Role of nuclear factor of activated T cells in chondrogenesis osteogenesis and osteochondroma formation.

PURPOSE: Nuclear factor of activated T cells (NFATc) are transcription factors that play a function in the immune response and in osteoclast differentiation. In the present work, we define the function of NFATc2 in chondrogenic and osteogenic cells. METHODS: Nfatc2loxP/loxP and Nfatc1loxP/loxP;Nfatc2loxP/loxP conditional mice were crossed with Prx1-Cre transgenics to inactivate Nfatc2 singly and with Nfatc1. Femurs and vertebrae were examined by microcomputed tomography (µCT) X-Ray images and histology and analyzed for the presence of osteochondromas. RESULTS: µCT demonstrated that Prx1-Cre;Nfatc2∆/∆ female mice had transient osteopenia and male mice did not have a cancellous or a cortical bone phenotype when compared to control mice. In contrast, the dual inactivation of Nfatc1 and Nfatc2 in Prx1-expressing cells resulted in cancellous osteopenia and small bones at 1 month of age in both sexes. Nfatc1;Nfatc2 deleted mice exhibited a ~ 50% decrease in bone volume and connectivity. Total bone area, periosteal and endocortical bone perimeters and femoral length were reduced indicating smaller bones. As the mice matured, the shortening of the femoral length persisted, but the osteopenic phenotype resolved and cancellous femoral bone of 4-month-old Nfatc1;Nfatc2 deleted mice was not different from controls although male mice had vertebral osteopenia. In addition, Nfatc1;Nfatc2 deleted mice displayed distortion of the distal metaphysis and, as they matured, the articular presence of mineralized tumors with the appearance of osteochondromas. CONCLUSION: Our studies reveal that NFATc1 and NFATc2 are necessary for optimal bone homeostasis and the suppression of osteochondroma formation.

Workplace-related risk of tick bites in military personnel stationed in Northern Germany.

PURPOSE: There are more than 500,000 employees in Germany alone who are at risk of being bitten by a tick at their workplace and thus also at risk of being infected with Borrelia burgdorferi s.l. or the tick-borne meningoencephalitis virus. So far, there are only a small number of studies on the risk of tick bites in Central Europe, in particular, for military personnel during relevant training activities. METHODS: During a total of 36 months of training in 2008/2009 and from 2012 to 2014, the number of tick bites and any resulting diseases of 1156 recruits under comparable conditions of exposure and prevention were documented based on their medical records. The incidence of tick bites was calculated with each recruit's individual exposure time for field training. RESULTS: There were a total of 66 tick bites during an exposure time of 317,059 h of field training (0.21 tick bites per 1000 h of training). The risk of tick bites was found to have a seasonal dependency. In 6 out of the 66 cases in which someone was bitten, the patients consulted a physician for a follow-up examination, and in one of these cases the bite resulted in an infection requiring treatment. CONCLUSIONS: It turns out that there is a rather low but relevant risk of being exposed to tick bites for military personnel during their field training. Under the given study conditions, months with a high risk of tick bites can be distinguished from months with a low risk of tick bites, which is of vital importance, in particular, for guidance and prevention.

[Lichen ruber planus : Better understanding, better treatment!] / Lichen ruber planus : Besser verstehen, besser behandeln!

Lichen ruber, also called lichen ruber planus or lichen planus (LP), is a noncontagious inflammatory skin disease. LP is the main representative and namesake of the group of lichenoid diseases, which are characterized by small papules often accompanied by severe itching. With 65% of cases, LP is primarily a disease of the mucous membranes. In 20% of the cases, the disease is found on the skin and mucous membranes; skin involvement alone is seen in only about 10% of cases. Cutaneous LP has a very favorable 1­year prognosis of almost 80% healing as opposed to the mucosa and the adnexal organs. Histologically, keratinocytes with vacuolar degeneration, leaving behind apoptotic Kamino bodies and the characteristic band-shaped lymphocytic infiltrate at the dermatoepithelial junction, are common to lichenoid diseases. The horny layer is firm and compact and the stratum granulosum is thickened as a correlate of the Wickham stripes. The molecular pathogenesis, still partially hypothetical, assumes trigger factors leading to the presentation of intrinsic or foreign antigens. The triggered inflammation becomes independent in the sense of a classical cell-mediated autoimmune disease. Other autoimmune diseases are often associated with LP. Classical anti-inflammatory-immunosuppressive therapeutic concepts dominate with systemic retinoids ranking first in the highest evidence class for cutaneous LP with limitations in treatment of both mucosal and adnexal LP. More recently, interesting and new complementary phototherapeutics have been identified.

[Postmenopausal lichen planopilaris also known as fibrosing frontotemporal alopecia Kossard : An evidence-oriented practical guide to treatment from the University of the Saarland, Hair Research Center of the Dr. Rolf M. Schwiete Foundation]. / Postmenopausaler Lichen planopilaris alias fibrosierende frontotemporale Alopezie Kossard : Ein evidenzorientierter "practical guide to treatment" des Haarforschungszentrums der Dr. Rolf M. Schwiete Stiftung an der Universität des Saarlandes.

Postmenopausal lichen planopilaris (PLPP), also known as fibrosing frontotemporal alopecia Kossard (FFAK), is a not uncommon inflammatory scalp disease affecting approximately 5% of patients at specialized hair centers. The overall incidence of sporadic occurrence is believed to be just under 1% in the older, predominantly female, general population. Since the disease is often undiagnosed, it is statistically likely to be underrepresented. It especially occurs in postmenopausal women who are in the 6th and 7th decade of life (90%), but also in about 10% of premenopausal women, and in men it is documented only in isolated cases. The result is a permanent scarring hair loss accentuated at the front hairline with backward movement towards the neck mostly accompanied by a typical loss of the eyebrows. The disease therefore often leads to significant mental distress and social anxiety in those affected. This is the basis for a compelling need to develop evidence-based therapeutic concepts. While numerous retrospective case series have characterized the phenomenology of FFAK very well, to date there are no randomized controlled trials on evidence-based therapy. Here, we present the Homburger Evidence-Oriented Therapy Algorithm, which is oriented along the available case series evidence: It may (1) serve as a therapy guide for practice and (2) can be used as a basis for working out reliable data based on study evidence. The article contains detailed practical information on photo documentation, biopsy and histological processing up to the practical implementation of, for example, intralesional steroid therapy as well as information on selection criteria for suitable systemic therapies.

Reproductive activity triggers accelerated male mortality and decreases lifespan: genetic and gene expression determinants in Drosophila.

Reproduction and aging evolved to be intimately associated. Experimental selection for early-life reproduction drives the evolution of decreased longevity in Drosophila whereas experimental selection for increased longevity leads to changes in reproduction. Although life history theory offers hypotheses to explain these relationships, the genetic architecture and molecular mechanisms underlying reproduction-longevity associations remain a matter of debate. Here we show that mating triggers accelerated mortality in males and identify hundreds of genes that are modulated upon mating in the fruit fly Drosophila melanogaster. Interrogation of genome-wide gene expression in virgin and recently mated males revealed coherent responses, with biological processes that are upregulated (testis-specific gene expression) or downregulated (metabolism and mitochondria-related functions) upon mating. Furthermore, using a panel of genotypes from the Drosophila Synthetic Population Resource (DSPR) as a source of naturally occurring genetic perturbation, we uncover abundant variation in longevity and reproduction-induced mortality among genotypes. Genotypes displayed more than fourfold variation in longevity and reproduction-induced mortality that can be traced to variation in specific segments of the genome. The data reveal individual variation in sensitivity to reproduction and physiological processes that are enhanced and suppressed upon mating. These results raise the prospect that variation in longevity and age-related traits could be traced to processes that coordinate germline and somatic function.

Induction of a NOTCH3 Lehman syndrome mutation in osteocytes causes osteopenia in male C57BL/6J mice.

Lateral Meningocele or Lehman Syndrome (LMS) is associated with NOTCH3 mutations causing deletions of the PEST domain and a gain-of-NOTCH3 function. We demonstrated that Notch3em1Ecan mice harboring Notch3 mutations analogous to those found in LMS are osteopenic because of enhanced bone resorption. To determine the contribution of specific cell lineages to the phenotype, we created a conditional-by-inversion (Notch3COIN) model termed Notch3em2Ecan in which Cre recombination generates a Notch3INV allele expressing a NOTCH3 mutant lacking the PEST domain. Germ line Notch3COIN inversion caused osteopenia and phenocopied the Notch3em1Ecan mutant, validating the model. To induce the mutation in osteocytes, smooth muscle and endothelial cells, Notch3COIN mice were bred with mice expressing Cre from the Dmp1, Sm22a and Cdh5 promoters, respectively, creating experimental mice harboring Notch3INV alleles in Cre-expressing cells and control littermates harboring Notch3COIN alleles. Notch3COIN inversion in osteocytes led to femoral and vertebral cancellous bone osteopenia, whereas Notch3COIN inversion in mural Sm22a or endothelial Cdh5-expressing cells did not result in a skeletal phenotype. In conclusion, introduction of the LMS mutation in osteocytes but not in vascular cells causes osteopenia and phenocopies Notch3em1Ecan global mutant mice.

Quality of life in thalassemia: a comparison of SF-36 results from the thalassemia longitudinal cohort to reported literature and the US norms.

Thalassemia is a chronic, inherited blood disorder, which, in its most severe form, causes life-threatening anemia. Advances in treatment have led to increased life expectancy however the need for chronic blood transfusions and chelation therapy remains a significant burden for patients. Our study compared health related quality of life (HRQOL) from the Thalassemia Clinical Research Network's (TCRNs) Thalassemia Longitudinal Cohort (TLC) study to US norms and assessed association with clinical variables. There were 264 patients over age 14 who completed the Medical Outcomes Study 36-Item Short Form Health Survey version 2 (SF36v2) baseline assessment. When compared to US norms, TLC patients had statistically significant (P < 0.05) worse HRQOL on five of the eight subscales (physical functioning, role-physical, general health, social functioning, and role-emotional) and on both summary scales (physical component summary and mental component summary). Women, older patients, and those with more disease complications and side effects from chelation reported lower HRQOL. In general, adolescents and adults with thalassemia report worse HRQOL than the US population, despite contemporary therapy. The SF-36 should become a standard instrument for assessing HRQOL in thalassemia to determine predictors of low HRQOL which may be better addressed by a multidisciplinary team.

Comparative transcriptome analysis of inner blood-retinal barrier and blood-brain barrier in rats.

Although retinal microvessels (RMVs) and brain microvessels (BMVs) are closely related in their developmental and share similar blood-neural barriers, studies have reported markedly different responses to stressors such as diabetes. Therefore, we hypothesized that RMVs and BMVs will display substantial differences in gene expression levels even though they are of the same embryological origin. In this study, both RMVs and BMVs were mechanically isolated from rats. Full retinal and brain tissue samples (RT, BT) were collected for comparisons. Total RNA extracted from these four groups were processed on Affymetrix rat 2.0 microarray Chips. The transcriptional profiles of these tissues were then analyzed. In the present paper we looked at differentially expressed genes (DEGs) in RMVs (against RT) and BMVs (against BT) using a rather conservative threshold value of ≥ ± twofold change and a false discovery rate corrected for multiple comparisons (p < 0.05). In RMVs a total of 1559 DEGs were found, of which 1004 genes were higher expressed in RMVs than in RT. Moreover, 4244 DEGs between BMVs and BT were identified, of which 1956 genes were ≥ twofold enriched in BMVs. Using these DEGs, we comprehensively analyzed the actual expression levels and highlighted their involvement in critical functional structures in RMVs and BMVs, such as junctional complex, transporters and signaling pathways. Our work provides for the first time the transcriptional profiles of rat RMVs and BMVs. These results may help to understand why retina and brain microvasculature show different susceptibilities to stressors, and they might even provide new insight for pharmacological interventions.

[The use of sunglasses during leisure time and work : Lack of prevention of sun-induced eye damage]. / Die Nutzung von Sonnenbrillen in Freizeit und Beruf : Defizite in der Prävention sonnenbedingter Augenschäden.

OBJECTIVE: This study aimed at collecting representative national data on the use of sunglasses on sunny summer days during leisure time or work as well as identifying population and professional groups with a pronounced lack of preventive measures to avoid sun-induced eye damage. MATERIAL AND METHODS: Within the representative National Cancer Aid Monitoring, data on the use of sunglasses during leisure time was assessed among 3000 individuals aged 14-45 years in 2015, as well as on the use during outdoor work among 485 workers aged 14-45 years in 2016. Associations between the use of sunglasses and sociodemographic characteristics were assessed with the χ2-test. Additionally, descriptive and bivariate methods were used to assess connections between the use of sunglasses at work and each professional group. RESULTS: While more than half of the general population normally or often wear sunglasses on a sunny summer day, only one third of outdoor workers do so. While approximately every seventh individual surveyed never wears sunglasses during leisure time, among outdoor workers it is one out of three. The use during leisure time increases with age. DISCUSSION: Use of sunglasses during work could be supported by targeted information on UV-induced eye damage by ophthalmologists and company physicians with additional support from accident insurances and employers. Concerning preventive measures occupational groups such as landscapers, farmers and bricklayers who are strongly exposed to sunlight but rarely wear sunglasses are important groups.

Identification of a new promoter upstream of the murine dihydrofolate reductase gene.

In vitro reactions identified a transcription initiation site located 740 nucleotides upstream of the dihydrofolate reductase translational start. Transcription from this site proceeded in the direction opposite to that of dihydrofolate reductase mRNA. Deletion mapping indicated that this new promoter can be separated from the dihydrofolate reductase promoter and that separation increased transcription at -740. Transcripts that initiate at -740 were also detected in cellular RNA, indicating that this is a bona fide transcription initiation site in vivo.

Statins and fibrates for preventing melanoma.

BACKGROUND: Effective treatment for advanced melanoma is lacking. While no drug therapy currently exists for prevention of melanoma, in vitro, case-control, and animal model evidence suggest that lipid-lowering medications, commonly taken for high cholesterol, might prevent melanoma. OBJECTIVES: To assess the effects of statin or fibrate lipid-lowering medications on melanoma outcomes. SEARCH STRATEGY: We searched the Cochrane Skin Group Specialised Register (February 2003), CENTRAL (The Cochrane Library Issue 1, 2005), MEDLINE (to March 2003), EMBASE (to September 2003), CANCERLIT (to October 2002), Web of Science (to May 2003), and reference lists of articles. We approached study investigators and pharmaceutical companies for additional information (published or unpublished studies). SELECTION CRITERIA: Trials involving random allocation of study participants, where experimental groups used statins or fibrates and participants were enrolled for at least four years of therapy. DATA COLLECTION AND ANALYSIS: Three authors screened 109 abstracts of articles with titles of possible relevance. We then thoroughly examined the full text of 72 potentially relevant articles. We requested unpublished melanoma outcomes data from the corresponding author of each qualifying trial. MAIN RESULTS: We identified 16 qualifying randomised controlled trials (RCTs) (seven statin, nine fibrate). Thirteen of these trials (involving 62,197 participants) provided data on incident melanomas (six statin, seven fibrate). A total of 66 melanomas were reported in groups receiving the experimental drug and 86 in groups receiving placebo or other control therapies. For statin trials this translated to an odds ratio of 0.90 (95% confidence interval 0.56 to 1.44) and for fibrate trials an odds ratio of 0.58 (95% confidence interval 0.19 to 1.82). Subgroup analyses failed to show statistically significant differences in melanoma outcomes by gender, melanoma occurrence after two years of participation in trial, stage or histology, or trial funding. Subgroup analysis by type of fibrate or statin also failed to show statistically significant differences, except for the statin subgroup analysis which showed reduced melanoma incidence for lovastatin, based on one trial only (odds ratio 0.52, 95% confidence interval 0.27 to 0.99). AUTHORS' CONCLUSIONS: The melanoma outcomes data collected in this review of RCTs of statins and fibrates does not exclude the possibility that these drugs prevent melanoma. There was a 10% and 42% reduction for participants on statins and fibrates, respectively, however these results were not statistically significant. Until further evidence is established, limiting exposure to ultraviolet radiation remains the most effective way to reduce the risk of melanoma.

Is patients' perception of time spent with the physician a determinant of ambulatory patient satisfaction?

BACKGROUND: Time management in ambulatory patient visits is increasingly critical. Do patients who perceive a longer visit with internists report increased satisfaction? METHODS: Prospective survey of 1486 consecutively encountered ambulatory visits to 16 primary care physicians (PCPs) in an academic primary care clinic. Patients were queried regarding demographics, health status, perception of time spent before and after ambulatory visits, whether the physician appeared rushed, and visit satisfaction. Physicians were queried regarding time spent, estimated patient satisfaction, and whether they felt rushed. RESULTS: In 69% of 1486 consecutive visits, patient previsit expectation of visit duration was 20 minutes or less. Patient and PCP postvisit estimates of time spent significantly exceeded patient previsit time expectation. Patients who estimated that they spent more time than expected with the PCP were significantly more satisfied with the visit. When patient postvisit estimate of time spent was less than the previsit expectation, visit satisfaction was significantly lower independent of time spent. Patient worry about health and lower self-perceived health status were significantly associated with patient expectation for longer visits. Primary care physicians felt rushed in 10% of encounters. Although PCPs estimated patient satisfaction was significantly lower when they felt rushed, patient satisfaction was identical when PCPs did and did not feel rushed. Patients indicated that PCPs appeared rushed in 3% of encounters, but this perception did not affect patient satisfaction. CONCLUSION: Perceived ambulatory visit duration and meeting or exceeding patient expectation of time needed to be spent with the physician are determinants of patient satisfaction in an ambulatory internal medicine practice.

Dilating effect of perivascularly applied potassium channel openers cromakalim and pinacidil in rat and cat pial arteries in situ.

OBJECTIVE: The aim was to test the vasodilator effect of perivascularly applied potassium channel openers cromakalim and pinacidil in pial arteries of cat and rat. METHODS: Using an open cranial window technique the diameter of pial arteries in the parietal cortex of rat and cat was recorded with an image splitting method. Employing micropuncture techniques, test compounds were dissolved in inert cerebrospinal fluid and infused into the perivascular space of individual arteries. RESULTS: Cromakalim (7 x 10(-18)-7 x 10(-13) M) induced concentration dependent dilatation of feline pial arteries with a maximum effect of 26.8(SEM 3.2)% at 7 x 10(-15) M. In rat arteries, cromakalim had a maximum effect of 32.1(4.97)% at 10(-15) M, while pinacidil (10(-10)-10(7) M) exerted a maximum dilatation of 29.5(3.3)% at 10(-8) M. The latter is consistent with previous findings in feline pial arteries. The sulphonylurea glibenclamide (10(-8)-10(-6) M) had no effect on the resting diameter of rat and cat pial arteries, indicating that their resting tone is not influenced by mechanisms involving ATP sensitive potassium channels. However, glibenclamide reduced the dilating effect of cromakalim and pinacidil in rat and cat in a dose dependent manner. CONCLUSIONS: A comparison with previously published data obtained in isolated middle cerebral and basilar arteries showed that potassium channel openers of the benzopyrane and cyanoguanidine type are much more potent in pial arteries than in peripheral arteries in situ or in isolated arteries of the circle of Willis and of peripheral vascular beds.

Inhibition of tumor growth, angiogenesis, and microcirculation by the novel Flk-1 inhibitor SU5416 as assessed by intravital multi-fluorescence videomicroscopy.

Vascular endothelial growth factor (VEGF) plays a fundamental role in mediating tumor angiogenesis and tumor growth. Here we investigate the direct effect of a novel small molecule inhibitor of the Flk-1-mediated signal transduction pathway of VEGF, SU5416, on tumor angiogenesis and microhemodynamics of an experimental glioblastoma by using intravital multifluorescence videomicroscopy. SU5416 treatment significantly suppressed tumor growth. In parallel, SU5416 demonstrated a potent antiangiogenic activity, resulting in a significant reduction of both the total and functional vascular density of the tumor microvasculature, which indicates an impaired vascularization as well as significant perfusion failure in treated tumors. This malperfusion was not compensated for by changes in vessel diameter or recruitment of nonperfused vessels. Analyses of the tumor microcirculation revealed significant microhemodynamic changes after angiogenesis blockage such as a higher red blood cell velocity and blood flow in remnant tumor vessels when compared with controls. Our results demonstrate that the novel antiangiogenic concept of targeting the tyrosine kinase of Flk-1/KDR by means of a small molecule inhibitor represents an efficient strategy to control growth and progression of angiogenesis-dependent tumors. This study provides insight into microvascular consequences of Flk-1/KDR targeting in vivo and may have important implications for the future treatment of angiogenesis-dependent neoplasms.

Stimulation of the growth hormone (GH)-insulin-like growth factor I axis by daily oral administration of a GH secretogogue (MK-677) in healthy elderly subjects.

Aging is associated with declining activity of the GH axis, possibly contributing to adverse body composition changes and increased incidence of cardiovascular disease. The stimulatory effects on the GH-insulin-like growth factor I (IGF-I) axis of orally administered MK-677, a GH-releasing peptide mimetic, were investigated. Thirty-two healthy subjects (15 women and 17 men, aged 64-81 yr) were enrolled in a randomized, double blind, placebo-controlled trial. They received placebo or 2, 10, or 25 mg MK-677, orally, once daily for 2 separate study periods of 14 and 28 days. At baseline and on day 14 of each study period, blood was collected every 20 min for 24 h to measure GH, PRL, and cortisol. Attributes of pulsatile GH release were assessed by 3 independent algorithms. MK-677 administration for 2 weeks increased GH concentrations in a dose-dependent manner, with 25 mg/day increasing mean 24-h GH concentration 97 +/- 23% (mean +/- SE; P < 0.05 vs. baseline). This increase was due to an enhancement of preexisting pulsatile GH secretion. GH pulse height and interpulse nadir concentrations increased significantly without significant changes in the number of pulses. With 25 mg/day MK-677 treatment, mean serum IGF-I concentrations increased into the normal range for young adults (141 +/- 21 microgram/L at baseline, 219 +/- 21 micrograms/L at 2 weeks, and 265 +/- 29 micrograms/L at 4 weeks; P < 0.05). MK-677 produced significant increases in fasting glucose (5.4 +/- 0.3 to 6.8 +/- 0.4 mmol/L at 4 weeks; P < 0.01 vs. baseline) and IGF-binding protein-3. Circulating cortisol concentrations did not change, and PRL concentrations increased 23%, but remained within the normal range. Once daily treatment of older people with oral MK-677 for up to 4 weeks enhanced pulsatile GH release, significantly increased serum GH and IGF-I concentrations, and, at a dose of 25 mg/day, restored serum IGF-I concentrations to those of young adults.

Analysis of acetylcholine-induced relaxation of rabbit isolated middle cerebral artery: effects of inhibitors of nitric oxide synthesis, Na,K-ATPase, and ATP-sensitive K channels.

The functional importance of membrane hyperpolarization through activation of ATP-sensitive K channels, or activation of the Na,K-ATPase, was investigated for acetylcholine (ACh)-induced relaxation of the rabbit isolated middle cerebral artery (MCA) precontracted with uridine triphosphate. Incubation with glibenclamide (1 microM), a known blocker of ATP-sensitive K channels, or precontraction with a high concentration of KCl (50 mM) had no effect on ACh-induced relaxation. Similarly, inhibition of the Na,K-ATPase with ouabain (10 microM) or incubation with a potassium-free solution had either no or only a small effect on ACh-induced relaxation. In contrast, NG-nitro-L-arginine (NOLAG) (1 to 10 microM), a structural analogue of L-arginine and an inhibitor of nitric oxide synthesis, produced concentration-dependent although apparently noncompetitive inhibition of ACh-induced relaxation. This inhibition was partially reversed by application of L-arginine (100 microM), a putative precursor for nitric oxide synthesis. It is concluded that membrane hyperpolarization induced by activation of ATP-sensitive K channels or Na,K-ATPase does not play a major functional role in ACh-induced relaxation of rabbit MCA. The potent inhibitory actions of NOLAG would suggest that the major mechanism of ACh-induced relaxation is by release of nitric oxide as in other cerebral and peripheral arteries.

Endothelin-induced contraction and relaxation of rat isolated basilar artery: effect of BQ-123.

In ring segments from rat basilar artery (BA) the endothelin (ET) peptides ET-1, ET-2, and ET-3 induced concentration-related contractions. The order of potency was ET-1 = ET-2 > ET-3, while no differences occurred in the maximum contraction. The selective ETA receptor antagonist, BQ-123 (10(-10)-10(-4) M) alone elicited a small contraction only at 10(-4) M. In the presence of BQ-123 (10(-7)-10(-5) M), the concentration-response curve for ET-1 was shifted to the right without any decrease in maximum contraction, indicating competitive inhibition of ET-1 binding to the ETA receptor by BQ-123. The pA2 value calculated for BQ-123 was 6.935; the slope of the regression curve was 0.734. In contrast to ET-1, the contractile action of ET-3 was abolished by 10(-5) M BQ-123. In segments precontracted with 10(-6) M serotonin, ET-3, but not ET-1, induced relaxation at low concentrations (10(-11)-10(-8) M), with maximum relaxation amounting to 17.8 +/- 14.7% of precontraction (mean +/- SD; n = 16). The relaxant action of ET-3 was abolished in vessels incubated with NG-nitro-L-arginine (10(-5) M), an inhibitor of nitric oxide synthase. These results indicate that the ET-induced contraction of the isolated rat BA involves activation of the ETA receptor. The ET-3-induced relaxation of precontracted rat BA is apparently mediated by release of nitric oxide from the endothelium.

Endothelin-3-induced relaxation of isolated rat basilar artery is mediated by an endothelial ETB-type endothelin receptor.

The endothelin (ET) receptor mediating relaxation of cerebral arteries was characterized using ring segments obtained from the rat basilar artery. Under resting tension, ET-3 (> 10(-8) M) but not the specific ETB receptor agonist IRL 1620 induced contraction. In ring segments precontracted with 3 x 10(-6) M prostaglandin (PG) F2 alpha, ET-3 (10(-12) - 10(-8) M) and IRL 1620 (10(-14) - 10(-6) M) induced concentration-related relaxation. IRL 1620 was more potent than ET-3, the pD2 (-log10EC50) values being 10.002 +/- 0.751 (mean +/- SD) for IRL 1620 and 8.836 +/- 0.415 for ET-3. Relaxation was abolished after preincubation with the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (10(-5) M) as well as in segments devoid of a functionally intact endothelium. At a concentration above 10(-8) M, ET-3 resulted in a further increase of PGF2 alpha-induced contraction that was not observed with IRL 1620. The presumably specific ETB receptor antagonist IRL 1038 (10(-7) - 3 x 10(-6) M) diminished or even abolished (3 x 10(-6) M) the relaxation induced by ET-3 or IRL 1620. IRL 1038 did not exert any vasomotor effect by itself, and it did not significantly affect ET-3-induced contraction. These results indicate that in the rat isolated basilar artery, the ET-3-induced relaxation is probably due to activation of an ETB-type receptor located on the endothelial cells and mediated by release of nitric oxide.

Characterization of angiogenesis and microcirculation of high-grade glioma: an intravital multifluorescence microscopic approach in the athymic nude mouse.

The current study follows angiogenesis and microcirculatory changes associated with malignant glioma growth by means of an intravital fluorescence microscopic approach, which allows for the direct and continuous visualization of the glioma microvasculature and its quantitative analysis. Fluorescently labeled C6 rat glioma cells (5 x 10(5)) were implanted into dorsal skinfold chamber preparations of athymic nude mice. Glioma growth, vascularization, microhemodynamics, vascular permeability, and leukocyte-endothelial cell interactions were simultaneously followed over a 22-day observation period using intravital epiillumination microscopy and a multifluorescent labeling technique. Analysis of the process of glioma vascularization revealed three stages with distinct microvascular characteristics: avascular stage (days 0 to 6), lag of glioma growth but initial glioma-induced angiogenesis within the host tissue in peritumoral areas; early vascular stage (days 6 to 14), glioma cell proliferation associated with a spatially homogeneous development of a glioma microvasculature; and late vascular stage (days 14 to 22), exponential tumor growth and expansion (> 400 mm3) with high vascular densities in the peritumoral region and reduced vascularization (microvascular perfusion) in the glioma center. Within the center, the functional vessel length per area correlated inversely with glioma size (P < 0.01). In the peritumoral region, functional vessel length per area was independent of glioma size, indicating persistent, high angiogenic activity throughout the observation period. Thus, the microvasculature of mature gliomas revealed a microvascular zonal division with a progressive reduction of the functional vessel length per area within the tumor center. The perfusion failure of individual microvessels within the glioma center was partly compensated by an increase of diameters (P < 0.05), and thus by an increase of blood flow in these functional microvessels (P < 0.05) over time. Histologic analysis demonstrated both expanding and infiltrating growth patterns, as well as focal necroses on day 22. These are the first data from repeated in vivo analysis of glioma growth, vascularization, and microcirculation.

Delayed loss of ETB receptor-mediated vasorelaxation after cold lesion of the rat parietal cortex.

The aim of this study was to investigate the involvement of endothelins (ET) in brain injury. The effect of ET was studied in the isolated basilar artery (BA) taken from control, sham-operated, and cold-lesioned rats. Cold lesion was induced by application of a precooled (-78 degrees C) copper cylinder (outer diameter 5 mm) for 60 seconds to the intact dura over the parietal cortex. After precontraction with prostaglandin (PG) F2alpha, ET-3 (10(-10) to 10(-8) mol/L) dilated BA with a pD2 (negative log of the half-maximal concentration) of 9.06+/-0.031 (mean +/- SD) and a maximal effect (Emax) of 1.64+/-1.0 mN at 3 x 10(-9) mol/L in sham-operated animals. This dilation was reduced 24 and 48 hours after cold lesion by 33% and 73%, respectively, at 3 x 10(-9) mol/L. The effects of acetylcholine (10(-8) to 10(-4) mol/L) and sodium nitroprusside (10(-3) mol/L) were unaltered. Activation of the ETB receptor in thoracic aorta by the specific agonist IRL 1620 also resulted in a reduced dilation (51% by 48 hours after cold lesion). Reverse transcriptase-polymerase chain reaction of the BA showed unaltered expression of mRNA for the ETB receptor after cold lesion whereas ETB immunoreactivity in BA and in its intraparenchymal arteries was reduced at 24 and 48 hours. In contrast to the reduction of ET-3-induced dilation, the constrictor effects of ET-1 and ET-3 were retained after cold lesion. Endothelin-1 (10(-12) to 10(-6) mol/L) dose-dependently contracted segments of untreated control BA segments under resting conditions with a pD2 of 8.03+/-0.22 and an Emax of 6.35+/-0.70 mN. Further evidence that the constrictor ability of BA was not influenced by cold lesion is given by the unaltered response to 124 mmol/L K+ and 10(-6) mol/L serotonin. We conclude that the ETB receptor of BA after cold lesion is downregulated specifically, apparently at the posttranscriptional level. Because the ETB-mediated dilation in thoracic aorta was also reduced, downregulation of the ETB receptor apparently is not restricted to cerebral arteries. The nitric oxide-cyclic guanosine monophosphate system in BA is, however, intact.