Lichen Sclerosis is Associated With a High Rate of Local Failure After Radio(chemo)therapy for Vulvar Cancer.

AIMS: Radio(chemo)therapy plays an important role in the treatment of vulvar cancer, either as postoperative treatment or as definitive treatment in patients who present with inoperable disease. Only limited data are available regarding outcome after modern state of the art radio(chemo)therapy and more information regarding prognostic factors are warranted. The aim of this study was to evaluate disease outcomes after radio(chemo)therapy in patients with vulvar cancer with special emphasis on the impact of lichen sclerosis on local control. MATERIALS AND METHODS: All consecutive patients (n = 109) from the western half of Denmark who were treated with definitive (n = 52) or postoperative (n = 57) radio(chemo)therapy between January 2013 and January 2020 were included. Local control, cause-specific survival and overall survival, as well as morbidity, were analysed using Kaplan-Meier statistics. Prognostic factors for local control were analysed in univariate and multivariate analysis. RESULTS: At a median follow-up of 35 (4-95) months, 46 (42.0%) patients were diagnosed with recurrence. Eighty per cent of the recurrences were located to the vulva region, leading to a 5-year local control of 58.9% (confidence interval 47.9-69.9). Cause-specific survival was 62.9% (confidence interval 53.1-72.7), whereas overall survival was 58.0% (confidence interval 47.6-68.5). Grade 3-4 morbidity was diagnosed in 10 (9%) patients. Lichen sclerosis (hazard ratio 3.89; confidence interval 1.93-7.79) was an independent risk factors for local recurrence. Patients without lichen sclerosis had a 5-year local control rate of 83.6% (confidence interval 67.2-99.0) and 62.6% (confidence interval 43.2-82.0) after postoperative and definitive radio(chemo)therapy, respectively. In patients with lichen sclerosis, the local control rate was 44.0% (confidence interval 19.3-69.0) and 17.6% (confidence interval 0-30.0) after postoperative and definitive radio(chemo)therapy, respectively. CONCLUSION: Radio(chemo)therapy plays an important role in the treatment of vulvar cancer. However, despite dose escalation, a substantial proportion of patients experienced local relapse. Pre-existing lichen sclerosis seems to have a significant impact on the risk of recurrence. This should influence surveillance programmes for these patients.

Risk of miscarriage following amniocentesis or chorionic villus sampling: systematic review of literature and updated meta-analysis.

OBJECTIVE: To estimate the procedure-related risk of miscarriage after amniocentesis and chorionic villus sampling (CVS) based on a systematic review of the literature and an updated meta-analysis. METHODS: A search of MEDLINE, EMBASE and The Cochrane Library was carried out to identify studies reporting complications following CVS or amniocentesis. Eligible for inclusion were large controlled studies reporting data for pregnancy loss prior to 24 weeks' gestation. Study authors were contacted when required to identify additional necessary data. Data for cases that had an invasive procedure and controls were inputted into contingency tables and the risk of miscarriage was estimated for each study. Summary statistics based on a random-effects model were calculated after taking into account the weighting for each study included in the systematic review. Procedure-related risk of miscarriage was estimated as a weighted risk difference from the summary statistics for cases and controls. Subgroup analyses were performed according to the similarity in risk levels for chromosomal abnormality between the invasive-testing and control groups. Heterogeneity was assessed using the I2 statistic. Egger's bias was estimated to assess reporting bias in published studies. RESULTS: The electronic search yielded 2943 potential citations, from which 12 controlled studies for amniocentesis and seven for CVS were selected for inclusion in the systematic review. A total of 580 miscarriages occurred following 63 723 amniocentesis procedures, resulting in a weighted risk of pregnancy loss of 0.91% (95% CI, 0.73-1.09%). In the control group, there were 1726 miscarriages in 330 469 pregnancies with a loss rate of 0.58% (95% CI, 0.47-0.70%). The weighted procedure-related risk of miscarriage following amniocentesis was 0.30% (95% CI, 0.11-0.49%; I2 = 70.1%). A total of 163 miscarriages occurred following 13 011 CVS procedures, resulting in a risk of pregnancy loss of 1.39% (95% CI, 0.76-2.02%). In the control group, there were 1946 miscarriages in 232 680 pregnancies with a loss rate of 1.23% (95% CI, 0.86-1.59%). The weighted procedure-related risk of miscarriage following CVS was 0.20% (95% CI, -0.13 to 0.52%; I2 = 52.7%). However, when studies including only women with similar risk profiles for chromosomal abnormality in the intervention and control groups were considered, the procedure-related risk for amniocentesis was 0.12% (95% CI, -0.05 to 0.30%; I2 = 44.1%) and for CVS it was -0.11% (95% CI, -0.29 to 0.08%; I2 = 0%). CONCLUSIONS: The procedure-related risks of miscarriage following amniocentesis and CVS are lower than currently quoted to women. The risk appears to be negligible when these interventions were compared to control groups of the same risk profile. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Riesgo de aborto después de una amniocentesis o una biopsia de vellosidades coriónicas: revisión sistemática de bibliografía y metaanálisis actualizado OBJETIVO: Estimar el riesgo de aborto relacionado con el procedimiento de la amniocentesis o la biopsia de vellosidades coriónicas (BVC) mediante una revisión sistemática de bibliografía y un metaanálisis actualizado. MÉTODOS: Se realizó una búsqueda en MEDLINE, EMBASE y The Cochrane Library para identificar estudios que reportaron sobre complicaciones después de una BVC o amniocentesis. Se consideraron elegibles para su inclusión los estudios controlados de gran tamaño que reportaron datos sobre la pérdida del embarazo antes de las 24 semanas de gestación. Se estableció contacto con los autores de los estudios cuando fue necesario para identificar datos adicionales necesarios. Se introdujeron en tablas de contingencia los datos de los casos que se sometieron a un procedimiento invasivo y controles y se estimó el riesgo de aborto para cada estudio. Las estadísticas resumen basadas en un modelo de efectos aleatorios se calcularon después de tener en cuenta la ponderación para cada estudio incluido en la revisión sistemática. El riesgo de aborto relacionado con cada procedimiento se estimó como una diferencia de riesgo ponderada de las estadísticas resumen para los casos y controles. Los análisis de subgrupos se realizaron de acuerdo con la similitud en los niveles de riesgo de anomalías cromosómicas entre los grupos de prueba invasiva y de control. La heterogeneidad se evaluó mediante el test estadístico I2 . Se estimó el sesgo de Egger para evaluar el sesgo de información reportada en los estudios publicados. RESULTADOS: La búsqueda electrónica arrojó 2943 citas potenciales, de las cuales se seleccionaron para su inclusión en la revisión sistemática 12 estudios controlados para la amniocentesis y siete para la BVC. Después de los 63723 procedimientos de amniocentesis sucedieron un total de 580 abortos, lo que resultó en un riesgo ponderado de pérdida de embarazo del 0,91% (IC 95%, 0,73-1,09%). En el grupo de control hubo 1726 abortos en 330469 embarazos, con una tasa de pérdida del 0,58% (IC 95%, 0,47-0,70%). El riesgo ponderado de aborto relacionado con el procedimiento de amniocentesis fue del 0,30% (IC 95%, 0,11-0,49%; I2  = 70,1%). Después de 13011 procedimientos de BVC se produjeron un total de 163 abortos, lo que resultó en un riesgo de pérdida de embarazo del 1,39% (IC 95%, 0,76-2,02%). En el grupo de control hubo 1946 abortos en 232680 embarazos, lo que supuso una tasa de pérdida del 1,23% (IC 95%, 0,86-1,59%). El riesgo ponderado de aborto relacionado con el procedimiento de BVC fue de 0,20% (IC 95%, -0,13-0,52%; I2  = 52,7%). Sin embargo, cuando se consideraron los estudios que incluyeron sólo mujeres con perfiles de riesgo similares para la anomalía cromosómica en los grupos de intervención y control, el riesgo relacionado con el procedimiento de la amniocentesis fue de 0,12% (IC 95%, -0,05-0,30%; I2  = 44.1%) y para el MVC fue de -0,11% (IC 95%, -0,29-0,08%; I2  = 0%). CONCLUSIONES: Los riesgos de aborto relacionados con el procedimiento de la amniocentesis y la BVC son menores que los actualmente mencionados a las mujeres. El riesgo parece ser insignificante cuando estas intervenciones se compararon con grupos de control del mismo perfil de riesgo.

Neurodevelopmental disorder in children believed to have isolated mild ventriculomegaly prenatally.

OBJECTIVE: To estimate the prevalence of specific neurodevelopmental disorders in children believed to have isolated mild ventriculomegaly (IMV) prenatally in the second trimester of pregnancy, in order to optimize the counseling process. METHODS: This was a nationwide registry-based study including all singleton pregnancies that had first- and second-trimester ultrasound scans in the period 1st January 2008 to 1st October 2014, identified in the Danish Fetal Medicine Database and local clinical databases in Denmark. All fetuses diagnosed prenatally with IMV (measurement of the atrium of the lateral ventricles, 10.0-15.0 mm) between 18 and 22 weeks' gestation were followed up in national patient registers until the age of 2-7 years. Information was obtained on the diagnoses of intellectual disability, cerebral palsy, autism spectrum disorder, epilepsy and impaired psychomotor development. Neurodevelopmental disorders were compared between those with postnatally confirmed IMV and a reference population of children in the same age range. RESULTS: Of a cohort of 292 046 fetuses, 133 were found to have apparent IMV on the second-trimester scan for fetal malformations. In 11 cases, long-term follow-up was not possible owing to termination of pregnancy, spontaneous miscarriage, neonatal death or loss to follow-up. Of the 122 liveborn children followed up until 2-7 years, 15 were identified as having an additional abnormality while 107 were confirmed postnatally to have IMV. Of these 107 children, the diagnosis of a neurodevelopmental disorder was registered in six (5.6%), corresponding to an odds ratio of 2.64 (95% CI, 1.16-6.02), as compared with the reference population. The diagnoses were autism spectrum disorder, epilepsy and impaired psychomotor development. None of these 107 children was diagnosed with intellectual disability or cerebral palsy. CONCLUSIONS: Our results show that a confirmed diagnosis of IMV was associated with an increased risk of a neurodevelopmental disorder, as compared with the reference population, but the absolute risk was low and there were no cases of intellectual disability or cerebral palsy. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Trastorno del desarrollo neurológico en fetos con sospecha de ventriculomegalia leve aislada prenatal OBJETIVO: Estimar la prevalencia de trastornos específicos del desarrollo neurológico en fetos con sospecha de ventriculomegalia leve aislada (IMV, por sus siglas en inglés) prenatal en el segundo trimestre del embarazo, a fin de optimizar el proceso de asesoramiento. MÉTODOS: Este estudio estuvo basado en un registro nacional que incluyó todos los embarazos con feto único a los que se les hizo ecografías en el primer y segundo trimestre entre el 1 de enero de 2008 y el 1 de octubre de 2014, identificados en la Base de Datos Danesa de Medicina Fetal y en las bases de datos clínicas locales en Dinamarca. Todos los fetos diagnosticados prenatalmente con IMV (por medición de la aurícula de los ventrículos laterales, 10,0-15,0 mm) entre las semanas de gestación 18 y 22 fueron monitoreados en los registros nacionales de pacientes hasta la edad de 2-7 años. Se obtuvo información sobre los diagnósticos de discapacidad intelectual, parálisis cerebral, trastornos del espectro autista, epilepsia y trastornos del desarrollo psicomotor. Se compararon los trastornos del desarrollo neurológico entre aquellos con IMV confirmada después del nacimiento y una población de referencia de niños en el mismo rango de edad. RESULTADOS: De una cohorte de 292 046 fetos, se encontró que 133 tenían IMV aparente en la ecografía del segundo trimestre realizada para detectar malformaciones fetales. El seguimiento a largo plazo no fue posible en 11 casos debido a la interrupción del embarazo, el aborto espontáneo, la muerte del recién nacido o el abandono del monitoreo. De los 122 niños nacidos vivos a los que se les dio seguimiento hasta los 2-7 años, se identificó a 15 con una anomalía adicional, mientras que a 107 se les confirmó postnatalmente que tenían IMV. De estos 107 niños, se registró el diagnóstico de un trastorno del desarrollo neurológico en seis (5,6%), lo que corresponde a una razón de momios de 2,64 (IC 95%: 1,16-6,02), en comparación con la población de referencia. Los diagnósticos fueron trastornos del espectro autista, epilepsia y trastornos del desarrollo psicomotor. Ninguno de estos 107 niños fue diagnosticado con discapacidad intelectual o parálisis cerebral. CONCLUSIONES: Nuestros resultados muestran que un diagnóstico confirmado de IMV se asoció con un mayor riesgo de trastorno del desarrollo neurológico, en comparación con la población de referencia, pero que el riesgo absoluto fue bajo y no hubo casos de discapacidad intelectual o parálisis cerebral.

Transvaginal sonographic cervical length in first and second trimesters in a low-risk population: a prospective study.

OBJECTIVES: To assess cervical length (CL) longitudinally between the first and second trimesters and to determine the proportion of women with short CL. The study also aimed to assess if women with short CL at 19-24 weeks' gestation could be identified at the time of combined first-trimester screening (cFTS) at 11-14 weeks' gestation, in order to determine the potential value of implementation of CL screening for prediction of preterm delivery in a Danish population. METHODS: This was a prospective longitudinal study of women with singleton pregnancy attending three University Hospitals in Denmark for combined first-trimester screening from 1 November 2013 to 1 December 2014. Exclusion criteria were multiple pregnancy, uterine anomaly, cerclage or progesterone treatment at inclusion. CL was measured on transvaginal sonography at 11-14 weeks (Cx1), 19-21 weeks (Cx2) and 23-24 weeks (Cx3), by trained operators as a straight line from external to internal os. Women with CL ≤ 25 mm were referred to a maternal-fetal medicine specialist for treatment according to a standardized management protocol. RESULTS: Of the 4904 eligible women, 3477 (71%) participated and had Cx1 recorded. Of those, 3232 (93.0%) had CL measured on all three scans. Median Cx1 was 37 mm, and median Cx2 and Cx3 were 40 mm. The proportion of women with CL ≤ 25 mm increased with gestational age, from 0.41% (95% CI, 0.19-0.62%) at Cx1 to 1.79% (95% CI, 1.34-2.24%) at Cx3. In total, the proportion of women with second-trimester CL (Cx2 or Cx3) ≤ 25 mm was 2.0% (n = 67), of which 38.8% (n = 26) were detected at 19-21 weeks. The probability of short CL between 19 and 24 weeks was greater for those with shorter first-trimester CL. It was nearly nine-fold higher for women with Cx1 ≤ 25 mm compared with Cx1 ≥ 35 mm (17% vs 2%). The performance of Cx1 for prediction of short second-trimester CL was 50% at a 10% false-positive rate. It was found that more than 1500 women would need to be screened for short CL at 19-21 weeks to prevent one case of spontaneous preterm delivery before 34 weeks in a population such as the one in this study. CONCLUSIONS: There is an association between first-trimester CL and risk of short cervix in the second trimester. Once short CL was observed, risk of preterm delivery was greatly increased. However, whether universal CL screening should be implemented in this low-risk population depends on cost-benefit analysis taking into account the low proportions of women with short CL and at risk for preterm delivery. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

Slit2/Robo4 Signaling: Potential Role of a VEGF-Antagonist Pathway to Regulate Luteal Permeability.

Introduction The corpus luteum (CL) is dependent on luteal vascular permeability, which is controlled by human chorionic gonadotropin (hCG) via vascular endothelial growth factor (VEGF). In this study we investigated the role of a potential VEGF antagonist pathway - Slit2/Robo4 - and its influence on endothelial cell adhesion. Materials and Methods Luteinized granulosa cells (LGCs) were stimulated with hCG in the absence or presence of a VEGF inhibitor. The expression of VEGF and Slit2 were measured. Human umbilical vein endothelial cells (HUVECs) were stimulated with Slit2 or VEGF, and gene expressions of cadherin 5 (CDH5) and claudin 5 (CLDN5) were measured. Following Robo4 knockdown, CDH5, CLDN5 and endothelial permeability were measured. Results Stimulation of human LGCs with hCG significantly increased VEGF while Slit2 expression was significantly suppressed. Inhibition of VEGF action after hCG stimulation did not change Slit2 suppression. Slit2 knockdown did not affect VEGF expression. While VEGF stimulation of HUVECs significantly suppressed CDH5 and CLDN5 gene expression, stimulation of HUVECs with Slit2 resulted in a significant increase in CDH5 and CLDN5. Robo4 knockdown was done, leading to downregulation of CDH5 and CLDN5 which resulted in significantly increased permeability. Conclusions Our results indicate the existence of a VEGF-antagonist pathway in the CL that decreases vascular permeability. During the functional life of the CL the pathway is suppressed by hCG. It is possible that stimulation of this pathway could be used to treat ovarian hyperstimulation syndrome.

Risk of fetal loss associated with invasive testing following combined first-trimester screening for Down syndrome: a national cohort of 147,987 singleton pregnancies.

OBJECTIVE: To assess prospectively the risk of fetal loss associated with chorionic villus sampling (CVS) and amniocentesis (AC) following combined first-trimester screening (cFTS) for Down syndrome. METHODS: This was a nationwide population-based study (Danish Fetal Medicine Database, 2008-2010) including 147,987 women with singleton pregnancy who underwent cFTS. Propensity score stratification was used to assess the risk of fetal loss with and without invasive testing. Analyses were performed between 3 and 21 days after cFTS for CVS and between 28 and 42 days after cFTS for AC. Results are reported as average risk differences with 95% CIs. RESULTS: The risks of miscarriage and stillbirth were not higher in women exposed to CVS or AC compared with unexposed women, independent of the analysis time-point. The average effect of CVS on risk of miscarriage was -0.08% (95% CI, -0.64; 0.47) at 3 days and -0.21% (95% CI, -0.58; 0.15) at 21 days after cFTS, while the effect on risk of stillbirth was -0.18% (95% CI, -0.50; 0.13) at 3 days and -0.27% (95% CI, -0.58; 0.04) at 21 days after cFTS. Regarding the effect of AC on risk of miscarriage, the analysis at 28 days after cFTS showed an average effect of 0.56% (95% CI, -0.21; 1.33), while the effect on risk of stillbirth was 0.09% (95% CI, -0.39; 0.58) at 42 days after cFTS. CONCLUSION: Neither CVS nor AC was associated with increased risk of miscarriage or stillbirth. These findings indicate that the procedure-related risk of CVS and AC is very low.

Regulation of Endothelial Permeability in the Corpus Luteum: A Review of the Literature.

The development of the human corpus luteum (yellow body) is dictated by a strictly controlled system of mutually communicating cells, the luteal steroid hormone-producing cells and endothelial cells. This cell-to-cell communication facilitates control of neoangiogenesis which is a prerequisite for the development of the corpus luteum and its function, the rapid release of large amounts of progesterone into the blood-vascular system. Preconditions for this process are the hormonal regulation of endothelial cell proliferation as well as of vascular permeability through LH and hCG. The morphological correlates of endothelial permeability are cell-to-cell adhesion molecules such as adherens junctions (AJ) and tight junctions (TJ) that open and close the gaps between mutually interacting, neighbouring endothelial cells like a "zip fastener". Various types of cell adhesion molecules have been detected in the corpus luteum such as occludin, claudin 1 and claudin 5 as well as VE-cadherin. It may be assumed that the regulation of AJ and TJ proteins is of particular importance for the permeability and thus for the function of the corpus luteum in early pregnancy since hCG treatment leads to a down-regulation of cell adhesion molecules in the luteal vessels. This effect is apparently mediated by VEGF. From a functional point of view, the hCG-dependent and VEGF-mediated down-regulation of cell adhesion molecules leads to a reduced transmissibility of cell-to-cell contacts and thus to an increased endothelial permeability. In this process the various cell adhesion molecules are not only directly regulated by VEGF but they also mutually interact and thus influence one another.

Molecular mechanisms of ovarian hyperstimulation syndrome: paracrine reduction of endothelial claudin 5 by hCG in vitro is associated with increased endothelial permeability.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a potentially life-threatening complication of ovarian stimulation associated with severe vascular hyperpermeability. Primary co-cultures of human luteinized granulosa cells (LGCs) and human umbilical vein endothelial cells (HUVECs) were used as a model of steroidgenic/endothelial cell interaction in OHSS. METHODS: hCG and the vascular endothelial growth factor (VEGF) inhibitor, Flt-1Fc, were added to co-cultures of LGCs and HUVECs separated by a micropore membrane. Endothelial permeability to labeled bovine serum albumin was measured and the expression of the endothelial cell-specific adhesion protein claudin 5 was investigated using immunocytochemistry and western blotting. RESULTS: The addition of hCG increased HUVEC permeability in the presence of LGCs (P < 0.05). hCG increased VEGF concentrations in both chambers of the co-culture system (P < 0.05). The increased permeability in the presence of LGCs and hCG was inhibited when VEGF was blocked by Flt-1Fc (P < 0.05). Endothelial membrane claudin 5 protein was reduced in the presence of hCG and LGCs, as measured by immunocytochemistry (P < 0.05) and western blotting (P < 0.05) and this reduction was inhibited by Flt-1Fc. hCG had no direct effects on endothelial cell claudin 5. CONCLUSIONS: For OHSS, this novel paradigm suggests that hCG can increase endothelial permeability by up-regulating VEGF in LGCs which causes reduction in endothelial claudin 5 expression.

Potential role of Renin-Angiotensin-system for tumor angiogenesis in receptor negative breast cancer.

OBJECTIVE: This study examined the potential role of Angiotensin II for the regulation of angiogenesis associated genes in receptor positive and negative human breast cancer. METHODS: Expression of different Renin-Angiotensin system (RAS) components in human breast cancer tissue was investigated using immunofluorescence, and in a receptor positive (MCF-7) and receptor negative (MDA-MB 468) breast cancer cell line by performing immunocytochemistry and RT-PCR. Both cell lines were stimulated with Angiotensin II and Angiotensin II receptor type 1 (At(1)R) blocker Candesartan, and gene expression of vascular endothelial growth factor (VEGF), Angiopoietin 1 and 2 (Ang-1 and Ang-2), tissue inhibitor of matrix metalloproteinases 1 (TIMP-1), and hypoxia inducible transcription factor 2alpha (HIF-2alpha) were quantified by TaqMan-Real-Time PCR analysis. RESULTS: RAS components, Angiotensinogen, Renin, Angiotensin I-converting enzyme (ACE), and At(1)R and At(2)R were expressed in hormone-receptor negative and positive human breast cancer tissue as well as in MDA-MB 468 and in MCF-7 human breast cancer cells. In addition, we found expression of VEGF, Ang-1, TIMP-1, and HIF-2alpha in both cell lines. However, only in receptor negative MDA-MB 468 cells, did Angiotensin II significantly increase gene expression of VEGF, HIF-2alpha, and TIMP-1. This effect was completely inhibited by Candesartan. CONCLUSION: In conclusion, it is hypothesized that Angiotensin II may be involved in regulation of tumor angiogenesis especially in receptor negative breast cancer by regulation of angiogenesis associated genes via At(1)R. These findings are the first evidence for targeting tumor angiogenesis by inhibition of At(1)R in receptor negative human breast cancer cells and may lead to new therapeutical anticancer strategies based upon inhibition of At(1)R.

Regulation of endothelial proliferation by the renin-angiotensin system in human umbilical vein endothelial cells.

This study was performed in order to evaluate the role of angiotensin II in physiological angiogenesis. Human umbilical vein endothelial cells (HUVEC) were stained for angiotensin II type 1 receptor (AGTR1) immunocytochemically and for gene expression of renin-angiotensin system (RAS) components. The regulation of the angiogenesis-associated genes vascular endothelial growth factor (VEGF) and angiopoietins (ANGPT1 and ANGPT2) were studied using quantitative RT-PCR. Furthermore, we examined the effect of angiotensin II on the proliferation of HUVEC using Ki-67 as well as BrdU immunocytochemistry and investigated whether the administration of the AGTR1 blocker candesartan or the VEGF antagonist FLT1-Fc could suppress the observed angiotensin II-dependent proangiogenic effect. AGTR1 was expressed in HUVEC and the administration of angiotensin II significantly increased the gene expression of VEGF and decreased the gene expression of ANGPT1. Since the expression of ANGPT2 was not affected significantly the ratio of ANGPT1/ANGPT2 was decreased. In addition, a significantly increased endothelial cell proliferation was observed after stimulation with angiotensin II, which was suppressed by the simultaneous administration of candesartan or the VEGF antagonist FLT1-Fc. These results indicate the potential capacity of angiotensin II in influencing angiogenesis by the regulation of angiogenesis-associated genes via AGTR1. Since VEGF blockade opposed the effect of angiotensin II on cell proliferation, it is hypothesised that VEGF mediates the angiotensin II-dependent effect in concert with the changes in angiopoietin expression. This is the first report of the RAS on the regulation of angiogenesis-associated genes in physiology.

Regulation of tight junction proteins occludin and claudin 5 in the primate ovary during the ovulatory cycle and after inhibition of vascular endothelial growth factor.

Ovarian follicular and corpus luteum development, including angiogenesis, are characterized by cell-cell rearrangements that may require dynamic changes in cell-cell adhesion. The present study investigates the expression of tight junction proteins occludin and claudin 5 during follicular and luteal development in the primate ovary and after inhibition of vascular endothelial growth factor (VEGF) by VEGF trap treatment. Occludin was localized to the plasma membrane of granulosa cells. During follicular development occludin staining decreased significantly (P < 0.05) and disappeared completely by the ovulatory stage. After inhibition of VEGF, occludin staining was significantly (P < 0.05) higher in the granulosa of secondary and tertiary follicles compared with controls. Claudin 5 was exclusively localized to the theca vasculature. A significant (P < 0.05) increase in staining was detected from the pre-antral to the antral and ovulatory stage. However, dual staining with CD31 revealed that within the theca endothelium the amount of claudin 5 remained constant during follicular development. Treatment with VEGF trap throughout the follicular phase revealed a lack of claudin 5 staining in the theca interna but no difference was observed in the remaining theca externa vasculature. In the corpus luteum, claudin 5 was also localized in the vasculature. Treatment with VEGF trap in the mid-luteal phase resulted in a significant increase in staining (P < 0.05). These results led us to hypothesize that tight junctions are involved in regulation of follicular growth, antrum transition and follicular angiogenesis which is compromised by VEGF inhibition. VEGF may influence luteal vascular permeability by regulation of the endothelial specific tight junction protein claudin 5.

Navigation within host tissues: cues for orientation of Diplostomum spathaceum (Trematoda) in fish towards veins, head and eye.

Cercariae of Diplostomum spathaceum penetrate the skin of fish, and then migrate along blood vessels and tissues towards the head and the eye-lens. We studied their orientation behaviour in tail fins of guppies and in chemical concentration gradients within agar-filled choice chambers. In fins, they entered veins and orientated cranially, independent of the blood flow and living cells. In choice chambers, they were attracted by a small molecular fraction of fish serum, D-glucose (at 1, 10, and 1000 microM), D-mannose, D-maltotriose and Cl-ions, whereas D-glucosamine repelled them (even at 1.0 nM). Amino acids were not attractive, but arginine in tetrapeptides attracted at concentrations as low as 1 microm and melatonin at 0.4-4.3 PM. We suggest a preliminary model for the behaviour of diplostomula in fish fins and attracting (+) or repelling (-) host cues: (1) migration towards deeper skin layers and avoidance of skin surface, cues: Cl-ions (+ and -), glucose (+), glucosamine (-), light radiation (-); (2) orientation in cranial direction, cue: Cl-ions (+); (3) localization of blood vessels, cues: glucose (+), arginine-residues (+); (4) localization of the retina, cue: melatonin (+). A comparison with the navigation mechanisms of tissue-migrating schistosomules and hookworm larvae reveals an enormous diversity of strategies.

Cream formulations protecting against cercarial dermatitis by Trichobilharzia.

Dermatitis caused by penetrating bird schistosome cercariae is an emerging global public health problem. Infections may be prevented by the use of topical formulations that inhibit cercarial skin penetration. We evaluated nine water resistant formulations by exposing treated arms of volunteers to Trichobilharzia szidati cercariae. Six formulations protected from cercarial invasion. However, after immersion of the treated skin in water (2 x 20 min), only two formulations offered full protection: (1) Safe Sea, a cream protecting against jelly fish, (2) niclosamide in water resistant sun protecting cream formulations at concentrations as low as 0.05%. In an in vitro system Safe Sea and a 0.1% niclosamide formulation caused a high damage rate in T. szidati (92% and 99% after 5 min; only niclosamide with lethal effect) but not in Schistosoma mansoni (1% and 72%; both formulations with lethal effect). However, a 1% niclosamide formulation damaged S. mansoni sufficiently (100% after 5 min) and might offer full penetration protection.

Cell junctional proteins in the human corpus luteum: changes during the normal cycle and after HCG treatment.

BACKGROUND: Regulation of tissue remodelling and ovarian permeability by intercellular adhesion complexes may be involved in normal and pathological ovarian function. Therefore, the occurrence, distribution and hormonal control of the adherens junction protein vascular endothelial cadherin (VE-cadherin) and the tight junction proteins occludin and claudin in the human corpus luteum (CL) were investigated. METHODS: CLs from patients undergoing hysterectomy for benign reasons were enucleated during early, mid- and late stages of the functional luteal phase and after HCG rescue in vivo. Immunostaining for occludin, claudins 1 and 5 and VE-cadherin was carried out on fixed tissue. Endothelial cells, granulosa lutein cells and theca lutein cells were identified by reference to serial sections immunostained for CD34, 17alpha-hydroxylase and 3beta-hydroxy-steroid-dehydrogenase. Quantitative analyses were performed using image analyses. RESULTS: Occludin was localized to the plasma membrane of granulosa lutein cells and endothelial cells but was absent in theca lutein cells. Claudin 1 was exclusively localized to the plasma membrane of steroidogenic cells. Claudin 5 and VE-cadherin were only present in endothelial cells. After HCG administration in vivo, adherens and tight junction proteins were significantly down-regulated (P < 0.05). CONCLUSIONS: The decrease of junctional proteins after HCG treatment suggests a hormonal control of tight and adherens junctions in the CL associated with tissue remodelling and an increase in luteal permeability during early pregnancy.

Steroid-responsive subacute polymyositis in an adult following respiratory syncytial virus infection.

The role of viral infections in the aetiopathogenesis of polymyositis remains speculative. We report a case of profound subacute polymyositis with incipient ventilatory failure following serologically confirmed infection by respiratory syncytial virus (RSV), with a dramatic and sustained response to pulse corticosteroid therapy. We suggest a possible autoimmune mechanism to account for this sequence of events.

Localization of mRNA for vascular endothelial growth factor (VEGF), angiopoietins and their receptors during the peri-implantation period and early pregnancy in marmosets (Callithrix jacchus).

Implantation of a blastocyst into a receptive endometrium is a prerequisite for successful pregnancy. Angiogenesis is a key event in this process but the mechanisms by which localized changes in vascular permeability and angiogenesis occur have yet to be elucidated. Vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 have been implicated as key players in vascular remodelling and placentation. Angiopoietins also appear to have a significant role in regulation of blood vessel growth, maturation and regression. The aim of this study was to describe the molecular regulation of angiogenesis in the first month of pregnancy in marmosets and to address the putative physiological roles for these factors. Uteri were studied at weeks 2, 3 and 4 of pregnancy and compared with late secretory non-pregnant endometrium. Implantation in marmosets occurs at day 11 of pregnancy; hence, these time points were chosen so that the peri-implantation period and very early pregnancy could be studied. mRNAs for VEGF, VEGFR-1 and VEGFR-2, angiopoietin 1, angiopoietin 2 and their receptor Tie-2 were localized and quantified by in situ hybridization. Endothelial cells were identified by CD31 immunocytochemistry. VEGF mRNA was present in all compartments except endothelial cells, and its expression generally increased throughout pregnancy except in upper zone glandular epithelium and luminal epithelium, where a decrease in expression was observed. VEGF receptor mRNAs were found in endothelial cells of the upper zones immediately surrounding glandular epithelium. Angiopoietin 1 mRNA was localized to glandular epithelium of the upper and lower zones throughout pregnancy, and increased in stroma at week 4. Expression of angiopoietin 2 mRNA was localized exclusively to endothelial cells of large luminal vessels and was higher in endometrium from marmosets at week 4 of pregnancy than in endometrium from all other stages. These data provide comprehensive evidence that VEGFR-1 and -2, and angiopoietin 1, angiopoietin 2 and Tie-2 interactions may be involved in the preparation of endometrium for implantation, remodelling of the maternal vasculature and trophoblast invasion during the peri-implantation period in this primate species.

Angiogenesis in the human corpus luteum: simulated early pregnancy by HCG treatment is associated with both angiogenesis and vessel stabilization.

BACKGROUND: This study examined changes in the luteal vasculature throughout the menstrual cycle and during simulated pregnancy with human chorionic gonadotrophin (HCG) in the human. METHODS: Endothelial cell and pericyte area were assessed by quantitative immunocytochemistry for CD34 and alpha-smooth muscle actin respectively, taking into consideration the dynamics of lutein cell hypertrophy and atrophy throughout the cycle and after HCG treatment. Endothelial cell proliferation was detected by Ki-67/CD34 dual staining and a proliferation index was obtained. The molecular regulation of angiogenesis was studied by examining changes in vascular endothelial growth factor (VEGF) immunostaining. RESULTS: The early luteal phase is associated with intense angiogenesis, as indicated by high endothelial cell proliferation, and by the mid-luteal phase a mature vasculature was apparent, as shown by maximal endothelial cell and pericyte areas. During the late luteal phase, decreased endothelial proliferation, endothelial cell and pericyte area indicated vascular regression. HCG treatment induced a second burst of total and endothelial cell proliferation and a concomitant increase in endothelial cell and pericyte areas. VEGF protein was expressed throughout the luteal phase and a significant increase was found after HCG treatment. CONCLUSION: Luteal rescue with HCG is associated with a second wave of angiogenesis and vascular stabilization.

Luteal angiogenesis: prevention and intervention by treatment with vascular endothelial growth factor trap(A40).

The possibility of stimulating or inhibiting paracrine factors regulating angiogenesis may lead to new approaches for the treatment of pathological conditions of the female reproductive tract. We examined the effects of a clinical candidate, a soluble truncated form of the Flt-1 receptor, vascular endothelial growth factor trap(A40) (VEGF trap), in a primate model to determine its ability to prevent the onset of luteal angiogenesis or intervene with the on-going process. Marmosets were treated from the day of ovulation until luteal day 3 (prevention regimen) or on luteal day 3 for 1 day (intervention regimen). Effects of VEGF inhibition were studied by obtaining a proliferation index using bromodeoxyuridine incorporation, quantifying endothelial cell area using CD31, and assessing luteal function by plasma progesterone. After both treatments, intense luteal endothelial proliferation was suppressed, a concomitant decrease in endothelial cell area confirmed the inhibition of vascular development, and a marked fall in plasma progesterone levels showed that luteal function was compromised. In situ hybridization was used to localize and quantify compensatory effects on the expression of angiogenic genes. VEGF messenger ribonucleic acid (mRNA) expression in luteal cells was increased, whereas expression of its receptor, Flt, was decreased. Inhibition of VEGF resulted in localized increased expression of angiopoietin-2 mRNA and its receptor, Tie-2. The results show that the VEGF trap can prevent luteal angiogenesis and inhibit the established process with resultant suppression of luteal function. Luteal Flt mRNA expression is dependent upon VEGF, and VEGF inhibition results in abortive increases in expression of VEGF, angiopoietin-2, and Tie-2.

Angiogenesis during follicular development in the primate and its inhibition by treatment with truncated Flt-1-Fc (vascular endothelial growth factor Trap(A40)).

The aims of this study were to 1) quantify changes in angiogenesis during follicular growth in a primate model; 2) investigate the molecular regulation using in situ hybridization of vascular endothelial growth factor (VEGF), its receptor, Flt-1, the angiopoietins (Ang-1 and Ang-2), and their receptor, Tie-2; 3) elucidate the role of VEGF in follicular angiogenesis by blocking its action by treatment with a soluble truncated form of the Flt-1 receptor, (VEGF Trap(A40)). Changes in angiogenesis were quantified using bromodeoxyuridine to obtain a proliferation index, and CD31 immunocytochemistry to visualize endothelial cell area. Percentage of proliferating endothelial cells was calculated by double labeling for bromodeoxyuridine and CD31. Vascularization was first observed in follicles containing four granulosa cell layers. A significant increase in proliferation in the thecal layer was observed from the early to late secondary stage, and dual staining showed that 25% of proliferating cells were of endothelial cell origin. VEGF messenger RNA (mRNA) was expressed in granulosa cells with an increase of grain density from late secondary to tertiary follicles. Ang-1 was weakly expressed in the theca of tertiary follicles. Ang-2 mRNA was not detected in any follicles. The mRNA for the Flt-1 and Tie-2 receptors was localized in endothelial cells of the theca. Unexpectedly, Tie-2 mRNA was also found in granulosa cells of early follicular stages and its translation was confirmed by immunocytochemistry. VEGF trap treatment for 3 days resulted in an 87% decrease of proliferation in the theca of secondary and tertiary follicles, a reduction in endothelial cell area and a marked decline in Flt-1 mRNA expression. Granulosa cell proliferation also decreased. These results show that onset and establishment of the follicle vasculature takes place early during follicular development. The ability of VEGF trap treatment to severely restrict follicular angiogenesis establishes that VEGF is the major regulator of this process in the primate ovary.