Current Practices of Medication Plans in Austrian Patients Undergoing Coronary Angiography: An In-Depth Analysis.

A complete medication plan (MPlan) increases medication safety and adherence and is crucial in care transitions. Countries that implemented a standardized MPlan reported benefits on patients' understanding and handling of their medication. Austria lacks such a standardization, with no available data on the issue. Objective: This study aimed to investigate the current state of all medication documentations (MDocs) at hospital admission in a population at high risk for polypharmacy in Austria. Methods: We enrolled 512 consecutive patients undergoing elective coronary angiography. Their MDocs and medications were recorded at admission. MDocs were categorized, whereby a MPlan was defined as a tabular list including medication name, dose, route, frequency and patient name. Results: Out of 485 patients, 55.1% had an MDoc (median number of drugs: 6, range 2-17), of whom 24.7% had unstructured documentation, 18.0% physicians' letters and 54.3% MPlans. Polypharmacy patients did not have a MDoc in 31.3%. Crucial information as the patients's name or the originator of the MDoc was missing in 31.1% and 20.4%, respectively. Patients with MDoc provided more comprehensive medication information (p = 0.019), although over-the-counter-medication was missing in 94.5% of MDocs. A discrepancy between the MPlan and current medication at admission existed in 64.4%. In total, only 10.7% of our patient cohort presented an MPlan that was in accordance with their current medication. Conclusion: The situation in Austria is far from a standardized MPlan generated in daily routine. Numerous MPlans do not represent the current medication and could pose a potential risk for the effectiveness and safety of pharmacotherapy.

Cervical vertebral body replacement using a modern in situ expandable and angulable corpectomy cage system: early clinical and radiological outcome.

PURPOSE: Vertebral body replacement (VBR) cages are commonly implanted to reconstruct the cervical vertebrae in cases of tumour, trauma, spondylodiscitis, and degeneration. Expandable cages have been widely used for this purpose; however, the lacking congruence at the implant-bone interface and consequent implant displacement were considered as a serious drawback of such systems. Aim of this study is to evaluate the early clinical and radiological outcome of a modern in situ not only expandable but also angulable cervical corpectomy cage system. METHODS: A total of 42 patients who underwent a single or multilevel cervical VBR procedure were included and retrospectively evaluated in this single-centre case series. The neurological status was assessed using American Spinal Injury Association (ASIA) score. Complications were categorized into surgical (including implant-associated) and general medical. Radiographic parameters included regional angulation, segmental height, and coronal alignment. RESULTS: Mean age was 59.5 ± 20.6 years. The recorded ASIA score improved postoperatively by 10 points (p  0.0001). Surgical including implant-associated complication rates were 19.05%. Radiographic evaluation showed a height gain of 11.2 mm (p < 0.0001), lordotic correction of 7° (p < 0.0001), and coronal alignment of 3° (p < 0.0001). At the last follow-up, loss of angulation correction of 1.9° (p  0.0002), subsidence of 1.92 mm (p  0.0006), and fusion rates of 68.42% were observed. CONCLUSIONS: The use of an in situ angulable and expandable cage system in cervical VBR seems to offer better results compared to conventional static or expandable cages regarding segmental height gain, lordotic correction, and clinical improvement as well as low complication and revision rates. Significant height gain in multilevel surgeries is associated with higher rates of implant-associated complications.

Improved method for surgical induction of chronic hypertension in mice.

Chronic hypertension can be induced in mice by one-kidney one-clip (1K1C) or two-kidney one-clip surgery, transgenic overexpression of angiotensinogen and renin, administration of deoxycorticosterone acetate-salt, supplying Nitro-L-arginine methyl-ester in the drinking water and Angiotensin-II infusion. Although each model has its own pros and cons, selection of a model that mimics human hypertensive disease accurately is essential to ensure rigor and reproducibility in hypertension research. 1K1C mice represent an efficient, budget-friendly, and translationally capable model; however, their use in preclinical research has remained largely hindered due to concerns about potential technical complexity and lack of reported information regarding procedure-related mortality rates. Here, we describe in detail an improved version of the 1K1C surgery in mice that has zero intraoperative mortality and excellent survival rates in a long-term setting and permits the development of stable chronic hypertension and its target organ complications. Key to this outcome is unilateral nephrectomy 1 week after renal artery clipping to decelerate the blood pressure (BP) increase, which allows the organism to adapt better to the BP rise. The technical and animal welfare improvements presented here may promote the acceptance of the 1K1C model.

Targeting neovascularization and respiration of tumor grafts grown on chick embryo chorioallantoic membranes.

Since growing tumors stimulate angiogenesis, via vascular endothelial growth factor (VEGF), angiogenesis inhibitors (AIs, blockers of the VEGF signaling pathway) have been introduced to cancer therapy. However, AIs often yielded only modest and short-lived gains in cancer patients and more invasive tumor phenotypes in animal models. Combining anti-VEGF strategies with lactate uptake blockers may boost both efficacy and safety of AIs. We assessed this hypothesis by using the ex ovo chorioallantoic membrane (CAM) assay. We show that AI-based monotherapy (Avastin®, AVA) increases tumor hypoxia in human CAM cancer cell xenografts and cell spread in human as well as canine CAM cancer cell xenografts. In contrast, combining AVA treatment with lactate importer MCT1 inhibitors (α-cyano-4-hydroxycinnamic acid (CHC) or AZD3965 (AZD)) reduced both tumor growth and cell dissemination of human and canine explants. Moreover, combining AVA+AZD diminished blood perfusion and tumor hypoxia in human explants. Thus, the ex ovo CAM assay as an easy, fast and cheap experimental setup is useful for pre-clinical cancer research. Moreover, as an animal-free experimental setup the CAM assay can reduce the high number of laboratory animals used in pre-clinical cancer research.

Blood Pressure Normalization-Independent Cardioprotective Effects of Endogenous, Physical Activity-Induced αCGRP (α Calcitonin Gene-Related Peptide) in Chronically Hypertensive Mice.

RATIONALE: αCGRP (α calcitonin gene-related peptide), one of the strongest vasodilators, is cardioprotective in hypertension by reducing the elevated blood pressure. OBJECTIVE: However, we hypothesize that endogenous, physical activity-induced αCGRP has blood pressure-independent cardioprotective effects in chronic hypertension. METHODS AND RESULTS: Chronically hypertensive (one-kidney-one-clip surgery) wild-type and αCGRP-/- sedentary or voluntary wheel running mice were treated with vehicle, αCGRP, or the αCGRP receptor antagonist CGRP8-37. Cardiac function and myocardial phenotype were evaluated echocardiographically and by molecular, cellular, and histological analysis, respectively. Blood pressure was similar among all hypertensive experimental groups. Endogenous αCGRP limited pathological remodeling and heart failure in sedentary, chronically hypertensive wild-type mice. In these mice, voluntary wheel running significantly improved myocardial phenotype and function, which was abolished by CGRP8-37 treatment. In αCGRP-/- mice, αCGRP treatment, in contrast to voluntary wheel running, improved myocardial phenotype and function. Specific inhibition of proliferation and myofibroblast differentiation of primary, murine cardiac fibroblasts by αCGRP suggests involvement of these cells in αCGRP-dependent blunting of pathological cardiac remodeling. CONCLUSIONS: Endogenous, physical activity-induced αCGRP has blood pressure-independent cardioprotective effects and is crucial for maintaining cardiac function in chronic hypertension. Consequently, inhibiting endogenous αCGRP signaling, as currently approved for migraine prophylaxis, could endanger patients with hypertension.

Expression of Hypoxia-Inducible Factor 1α (HIF-1α) and Genes of Related Pathways in Altered Gravity.

Immune system deterioration in space represents a major risk, which has to be mitigated for exploration-class missions into the solar system. Altered gravitational forces have been shown to regulate adaptation processes in cells of the immune system, which are important for appropriate risk management, monitoring and development of countermeasures. T lymphocytes and cells of the monocyte-macrophage system are highly migratory cell types that frequently encounter a wide range of oxygen tensions in human tissues and in hypoxic areas, even under homeostatic conditions. Hypoxia-inducible factor 1 and 2 (HIF's) might have an important role in activation of T cells and cells of the monocyte-macrophages system. Thus, we investigated the regulation of HIF-dependent and, therefore, hypoxia-signaling systems in both cell types in altered gravity and performed transcript and protein analysis from parabolic flight and suborbital ballistic rocket experiments. We found that HIF-1α and HIF-1-dependent transcripts were differently regulated in altered gravity, whereas HIF-1α-dependent gene expression adapted after 5 min microgravity. Inter-platform comparisons identified PDK1 as highly responsive to gravitational changes in human U937 myelomonocytic cells and in Jurkat T cells. We suggest HIF-1 as a potential pharmacological target for counteracting immune system deterioration during space flight.

Aquaporin 1 controls the functional phenotype of pulmonary smooth muscle cells in hypoxia-induced pulmonary hypertension.

Vascular remodelling in hypoxia-induced pulmonary hypertension (PH) is driven by excessive proliferation and migration of endothelial and smooth muscle cells. The expression of aquaporin 1 (AQP1), an integral membrane water channel protein involved in the control of these processes, is tightly regulated by oxygen levels. The role of AQP1 in the pathogenesis of PH, however, has not been directly addressed so far. This study was designed to characterize expression and function of AQP1 in pulmonary vascular cells from human arteries and in the mouse model of hypoxia-induced PH. Exposure of human pulmonary vascular cells to hypoxia significantly induced the expression of AQP1. Similarly, levels of AQP1 were found to be upregulated in lungs of mice with hypoxia-induced PH. The functional role of AQP1 was further tested in human pulmonary artery smooth muscle cells demonstrating that depletion of AQP1 reduced proliferation, the migratory potential, and, conversely, increased apoptosis of these cells. This effect was associated with higher expression of the tumour suppressor gene p53. Using the mouse model of hypoxia-induced PH, application of GapmeR inhibitors targeting AQP1 abated the hypoxia-induced upregulation of AQP1 and, of note, reversed PH by decreasing both right ventricular pressure and hypertrophy back to the levels of control mice. Our data suggest an important functional role of AQP1 in the pathobiology of hypoxia-induced PH. These results offer novel insights in our pathogenetic understanding of the disease and propose AQP1 as potential therapeutic in vivo target.

Analysis of hypoxia-induced noncoding RNAs reveals metastasis-associated lung adenocarcinoma transcript 1 as an important regulator of vascular smooth muscle cell proliferation.

Vascular remodeling, a pathogenic hallmark in pulmonary hypertension, is mainly driven by a dysbalance between proliferation and apoptosis of human pulmonary artery smooth muscle cells. It has previously been shown that microRNAs are involved in the pathogenesis of pulmonary hypertension. However, the role of long noncoding RNAs has not been evaluated. long noncoding RNA expression was quantified in human pulmonary artery smooth muscle cells using PCR arrays and quantitative PCR. Knockdown of genes was performed by transfection of siRNA or GapmeR. Proliferation and migration were measured using BrdU incorporation and wound healing assays. The mouse model of hypoxia-induced PH was used to determine the physiological meaning of identified long noncoding RNAs. The expression of 84 selected long noncoding RNAs was assessed in hypoxic human pulmonary artery smooth muscle cells and the levels of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) were significantly increased. Depletion of hypoxia-inducible factor 1α abolished the hypoxia-induced upregulation of metastasis-associated lung adenocarcinoma transcript 1 expression. Silencing of MALAT1 significantly decreased proliferation and migration of human pulmonary artery smooth muscle cells. In vivo, MALAT1 expression was significantly increased in lungs of hypoxic mice. Of note, targeting of MALAT1 by GapmeR ameliorated heart hypertrophy in mice with pulmonary hypertension. This is the first report on functional characterization of MALAT1 in the pulmonary vasculature. Our data provide evidence that MALAT1 expression is significantly increased by hypoxia, probably by hypoxia-inducible factor 1α. Intervention experiments confirmed that MALAT1 regulates the proliferative phenotype of smooth muscle cells and silencing of MALAT1 reduced heart hypertrophy in mice with pulmonary hypertension. These data indicate a potential role of MALAT1 in the pathogenesis of pulmonary hypertension. Impact statement Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long noncoding RNA that mediates several biological processes. In the context of vascular biology, MALAT1 has been shown to be inducible by hypoxia and to control cell proliferation. These processes are of major importance for the pathophysiology of hypoxia-induced pulmonary hypertension (PH). Until now, the physiological role of MALAT1 in PH remains unclear. By using smooth muscle cells and by employing an established PH mouse model, we provide evidence that hypoxia induces MALAT1 expression. Moreover, depletion of MALAT1 inhibited migration and proliferation of smooth muscle cells, probably by the induction of cyclin-dependent kinase inhibitors. Of note, MALAT1 was significantly increased in mice exposed to hypoxia and silencing of MALAT1 ameliorated heart hypertrophy in mice with hypoxia-induced PH. Since vascular remodeling and right heart failure as a consequence of pulmonary pressure overload is a major problem in PH, these data have implications for our pathogenetic understanding.

Nogo-A regulates vascular network architecture in the postnatal brain.

Recently, we discovered a new role for the well-known axonal growth inhibitory molecule Nogo-A as a negative regulator of angiogenesis in the developing central nervous system. However, how Nogo-A affected the three-dimensional (3D) central nervous system (CNS) vascular network architecture remained unknown. Here, using vascular corrosion casting, hierarchical, synchrotron radiation µCT-based network imaging and computer-aided network analysis, we found that genetic ablation of Nogo-A significantly increased the three-dimensional vascular volume fraction in the postnatal day 10 (P10) mouse brain. More detailed analysis of the cerebral cortex revealed that this effect was mainly due to an increased number of capillaries and capillary branchpoints. Interestingly, other vascular parameters such as vessel diameter, -length, -tortuosity, and -volume were comparable between both genotypes for non-capillary vessels and capillaries. Taken together, our three-dimensional data showing more vessel segments and branchpoints at unchanged vessel morphology suggest that stimulated angiogenesis upon Nogo-A gene deletion results in the insertion of complete capillary micro-networks and not just single vessels into existing vascular networks. These findings significantly enhance our understanding of how angiogenesis, vascular remodeling, and three-dimensional vessel network architecture are regulated during central nervous system development. Nogo-A may therefore be a potential novel target for angiogenesis-dependent central nervous system pathologies such as brain tumors or stroke.

Featured Article: microRNA-125a in pulmonary hypertension: Regulator of a proliferative phenotype of endothelial cells.

Vascular remodeling due to excessive proliferation of endothelial and smooth muscle cells is a hallmark feature of pulmonary hypertension. microRNAs (miRNAs) are a class of small, non-coding RNA fragments that have recently been associated with remodeling of pulmonary arteries, in particular by silencing the bone morphogenetic protein receptor type II (BMPR2). Here we identified a novel pathway involving the concerted action of miR-125a, BMPR2 and cyclin-dependent kinase inhibitors (CDKN) that controls a proliferative phenotype of endothelial cells. An in silico approach predicted miR-125a to target BMPR2. Functional inhibition of miR-125a resulted in increased proliferation of these cells, an effect that was found accompanied by upregulation of BMPR2 and reduced expression of the tumor suppressors CDKN1A (p21) and CDKN2A (p16). These data were confirmed in experimental pulmonary hypertension in vivo. Levels of miR-125a were elevated in lung tissue of hypoxic animals that develop pulmonary hypertension. In contrast, circulating levels of miR-125a were found to be lower in mice with pulmonary hypertension as compared to control mice. Similar findings were observed in a small cohort of patients with precapillary pulmonary hypertension. These translational data emphasize the pathogenetic role of miR-125a in pulmonary vascular remodeling.

The hypoxia-induced microRNA-130a controls pulmonary smooth muscle cell proliferation by directly targeting CDKN1A.

Excessive proliferation of human pulmonary artery smooth muscle cells (HPASMC) is one of the major factors that trigger vascular remodeling in hypoxia-induced pulmonary hypertension. Several studies have implicated that hypoxia inhibits the tumor suppressor p21 (CDKN1A). However, the precise mechanism is unknown. The mouse model of hypoxia-induced PH and in vitro experiments were used to assess the impact of microRNAs (miRNAs) on the expression of CDKN1A. In these experiments, the miRNA family miR-130 was identified to regulate the expression of CDKN1A. Transfection of HPASMC with miR-130 decreased the expression of CDKN1A and, in turn, significantly increased smooth muscle proliferation. Conversely, inhibition of miR-130 by anti-miRs and seed blockers increased the expression of CDKN1A. Reporter gene analysis proved a direct miR-130-CDKN1A target interaction. Exposure of HPASMC to hypoxia was found to induce the expression of miR-130 with concomitant decrease of CDKN1A. These findings were confirmed in the mouse model of hypoxia-induced pulmonary hypertension showing that the use of seed blockers against miR-130 restored the expression of CDKN1A. These data suggest that miRNA family miR-130 plays an important role in the repression of CDKN1A by hypoxia. miR-130 enhances hypoxia-induced smooth muscle proliferation and might be involved in the development of right ventricular hypertrophy and vascular remodeling in pulmonary hypertension.

Alternatively spliced tissue factor is not sufficient for embryonic development.

Tissue factor (TF) triggers blood coagulation and is translated from two mRNA splice isoforms, encoding membrane-anchored full-length TF (flTF) and soluble alternatively-spliced TF (asTF). The complete knockout of TF in mice causes embryonic lethality associated with failure of the yolk sac vasculature. Although asTF plays roles in postnatal angiogenesis, it is unknown whether it activates coagulation sufficiently or makes previously unrecognized contributions to sustaining integrity of embryonic yolk sac vessels. Using gene knock-in into the mouse TF locus, homozygous asTF knock-in (asTFKI) mice, which express murine asTF in the absence of flTF, exhibited embryonic lethality between day 9.5 and 10.5. Day 9.5 homozygous asTFKI embryos expressed asTF protein, but no procoagulant activity was detectable in a plasma clotting assay. Although the α-smooth-muscle-actin positive mesodermal layer as well as blood islands developed similarly in day 8.5 wild-type or homozygous asTFKI embryos, erythrocytes were progressively lost from disintegrating yolk sac vessels of asTFKI embryos by day 10.5. These data show that in the absence of flTF, asTF expressed during embryonic development has no measurable procoagulant activity, does not support embryonic vessel stability by non-coagulant mechanisms, and fails to maintain a functional vasculature and embryonic survival.

A novel in vivo vascular imaging approach for hierarchical quantification of vasculature using contrast enhanced micro-computed tomography.

The vasculature of body tissues is continuously subject to remodeling processes originating at the micro-vascular level. The formation of new blood vessels (angiogenesis) is essential for a number of physiological and pathophysiological processes such as tissue regeneration, tumor development and the integration of artificial tissues. There are currently no time-lapsed in vivo imaging techniques providing information on the vascular network at the capillary level in a non-destructive, three-dimensional and high-resolution fashion. This paper presents a novel imaging framework based on contrast enhanced micro-computed tomography (micro-CT) for hierarchical in vivo quantification of blood vessels in mice, ranging from largest to smallest structures. The framework combines for the first time a standard morphometric approach with densitometric analysis. Validation tests showed that the method is precise and robust. Furthermore, the framework is sensitive in detecting different perfusion levels after the implementation of a murine ischemia-reperfusion model. Correlation with both histological data and micro-CT analysis of vascular corrosion casts confirmed accuracy of the method. The newly developed time-lapsed imaging approach shows high potential for in vivo monitoring of a number of different physiological and pathological conditions in angiogenesis and vascular development.

Decreased stability of erythroblastic islands in integrin ß3-deficient mice.

Erythroblasts proliferate and differentiate in hematopoietic organs within erythroblastic islands (EI) composed of erythropoietic progenitor cells attached to a central macrophage. This cellular interaction crucially involves the erythroid intercellular adhesion molecule-4 (ICAM-4) and αv integrin. Because integrins are biologically active as α/ß heterodimers, we asked whether ß3 could be a heterodimerization partner of αv integrin in EIs. To this end we compared stress erythropoiesis driven by two different mechanisms, namely that of integrin ß3-deficient (ß3(-/-)) mice that exhibit impaired hemostasis due to platelet dysfunction with that of systemically erythropoietin-overexpressing (tg6) mice. While compared to the respective wild type (wt) controls ß3(-/-) mice had much less erythropoietic stimulation than tg6 mice ß3(-/-) blood contained more erythrocytes of a lower maturity stage. Unexpectedly, membranes of peripheral erythrocytes from ß3(-/-) mice (but not those from either wt control or from tg6 mice) contained calnexin, a chaperone that is normally completely lost during terminal differentiation of reticulocytes prior to their release into the circulation. In contrast to erythropoietin-overexpressing mice, the erythropoietic subpopulations representing orthochromatic erythroblasts and premature reticulocytes as well as the number of cells per EI were reduced in ß3(-/-) bone marrow. In conclusion, absence of integrin ß3 impairs adhesion of the latest erythroid developmental stage to the central macrophage of EIs resulting in preterm release of abnormally immature erythrocytes into the circulation.

Liver iron modulates hepcidin expression during chronically elevated erythropoiesis in mice.

UNLABELLED: The liver-derived peptide hepcidin controls the balance between iron demand and iron supply. By inhibiting the iron export activity of ferroportin, hepcidin modulates iron absorption and delivery from the body's stores. The regulation of hepcidin, however, is not completely understood and includes a variety of different signals. We studied iron metabolism and hepcidin expression in mice constitutively overexpressing erythropoietin (Epo) (Tg6 mice), which leads to excessive erythropoiesis. We observed a very strong down-regulation of hepcidin in Tg6 mice that was accompanied by a strong increase in duodenal expression of ferroportin and divalent metal tranporter-1, as well as enhanced duodenal iron absorption. Despite these compensatory mechanisms, Tg6 mice displayed marked circulating iron deficiency and low levels of iron in liver, spleen, and muscle. To elucidate the primary signal affecting hepcidin expression during chronically elevated erythropoiesis, we increased iron availability by either providing iron (thus further increasing the hematocrit) or reducing erythropoiesis-dependent iron consumption by means of splenectomy. Both treatments increased liver iron and up-regulated hepcidin expression and the BMP6/SMAD pathway despite continuously high plasma Epo levels and sustained erythropoiesis. This suggests that hepcidin expression is not controlled by erythropoietic signals directly in this setting. Rather, these results indicate that iron consumption for erythropoiesis modulates liver iron content, and ultimately BMP6 and hepcidin. Analysis of the BMP6/SMAD pathway targets showed that inhibitor of DNA binding 1 (ID1) and SMAD7, but not transmembrane serine protease 6 (TMPRSS6), were up-regulated by increased iron availability and thus may be involved in setting the upper limit of hepcidin. CONCLUSION: We provide evidence that under conditions of excessive and effective erythropoiesis, liver iron regulates hepcidin expression through the BMP6/SMAD pathway.

Assessment of postsurgical distress and pain in laboratory mice by nest complexity scoring.

Preliminary studies have suggested a correlation between postsurgical pain and nest building behaviour in laboratory mice. However, there is no standardized measure for estimating pain by means of nest building performance. Here, we investigated nest building under various conditions, and scored nest complexity to assess postsurgical pain. Mice of both sexes, different strains [C57BL/6J, DBA/2J, and B6D2-Tg(Pr-mSMalphaActin)V5rCLR-25], and kept under different housing conditions, showed no differences in their latency to use the offered nest material. Healthy female C57BL/6J mice were engaged 4.3% of the day with nest building and showed three peaks of this behaviour: in the beginning and middle of the light phase, and in the second half of the dark phase. For assessment of postsurgical pain, female C57BL/6J mice underwent a sham embryo transfer +/− different doses of the analgesic carprofen or control treatment. Nest complexity scoring at 9 h after the experimental treatments (i.e. at the end of the light phase) resulted in less than 10% of animals with noticeably manipulated nest material (nestlet) after surgery and more than 75% of healthy mice having built identifiable-to-complex nests or had noticeably manipulated nestlets, while animals after anaesthesia-only showed intermediate nest complexity. Carprofen analgesia resulted in no (5 mg/kg) or only slight (50 mg/kg) improvement of nest complexity after surgery. Thus, nest complexity scoring can be incorporated into daily laboratory routine and can be used in mice as a sensitive tool for detecting reduced wellbeing and general condition, but probably not for determining the efficacy of pain treatment.

Nogo-A is a negative regulator of CNS angiogenesis.

Nogo-A is an important axonal growth inhibitor in the adult and developing CNS. In vitro, Nogo-A has been shown to inhibit migration and cell spreading of neuronal and nonneuronal cell types. Here, we studied in vivo and in vitro effects of Nogo-A on vascular endothelial cells during angiogenesis of the early postnatal brain and retina in which Nogo-A is expressed by many types of neurons. Genetic ablation or virus-mediated knock down of Nogo-A or neutralization of Nogo-A with an antibody caused a marked increase in the blood vessel density in vivo. In culture, Nogo-A inhibited spreading, migration, and sprouting of primary brain microvascular endothelial cells (MVECs) in a dose-dependent manner and induced the retraction of MVEC lamellipodia and filopodia. Mechanistically, we show that only the Nogo-A-specific Delta 20 domain exerts inhibitory effects on MVECs, but the Nogo-66 fragment, an inhibitory domain common to Nogo-A, -B, and -C, does not. Furthermore, the action of Nogo-A Delta 20 on MVECs required the intracellular activation of the Ras homolog gene family, member A (Rho-A)-associated, coiled-coil containing protein kinase (ROCK)-Myosin II pathway. The inhibitory effects of early postnatal brain membranes or cultured neurons on MVECs were relieved significantly by anti-Nogo-A antibodies. These findings identify Nogo-A as an important negative regulator of developmental angiogenesis in the CNS. They may have important implications in CNS pathologies involving angiogenesis such as stroke, brain tumors, and retinopathies.

A new method for repeated drug infusion into the femoral artery of mice.

Intraarterial chemotherapy (IAC) is considered effective for the treatment of solid tumors with high local doses of systemically toxic chemotherapeutics. Osteosarcoma, which is often located in the extremities, is a potential target for IAC. However, the efficacy of this treatment modality has varied, and standardized protocols are difficult to establish due to tumor heterogeneity and the limited numbers of patients available for clinical trials. Reproducible experimental models are needed to further investigate IAC in osteosarcoma. Here, we describe a new microsurgical technique for repeated infusion of drugs into the mouse femoral artery for local treatment of experimental intratibial metastasizing osteosarcoma. We successfully achieved 5 catheterizations at 3-d intervals in 70% of the mice tested. Laser speckle imaging indicated a maximal 50% reduction in blood flow around the ankle region of catheterized legs infused with 0.5 mg/kg cisplatin. However, blood flow in the front feet was affected only minimally. Histologic examination of catheterized arteries of saline control or cisplatin-treated mice showed circular fibrinoid media necrosis and partial thrombosis, but total occlusion of the arteries was not observed. The method we describe for repeated transient catheterization of the mouse femoral artery likely will be useful in future studies comparing the efficacies of intraarterial and systemic cisplatin treatment of intratibial metastasizing osteosarcoma in mice under standardized conditions.

Dynamics of polyploid formation and establishment in the allotetraploid rock fern Asplenium majoricum.

BACKGROUND AND AIMS: Successful establishment of newly formed polyploid species depends on several interlinked genetic and ecological factors. These include genetic diversity within and among individuals, chromosome behaviour and fertility, novel phenotypes resulting from novel genomic make-up and expression, intercytotypic and interspecific competition, and adaptation to distinct habitats. The allotetraploid rock fern Asplenium majoricum is known from one small population in Valencia, Spain, and several larger populations on the Balearic island of Majorca. In Valencia, it occurs sympatrically with its diploid parents, A. fontanum subsp. fontanum and A. petrarchae subsp. bivalens, and their diploid hybrid A. × protomajoricum. This highly unusual situation allowed the study of polyploid genetic diversity and its relationship to the formation and establishment of nascent polyploid lineages. METHODS: Genetic variation for isozyme and chloroplast DNA markers was determined for A. majoricum and A. × protomajoricum sampled thoroughly from known sites in Majorca and Valencia. Results were compared with variation determined previously for the diploid parent taxa. KEY RESULTS: A highly dynamic system with recurring diploid hybrid and allotetraploid formation was discovered. High diversity in the small Valencian A. majoricum population indicates multiple de novo origins from diverse parental genotypes, but most of these lineages become extinct without becoming established. The populations on Majorca most probably represent colonization(s) from Valencia rather than an in situ origin. Low genetic diversity suggests that this colonization may have occurred only once. CONCLUSIONS: There is a striking contrast in success of establishment of the Majorcan and Valencian populations of A. majoricum. Chance founding of populations in a habitat where neither A. fontanum subsp. fontanum nor A. petrarchae subsp. bivalens occurs appears to have been a key factor enabling the establishment of A. majoricum on Majorca. Successful establishment of this polyploid is probably dependent on geographic isolation from diploid progenitor competition.

Impaired ventilatory acclimatization to hypoxia in female mice overexpressing erythropoietin: unexpected deleterious effect of estradiol in carotid bodies.

Apart from enhancing the production of red blood cells, erythropoietin (Epo) alters the ventilatory response when oxygen supply is reduced. We recently demonstrated that Epo's beneficial effect on the ventilatory response to acute hypoxia is sex dependent, with female mice being better able to cope with reduced oxygenation. In the present work, we hypothesized that ventilatory acclimatization to chronic hypoxia (VAH) in transgenic female mice (Tg6) harboring high levels of Epo in the brain and blood will also be improved compared with wild-type (WT) animals. Surprisingly, VAH was blunted in Tg6 female mice. To define whether this phenomenon had a central (brain stem respiratory centers) and/or peripheral (carotid bodies) origin, a bilateral transection of carotid sinus nerve (chemodenervation) was performed. This procedure allowed the analysis of the central response in the absence of carotid body information. Interestingly, chemodenervation restored the VAH in Tg6 mice, suggesting that carotid bodies were responsible for the blunted response. Coherently with this observation, the sensitivity to oxygen alteration in arterial blood (Dejour test) after chronic hypoxia was lower in transgenic carotid bodies compared with the WT control. As blunted VAH occurred in female but not male transgenic mice, the involvement of sex female steroids was obvious. Indeed, measurement of sexual female hormones revealed that the estradiol serum level was 4 times higher in transgenic mice Tg6 than in WT animals. While ovariectomy decreased VAH in WT females, this treatment restored VAH in Tg6 female mice. In line with this observation, injections of estradiol in ovariectomized Tg6 females dramatically reduced the VAH. We concluded that during chronic hypoxia, estradiol in carotid bodies suppresses the Epo-mediated elevation of ventilation. Considering the increased application of recombinant Epo for a variety of disorders, our data imply the need to take the patient's hormonal status into consideration.