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Yes-associated protein (YAP), a transcription coactivator, is the major downstream effector of the Hippo pathway, which plays a critical role in organ size control and cancer development. However, how YAP is regulated by extracellular stimuli in tumorigenesis remains incompletely understood. Netrin-1, a laminin-related secreted protein, displays proto-oncogenic activity in cancers. Nonetheless, the downstream signaling mediating its oncogenic effects is not well defined. Here we show that netrin-1 via its transmembrane receptors, deleted in colorectal cancer and uncoordinated-5 homolog, up-regulates YAP expression, escalating YAP levels in the nucleus and promoting cancer cell proliferation and migration. Inactivating netrin-1, deleted in colorectal cancer, or uncoordinated-5 homolog B (UNC5B) decreases YAP protein levels, abrogating cancer cell progression by netrin-1, whereas knockdown of mammalian STE20-like protein kinase 1/2 (MST1/2) or large tumor suppressor kinase 1/2 (Lats1/2), two sets of upstream core kinases of the Hippo pathway, has no effect in blocking netrin-1-induced up-regulation of YAP. Netrin-1 stimulates phosphatase 1A to dephosphorylate YAP, which leads to decreased ubiquitination and degradation, enhancing YAP accumulation and signaling. Hence, our findings support that netrin-1 exerts oncogenic activity through YAP signaling, providing a mechanism coupling extracellular signals to the nuclear YAP oncogene. [ABSTRACT FROM AUTHOR]

Cerebral cavernous malformations are common vascular lesions of the central nervous system that predispose to seizures, focal neurologic deficits and potentially fatal hemorrhagic stroke. Human genetic studies have identified three genes associated with the disease and biochemical studies of these proteins have identified interaction partners and possible signaling pathways. A variety of animal models of CCM have been described to help translate the cellular and biochemical insights into a better understanding of disease mechanism. In this minireview, we discuss the contributions of animal models to our growing understanding of the biology of cavernous malformations, including the elucidation of the cellular context of CCM protein actions and the in vivo confirmation of abnormal endothelial cell–cell interactions. Challenges and progress towards developing a faithful model of CCM biology are reviewed. [ABSTRACT FROM AUTHOR]

Nerves and blood vessels often follow parallel trajectories as they course through the body to their distal targets. Proteins that regulate the process of axon guidance have likewise been shown to play a crucial role in blood vessel migration. With the recent description of the endothelial tip cell as an analog of the axonal growth cone, the nerve–vessel analogy seems complete. Notwithstanding these considerable similarities, one crucial difference remains between neural and vascular guidance. While a navigating axon is but a single cell, a sprouting vessel is composed of multiple cells that must be co-ordinately regulated. Recent studies of the Dll4-Notch1 signaling pathway have provided valuable insight into how the vasculature accomplishes this crucial task. [Copyright &y& Elsevier]

Deals with a study which assessed the role of transesophageal echocardiography in the diagnosis of patients with suspected infective endocarditis. Methods; Results; Discussion.

Reports that loss-of-function mutations in the human endoglin gene ENG causes hereditary hemorrhagic telangiectasis (HHT1), a disease characterized by vascular malformations. Shows that by gestational day 11.5 mice lacking endoglin die from defective vascular development; Vasculogenesis unaffected in mice lacking transforming growth factor-beta (TGF-beta); Results of loss of endoglin; Need for endoglin for angiogenesis.

Presents research which defined the role of elastin in arterial development and disease by generating mice that lack elastin. Previous thoughts about elastin's role; How the generated mice die; Developments similar to atherosclerosis; Traits of mice; Disruption of elastin and obstructive arterial disease; Elastin's regulatory function during arterial development.

Blood vessel abnormalization alters cancer cell metabolism and promotes cancer dissemination and metastasis. However, the biological features of the abnormalized blood vessels that facilitate cancer progression and whether they can be targeted therapeutically have not been fully investigated. Here, we found that an axon guidance molecule, fibronectin leucine-rich transmembrane protein 2 (FLRT2), is expressed preferentially in abnormalized vessels of advanced colorectal cancers in humans and that its expression correlates negatively with long-term survival. Endothelial cell-specific deletion of Flrt2 in mice selectively pruned abnormalized vessels, resulting in a unique metabolic state termed "oxygen-glucose uncoupling," which suppressed tumor metastasis. Moreover, Flrt2 deletion caused an increase in the number of mature vessels, resulting in a significant increase in the antitumor effects of immune checkpoint blockers. Mechanistically, we found that FLRT2 forms noncanonical interendothelial adhesions that safeguard against oxidative stress through homophilic binding. Together, our results demonstrated the existence of tumor-specific interendothelial adhesions that enable abnormalized vessels to facilitate cancer aggressiveness. Targeting this type of adhesion complex could be a safe and effective therapeutic option to suppress cancer progression. [ABSTRACT FROM AUTHOR]

Study Type - Aetiology (case control) Level of Evidence 3b What's known on the subject? and What does the study add? As one of the major components of the extracellular matrix, elastic fibres are believed to enhance tissue compliance. However, the role of elastic fibres in normal bladder function and dysfunction remains speculative. Although transgenic mice overexpressing elastin showed increased bladder compliance, the findings in patients with non-compliant bladders are inconsistent. Using transgenic elastin-deficient mouse models, this study provides the first direct evidence that sufficient elastin content is critical for healthy bladder function, and elastin is involved in the detrusor response to partial bladder outlet obstruction. OBJECTIVE To examine functional and molecular changes of the bladders from elastin-haploinsufficient mice ( Eln+/−) at baseline as well as in response to partial bladder outlet obstruction (pBOO)., MATERIALS AND METHODS Female Eln+/− and wild type ( Wt) mice (3-4 months old) were studied., The bladder elastin content was quantified by measuring desmosine., Mice were divided into two groups to undergo surgery to create pBOO or to undergo sham surgery. Three days after surgery, bladder function was evaluated by in vivo cystometry, and the contractile response of bladder strips exposed to electrical field stimulation (EFS) and carbachol was examined by ex vivo myography., RESULTS The Eln+/−-sham mice had a 33.6% decrease in bladder elastin compared with Wt-sham mice., Cystometry showed significantly decreased bladder compliance and capacity in Eln+/−-sham vs Wt-sham mice; pBOO increased bladder compliance and capacity to a greater extent in Eln+/− mice compared with Wt mice., Bladder strips from Eln+/−-sham mice showed a significantly heightened contractile response to both EFS and carbachol compared with Wt-sham mice., A significantly increased contractile response to carbachol was detected in Wt-pBOO vs Wt-sham but not between Eln+/−-pBOO and Eln+/−-sham mice., CONCLUSION The results that elastin-deficient mice had decreased bladder compliance and capacity and increased bladder contractility; and that Wt-pBOO mice showed an enhanced contractile response to carbachol, but Eln+/−-pBOO mice did not, suggest that elastin is critical for normal bladder function and is involved in bladder response to pBOO. [ABSTRACT FROM AUTHOR]

Netrins were initially identified as secreted ligands regulating axon guidance and migration through interaction with canonical receptors. Netrins were then shown to be necessary for development of a range of tissues, including lung, mammary gland, and the vasculature. While new netrin receptors, as well as alternative ligands for classical netrin receptors, were described in the neuronal and epithelial fields, there was a singular focus on canonical netrin receptors in the vascular system, leading to controversy on netrin function and the nature of receptor-mediated netrin signaling in the endothelium. Here, we summarize the current state of knowledge on netrin ligands and receptors and discuss questions, controversies, and perspectives surrounding netrin functions and receptor identity in the vasculature. [ABSTRACT FROM AUTHOR]

Mice heterozygous for the elastin gene (ELN+/-) show unique cardiovascular properties, including increased blood pressure and smaller, thinner arteries with an increased number of lamellar units. Some of these properties are also observed in humans with supravalvular aortic stenosis, a disease caused by functional heterozygosity of the elastin gene. The arterial geometry in ELN+/- mice is contrary to the increased thickness that would be expected in an animal demonstrating hypertensive remodeling. To determine whether this is due to a decreased capability for cardiovascular remodeling or to a novel adaptation of the ELN+/- cardiovascular system, we increased blood pressure in adult ELN+/+ and ELN+/- mice using the two-kidney, one-clip Goldblatt model of hypertension. Successfully clipped mice have a systolic pressure increase of at least 15 mmHg over sham-operated animals. ELN+/+ and ELN+/--clipped mice show significant increases over sham-operated mice in cardiac weight, arterial thickness, and arterial cross-sectional area with no changes in lamellar number. There are no significant differences in most mechanical properties with clipping in either genotype. These results indicate that ELN+/+ and ELN+/- hearts and arteries remodel similarly in response to adult induced hypertension. Therefore, the cardiovascular properties of ELN+/- mice are likely due to developmental remodeling in response to altered hemodynamics and reduced elastin levels. [ABSTRACT FROM AUTHOR]

Several characteristic morphological and functional differences distinguish arteries from veins. It was thought that hemodynamic forces shaped these differences; however, increasing evidence suggests that morphogenetic programs play a central role in blood vessel differentiation. Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia characterized by the inappropriate fusion of arterioles with venules. The genes implicated in this disease, ALK1 and endoglin, may be involved in defining the fundamental boundaries between arteries and veins. We previously showed that mice lacking Alk1 lost structural, molecular, and functional distinctions between arteries and veins [Nat. Genet. 26 (2000), 328]. Here, we report that mice lacking endoglin develop arterial–venous malformations and fail to confine intraembryonic hematopoiesis to arteries. In contrast to Alk1 mutants, endoglin mutants do not show profound vessel dilation or downregulation of arterial ephrinB2. Finally, our data indicate that a failure in cardiac cushion formation observed in both strains may be secondary to the peripheral vasculature defect. The phenotypic similarities, yet reduced severity, implicates endoglin as an accessory coreceptor that specifically modulates Alk1 signaling. We propose that endoglin and Alk1 are necessary for the maintenance of distinct arterial–venous vascular beds and that attenuation of the Alk1 signaling pathway is the precipitating event in the etiology of HHT. [Copyright &y& Elsevier]

Elastin is synthesized and secreted by vascular smooth muscle cells and is the major extracellular matrix component deposited in the arterial wall. When last reviewed by this journal in 1994, the link between elastin and a rare occlusive vascular disease had just been established. Since that time, it has become increasingly clear that elastin is a critical autocrine factor that maintains vascular homeostasis through a combination of biomechanical support and biologic signaling. This review examines the complexity of elastin–smooth muscle cell interactions, and how new insights may impact understanding of the pathogenesis and treatment of vascular disease. [Copyright &y& Elsevier]

The vascular system matures during embryonic development to form a stable, well-organized tubular network. In vivo data have established that the extracellular matrix (ECM) is crucial in providing structural support to the vascular system. In vitro studies are defining the involvement of ECM–smooth-muscle cell signaling in establishing and maintaining the mature tubular structure. However, correlating cell signaling with established structural functions for the ECM and determining the relative importance of these two roles in vivo is often difficult. Here, we examine human genetics, murine gene targeting and cell biology to better understand the relationship between structural and signaling roles for the ECM in vascular morphogenesis and disease. [Copyright &y& Elsevier]

The mature circulatory system is comprised of two parallel, yet distinct, vascular networks that carry blood to and from the heart. Studies have suggested that endothelial tubes are specified as arteries and veins at the earliest stages of angiogenesis, before the onset of circulation. To understand the molecular basis for arterial-venous identity, we have focused our studies on a human vascular dysplasia, hereditary haemorrhagic telangiectasia (HHT), wherein arterial and venous beds fail to remain distinct. Genetic studies have demonstrated that HHT can be caused by loss-of-function mutations in the gene encoding activin receptor-like kinase-1 (ACVRL1; ref. 5). ACVRL1 encodes a type I receptor for the TGF-β superfamily of growth factors. At the earliest stage of vascular development, mice lacking Acvrl1 develop large shunts between arteries and veins, downregulate arterial Efnb2 and fail to confine intravascular haematopoiesis to arteries. These mice die by mid-gestation with severe arteriovenous malformations resulting from fusion of major arteries and veins. The early loss of anatomical, molecular and functional distinctions between arteries and veins indicates that Acvrl1 is required for developing distinct arterial and venous vascular beds. [ABSTRACT FROM AUTHOR]

New Findings: What is the central question of this study?Greater large artery stiffness is associated with dysfunctional resistance artery vasodilatory responses, impaired memory and greater risk of Alzheimer's disease. However, it is unknown whether stiffer large arteries affect cerebral and skeletal muscle feed artery responses to vasoconstrictors.What is the main finding and its importance?In a mouse model with greater large artery stiffness (Eln+/−), we find an exacerbated vasoconstrictor response to angiotensin II in cerebral arteries, but not skeletal muscle feed arteries, thus implicating altered cerebral artery angiotensin II responsiveness in the poor brain outcomes associated with greater large artery stiffness. Greater stiffness of the large elastic arteries is associated with end‐organ damage and dysfunction. At the same time, resistance artery vasoconstrictor responsiveness influences vascular tone and organ blood flow. However, it is unknown whether large elastic artery stiffness modulates the responsiveness to vasoconstrictors in resistance arteries of the cerebral or skeletal muscle circulations. We previously described the elastin haploinsufficient (Eln+/−) mouse as a model with greater aortic stiffness, but with similar cerebral and skeletal muscle feed artery stiffness to wild‐type (Eln+/+) mice. Here, we used this model to examine the relationship between large elastic artery stiffness and resistance artery vasoconstrictor responses. In middle cerebral arteries (MCAs), vasoconstriction in response to angiotensin II (Ang II) was ∼40% greater in Eln+/− compared with Eln+/+ mice (P = 0.02), and this group difference was ameliorated by losartan, indicating a role for Ang II type 1 receptors (AT1Rs). In gastrocnemius feed arteries, Eln+/− and Eln+/+ mice did not differ in the response to Ang II. In addition, the vasoconstrictor responses to noradrenaline, endothelin‐1 and potassium chloride were not different between Eln+/− and Eln+/+ mice for either MCAs or gastrocnemius feed arteries. The MCA AT1R gene expression did not differ between groups, whereas Ang II type 2 receptor gene expression was ∼50% lower in MCAs from Eln+/−versus Eln+/+ mice (P = 0.01). In conclusion, greater large elastic artery stiffness is associated with an exacerbated vasoconstriction response to Ang II in cerebral arteries, but is not associated with the responses to other vasoconstrictors in either cerebral or skeletal muscle feed arteries. [ABSTRACT FROM AUTHOR]

The activation of the small GTPase ARF6 has been implicated in promoting several pathological processes related to vascular instability and tumor formation, growth, and metastasis. ARF6 also plays a vital role during embryonic development. Recent studies have suggested that ARF6 carries out these disparate functions primarily by controlling protein trafficking within the cell. ARF6 helps direct proteins to intracellular or extracellular locations where they function in normal cellular responses during development and in pathological processes later in life. This transport of proteins is accomplished through a variety of mechanisms, including endocytosis and recycling, microvesicle release, and as yet uncharacterized processes. This Commentary will explore the functions of ARF6, while focusing on the role of this small GTPase in development and postnatal physiology, regulating barrier function and diseases associated with its loss, and tumor formation, growth, and metastasis. [ABSTRACT FROM AUTHOR]

The devastating sequelae of diabetes mellitus include microvascular permeability, which results in retinopathy. Despite clinical and scientific advances, there remains a need for new approaches to treat retinopathy. Here, we have presented a possible treatment strategy, whereby targeting the small GTPase ARF6 alters VEGFR2 trafficking and reverses signs of pathology in 4 animal models that represent features of diabetic retinopathy and in a fifth model of ocular pathological angiogenesis. Specifically, we determined that the same signaling pathway utilizes distinct GEFs to sequentially activate ARF6, and these GEFs exert distinct but complementary effects on VEGFR2 trafficking and signal transduction. ARF6 activation was independently regulated by 2 different ARF GEFs - ARNO and GEP100. Interaction between VEGFR2 and ARNO activated ARF6 and stimulated VEGFR2 internalization, whereas a VEGFR2 interaction with GEP100 activated ARF6 to promote VEGFR2 recycling via coreceptor binding. Intervening in either pathway inhibited VEGFR2 signal output. Finally, using a combination of in vitro, cellular, genetic, and pharmacologic techniques, we demonstrated that ARF6 is pivotal in VEGFR2 trafficking and that targeting ARF6-mediated VEGFR2 trafficking has potential as a therapeutic approach for retinal vascular diseases such as diabetic retinopathy. [ABSTRACT FROM AUTHOR]

Key points Endothelial cells employ transient receptor potential isoform 4 (TRPV4) channels to sense ambient mechanical and chemical stimuli., In retinal microvascular endothelial cells, TRPV4 channels regulate calcium homeostasis, cytoskeletal signalling and the organization of adherens junctional contacts., Intracellular calcium increases induced by TRPV4 agonists include a significant contribution from calcium release from internal stores., Activation of TRPV4 channels regulates retinal endothelial barriers in vitro and in vivo., TRPV4 sensing may provide a feedback mechanism between sensing shear flow and eicosanoid modulators, vascular permeability and contractility at the inner retinal endothelial barrier., Abstract The identity of microvascular endothelial (MVE) mechanosensors that sense blood flow in response to mechanical and chemical stimuli and regulate vascular permeability in the retina is unknown. Using immunohistochemistry, calcium imaging, electrophysiology, impedance measurements and vascular permeability assays, we show that the transient receptor potential isoform 4 (TRPV4) plays a major role in Ca2+/cation signalling, cytoskeletal remodelling and barrier function in retinal microvasculature in vitro and in vivo. Human retinal MVE cells (HrMVECs) predominantly expressed Trpv1 and Trpv4 transcripts, and TRPV4 was broadly localized to the plasma membrane of cultured cells and intact blood vessels in the inner retina. Treatment with the selective TRPV4 agonist GSK1016790A (GSK101) activated a nonselective cation current, robustly elevated [Ca2+]i and reversibly increased the permeability of MVEC monolayers. This was associated with disrupted organization of endothelial F-actin, downregulated expression of occludin and remodelling of adherens contacts consisting of vascular endothelial cadherin (VE-cadherin) and β-catenin. In vivo, GSK101 increased the permeability of retinal blood vessels in wild type but not in TRPV4 knockout mice. Agonist-evoked effects on barrier permeability and cytoskeletal reorganization were antagonized by the selective TRPV4 blocker HC 067047. Human choroidal endothelial cells expressed lower TRPV4 mRNA/protein levels and showed less pronounced agonist-evoked calcium signals compared to MVECs. These findings indicate a major role for TRPV4 in Ca2+ homeostasis and barrier function in human retinal capillaries and suggest that TRPV4 may differentially contribute to the inner vs. outer blood-retinal barrier function. [ABSTRACT FROM AUTHOR]

The placental labyrinth is the interface for gas and nutrient exchange between the embryo and the mother; hence its proper development is essential for embryogenesis. However, the molecular mechanism underlying development of the placental labyrinth, particularly in terms of its endothelial organization, is not well understood. Here, we determined that fibronectin leucine-rich transmembrane protein 2 (FLRT2), a repulsive ligand of the UNC5 receptor family for neurons, is unexpectedly expressed in endothelial cells specifically in the placental labyrinth. Mice lacking FLRT2 in endothelial cells exhibited embryonic lethality at mid-gestation, with systemic congestion and hypoxia. Although they lacked apparent deformities in the embryonic vasculature and heart, the placental labyrinths of these embryos exhibited aberrant alignment of endothelial cells, which disturbed the feto-maternal circulation. Interestingly, this vascular deformity was related to endothelial repulsion through binding to the UNC5B receptor. Our results suggest that the proper organization of the placental labyrinth depends on coordinated inter-endothelial repulsion, which prevents uncontrolled layering of the endothelium. [ABSTRACT FROM AUTHOR]

Background: The number of centers with left ventricular assist device (LVAD) research programs focused on cardiac recovery is very small. Therefore, this phenomenon has been reported in real-world multi-center registries as a rare event.Objectives: This study evaluated the incidence of cardiac recovery with an a priori LVAD implantation strategy of bridge-to-recovery (BTR) and constructed a recovery predictive model.Methods: The study included LVAD recipients registered in the Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS). Cardiac recovery was evaluated in BTR and non-BTR patients. A weighted score was derived and externally validated in patients of the Utah Cardiac Recovery (UCAR) program.Results: Of 15,138 INTERMACS patients, cardiac recovery occurred in 192 (1.3%). The incidence of recovery was 11.2% (n = 14) in BTR compared with 1.2% (n = 178) in non-BTR patients (p < 0.0001). Independent predictors of recovery included: age <50 years, non-ischemic cardiomyopathy, time from cardiac diagnosis <2 years, absence of ICD, creatinine ≤1.2 mg/dl, and LVEDD <6.5 cm (c-index: 0.85; p < 0.0001). A weighted score termed I-CARS, effectively stratified patients based on their probability of recovery. I-CARS was validated in the UCAR cohort (n = 190) with good performance (AUC: 0.94; 95% CI: 0.91 to 0.98). One-year survival after LVAD explantation, available in INTERMACS for 21 (11%) patients, was 86%.Conclusions: The incidence of cardiac recovery is higher in patients implanted with an a priori BTR strategy. We developed a simple tool to help identify patients in whom recovery is feasible. In BTR patients with favorable characteristics, I-CARS suggests a 24% probability of successful LVAD explantation. Large-scale studies to better address post-explantation outcomes are warranted. [ABSTRACT FROM AUTHOR]

Adipocytes are differentiated by various transcriptional cascades integrated on the master regulator, Pparγ. To discover new genes involved in adipocyte differentiation, preadipocytes were treated with three newly identified pro-adipogenic small molecules and GW7845 (a Pparγ agonist) for 24 hours and transcriptional profiling was analyzed. Four genes, Peroxisome proliferator-activated receptor γ (Pparγ), human complement factor D homolog (Cfd), Chemokine (C-C motif) ligand 9 (Ccl9), and GIPC PDZ Domain Containing Family Member 2 (Gipc2) were induced by at least two different small molecules but not by GW7845. Cfd and Ccl9 expressions were specific to adipocytes and they were altered in obese mice. Small hairpin RNA (shRNA) mediated knockdown of Cfd in preadipocytes inhibited lipid accumulation and expression of adipocyte markers during adipocyte differentiation. Overexpression of Cfd promoted adipocyte differentiation, increased C3a production, and led to induction of C3a receptor (C3aR) target gene expression. Similarly, treatments with C3a or C3aR agonist (C4494) also promoted adipogenesis. C3aR knockdown suppressed adipogenesis and impaired the pro-adipogenic effects of Cfd, further suggesting the necessity for C3aR signaling in Cfd-mediated pro-adipogenic axis. Together, these data show the action of Cfd in adipogenesis and underscore the application of small molecules to identify genes in adipocytes. [ABSTRACT FROM AUTHOR]

Mutations in CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation in humans. Mouse models of CCM disease have been established by deleting Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. However, the full value of these animal models is limited by the lack of an accurate and quantitative method to assess lesion burden and progression. In the present study we have established a refined and detailed contrast enhanced X-ray micro-CT method to measure CCM lesion burden in mouse brains. As this study utilized a voxel dimension of 9.5μm (leading to a minimum feature size of approximately 25μm), it is therefore sufficient to measure CCM lesion volume and number globally and accurately, and provide high-resolution 3-D mapping of CCM lesions in mouse brains. Using this method, we found loss of Ccm1 or Ccm2 in neonatal endothelium confers CCM lesions in the mouse hindbrain with similar total volume and number. This quantitative approach also demonstrated a rescue of CCM lesions with simultaneous deletion of one allele of Mekk3. This method would enhance the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases. [ABSTRACT FROM AUTHOR]

Chromatin structure is determined by nucleosome positioning, histone modifications, and DNA methylation. How chromatin modifications are coordinately altered under pathological conditions remains elusive. Here we describe a stress-activated mechanism of concerted chromatin modification in the heart. In mice, pathological stress activates cardiomyocytes to express Brg1 (nucleosome-remodeling factor), G9a/Glp (histone methyltransferase), and Dnmt3 (DNA methyltransferase). Once activated, Brg1 recruits G9a and then Dnmt3 to sequentially assemble repressive chromatin—marked by H3K9 and CpG methylation—on a key molecular motor gene ( Myh6 ), thereby silencing Myh6 and impairing cardiac contraction. Disruption of Brg1, G9a or Dnmt3 erases repressive chromatin marks and de-represses Myh6 , reducing stress-induced cardiac dysfunction. In human hypertrophic hearts, BRG1–G9a/GLP–DNMT3 complex is also activated; its level correlates with H3K9/CpG methylation, Myh6 repression, and cardiomyopathy. Our studies demonstrate a new mechanism of chromatin assembly in stressed hearts and novel therapeutic targets for restoring Myh6 and ventricular function. The stress-induced Brg1–G9a–Dnmt3 interactions and sequence of repressive chromatin assembly on Myh6 illustrates a molecular mechanism by which the heart epigenetically responds to environmental signals. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel. [ABSTRACT FROM AUTHOR]

Motivation: The Cancer Genome Atlas (TCGA) RNA-Sequencing data are used widely for research. TCGA provides 'Level 3' data, which have been processed using a pipeline specific to that resource. However, we have found using experimentally derived data that this pipeline produces geneexpression values that vary considerably across biological replicates. In addition, some RNA-Sequencing analysis tools require integer-based read counts, which are not provided with the Level 3 data. As an alternative, we have reprocessed the data for 9264 tumor and 741 normal samples across 24 cancer types using the Rsubread package. We have also collated corresponding clinical data for these samples. We provide these data as a community resource. Results: We compared TCGA samples processed using either pipeline and found that the Rsubread pipeline produced fewer zero-expression genes and more consistent expression levels across replicate samples than the TCGA pipeline. Additionally, we used a genomic-signature approach to estimate HER2 (ERBB2) activation status for 662 breast-tumor samples and found that the Rsubread data resulted in stronger predictions of HER2 pathway activity. Finally, we used data from both pipelines to classify 575 lung cancer samples based on histological type. This analysis identified various non-coding RNA that may influence lung-cancer histology. Availability and implementation: The RNA-Sequencing and clinical data can be downloaded from Gene Expression Omnibus (accession number GSE62944). Scripts and code that were used to process and analyze the data are available from https://github.com/srp33/TCGA_RNASeq_Clinical. [ABSTRACT FROM AUTHOR]

Vitamin D is a known modulator of inflammation. Native dietary vitamin D3 is thought to be bio-inactive, and beneficial vitamin D3 effects are thought to be largely mediated by the metabolite 1,25(OH)2D3. Reduced serum levels of the most commonly measured precursor metabolite, 25(OH)D3, is linked to an increased risk of multiple inflammatory diseases, including: cardiovascular disease, arthritis, multiple sclerosis, and sepsis. Common to all of these diseases is the disruption of endothelial stability and an enhancement of vascular leak. We previously performed an unbiased chemical suppressor screen on a genetic model of vascular instability, and identified cholecalciferol (D3, dietary Vitamin D3) as a factor that had profound and immediate stabilizing and therapeutic effects in that model. In this manuscript we show that the presumed inactive sterol, D3, is actually a potent and general mediator of endothelial stability at physiologically relevant concentrations. We further demonstrate that this phenomenon is apparent in vitamin D3 metabolites 25(OH)D3 and 1,25(OH)2D3, and that the effects are independent of the canonical transcription-mediated vitamin D pathway. Our data suggests the presence of an alternative signaling modality by which D3 acts directly on endothelial cells to prevent vascular leak. The finding that D3 and its metabolites modulate endothelial stability may help explain the clinical correlations between low serum vitamin D levels and the many human diseases with well-described vascular dysfunction phenotypes. [ABSTRACT FROM AUTHOR]

Key points Increased large artery stiffness is a hallmark of arterial dysfunction with advancing age and is also present in other disease conditions such as diabetes. Increased large artery stiffness is correlated with resistance artery dysfunction in humans., Using a mouse model of altered arterial elastin content, this is the first study to examine the cause-and-effect relationship between large artery stiffness and peripheral resistance artery function., Our results indicate that mice with genetically greater large artery stiffness have impaired cerebral artery endothelial function, but generally preserved skeletal muscle feed artery endothelial function. The mechanisms for impaired cerebral artery endothelial function are reduced nitric oxide bioavailability and increased oxidative stress., These findings suggest that interventions that target large artery stiffness may be important to reduce disease risk associated with cerebral artery dysfunction in conditions such as advancing age., Abstract Advancing age as well as diseases such as diabetes are characterized by both increased large artery stiffness and impaired peripheral artery function. It has been hypothesized that greater large artery stiffness causes peripheral artery dysfunction; however, a cause-and-effect relationship has not previously been established. We used elastin heterozygote mice ( Eln+/-) as a model of increased large artery stiffness without co-morbidities unrelated to the large artery properties. Aortic stiffness, measured by pulse wave velocity, was ∼35% greater in Eln+/- mice than in wild-type ( Eln+/+) mice ( P = 0.04). Endothelium-dependent dilatation (EDD), assessed by the maximal dilatation to acetylcholine, was ∼40% lower in Eln+/- than Eln+/+ mice in the middle cerebral artery (MCA, P < 0.001), but was similar between groups in the gastrocnemius feed arteries (GFA, P = 0.79). In the MCA, EDD did not differ between groups after incubation with the nitric oxide (NO) synthase inhibitor Nω-nitro- l-arginine methyl ester ( P > 0.05), indicating that lower NO bioavailability contributed to the impaired EDD in Eln+/- mice. Superoxide production and content of the oxidative stress marker nitrotyrosine was higher in MCAs from Eln+/− compared with Eln+/+ mice ( P < 0.05). In the MCA, after incubation with the superoxide scavenger TEMPOL, maximal EDD improved by ∼65% in Eln+/- ( P = 0.002), but was unchanged in Eln+/+ mice ( P = 0.17). These results indicate that greater large artery stiffness has a more profound effect on endothelial function in cerebral arteries compared with skeletal muscle feed arteries. Greater large artery stiffness can cause cerebral artery endothelial dysfunction by reducing NO bioavailability and increasing oxidative stress. [ABSTRACT FROM AUTHOR]

Background: In animal models of heterotopic transplantation, mechanical unloading of the normal, nonhypertrophic heart results in atrophy. Primarily on the basis of these animal data, the notion that chronic left ventricular assist device (LVAD)-induced unloading will result in atrophy has dominated the clinical heart failure field, and anti-atrophic drugs have been used to enhance the cardiac recovery potential observed in some LVAD patients. However, whether unloading-induced atrophy in experimental normal heart models applies to failing and hypertrophic myocardium in heart failure patients unloaded by continuous-flow LVADs has not been studied.Objectives: The study examined whether mechanical unloading by continuous-flow LVAD leads to myocardial atrophy.Methods: We prospectively examined myocardial tissue and hemodynamic and echocardiographic data from 44 LVAD patients and 18 untransplanted normal donors.Results: Cardiomyocyte size (cross-sectional area) decreased after LVAD unloading from 1,238 ± 81 μm(2) to 1,011 ± 68 μm(2) (p = 0.001), but not beyond that of normal donor hearts (682 ± 56 μm(2)). Electron microscopy ultrastructural evaluation, cardiomyocyte glycogen content, and echocardiographic assessment of myocardial mass and left ventricular function also did not suggest myocardial atrophy. Consistent with these findings, t-tubule morphology, cytoplasmic penetration, and distance from the ryanodine receptor were not indicative of ongoing atrophic remodeling during LVAD unloading. Molecular analysis revealed no up-regulation of proatrophic genes and proteins of the ubiquitin proteasome system.Conclusions: Structural, ultrastructural, microstructural, metabolic, molecular, and clinical functional data indicated that prolonged continuous-flow LVAD unloading does not induce hypertrophy regression to the point of atrophy and degeneration. These findings may be useful in designing future investigations that combine LVAD unloading and pharmaceutical therapies as a bridge to recovery of the failing heart. [ABSTRACT FROM AUTHOR]

Background In animal models of heterotopic transplantation, mechanical unloading of the normal, nonhypertrophic heart results in atrophy. Primarily on the basis of these animal data, the notion that chronic left ventricular assist device (LVAD)-induced unloading will result in atrophy has dominated the clinical heart failure field, and anti-atrophic drugs have been used to enhance the cardiac recovery potential observed in some LVAD patients. However, whether unloading-induced atrophy in experimental normal heart models applies to failing and hypertrophic myocardium in heart failure patients unloaded by continuous-flow LVADs has not been studied. Objectives The study examined whether mechanical unloading by continuous-flow LVAD leads to myocardial atrophy. Methods We prospectively examined myocardial tissue and hemodynamic and echocardiographic data from 44 LVAD patients and 18 untransplanted normal donors. Results Cardiomyocyte size (cross-sectional area) decreased after LVAD unloading from 1,238 ± 81 μm 2 to 1,011 ± 68 μm 2 (p = 0.001), but not beyond that of normal donor hearts (682 ± 56 μm 2 ). Electron microscopy ultrastructural evaluation, cardiomyocyte glycogen content, and echocardiographic assessment of myocardial mass and left ventricular function also did not suggest myocardial atrophy. Consistent with these findings, t-tubule morphology, cytoplasmic penetration, and distance from the ryanodine receptor were not indicative of ongoing atrophic remodeling during LVAD unloading. Molecular analysis revealed no up-regulation of proatrophic genes and proteins of the ubiquitin proteasome system. Conclusions Structural, ultrastructural, microstructural, metabolic, molecular, and clinical functional data indicated that prolonged continuous-flow LVAD unloading does not induce hypertrophy regression to the point of atrophy and degeneration. These findings may be useful in designing future investigations that combine LVAD unloading and pharmaceutical therapies as a bridge to recovery of the failing heart. [ABSTRACT FROM AUTHOR]

The proteasome inhibiter bortezomib has been successfully used to treat patients with relapsed multiple myeloma; however, many of these patients become thrombocytopenic, and it is not clear how the proteasome influences platelet production. Here we determined that pharmacologic inhibition of proteasome activity blocks proplatelet formation in human and mouse megakaryocytes. We also found that megakaryocytes isolated from mice deficient for PSMC1, an essential subunit of the 26S proteasome, fail to produce proplatelets. Consistent with decreased proplatelet formation, mice lacking PSMC1 in platelets (Psmc1fl/fl Pf4-Cre mice) exhibited severe thrombocytopenia and died shortly after birth. The failure to produce proplatelets in proteasome-inhibited megakaryocytes was due to upregulation and hyperactivation of the small GTPase, RhoA, rather than NF-κB, as has been previously suggested. Inhibition of RhoA or its downstream target, Rho-associated protein kinase (ROCK), restored megakaryocyte proplatelet formation in the setting of proteasome inhibition in vitro. Similarly, fasudil, a ROCK inhibitor used clinically to treat cerebral vasospasm, restored platelet counts in adult mice that were made thrombocytopenic bytamoxifen-induced suppression of proteasome activity in megakaryocytes and platelets (Psmc1fl/fl Pdgf-Cre-ER mice). These results indicate that proteasome function is critical forthrombopoiesis, and suggest inhibition of RhoA signalingas a potential strategy to treat thrombocytopenia in bortezomib-treated multiple myeloma patients. [ABSTRACT FROM AUTHOR]

The vascular endothelium responds to infection by destabilizing endothelial cell-cell junctions to allow fluid and cells to pass into peripheral tissues, facilitating clearance of infection and tissue repair. During sepsis, endotoxin and other proinflammatory molecules induce excessive vascular leak, which can cause organ dysfunction, shock, and death. Current therapies for sepsis are limited to antibiotics and supportive care, which are often insufficient to reduce morbidity and prevent mortality. Previous attempts at blocking inflammatory cytokine responses in humans proved ineffective at reducing the pathologies associated with sepsis, highlighting the need for a new therapeutic strategy. The small GTPase ARF6 is activated by a MyD88-ARNO interaction to induce vascular leak through disruption of endothelial adherens junctions. In this study, we show that the MyD88-ARNO-ARF6-signaling axis is responsible for LPS-induced endothelial permeability and is a destabilizing convergence point used by multiple inflammatory cues. We also show that blocking ARF6 with a peptide construct of its N terminus is sufficient to reduce vascular leak and enhance survival during endotoxic shock, without inhibiting the host cytokine response. Our data highlight the therapeutic potential of blocking ARF6 and reducing vascular leak for the treatment of inflammatory conditions, such as endotoxemia. [ABSTRACT FROM AUTHOR]

Background Signaling through vascular endothelial growth factor C (VEGF-C) and VEGF receptor 3 (VEGFR-3) plays a central role in lymphangiogenesis and the metastasis of several cancers via the lymphatics. Recently, the Slit2/Robo4 pathway has been recognized as a modulator of vascular permeability and integrity. Signaling via the Robo receptor inhibits VEGF-mediated effects; however, its effects on lymphatic endothelial cell function have not been well characterized. Results We found that pretreatment with Slit2N, an active fragment of Slit2, inhibited VEGF-C-mediated lung-derived lymphatic endothelial cell (L-LEC) proliferation, migration, and in vitro tube formation. Slit2N induced the internalization of VEGFR-3, which blocked its activation, and inhibited the activation of the PI3K/Akt pathway by VEGF-C in L-LECs. Moreover, we found that inhibition of VEGF-C-induced effects by Slit2N was Robo4-dependent. Conclusion These results indicate that Slit2N/Robo4 modulates several key cellular functions, which contribute to lymphangiogenesis, and identify this ligand-receptor pair as a potential therapeutic target to inhibit lymphatic metastasis of VEGF-C-overexpressing cancers and manage lymphatic dysfunctions characterized by VEGF-C/VEGFR-3 activation. Cellular signaling initiated by the binding of vascular endothelial growth factor C (VEGF-C) to the receptor, VEGFR-3, is central to the growth of lymphatic channels, their constituent endothelial cells, and the spread of several types of cancer via the lymphatic system. Signaling through the ligand-receptor pair, Slit2/Robo4, modulates the permeability and integrity of vascular endothelium, the cells that line blood vessels. The Slit2/Robo pathway inhibits cellular effects induced by VEGF; however, its effects on lymphatic channels and lymphatic endothelium have not been fully studied. We found that pretreating lung-derived lymphatic endothelial cells (L-LECs) with Slit2N, an active fragment of the protein Slit2, inhibited VEGF-C-induced functions critical for the formation of the lymphatics, i.e. lymphangiogenesis. Specifically, it blocked the growth and migration of L-LECs, and the formation of tube-like structures on a gelatinous matrix. Slit2N induced the internalization VEGR-3, which blocked its activation, and inhibited signaling through PI3K/Akt, a pathway that modulates diverse cellular processes, including cell growth and cancer progression. In addition, we found that inhibition of VEGF-C-induced effects by Slit2N required sufficient levels of Robo4, indicating that Robo4 is the receptor to which Slit2N binds to inhibit the aforementioned effects. These results indicate that Slit2N/Robo4 modulates key cellular functions that contribute to lymphangiogenesis, and identify this ligand-receptor pair as a potential drug target to inhibit cancer metastasis via the lymphatic system and to treat other lymphatic pathologies characterized by abnormal VEGF-C/VEGFR-3 signaling. [ABSTRACT FROM AUTHOR]

Abstract: Cationic Rh(II) complexes are able to catalyze the regioselective hydroamination of propargyl ureas in a 6-endo fashion. This transformation permits access to interesting substitution patterns of dihydropyrimidines, which have found use as nucleotide exchange factor inhibitors. [Copyright &y& Elsevier]

Objectives: This study sought to prospectively investigate the longitudinal effects of continuous-flow left ventricular assist device (LVAD) unloading on myocardial structure and systolic and diastolic function. Background: The magnitude, timeline, and sustainability of changes induced by continuous-flow LVAD on the structure and function of the failing human heart are unknown. Methods: Eighty consecutive patients with clinical characteristics consistent with chronic heart failure requiring implantation of a continuous-flow LVAD were prospectively enrolled. Serial echocardiograms (at 1, 2, 3, 4, 6, 9, and 12 months) and right heart catheterizations were performed after LVAD implant. Cardiac recovery was assessed on the basis of improvement in systolic and diastolic function indices on echocardiography that were sustained during LVAD turn-down studies. Results: After 6 months of LVAD unloading, 34% of patients had a relative LV ejection fraction increase above 50% and 19% of patients, both ischemic and nonischemic, achieved an LV ejection fraction ≥40%. LV systolic function improved as early as 30 days, the greatest degree of improvement was achieved by 6 months of mechanical unloading and persisted over the 1-year follow up. LV diastolic function parameters also improved as early as 30 days after LVAD unloading, and this improvement persisted over time. LV end-diastolic and end-systolic volumes decreased as early as 30 days after LVAD unloading (113 vs. 77 ml/m2, p < 0.01, and 92 vs. 60 ml/m2, p < 0.01, respectively). LV mass decreased as early as 30 days after LVAD unloading (114 vs. 95 g/m2, p < 0.05) and continued to do so over the 1-year follow-up but did not reach values below the normal reference range, suggesting no atrophic remodeling after prolonged LVAD unloading. Conclusions: Continuous-flow LVAD unloading induced in a subset of patients, both ischemic and nonischemic, early improvement in myocardial structure and systolic and diastolic function that was largely completed within 6 months, with no evidence of subsequent regression. [Copyright &y& Elsevier]

Background:--Survival of patients on left ventricular assist devices (LVADs) has improved. We examined the differences in risk of adverse outcomes between LVAD-supported and medically managed candidates on the heart transplant waiting list. Methods and Results:--We analyzed mortality and morbidity in 33073 heart transplant candidates registered on the United Network for Organ Sharing (UNOS) waiting list between 1999 and 2011. Five groups were selected: patients without LVADs in urgency status 1A, IB, and 2; patients with pulsatile-flow LVADs; and patients with continuous-flow LVADs. Outcomes in patients requiring biventricular assist devices, total artificial heart, and temporary VADs were also analyzed. Two eras were defined on the basis of the approval date of the first continuous-flow LVAD for bridge to transplantation in the United States (2008). Mortality was lower in the current compared with the first era (2.1%/mo versus 2.9%/mo; P<0.0001). In the first era, mortality of pulsatile-flow LVAD patients was higher than in status 2 (hazard ratio [HR], 2.15; P<0.0001) and similar to that in status IB patients (HR, 1.04; P=0.61). In the current era, patients with continuous-flow LVADs had mortality similar to that of status 2 (HR, 0.80; P=0.12) and lower mortality compared with status lAand IB patients (HR, 0.24 and 0.47; P<0.0001 for both comparisons). However, status upgrade for LVAD-related complications occurred frequently (28%) and increased the mortality risk (HR, 1.75; P=0.001). Mortality was highest in patients with biventricular assist devices (HR, 5.00; P<0.0001) and temporary VADs (HR, 7.72; P<0.0001). Conclusions:--Mortality and morbidity on the heart transplant waiting list have decreased. Candidates supported with contemporary continuous-flow LVADs have favorable waiting list outcomes; however, they worsen significantly once a serious LVAD-related complication occurs. Transplant candidates requiring temporary and biventricular support have the highest risk of adverse outcomes. These results may help to guide optimal allocation of donor hearts. [ABSTRACT FROM AUTHOR]

The innate immune response is essential for combating infectious disease. Macrophages and other cells respond to infection by releasing cytokines, such as interleukin-1? (IL-1?), which in turn activate a well-described, myeloid-differentiation factor 88 (MYD88)-mediated, nuclear factor-?B (NF-?B)-dependent transcriptional pathway that results in inflammatory-cell activation and recruitment. Endothelial cells, which usually serve as a barrier to the movement of inflammatory cells out of the blood and into tissue, are also critical mediators of the inflammatory response. Paradoxically, the cytokines vital to a successful immune defence also have disruptive effects on endothelial cell-cell interactions and can trigger degradation of barrier function and dissociation of tissue architecture. The mechanism of this barrier dissolution and its relationship to the canonical NF-?B pathway remain poorly defined. Here we show that the direct, immediate and disruptive effects of IL-1? on endothelial stability in a human in vitro cell model are NF-?B independent and are instead the result of signalling through the small GTPase ADP-ribosylation factor 6 (ARF6) and its activator ARF nucleotide binding site opener (ARNO; also known as CYTH2). Moreover, we show that ARNO binds directly to the adaptor protein MYD88, and thus propose MYD88-ARNO-ARF6 as a proximal IL-1? signalling pathway distinct from that mediated by NF-?B. Finally, we show that SecinH3, an inhibitor of ARF guanine nucleotide-exchange factors such as ARNO, enhances vascular stability and significantly improves outcomes in animal models of inflammatory arthritis and acute inflammation. [ABSTRACT FROM AUTHOR]

Cell-mediated transmission and dissemination of sexually-acquired human immunodeficiency virus 1 (HIV-1) in the host involves the migration of immature dendritic cells (iDCs). iDCs migrate in response to the HIV-1 envelope protein, gp120, and inhibiting such migration may limit the mucosal transmission of HIV-1. In this study, we elucidated the mechanism of HIV-1-gp120-induced transendothelial migration of iDCs. We found that gp120 enhanced the binding of Wiskott-Aldrich Syndrome protein (WASp) and the Actin-Related Protein 2/3 (Arp2/3) complex with β-actin, an interaction essential for the proper formation of podosomes, specialized adhesion structures required for the migration of iDCs through different tissues. We further identified Leukocyte-Specific Protein 1 (LSP1) as a novel component of the WASp-Arp2/3-β-actin complex. Pretreating iDCs with an active fragment of the secretory glycoprotein Slit2 (Slit2N) inhibited HIV-1-gp120- mediated migration and podosome formation, by inducing the cognate receptor Roundabout 1 (Robo1) to bind to and sequester WASp and LSP1 from β-actin. Slit2N treatment also inhibited Src signaling and the activation of several downstream molecules, including Rac1, Pyk2, paxillin, and CDC42, a major regulator of podosome formation. Taken together, our results support a novel mechanism by which Slit2/Robo1 may inhibit the HIV-1-gp120-induced migration of iDCs, thereby restricting dissemination of HIV-1 from mucosal surfaces in the host. [ABSTRACT FROM AUTHOR]

Summary: We show that R-Ras, a small GTPase of the Ras family, is essential for the establishment of mature, functional blood vessels in tumors. The genetic disruption of R-Ras severely impaired the maturation processes of tumor vessels in mice. Conversely, the gain of function of R-Ras improved vessel structure and blood perfusion and blocked plasma leakage by enhanced endothelial barrier function and pericyte association with nascent blood vessels. Thus, R-Ras promotes normalization of the tumor vasculature. These findings identify R-Ras as a critical regulator of vessel integrity and function during tumor vascularization. [ABSTRACT FROM AUTHOR]

The article discusses a study on the use of Left ventricular (LV) assist devices (LVADs) as a tool for end-stage heart failure (HF) patients to heart transplantation or as a permanent therapy. It mentions that increase in clinical use of LVADs presents a key opportunity for in-depth investigations of the biology of the failing human heart. It also informs that trough biological and clinical outcomes it is possible to identify new therapeutic strategies of myocardial recovery and regeneration.

Fibroblast Growth Factor receptor (FGFR) activity plays crucial roles in tumor growth and patient survival. However, FGF (Fibroblast Growth Factor) signaling as a target for cancer therapy has been under-investigated compared to other receptor tyrosine kinases. Here, we studied the effect of FGFR signaling inhibition on tumor growth, metastasis and lymphangiogenesis by expressing a dominant negative FGFR (FGFR-2DN) in an orthotopic mouse mammary 66c14 carcinoma model. We show that FGFR-2DN-expressing 66c14 cells proliferate in vitro slower than controls. 66c14 tumor outgrowth and lung metastatic foci are reduced in mice implanted with FGFR-2DN-expressing cells, which also exhibited better overall survival. We found 66c14 cells in the lumen of tumor lymphatic vessels and in lymph nodes. FGFR-2DNexpressing tumors exhibited a decrease in VEGFR-3 (Vascular Endothelial Growth Factor Receptor-3) or podoplanin-positive lymphatic vessels, an increase in isolated intratumoral lymphatic endothelial cells and a reduction in VEGF-C (Vascular Endothelial Growth Factor-C) mRNA expression. FGFs may act in an autocrine manner as the inhibition of FGFR signaling in tumor cells suppresses VEGF-C expression in a COX-2 (cyclooxygenase-2) or HIF1-α (hypoxia-inducible factor-1 α) independent manner. FGFs may also act in a paracrine manner on tumor lymphatics by inducing expression of prolymphangiogenic molecules such as VEGFR-3, integrin a9, prox1 and netrin-1. Finally, in vitro lymphangiogenesis is impeded in the presence of FGFR-2DN 66c14 cells. These data confirm that both FGF and VEGF signaling are necessary for the maintenance of vascular morphogenesis and provide evidence that targeting FGFR signaling may be an interesting approach to inhibit tumor lymphangiogenesis and metastatic spread. [ABSTRACT FROM AUTHOR]

Dissemination of HIV in the host involves transit of the virus and virus-infected cells across the lymphatic endothelium. HIV may alter lymphatic endothelial permeability to foster dissemination, but the mechanism is largely unexplored. Using a primary human lymphatic endothelial cell model, we found that HIV-1 envelope protein gp120 induced lymphatic hyperpermeability by disturbing the normal function of Robo4, a novel regulator of endothelial permeability. HIV-1 gp120 induced fibronectin expression and integrin a5b1 phosphorylation, which led to the complexing of these three proteins, and their subsequent interaction with Robo4 through its fibronectin type III repeats. Moreover, pretreatment with an active N-terminus fragment of Slit2, a Robo4 agonist, protected lymphatic endothelial cells from HIV-1 gp120-induced hyperpermeability by inhibiting c-Src kinase activation. Our results indicate that targeting Slit2/Robo4 signaling may protect the integrity of the lymphatic barrier and limit the dissemination of HIV in the host. [ABSTRACT FROM AUTHOR]

Cerebral cavernous malformations (CCMs) are a common type of vascular malformation in the brain that are a major cause of hemorrhagic stroke. This condition has been independently linked to 3 separate genes: Krev1 interaction trapped (KRIT1), Cerebral cavernous malformation 2 (CCM2), and Programmed cell death 10 (PDCD10). Despite the commonality in disease pathology caused by mutations in these 3 genes, we found that the loss of Pdcd10 results in significantly different developmental, cell biological, and signaling phenotypes from those seen in the absence of Ccm2 and Krit1. PDCD10 bound to germinal center kinase III (GCKIII) family members, a subset of serine-threonine kinases, and facilitated lumen formation by endothelial cells both in vivo and in vitro. These findings suggest that CCM may be a common tissue manifestation of distinct mechanistic pathways. Nevertheless, loss of heterozygosity (LOH) for either Pdcd10 or Ccm2 resulted in CCMs in mice. The murine phenotype induced by loss of either protein reproduced all of the key clinical features observed in human patients with CCM, as determined by direct comparison with genotype-specific human surgical specimens. These results suggest that CCM may be more effectively treated by directing therapies based on the underlying genetic mutation rather than treating the condition as a single clinical entity. [ABSTRACT FROM AUTHOR]

Objectives: This study investigates alterations in myocardial microvasculature, fibrosis, and hypertrophy before and after mechanical unloading of the failing human heart. Background: Recent studies demonstrated the pathophysiologic importance and significant mechanistic links among microvasculature, fibrosis, and hypertrophy during the cardiac remodeling process. The effect of left ventricular assist device (LVAD) unloading on cardiac endothelium and microvasculature is unknown, and its influence on fibrosis and hypertrophy regression to the point of atrophy is controversial. Methods: Hemodynamic data and left ventricular tissue were collected from patients with chronic heart failure at LVAD implant and explant (n = 15) and from normal donors (n = 8). New advances in digital microscopy provided a unique opportunity for comprehensive whole-field, endocardium-to-epicardium evaluation for microvascular density, fibrosis, cardiomyocyte size, and glycogen content. Ultrastructural assessment was done with electron microscopy. Results: Hemodynamic data revealed significant pressure unloading with LVAD. This was accompanied by a 33% increase in microvascular density (p = 0.001) and a 36% decrease in microvascular lumen area (p = 0.028). We also identified, in agreement with these findings, ultrastructural and immunohistochemical evidence of endothelial cell activation. In addition, LVAD unloading significantly increased interstitial and total collagen content without any associated structural, ultrastructural, or metabolic cardiomyocyte changes suggestive of hypertrophy regression to the point of atrophy and degeneration. Conclusions: The LVAD unloading resulted in increased microvascular density accompanied by increased fibrosis and no evidence of cardiomyocyte atrophy. These new insights into the effects of LVAD unloading on microvasculature and associated key remodeling features might guide future studies of unloading-induced reverse remodeling of the failing human heart. [Copyright &y& Elsevier]

Formation of the vascular system within organs requires the balanced action of numerous positive and negative factors secreted by stromal and epithelial cells. Here, we used a genetic approach to determine the role of SLITs in regulating the growth and organization of blood vessels in the mammary gland. We demonstrate that vascularization of the gland is not affected by loss of Slit expression in the epithelial compartment. Instead, we identify a stromal source of SLIT, mural cells encircling blood vessels, and show that loss of Slit in the stroma leads to elevated blood vessel density and complexity. We examine candidate SLIT receptors, Robol and Robo4, and find that increased vessel angiogenesis is phenocopied by loss of endothelial-specific Robo4, as long as it is combined with the presence of an angiogenic stimulus such as preneoplasia or pregnancy. In contrast, loss of Robol does not affect blood vessel growth. The enhanced growth of blood vessels in Robo4 endothelium is due to activation of vascular endothelial growth factor (VEGF)-R2signaling through the Src and FAK kinases. Thus, our studies present a genetic dissection of SLIT/ROBO signaling during organ development. We identify a stromal, rather than epithelial. source of SLITs that inhibits blood vessel growth by signaling through endotheliál ROBO4 to down-regulate VEGF/ VEGFR2 signaling. [ABSTRACT FROM AUTHOR]

Right ventricular failure (RVF) after left ventricular assist device (LVAD) implantation appears to be associated with increased mortality. However, the determination of which patients are at greater risk of developing postoperative RVF remains controversial and relatively unknown. We sought to determine the preoperative risk factors for the development of RVF after LVAD implantation. The data were obtained for 175 consecutive patients who had received an LVAD. RVF was defined by the need for inhaled nitric oxide for ≥48 hours or intravenous inotropes for >14 days and/or right ventricular assist device implantation. An RVF risk score was developed from the β coefficients of the independent variables from a multivariate logistic regression model predicting RVF. Destination therapy (DT) was identified as the indication for LVAD implantation in 42% of our patients. RVF after LVAD occurred in 44% of patients (n = 77). The mortality rates for patients with RVF were significantly greater at 30, 180, and 365 days after implantation compared to patients with no RVF. By multivariate logistic regression analysis, 3 preoperative factors were significantly associated with RVF after LVAD implantation: (1) a preoperative need for intra-aortic balloon counterpulsation, (2) increased pulmonary vascular resistance, and (3) DT. The developed RVF risk score effectively stratified the risk of RV failure and death after LVAD implantation. In conclusion, given the progressively growing need for DT, the developed RVF risk score, derived from a population with a large percentage of DT patients, might lead to improved patient selection and help stratify patients who could potentially benefit from early right ventricular assist device implantation. [Copyright &y& Elsevier]

Background: A number of RNA viruses cause viral hemorrhagic fever (VHF), in which proinflammatory mediators released from infected cells induce increased permeability of the endothelial lining of blood vessels, leading to loss of plasma volume, hypotension, multi-organ failure, shock and death. The optimal treatment of VHF should therefore include both the use of antiviral drugs to inhibit viral replication and measures to prevent or correct changes in vascular function. Although rodent models have been used to evaluate treatments for increased vascular permeability (VP) in bacterial sepsis, such studies have not been performed for VHF. Results: Here, we use an established model of Pichinde virus infection of hamsters to demonstrate how changes in VP can be detected by intravenous infusion of Evans blue dye (EBD), and compare those measurements to changes in hematocrit, serum albumin concentration and serum levels of proinflammatory mediators. We show that EBD injected into sick animals in the late stage of infection is rapidly sequestered in the viscera, while in healthy animals it remains within the plasma, causing the skin to turn a marked blue color. This test could be used in live animals to detect increased VP and to assess the ability of antiviral drugs and vasoactive compounds to prevent its onset. Finally, we describe a multiplexed assay to measure levels of serum factors during the course of Pichinde arenavirus infection and demonstrate that viremia and subsequent increase in white blood cell counts precede the elaboration of inflammatory mediators, which is followed by increased VP and death. Conclusions: This level of model characterization is essential to the evaluation of novel interventions designed to control the effects of virus-induced hypercytokinemia on host vascular function in VHF, which could lead to improved survival. [ABSTRACT FROM AUTHOR]

Slit–Roundabout (Robo) signalling has a well-understood role in axon guidance. Unlike in the nervous system, however, Slit-dependent activation of an endothelial-specific Robo, Robo4, does not initiate a guidance program. Instead, Robo4 maintains the barrier function of the mature vascular network by inhibiting neovascular tuft formation and endothelial hyperpermeability induced by pro-angiogenic factors. In this study, we used cell biological and biochemical techniques to elucidate the molecular mechanism underlying the maintenance of vascular stability by Robo4. Here, we demonstrate that Robo4 mediates Slit2-dependent suppression of cellular protrusive activity through direct interaction with the intracellular adaptor protein paxillin and its paralogue, Hic-5. Formation of a Robo4–paxillin complex at the cell surface blocks activation of the small GTPase Arf6 and, consequently, Rac by recruitment of Arf-GAPs (ADP-ribosylation factor- directed GTPase-activating proteins) such as GIT1. Consistent with these in vitro studies, inhibition of Arf6 activity in vivo phenocopies Robo4 activation by reducing pathologic angiogenesis in choroidal and retinal vascular disease and VEGF-165 (vascular endothelial growth factor-165)-induced retinal hyperpermeability. These data reveal that a Slit2–Robo4–paxillin–GIT1 network inhibits the cellular protrusive activity underlying neovascularization and vascular leak, and identify a new therapeutic target for ameliorating diseases involving the vascular system. [ABSTRACT FROM AUTHOR]

Left ventricular assist device (LVAD) implantation before heart transplantation has been associated with formation of antibodies directed against human leukocyte antigens (HLA), often referred to as sensitization. This study investigated whether prior sensitization or LVAD type affected the degree of post-implantation sensitization. The records of consecutive HeartMate (HM) I and HM II LVAD patients were reviewed. Panel reactive antibody (PRA) was assessed before LVAD implantation and biweekly thereafter. Sensitization was defined as PRA > 10%, and high-degree sensitization was defined as PRA > 90%. An HM LVAD was implanted in 64 patients, and 11 received a HM II LVAD as a bridge to transplant. Ten HM I patients (16%) were sensitized before LVAD implantation (HM I-S), and 54 (84%) were not (HM I-Non-S). Nine HM I-S patients (90%) became highly sensitized (PRA > 90%) compared with 9 HM I-Non-S patients (16.7%; p < 0.001). The PRA remained elevated (> 90%) in 8 of the 9 (88.9%) highly sensitized HM I-S patients vs 5 of the 9 (55.6%) HM I-Non-S highly sensitized patients. The PRA levels in the rest of the HM I-S highly sensitized patients declined from 93% ± 4% to 55% ± 15% (p = 0.01). Among the 11 HM II patients, 1 (9%) was sensitized before LVAD implantation (PRA, 40%) and the PRA moderately increased to 80%. No other HM II patient became sensitized after implantation. Thus, 1 of 11 (9%) HM II patients became sensitized compared with 29 of 64 (45%) HM I patients (p = 0.04). Pre-sensitized patients are at higher risk for becoming and remaining highly HLA-allosensitized after LVAD implantation. The HeartMate II LVAD appears to cause less sensitization than HeartMate I. [Copyright &y& Elsevier]

A number of nuclear complexes modify chromatin structure and operate as functional units. However, the in vivo role of each component within the complexes is not known. ATP-dependent chromatin remodeling complexes form several types of protein complexes, which reorganize chromatin structure cooperatively with histone modifiers. Williams syndrome transcription factor (WSTF) was biochemically identified as a major subunit, along with 2 distinct complexes: WINAC, a SWI/SNF-type complex, and WICH, an ISWI-type complex. Here, WSTF-/- mice were generated to investigate its function in chromatin remodeling in vivo. Loss of WSTF expression resulted in neonatal lethality, and all WSTF-/- neonates and ≈10% of WSTF+/- neonates suffered cardiovascular abnormalities resembling those found in autosomal-dominant Williams syndrome patients. Developmental analysis of WSTF-/- embryos revealed that Gja5 gene regulation is aberrant from E9.5, conceivably because of inappropriate chromatin reorganization around the promoter regions where essential cardiac transcription factors are recruited. In vitro analysis in WSTF-/- mouse embryonic fibroblast (MEF) cells also showed impaired transactivation functions of cardiac transcription activators on the Gja5 promoter, but the effects were reversed by overexpression of WINAC components. Likewise in WSTF-/- MEF cells, recruitment of Snf2h, an ISWI ATPase, to PCNA and cell survival after DNA damage were both defective, but were ameliorated by overexpression of WICH components. Thus, the present study provides evidence that WSTF is shared and is a functionally indispensable subunit of the WICH complex for DNA repair and the WINAC complex for transcriptional control. [ABSTRACT FROM AUTHOR]

Cerebral cavernous malformation (CCM) is a common vascular dysplasia that affects both systemic and central nervous system blood vessels. Loss of function mutations in the CCM2 gene cause CCM. Here we show that targeted disruption of Ccm2 in mice results in failed lumen formation and early embryonic death through an endothelial cell autonomous mechanism. We show that CCM2 regulates endothelial cytoskeletal architecture, cell-to-cell interactions and lumen formation. Heterozygosity at Ccm2, a genotype equivalent to that in human CCM, results in impaired endothelial barrier function. On the basis of our biochemical studies indicating that loss of CCM2 results in activation of RHOA GTPase, we rescued the cellular phenotype and barrier function in heterozygous mice with simvastatin, a drug known to inhibit Rho GTPases. These data offer the prospect for pharmacological treatment of a human vascular dysplasia with a widely available and safe drug. [ABSTRACT FROM AUTHOR]