EV71:TLLcho virus murine model of enterovirus A71 neurological disease does not exhibit neurogenic pulmonary oedema.

Small animal models play an important role in investigating and revealing the molecular determinants and mechanisms underlying neuro-virulence of enterovirus A71 (EV-A71). In our previous study, we successfully developed two mouse cell-line replication competent EV-A71 strains (EV71:TLLm and EV71:TLLmv) which were capable of inducing neuro-invasion in BALB/c mice. The more virulent EV71:TLLmv exhibited ability to induce acute encephalomyelitis accompanied by neurogenic pulmonary oedema. EV71:TLLcho virus strain was generated from EV71:TLLm by a series of passages in CHO-K1 cells. EV71:TLLcho demonstrated a broader range of infectivity across various mammalian cell lines and exhibited complete cytopathic effects (CPE) within 48 hours post-inoculation in comparison to EV71:TLLm or EV71:TLLmv. EV71:TLLcho consistently yielded higher levels of viral replication at all time points examined. In comparison to EV71:TLLm, EV71:TLLcho consistently induced more severe disease and increased mortality in one-week old BALB/c mice. However, unlike mice challenged with EV71:TLLmv, none of the mice challenged with EV71:TLLcho progressed to severe acute encephalomyelitis and developed neurogenic pulmonary oedema.

Characterization of a hepatitis C virus-like particle vaccine produced in a human hepatocyte-derived cell line.

An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8+ and class II CD4+ T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm-3. The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8+ T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8+ T-cell responses and will have important implications for the development of a preventative vaccine for HCV.

Portable lysis apparatus for rapid single-step DNA extraction of Bacillus subtilis.

AIMS: To demonstrate and characterize a portable lysis apparatus for rapid single-step bacterial DNA extraction. METHODS AND RESULTS: Our portable lysis apparatus employed a novel design consisting of an annular piezo-element with perforated diaphragm. Using Bacillus subtilis as target bacteria, our portable lysis apparatus was able to achieve a normalized percent lysis as high as 66% within 30 s. This is comparable to that by microprobe ultrasonication and almost 7 times higher than that by conventional bead beating. The effect from adding glass beads was predictable. However, the results from the addition of sodium dodecyl sulphate (SDS) were counter-intuitive because a further increase from 0·5 to 1% concentration reduced the lysis performance. The portable lysis apparatus is also at least 1·5-5 times more power efficient than microprobe ultrasonication. CONCLUSIONS: Our portable lysis apparatus is capable of rapidly extracting bacterial DNA and is more power efficient than microprobe ultrasonication. The addition of glass beads or SDS concentration (up to 0·5%) improves its performance. SIGNIFICANCE AND IMPACT OF THE STUDY: The portable lysis apparatus provides a standalone, rapid, low cost and power efficient way of obtaining genomic constituents prior to a variety of bioassays used in the field of environmental, biomedical and other applied microbiology.

The paradox of extreme high-altitude migration in bar-headed geese Anser indicus.

Bar-headed geese are renowned for migratory flights at extremely high altitudes over the world's tallest mountains, the Himalayas, where partial pressure of oxygen is dramatically reduced while flight costs, in terms of rate of oxygen consumption, are greatly increased. Such a mismatch is paradoxical, and it is not clear why geese might fly higher than is absolutely necessary. In addition, direct empirical measurements of high-altitude flight are lacking. We test whether migrating bar-headed geese actually minimize flight altitude and make use of favourable winds to reduce flight costs. By tracking 91 geese, we show that these birds typically travel through the valleys of the Himalayas and not over the summits. We report maximum flight altitudes of 7290 m and 6540 m for southbound and northbound geese, respectively, but with 95 per cent of locations received from less than 5489 m. Geese travelled along a route that was 112 km longer than the great circle (shortest distance) route, with transit ground speeds suggesting that they rarely profited from tailwinds. Bar-headed geese from these eastern populations generally travel only as high as the terrain beneath them dictates and rarely in profitable winds. Nevertheless, their migration represents an enormous challenge in conditions where humans and other mammals are only able to operate at levels well below their sea-level maxima.

Patterns and risk factors for locoregional failures after mastectomy for breast cancer: an International Breast Cancer Study Group report.

BACKGROUND: Rates and risk factors of local, axillary and supraclavicular recurrences can guide patient selection and target for postmastectomy radiotherapy (PMRT). PATIENTS AND METHODS: Local, axillary and supraclavicular recurrences were evaluated in 8106 patients enrolled in 13 randomized trials. Patients received chemotherapy and/or endocrine therapy and mastectomy without radiotherapy. Median follow-up was 15.2 years. RESULTS: Ten-year cumulative incidence for chest wall recurrence of >15% was seen in patients aged <40 years (16.1%), with ≥4 positive nodes (16.5%) or 0-7 uninvolved nodes (15.1%); for supraclavicular failures >10%: ≥4 positive nodes (10.2%); for axillary failures of >5%: aged <40 years (5.1%), unknown primary tumor size (5.2%), 0-7 uninvolved nodes (5.2%). In patients with 1-3 positive nodes, 10-year cumulative incidence for chest wall recurrence of >15% were age <40, peritumoral vessel invasion or 0-7 uninvolved nodes. Age, number of positive nodes and number of uninvolved nodes were significant parameters for each locoregional relapse site. CONCLUSION: PMRT to the chest wall and supraclavicular fossa is supported in patients with ≥4 positive nodes. With 1-3 positive nodes, chest wall PMRT may be considered in patients aged <40 years, with 0-7 uninvolved nodes or with vascular invasion. The findings do not support PMRT to the dissected axilla.

TROG 14.04: Multicentre Study of Feasibility and Impact on Anxiety of DIBH in Breast Cancer Patients.

AIMS: The aim of TROG 14.04 was to assess the feasibility of deep inspiration breath hold (DIBH) and its impact on radiation dose to the heart in patients with left-sided breast cancer undergoing radiotherapy. Secondary end points pertained to patient anxiety and cost of delivering a DIBH programme. MATERIALS AND METHODS: The study comprised two groups - left-sided breast cancer patients engaging DIBH and right-sided breast cancer patients using free breathing through radiotherapy. The primary end point was the feasibility of DIBH, defined as left-sided breast cancer patients' ability to breath hold for 15 s, decrease in heart dose in DIBH compared with the free breathing treatment plan and reproducibility of radiotherapy delivery using mid-lung distance (MLD) assessed on electronic portal imaging as the surrogate. The time required for treatment delivery, patient-reported outcomes and resource requirement were compared between the groups. RESULTS: Between February and November 2018, 32 left-sided and 30 right-sided breast cancer patients from six radiotherapy centres were enrolled. Two left-sided breast cancer patients did not undergo DIBH (one treated in free breathing as per investigator choice, one withdrawn). The mean heart dose was reduced from 2.8 Gy (free breathing) to 1.5 Gy (DIBH). Set-up reproducibility in the first week of treatment assessed by MLD was 1.88 ± 1.04 mm (average ± 1 standard deviation) for DIBH and 1.59 ± 0.93 mm for free breathing patients. Using a reproducibility cut-off for MLD of 2 mm (1 standard deviation) as per study protocol, DIBH was feasible for 67% of DIBH patients. Radiotherapy delivery using DIBH took about 2 min longer than for free breathing. Anxiety was not significantly different in DIBH patients and decreased over the course of treatment in both groups. CONCLUSION: Although DIBH was shown to require about 2 min longer per treatment slot, it has the potential to reduce heart dose in left-sided breast cancer patients by nearly a half, provided careful assessment of breath hold reproducibility is carried out.

Three-dimensional reference and stereotactic atlas of human cerebrovasculature from 7Tesla.

Although there exist some reference and stereotactic anatomical human brain atlases, there is no equivalent cerebrovascular atlas. A 3D reference atlas of the human cerebrovasculature that is interactive, stereotactic, very detailed, completely parcellated, fully labeled, extendable, dissectible, deformable, and user friendly, is needed in education, training, research, and clinical applications. We constructed a sophisticated and very detailed cerebrovascular atlas with 1325 vascular segments, the smallest of 80 µm in diameter. The atlas was created from multiple 3 and 7T scans by employing tools and methods developed in our lab. In contrast to a sort of "sampled" vascular geometry cited in the literature, we provide information about the "continuous" geometry of the vascular model measurable in 3D at every location and along any vascular segment for both hemispheres. This cerebrovascular atlas was validated taking into account domain knowledge from various fields including angiography, neurosurgery, neuroradiology, (clinical) neuroanatomy, and terminology. Validation was considered as a confirmation of the created vascular model to a typical (conventional) cerebrovascular system with nearly average dimensions. It was done in terms of vessel subdivision, origin, course, surrounding anatomy, patterns, length, diameter, and branches. This validation demonstrates that the constructed cerebrovascular model generally represents a normal, typical cerebrovasculature with its dimensions close to average. This 7T atlas along with the vascular model is a rich source of knowledge about the human cerebrovasculature. The atlas is applicable in education, research, and clinical applications; it has already been applied in deep brain stimulation.

The utility of postoperative radiotherapy in intermediate-risk oral squamous cell carcinoma.

The effectiveness of postoperative radiotherapy (PORT) in improving outcomes remains debatable for oral squamous cell carcinoma (OSCC) patients with pathological intermediate-risk factors (IRFs) after surgery. A retrospective analysis was conducted on 432 intermediate-risk OSCC patients defined by histological reporting of close margin (<5mm), early nodal disease (pN1), depth of invasion/tumour thickness ≥5mm, perineural invasion, and/or lymphovascular invasion. Outcomes measured were disease-free survival (DFS), disease-specific survival (DSS), and overall survival (OS). PORT was associated with an improvement in 5-year DFS on univariable analysis (80% vs 71%; P=0.044), but this did not remain significant on multivariable analysis. PORT was not associated with differences in DSS or OS. The surgical salvage rate was similar in the PORT and surgery-only groups (41% vs 47%; P=0.972). Perineural invasion was found to be an independent predictor of inferior DSS (hazard ratio (HR) 2.19), DFS (HR 1.89), and OS (HR 1.97). Significantly worse outcomes were observed for patients with ≥4 concurrent IRFs. The application of PORT was associated with lower rates of recurrence, but the benefit was less apparent on mortality. Patients with perineural invasion and multiple concurrent IRFs were found to be at greatest risk, representing a subset of intermediate-risk OSCC patients who may benefit from PORT.

Determining tolerance levels for quality assurance of 3D printed bolus for modulated arc radiotherapy of the nose.

Given the existing literature on the subject, there is obviously a need for specific advice on quality assurance (QA) tolerances for departments using or implementing 3D printed bolus for radiotherapy treatments. With a view to providing initial suggested QA tolerances for 3D printed bolus, this study evaluated the dosimetric effects of changes in bolus geometry and density, for a particularly common and challenging clinical situation: specifically, volumetric modulated arc therapy (VMAT) treatment of the nose. Film-based dose verification measurements demonstrated that both the AAA and the AXB algorithms used by the Varian Eclipse treatment planning system (Varian Medical Systems, Palo Alto, USA) were capable of providing sufficiently accurate dose calculations to allow this planning system to be used to evaluate the effects of bolus errors on dose distributions from VMAT treatments of the nose. Thereafter, the AAA and AXB algorithms were used to calculate the dosimetric effects of applying a range of simulated errors to the design of a virtual bolus, to identify QA tolerances that could be used to avoid clinically significant effects from common printing errors. Results were generally consistent, whether the treatment target was superficial and treated with counter-rotating coplanar arcs or more-penetrating and treated with noncoplanar arcs, and whether the dose was calculated using the AAA algorithm or the AXB algorithm. The results of this study suggest the following QA tolerances are advisable, when 3D printed bolus is fabricated for use in photon VMAT treatments of the nose: bolus relative electron density variation within [Formula: see text] (although an action level at [Formula: see text] may be permissible); bolus thickness variation within [Formula: see text] mm (or 0.5 mm variation on opposite sides); and air gap between bolus and skin [Formula: see text] mm. These tolerances should be investigated for validity with respect to other treatment modalities and anatomical sites. This study provides a set of baselines for future comparisons and a useful method for identifying additional or alternative 3D printed bolus QA tolerances.

Thermoluminescence dosimetry for skin dose assessment during intraoperative radiotherapy for early breast cancer.

Dosimetry for intraoperative radiotherapy (IORT) after wide local excision for breast cancer using a 50 kV X-ray needle (Intrabeam) was performed in vivo using thermoluminescence dosimetry. Eight LiF:Mg,Ti chips were placed on the skin around the incision site after wide local excision while the tumour bed was irradiated to a prescribed dose of 5 Gy 10 mm from the applicator surface. The maximum and mean measured skin dose for 57 patients ranged from 0.64 to 7.1 Gy and 0.56 to 4.78 Gy, respectively, reflecting different tissue thicknesses overlying the applicator. The average maximum dose of 2.93+/-1.46 Gy was below the threshold for severe radiation skin toxicity.

A spreadsheet to determine the volume ratio for target and breast in partial breast irradiation.

The technical feasibility of Partial Breast Irradiation (PBI) using external beam radiotherapy depends on the ratio between the evaluation planning target volume (PTV_eval) and the whole breast volume ("PBI volume ratio" = PVR). We aimed to develop a simple method to determine PVR using measurements performed at the time of the planning CT scan. A PVR calculation tool was developed using a Microsoft Excel spreadsheet to determine the PTV from three orthogonal dimensions of the seroma cavity and a given margin on the CT scans. The breast volume is estimated from the separation and breast height in five equally spaced CT slices. The PTV_eval and whole breast volume were determined for 29 patients from two centres using the spreadsheet calculation tool and compared to volumes delineated on computerised treatment planning systems. Both the PTV_eval and whole breast volumes were underestimated by approximately 25% using the spreadsheet. The resulting PVRs were 1.05 +/- 0.35 (mean +/- 1SD) times larger than the ones determined from planning. Estimations of the PVR using the calculation tool were achievable in around 5 minutes at the time of CT scanning and allow a prompt decision on the suitability of the patients for PBI.

Tracking the mental health of a nation: prevalence and correlates of mental disorders in the second Singapore mental health study.

AIMS: The second Singapore Mental Health Study (SMHS) - a nationwide, cross-sectional, epidemiological survey - was initiated in 2016 with the intent of tracking the state of mental health of the general population in Singapore. The study employed the same methodology as the first survey initiated in 2010. The SMHS 2016 aimed to (i) establish the 12-month and lifetime prevalence and correlates of major depressive disorder (MDD), dysthymia, bipolar disorder, generalised anxiety disorder (GAD), obsessive compulsive disorder (OCD) and alcohol use disorder (AUD) (which included alcohol abuse and dependence) and (ii) compare the prevalence of these disorders with reference to data from the SMHS 2010. METHODS: Door-to-door household surveys were conducted with adult Singapore residents aged 18 years and above from 2016 to 2018 (n = 6126) which yielded a response rate of 69.0%. The subjects were randomly selected using a disproportionate stratified sampling method and assessed using World Health Organization Composite International Diagnostic Interview version 3.0 (WHO-CIDI 3.0). The diagnoses of lifetime and 12-month selected mental disorders including MDD, dysthymia, bipolar disorder, GAD, OCD, and AUD (alcohol abuse and alcohol dependence), were based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria. RESULTS: The lifetime prevalence of at least one mood, anxiety or alcohol use disorder was 13.9% in the adult population. MDD had the highest lifetime prevalence (6.3%) followed by alcohol abuse (4.1%). The 12-month prevalence of any DSM-IV mental disorders was 6.5%. OCD had the highest 12-month prevalence (2.9%) followed by MDD (2.3%). Lifetime and 12-month prevalence of mental disorders assessed in SMHS 2016 (13.8% and 6.4%) was significantly higher than that in SMHS 2010 (12.0% and 4.4%). A significant increase was observed in the prevalence of lifetime GAD (0.9% to 1.6%) and alcohol abuse (3.1% to 4.1%). The 12-month prevalence of GAD (0.8% vs. 0.4%) and OCD (2.9% vs. 1.1%) was significantly higher in SMHS 2016 as compared to SMHS 2010. CONCLUSIONS: The high prevalence of OCD and the increase across the two surveys needs to be tackled at a population level both in terms of creating awareness of the disorder and the need for early treatment. Youth emerge as a vulnerable group who are more likely to be associated with mental disorders and thus targeted interventions in this group with a focus on youth friendly and accessible care centres may lead to earlier detection and treatment of mental disorders.

The Singaporean public beliefs about the causes of mental illness: results from a multi-ethnic population-based study.

AIMS: To identify the common causal beliefs of mental illness in a multi-ethnic Southeast Asian community and describe the sociodemographic associations to said beliefs. The factor structure to the causal beliefs scale is explored. The causal beliefs relating to five different mental illnesses (alcohol abuse, depression, obsessive-compulsive disorder (OCD), dementia and schizophrenia) and desire for social distance are also investigated. METHODS: Data from 3006 participants from a nationwide vignette-based study on mental health literacy were analysed using factor analysis and multiple logistic regression to address the aims. Participants answered questions related to sociodemographic information, causal beliefs of mental illness and their desire for social distance towards those with mental illness. RESULTS: Physical causes, psychosocial causes and personality causes were endorsed by the sample. Sociodemographic differences including ethnic, gender and age differences in causal beliefs were found in the sample. Differences in causal beliefs were shown across different mental illness vignettes though psychosocial causes was the most highly attributed cause across vignettes (endorsed by 97.9% of respondents), followed by personality causes (83.5%) and last, physical causes (37%). Physical causes were more likely to be endorsed for OCD, depression and schizophrenia. Psychosocial causes were less often endorsed for OCD. Personality causes were less endorsed for dementia but more associated with depression. CONCLUSIONS: The factor structure of the causal beliefs scale is not entirely the same as that found in previous research. Further research on the causal beliefs endorsed by Southeast Asian communities should be conducted to investigate other potential causes such as biogenetic factors and spiritual/supernatural causes. Mental health awareness campaigns should address causes of mental illness as a topic. Lay beliefs in the different causes must be acknowledged and it would be beneficial for the public to be informed of the causes of some of the most common mental illnesses in order to encourage help-seeking and treatment compliance.

Immunological responses following administration of a genotype 1a/1b/2/3a quadrivalent HCV VLP vaccine.

The significant public health problem of Hepatitis C virus (HCV) has been partially addressed with the advent of directly acting antiviral agents (DAAs). However, the development of an effective preventative vaccine would have a significant impact on HCV incidence and would represent a major advance towards controlling and possibly eradicating HCV globally. We previously reported a genotype 1a HCV viral-like particle (VLP) vaccine that produced neutralizing antibodies (NAb) and T cell responses to HCV. To advance this approach, we produced a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine to produce broader immune responses. We show that this quadrivalent vaccine produces antibody and NAb responses together with strong T and B cell responses in vaccinated mice. Moreover, selective neutralizing human monoclonal antibodies (HuMAbs) targeting conserved antigenic domain B and D epitopes of the E2 protein bound strongly to the HCV VLPs, suggesting that these critical epitopes are expressed on the surface of the particles. Our findings demonstrate that a quadrivalent HCV VLP based vaccine induces broad humoral and cellular immune responses that will be necessary for protection against HCV. Such a vaccine could provide a substantial addition to highly active antiviral drugs in eliminating HCV.

Patterns of rapd markers and heavy metal concentrations in Perna viridis (L.), collected from metal-contaminated and uncontaminated coastal waters: are they correlated with each other?

Genetic variation due to heavy metal contamination has always been an interesting topic of study. Because of the numerous contaminants being found in coastal and intertidal waters, there is always much discussion and argument as to which contaminant(s) caused the variations in the genetic structures of biomonitors. This study used a Single Primer Amplification Reaction (SPAR) technique namely Random Amplified Polymorphic DNA (RAPD) to determine the genetic diversity of the populations of the green-lipped mussel Perna viridis collected from a metal-contaminated site at Kg. Pasir Puteh and those from four relatively' uncontaminated sites (reference sites). Heavy metal levels (Cd, Cu, Pb and Zn) were also measured in the soft tissues and byssus of the mussels from all the sites. Cluster analyses employing UPGMA done based on the RAPD makers grouped the populations into two major clusters; the Bagan Tiang, Pantai Lido, Pontian and Kg. Pasir Puteh populations were in one cluster, while the Sg. Belungkor population clustered by itself. This indicated that the genetic diversity based on bands resulting from the use of all four RAPD primers on P. viridis did not indicate its potential use as a biomarker of heavy metal pollution in coastal waters. However, based on a correlation analysis between a particular metal and a band resulting from a specific RAPD primer revealed some significant (P < 0.01) correlations between the primers and the heavy metal concentrations in the byssus and soft tissues. Thus, the correlation between a particular metal and the bands resulting from the use of a specific RAPD primer on P. viridis could be used as biomonitoring tool of heavy metal pollution.

Genetic diversity of enterovirus 71 isolated from cases of hand, foot and mouth disease in the 1997, 2000 and 2005 outbreaks, Peninsular Malaysia.

All known field isolates of enterovirus 71 (EV71) can be divided into three distinct genogroups (A, B, C) and 10 subgenogroups (A, B1-5, C1-4) based on VP1 gene sequences. We examined VP1 gene sequences of 10, 12 and 11 EV71 strains isolated in peninsular Malaysia during the outbreaks of hand, foot and mouth disease in 1997, 2000 and 2005 respectively. Four EV71 strains isolated in the hand, foot and mouth disease outbreak of 2006 in Sarawak (Malaysian Borneo) were included to describe their genetic relationship. Four subgenogroups (C1, C2, B3 and B4) of EV71 co-circulated and caused the outbreak of hand, foot and mouth disease in peninsular Malaysia in 1997. Two subgenogroups (C1 and B4) were noted to cause the outbreak in 2000. In the 2005 outbreak, besides EV71 strains of subgenogroup C1, EV71 strains belonged to subgenogroup B5 were isolated but formed a cluster which was distinct from EV71 strains of the subgenogroup B5 isolated in 2003. The four EV71 strains isolated from clinical specimens of patients with hand, foot and mouth disease in the Sarawak outbreak in early 2006 also belonged to subgenogroup B5. Phylogenetic analysis of the VP1 gene sequences showed that the four Sarawak EV71 isolates belonged to the same cluster as the EV71 strains that were isolated in peninsular Malaysia as early as May 2005. The data suggested that the EV71 strains causing the outbreak in Sarawak could have originated from peninsular Malaysia.

Growth-dependent factors in the regulation of aminoacyl-tRNA synthetase activities of Tetrahymena pyriformis.

Changes in phenylalanyl-tRNA synthetase (L-phenylalanine : tRNAPhe ligase, EC 6.1.1.20) and leucyl-tRNA synthetase (L-leucine : tRNALeu ligase. EC 6.1.1.4) activities were studied during the growth cycle of Tetrahymena pyriformis. High levels of charged tRNA observed during exponential growth were associated with elevated aminoacyl-tRNA synthetase activities. Low levels of charges tRNA in the stationary phase culture were associated with decreased aminoacyl-tRNA synthethase activities together with a concomitant accumulation of factor(s) which inhibited the enzyme activities. The inhibitory factor(s) has been partially purified and evidence is presented to rule out RNA, RNAases, proteases and ATPases as the responsible inhibitory factor(s) of the aminoacyl-tRNA synthetases.

Upregulation of vascular endothelial growth factor by angiotensin II in rat heart endothelial cells.

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and a vascular permeability factor. In this study we found that the addition of angiotensin II (AII) to rat heart endothelial cells induced VEGF mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of AII and decreased after 4 h. The induction was superinduced by cycloheximide and blocked by actinomycin D. Losartan, an AT1 receptor antagonist, abolished the induction of VEGF mRNA by AII, whereas PD 123319, an AT2 receptor antagonist, had no effect on VEGF mRNA induction. H7, a protein kinase C inhibitor, blocked the induction. RT-PCR experiments showed two mRNA species (VEGF 120 and VEGF 164) in these cells and both species were stimulated by AII. Transient transfection experiment showed that VEGF promoter activity was increased 2.2-fold upon AII stimulation. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-kappa B. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 8 h after AII stimulation. Our results demonstrate for the first time that the upregulation of VEGF by AII may play a significant role in AII-induced hyperpermeability.

Regulation of endothelin-1 mRNA by angiotensin II in rat heart endothelial cells.

In a series of experiments carried out in cultured endothelial cells derived from rat hearts (RHE), angiotensin II (AII) is shown to stimulate preproendothelin-1 mRNA in a dose- and time-dependent manner. The induction of preproendothelin-1 mRNA is rapid, reaching a maximal level 1 h after the addition of AII (1 x 10(-8) M). The mRNA levels decline rapidly to basal levels in 4 h. The addition of Losartan (Dup 753; 1 x 10(-6) M), an AII receptor (type I) antagonist, blocks the AII effect. Calphostin C, a potent protein kinase C inhibitor, is able to abolish this effect of AII suggesting that the induction of preproendothelin-1 mRNA is mediated by a protein kinase C-dependent pathway. Since endothelial cells line the inner surface of the myocardium and blood vessels and sense the rise of AII associated with renovascular hypertension at the endothelial surface, these data suggest that endothelin which is produced by RHE cells in response to AII could be an important mediator which may play a role in modulating gene expression in AII-mediated cardiac hypertrophy.

Angiotensin II induces TGF-beta 1 production in rat heart endothelial cells.

Angiotensin II (AII) was found to stimulate TGF-beta 1 gene expression in rat heart endothelial cells in a dose- and time-dependent manner. The maximal induction of TGF-beta 1 mRNA was achieved by 6 h after the addition of AII. This induction was blocked by losartan, an AT1 receptor antagonist and by calphostin C, a protein kinase C inhibitor. Addition of actinomycin D and cycloheximide abolished the induction. TGF-beta 1 promoter activities were stimulated 5-fold by AII. TGF-beta 1 secreted by the rat heart endothelial cells in response to AII was in a latent form and could be activated by mild heat treatment. These results suggest that AII stimulates TGF-beta 1 production by a protein kinase C-dependent pathway which is dependent upon de novo RNA synthesis and protein synthesis. Since endothelial cells line the blood vessels and sense the rise in AII associated with hypertension, the release of TGF-beta 1 by these cells may provide the initial trigger leading to cardiac fibrosis in angiotensin-renin-dependent hypertension.