity increased to 4 units during hypoxia, much higher than the 0.7 units seen in aerobic condition at 6 h. This activity was markedly inhibited by calpain-specific inhibitor I ( n -acetyl-leucyle-leucyle-norleucinal). β-Adrenergic blocking agents and calcium antagonists suppressed the calpain activity and decreased cell death during hypoxia. On the other hand, α-adrenergic blocking agents did not affect calpain activity and cell death under hypoxic conditions. These results prove that β-adrenergic blocking agents and calcium antagonists prevent protein degradation during hypoxic cell injury.

We observed that in hypoxic myocardial cells prostacyclin and arachidonic acid release increased and that during hypoxia phospholipid degradation also occurred. In order to clarify the mechanism of phospholipid degradation, we determined the activity of phospholipases A 2 and C. We found that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were markedly decreased and that lysophosphatidylcholine and lysophosphatidylethanolamine were increased. In contrast, there was only slight phosphatidylinositol degradation and no lysophosphatidylinositol elevation was observed. These results show that phospholipase A 2 was activated in hypoxic myocytes and had substrate specificity towards PC and PE. To study phospholipase C activity, membrane phospholipids were labeled with [ 3 H]choline, [ 3 H]inositol or [ 3 H]ethanolamine. The release of inositol was observed, but neither choline nor ethanolamine was released. In hypoxia, myocardial-cell phospholipase C has high substrate specificity towards phosphatidylinositol. The activation of phospholipases is closely related to the intracellular Ca 2+ concentration; it is thought that inositol polyphosphatides may regulate intracellular Ca 2+ . We determined how Ca 2+ influx occurs in hypoxia. β-Adrenergic blockade and Ca 2+ antagonists markedly suppressed Ca 2+ influx, phospholipase A 2 activity, phospholipase C activity and cell death. However, the α 1 -adrenergic blockade was less effective in suppressing these phenomena. These results suggest that in hypoxic myocardial cells Ca 2+ influx mediated by β-adrenergic stimulation activates phospholipases A 2 and C, and that phospholipid degradation and prostacyclin release then occur.