Aberrant Angiogenesis Research Articles

Neonatal Platelet Transfusions and Future Areas of Research.

Thrombocytopenia affects approximately one fourth of neonates admitted to neonatal intensive care units, and prophylactic platelet transfusions are commonly administered to reduce bleeding risk. However, there are few evidence-based guidelines to inform clinicians' decision-making process. Developmental differences in hemostasis and differences in underlying disease processes make it difficult to apply platelet transfusion practices from other patient populations to neonates. Thrombocytopenia is a risk factor for common preterm complications such as intraventricular hemorrhage; however, a causal link has not been established, and platelet transfusions have not been shown to reduce risk of developing intraventricular hemorrhage. Platelet count frequently drives the decision of whether to transfuse platelets, although there is little evidence to demonstrate what a safe platelet nadir is in preterm neonates. Current clinical assays of platelet function often require large sample volumes and are not valid in the setting of thrombocytopenia; however, evaluation of platelet function and/or global hemostasis may aid in the identification of neonates who are at the highest risk of bleeding. Although platelets' primary role is in establishing hemostasis, platelets also carry pro- and antiangiogenic factors in their granules. Aberrant angiogenesis underpins common complications of prematurity including intraventricular hemorrhage and retinopathy of prematurity. In addition, platelets play an important role in host immune defenses. Infectious and inflammatory conditions such as sepsis and necrotizing enterocolitis are commonly associated with late-onset thrombocytopenia in neonates. Severity of thrombocytopenia is correlated with mortality risk. The nature of this association is unclear, but preclinical data suggest that thrombocytopenia contributes to mortality rather than simply being a proxy for disease severity. Neonates are a distinct patient population in whom thrombocytopenia is common. Their unique physiology and associated complications make the risks and benefits of platelet transfusions difficult to understand. The goal of this review was to highlight research areas that need to be addressed to better understand the risks and benefits of platelet transfusions in neonates. Specifically, it will be important to identify neonates at risk of bleeding who would benefit from a platelet transfusion and to determine whether platelet transfusions either abrogate or exacerbate common neonatal complications such as sepsis, chronic lung disease, necrotizing enterocolitis, and retinopathy of prematurity.

Increased tumor response to neoadjuvant therapy among rectal cancer patients taking angiotensin-converting enzyme inhibitors or angiotensin receptor blockers.

Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) are commonly used antihypertensive medications that have been reported to affect aberrant angiogenesis and the dysregulated inflammatory response. Because of such mechanisms, it was hypothesized that these medications might affect the tumor response to neoadjuvant radiation in patients with rectal cancer. One hundred fifteen patients who were treated with neoadjuvant radiation at the University of Wisconsin (UW) between 1999 and 2012 were identified. Univariate analyses were performed with anonymized patient data. In a second independent data set, 186 patients with rectal cancer who were treated with neoadjuvant radiation at the Queen's Medical Center of the University of Hawaii (UH) between 1995 and 2010 were identified. These data were independently analyzed as before. Multivariate analyses were performed with aggregate data. Among patients taking ACEIs/ARBs in the UW data set, a significant 3-fold increase in the rate of pathologic complete response (pCR) to neoadjuvant therapy (52% vs 17%, P = .001) was observed. This finding was confirmed in the UH data set, in which a significant 2-fold-increased pCR rate (24% vs 12%, P = .03) was observed. Identified patient and treatment characteristics were otherwise balanced between patients taking and not taking ACEIs/ARBs. No significant effect was observed on pCR rates with other medications, including statins, metformin, and aspirin. Multivariate analyses of aggregate data identified ACEI/ARB use as a strong predictor of pCR (odds ratio, 4.02; 95% confidence interval, 2.06-7.82; P < .001). The incidental use of ACEIs/ARBs among patients with rectal cancer is associated with a significantly increased rate of pCR after neoadjuvant treatment. Cancer 2016;122:2487-95. © 2016 American Cancer Society.

Dimethylarginine dimethylaminohydrolase-2 deficiency promotes vascular regeneration and attenuates pathological angiogenesis

Ischemia-induced angiogenesis is critical for tissue repair, but aberrant neovascularization in the retina causes severe sight impairment. Nitric oxide (NO) has been implicated in neovascular eye disease because of its pro-angiogenic properties in the retina. Nitric oxide production is inhibited endogenously by asymmetric dimethylarginines (ADMA and L-NMMA) which are metabolized by dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2. The aim of this study was to determine the roles of DDAH1, DDAH2, ADMA and L-NMMA in retinal ischemia-induced angiogenesis. First, DDAH1, DDAH2, ADMA and L-NMMA levels were determined in adult C57BL/6J mice. The results obtained revealed that DDAH1 was twofold increased in the retina compared to the brain and the choroid. DDAH2 expression was approximately 150 fold greater in retinal and 70 fold greater in choroidal tissue compared to brain tissue suggesting an important tissue-specific role for DDAH2 in the retina and choroid. ADMA and L-NMMA levels were similar in the retina and choroid under physiological conditions. Next, characterization of DDAH1+/− and DDAH2−/− deficient mice by in vivo fluorescein angiography, immunohistochemistry and electroretinography revealed normal neurovascular function compared with wildtype control mice. Finally, DDAH1+/− and DDAH2−/− deficient mice were studied in the oxygen-induced retinopathy (OIR) model, a model used to emulate retinal ischemia and neovascularization, and VEGF and ADMA levels were quantified by ELISA and liquid chromatography tandem mass spectrometry. In the OIR model, DDAH1+/− exhibited a similar phenotype compared to wildtype controls. DDAH2 deficiency, in contrast, resulted in elevated retinal ADMA which was associated with attenuated aberrant angiogenesis and improved vascular regeneration in a VEGF independent manner. Taken together this study suggests, that in retinal ischemia, DDAH2 deficiency elevates ADMA, promotes vascular regeneration and protects against aberrant angiogenesis. Therapeutic inhibition of DDAH2 may therefore offer a potential therapeutic strategy to protect sight by promoting retinal vascular regeneration and preventing pathological angiogenesis.

Current screening and treatments in retinopathy of prematurity in the US.

Retinopathy of prematurity (ROP) is a complex disease characterized by an aberrant developmental retinal angiogenesis in preterm infants and can carry significant visual morbidity, including retinal detachment and blindness. Though large scale, randomized clinical trials have improved our understanding of the pathophysiology and progression of the disease, the management of ROP remains a challenge for ophthalmologists. This review addresses the up-to-date screening approach, diagnosis, and treatment guidelines for ROP in the US.

Anti-Angiogenic Therapy: Strategies to Develop Potent VEGFR-2 Tyrosine Kinase Inhibitors and Future Prospect.

Tumor angiogenesis has always been a major gap for effective cancer therapy. Interruption of aberrant angiogenesis by specific inhibitors targeting receptor tyrosine kinases (RTKs) has been of great interests to medicinal chemists. Among the factors that are involved in tumor angiogenesis, vascular endothelial growth factor receptor-2 (VEGFR-2) is validated as the most closely related factor which can drive angiogenesis through binding with its natural ligand VEGF. The well-validated VEGF-driven VEGFR-2 signaling pathway can stimulate many endothelial responses, including increasing vessel permeability and enhancing endothelial cell proliferation, migration and differentiation. Consequently, circumventing angiogenesis by VEGFR-2 inhibitors represents a promising strategy for counteracting various VEGFR-2-mediated disorders as well as drug resistance. Over the past decades, a considerable number of novel small molecular VEGFR-2 inhibitors have been exploited with diverse chemical scaffolds. Especially, recent frequently launched inhibitors have declared their research values and therapeutic potentials in oncology. Still, the antiangiogenesis based treatment remains an ongoing challenge. In this review, a comprehensive retrospective of newly emerged VEGFR-2 inhibitors have been summarized, with the emphasis on the structure-activity relationship (SAR) investigation, and also binding patterns of representative inhibitors with biotargets. On the basis of all of this information, varied strategies for developing potent VEGFR-2 inhibitors and the future prospect of the clinical application of antiangiogenic inhibitors are discussed hereby.

0297 : Targeting frizzled7 in endothelial cells: a novel approach to treat aberrant angiogenesis in proliferative retinopathy

Blinding diseases including retinopathy of prematurity and proliferative diabetic retinopathy are the consequence of pathological angiogenesis in the retina. A better understanding of the mechanisms involved in this abnormal proliferation of neovessels appears to be essential to develop drugs against new molecular targets. Recently, we have evidenced that Fzd7 receptor, a member of the Wnt/Fzd family, is required for retinal postnatal angiogenesis. This study aimed to investigate the role of Fzd7 during aberrant angiogenesis in a model of ischemic retinopathy. We used mice model of OIR to explore the involvement of Fzd7 during initial vaso-obliteration (VO) and subsequent neovascularization (NV) phases. By transgenic approaches, we observed that deletion of fzd7 in the endothelium ( fzd7 ECKO ) resulted in increased retinal tissue sensitivity to hypoxia during the VO phase of OIR. Moreover, fzd7 deletion in EC reduced the ectopic growth of pathologic neovessels into the vitreous during the NV phase (P17) demonstrating that Fzd7 was involved in pathological NV formation in mice retina during OIR. To determine how Fzd7 may regulate ischemic retinopathy, we explored canonical β-catenin and Notch signaling during OIR and showed that transcript expressions of lef1, axin2 and partners of Notch signaling were strongly decreased in fzd7 ECKO retina mice as compared to control. To evaluate therapeutic potential of Fzd7 blocking, we experimented 2 strategies: a monoclonal antibody (MabFzd7) and a soluble Fzd7 receptor (CRD domain). In vitro both MabFzd7 and Fzd7CRD decreased the activity of β-catenin reporter gene induced by Wnt3A in EC. In vivo intravitreal microinjection of MabFzd7 or intraperitoneal injection of Fzd7CRD in mice at P12 decreased neovessels in the treated eye compared to the control eye at P17. By controlling development of abnormal vascularization, Fzd7 could be a new target to develop promising anti-angiogenic drugs in the treatment of ischemic retinopathies. The author hereby declares no conflict of interest

Current management of glaucoma and vascular occlusive disease.

This article summarizes recent findings pertaining to advancements in the treatment of glaucomas secondary to vascular occlusive disease to maximize visual outcomes. Retinal ischemia leads to a local increase in transcription of proteins responsible for aberrant angiogenesis and subsequent neovascular glaucoma. Antivascular endothelial growth factor (VEGF) therapy is helpful in the management of this condition. Although bevacizumab and ranibizumab offer relatively short-term effects, preliminary studies suggest that aflibercept may allow for longer term treatment. Preoperative anti-VEGF injection therapy improves outcomes after trabeculectomy and glaucoma drainage implant surgeries. The treatments for vascular occlusive disease may lead to intraocular pressure elevation and subsequent glaucoma. Aflibercept appears to be a safer agent in this regard. Prompt diagnosis and management of glaucomas associated with vascular occlusive disease are required to allow for the best possible outcome. Novel anti-VEGF agents, particularly aflibercept, should be strongly considered in the management of these conditions.

Long-term safety and stability of angiogenesis induced by balanced single-vector co-expression of PDGF-BB and VEGF164 in skeletal muscle.

Therapeutic angiogenesis by growth factor delivery is an attractive treatment strategy for ischemic diseases, yet clinical efficacy has been elusive. The angiogenic master regulator VEGF-A can induce aberrant angiogenesis if expressed above a threshold level. Since VEGF remains localized in the matrix around expressing cells, homogeneous dose distribution in target tissues is required, which is challenging. We found that co-expression of the pericyte-recruiting factor PDGF-BB at a fixed ratio with VEGF from a single bicistronic vector ensured normal angiogenesis despite heterogeneous high VEGF levels. Taking advantage of a highly controlled gene delivery platform, based on monoclonal populations of transduced myoblasts, in which every cell stably produces the same amount of each factor, here we rigorously investigated a) the dose-dependent effects, and b) the long-term safety and stability of VEGF and PDGF-BB co-expression in skeletal muscle. PDGF-BB co-expression did not affect the normal angiogenesis by low and medium VEGF doses, but specifically prevented vascular tumors by high VEGF, yielding instead normal and mature capillary networks, accompanied by robust arteriole formation. Induced angiogenesis persisted unchanged up to 4 months, while no tumors appeared. Therefore, PDGF-BB co-expression is an attractive strategy to improve safety and efficacy of therapeutic angiogenesis by VEGF gene delivery.

The Pathophysiology of Moyamoya Disease: An Update.

Moyamoya disease (MMD) is a unique cerebrovascular disease characterized by the progressive stenosis of large intracranial arteries and a hazy network of basal collaterals called moyamoya vessels. Because the etiology of MMD is unknown, its diagnosis is based on characteristic angiographic findings. Re-vascularization techniques (e.g., bypass surgery) are used to restore perfusion, and are the primary treatment for MMD. There is no specific treatment to prevent MMD progression. This review summarizes the recent advances in MMD pathophysiology, including the genetic and circulating factors related to disease development. Genetic and environmental factors may play important roles in the development of the vascular stenosis and aberrant angiogenesis in complex ways. These factors include the related changes in circulating endothelial/smooth muscle progenitor cells, cytokines related to vascular remodeling and angiogenesis, and endothelium, such as caveolin which is a plasma membrane protein. With a better understanding of MMD pathophysiology, nonsurgical approaches targeting MMD pathogenesis may be available to stop or slow the progression of this disease. The possible strategies include targeting growth factors, retinoic acid, caveolin-1, and stem cells.

MicroRNAs modulating angiogenesis: miR-129-1 and miR-133 act as angio-miR in HUVECs.

The sprouting of new blood vessels by angiogenesis is critical in vascular development and homeostasis. Aberrant angiogenesis leads to enormous pathological conditions such as ischemia and cancer. MicroRNAs (also known as miRNAs or miRs) play key roles in regulation of a range of cellular processes by posttranscriptional suppression of their target genes. Recently, new studies have indicated that miRNAs are involved in certain angiogenic settings and signaling pathways use these non-coding RNAs to promote or suppress angiogenic processes. Herein, VEGFR2 and FGFR1 were identified as miR-129-1 and miR-133 targets using bioinformatic algorithms, respectively. Afterwards, using luciferase reporter assay and gene expression analysis at both mRNA and protein levels, VEGFR2 and FGFR1 were validated as miR-129-1 and miR-133 targets. In addition, we showed that miR-129-1 and miR-133 suppress angiogenesis properties such as proliferation rate, cell viability, and migration activity of human umbilical vein endothelial cells (HUVEC) in vitro. We conclude that these miRNAs can suppress key factors of angiogenesis by directly targeting them. These results have important therapeutic implications for a variety of diseases involving deregulation of angiogenesis, including cancer.

Down regulation of Jag-1 in VSMCs contributes to impaired angiogenesis under high glucose condition: Experimental study using aortic rings of rats.

The major cause of diabetes-related mortality is the complications involving aberrant angiogenesis. To understand the underlying mechanisms of such altered-angiogenesis in diabetes, examining the interaction between endothelial cells (ECs) and neighboring smooth muscle cells (VSMCs) rather than mainly focusing on EC might provide us useful information. Thus, in the present study, we examined the effect of high glucose on the expression of Jag1, one of the key trans-activating ligands of Notch receptors known to be involved in EC-SMC interaction, as well as angiogenic process, in vascular smooth muscle cells (VSMCs) to elucidate possible role of EC-VSMC interaction in diabetes-related angiopathy. Our data indicate that high glucose condition decreases the expression of Jag1 in VSMCs possibly by increasing Jag1-targeting micro RNAs (miRNAs) such as miR-21, and exogenous Jag1-simulating peptides increase proliferation and migration of ECs under high glucose condition in vitro. Ex vivo study using aortic rings from normal and streptozotocin (STZ)-treated diabetic mouse demonstrated that exogenous Jag1-simulating peptides increases EC sprouting of aortic rings from diabetic mouse under high glucose condition. Our data suggest that EC-VSMC interaction is altered under high glucose condition and restoring EC-VSMC interaction can be a feasible therapeutic target for treating diabetes-related angiopathy.

Culture dimensionality controls CD44 alternative splicing

Event Abstract Back to Event Culture dimensionality controls CD44 alternative splicing Cristiana Branco Da Cunha1, 2, 3, 4, Darinka D. Klumpers1, 2, 5, Sandeep T. Koshy1, 2, 6, James C. Weaver2, Ovijit Chaudhuri7, Raquel Seruca3, Fátima Carneiro3, 4, Pedro L. Granja3, 8, 9 and David J. Mooney1, 2 1 Harvard University, School of Engineering and Applied Sciences, United States 2 Harvard University, Wyss Institute for Biologically Inspired Engineering, United States 3 Universidade do Porto, Instituto de Investigação e Inovação em Saúde (i3S), Portugal 4 Universidade do Porto, Faculdade de Medicina/Hospital S. João, Portugal 5 VU University Medical Center, Department Orthopedic Surgery, Research Institute MOVE, Netherlands 6 Harvard University-MIT, Division of Health Sciences and Technology, United States 7 Stanford University, Department of Mechanical Engineering, United States 8 Faculdade de Engenharia da Universidade do Porto, Departamento de Engenharia Metalúrgica e Materiais, Portugal 9 Universidade do Porto, Instituto de Ciências Biomédicas Abel Salazar, Portugal CD44 and its alternative spliced variants have been broadly associated with cancer, from resistance to oxidative stress to metastatic behavior. In the context of gastric cancer (GC) we have previously shown that de novo expression of CD44 variant 6 (CD44v6) accompanies malignant progression. Two-dimensional (2D) cell culture is commonly used to study cancer in vitro, but fails to mimic key architectural and physical features of the tumor microenvironment. This study determined how more physiologically relevant three-dimensional (3D) culture impacts CD44 alternative splicing in epithelial GC cells. In 3D culture, GC cells gradually lost expression of the standard CD44 isoform (CD44s), while simultaneously gaining CD44v6 expression that was virtually absent in 2D monolayers. This switch in CD44 spliced isoforms was reversible, accelerated by nutrient shortage and delayed with lower initial cell densities, suggesting that it is a stress-induced response. Loss of CD44s in 3D culture was further shown to be dependent on the matrix mechanical properties and accompanied by the upregulation of key epithelial-mesenchymal transition (EMT) markers, such as SNAIL and ZEB1, along with several genes involved in aberrant metabolic pathways, cellular stress, angiogenesis and antisenescense cellular programs. Human gastric premalignant and malignant lesions revealed that 3D cultures, but not 2D monolayers, were able to capture aberrant CD44 splicing patterns observed in vivo. Altogether, these findings suggest that cells modulate CD44 expression in response to culture dimensionality and physical cues, and could translate into clinical strategies to locally modulate CD44 expression in GC, the 4th most common cancer in the world. Keywords: Extracellular Matrix, Clinical relevance, mechanical property, matrix-cell interaction Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials and cellular signaling Citation: Branco Da Cunha C, Klumpers DD, Koshy ST, Weaver JC, Chaudhuri O, Seruca R, Carneiro F, Granja PL and Mooney DJ (2016). Culture dimensionality controls CD44 alternative splicing. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00598 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Cristiana Branco Da Cunha Darinka D Klumpers Sandeep T Koshy James C Weaver Ovijit Chaudhuri Raquel Seruca Fátima Carneiro Pedro L Granja David J Mooney Google Cristiana Branco Da Cunha Darinka D Klumpers Sandeep T Koshy James C Weaver Ovijit Chaudhuri Raquel Seruca Fátima Carneiro Pedro L Granja David J Mooney Google Scholar Cristiana Branco Da Cunha Darinka D Klumpers Sandeep T Koshy James C Weaver Ovijit Chaudhuri Raquel Seruca Fátima Carneiro Pedro L Granja David J Mooney PubMed Cristiana Branco Da Cunha Darinka D Klumpers Sandeep T Koshy James C Weaver Ovijit Chaudhuri Raquel Seruca Fátima Carneiro Pedro L Granja David J Mooney Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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The Microenvironment of Lung Cancer and Therapeutic Implications.

The tumor microenvironment (TME) represents a milieu that enables tumor cells to acquire the hallmarks of cancer. The TME is heterogeneous in composition and consists of cellular components, growth factors, proteases, and extracellular matrix. Concerted interactions between genetically altered tumor cells and genetically stable intratumoral stromal cells result in an "activated/reprogramed" stroma that promotes carcinogenesis by contributing to inflammation, immune suppression, therapeutic resistance, and generating premetastatic niches that support the initiation and establishment of distant metastasis. The lungs present a unique milieu in which tumors progress in collusion with the TME, as evidenced by regions of aberrant angiogenesis, acidosis and hypoxia. Inflammation plays an important role in the pathogenesis of lung cancer, and pulmonary disorders in lung cancer patients such as chronic obstructive pulmonary disease (COPD) and emphysema, constitute comorbid conditions and are independent risk factors for lung cancer. The TME also contributes to immune suppression, induces epithelial-to-mesenchymal transition (EMT) and diminishes efficacy of chemotherapies. Thus, the TME has begun to emerge as the "Achilles heel" of the disease, and constitutes an attractive target for anti-cancer therapy. Drugs targeting the components of the TME are making their way into clinical trials. Here, we will focus on recent advances and emerging concepts regarding the intriguing role of the TME in lung cancer progression, and discuss future directions in the context of novel diagnostic and therapeutic opportunities.

The Propagation of Hemophilic Arthropathy - the Role of Abnormal Angiogenesis and Vascular Remodeling in Recurrent Joint Bleeding in Adults with Hemophilia

Neoangiogenesis and vascular remodeling in the hypertrophic synovium of arthropathic hemophilic joints may disturb vascular integrity and contribute to repeated bleeding (A von Drygalski, et al. Vascular Remodeling Underlies Re-Bleeding in Hemophilic Arthropathy; ASH 2015; Abstract ID 78677). However, it is difficult to distinguish the importance of the contribution of abnormal vascular structures to hemophilic joint bleeding from the contribution of low clotting factor levels. This is because most prophylactic clotting factor replacement strategies are designed to avoid trough levels below 1-5%, but not to fully correct the factor deficiency.This report reveals the importance of aberrant angiogenesis for joint bleeding in two hemophilia patients whose clotting factor activity levels were constantly normal or near normal. The first patient had severe Hemophilia A with normalized Factor (f)VIII activity levels after liver transplantation 6 years previously. Abnormal vascularization of elbows, knees and ankles (absent bleeding) was detected by musculoskeletal ultrasound and Power Doppler (PD). Three months after the baseline exam the patient experienced severe elbow bleeding associated with new vascular changes on PD. These changes were characterized by pronounced confluent, patchy vascular signals and numerous enlarged and thickened vessels by angiography consistent with acute and chronic vascular remodeling. Bleeding could not be controlled by increasing fVIII activity to supra-normal levels, and ultimately required synovial embolization. The second patient has severe Hemophilia B and presented shortly after left knee joint replacement with fluctuating knee bleeding for many months associated with pronounced dynamic vascular changes on PD. Neither bleeding nor vascular changes were prevented by daily fIX treatment which maintained near normal plasma fIX activity levels. Within one year, additional chronic bleeding with vascular changes developed in the left elbow and left ankle and remained unresponsive to intense factor replacement.These cases provide several insights. First, vascular changes in hemophilic arthropathy persist and evolve in dynamic fashion even in the presence of normal clotting factor activities. Second, vascular disturbance and leakiness contributed to bleeding independent of clotting factor activity levels as exemplified by the first patient and as suggested by the second patient. Third, the sequence of bleeding events in the second patient suggests that at least some of the observed vascular abnormalities are mediated systemically. These observations provide clinical evidence in support of the hypothesis that neoangiogenesis and vascular remodeling contribute to hemophilic joint bleeding and should lead to new studies of the pathophysiology of vascular remodeling in such joints. Targeting angiogenesis may emerge as a new treatment strategy to enhance the effectiveness of clotting factor replacement hemophilic arthropathy. DisclosuresChang:Biogen: Research Funding. Rose:Sirtex Medical: Equity Ownership, Honoraria; XLSciTech: Consultancy; Surefire Medical: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Embolx: Consultancy. von Drygalski:Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Research Funding; Hematherix Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

The Intracellular Loop 2 F328S Frizzled-4 Mutation Implicated in Familial Exudative Vitreoretinopathy Impairs Dishevelled Recruitment.

Familial exudative vitreoretinopathy (FEVR) is a disease state characterized by aberrant retinal angiogenesis. Norrin-induced activation of Frizzled-4 (Fz4) has a major role in regulating beta-catenin levels in the eye that, in turn, modulate the blood retina barrier (BRB). Here we gain insight on the basis of the pathology of a FEVR implicated F328S Fz4 mutant by study. The receptor exhibits a substantially reduced ability to activate Lef/Tcf-dependent transcription. This impaired activation correlates with a decreased ability to stabilize and recruit Dishevelled-2 (Dvl2) to the cell surface. Aromaticity at position 328 of the intracellular loop 2 (iloop2) is revealed similarly as a prerequisite for Dvl2 recruitment to the Fz4. This aromaticity at 328 enables normal Norrin-induced canonical activation. The corresponding position in iloop2 of other Frizzleds likely functions in Dvl recruitment.

CDKN2B Regulates TGF β Signaling and Smooth Muscle Cell Investment of Hypoxic Neovessels

Genetic variation at the chromosome 9p21 cardiovascular risk locus has been associated with peripheral artery disease, but its mechanism remains unknown. To determine whether this association is secondary to an increase in atherosclerosis, or it is the result of a separate angiogenesis-related mechanism. Quantitative evaluation of human vascular samples revealed that carriers of the 9p21 risk allele possess a significantly higher burden of immature intraplaque microvessels than carriers of the ancestral allele, irrespective of lesion size or patient comorbidity. To determine whether aberrant angiogenesis also occurs under nonatherosclerotic conditions, we performed femoral artery ligation surgery in mice lacking the 9p21 candidate gene, Cdkn2b. These animals developed advanced hindlimb ischemia and digital autoamputation, secondary to a defect in the capacity of the Cdkn2b-deficient smooth muscle cell to support the developing neovessel. Microarray studies identified impaired transforming growth factor β (TGFβ) signaling in cultured cyclin-dependent kinase inhibitor 2B (CDKN2B)-deficient cells, as well as TGFβ1 upregulation in the vasculature of 9p21 risk allele carriers. Molecular signaling studies indicated that loss of CDKN2B impairs the expression of the inhibitory factor, SMAD-7, which promotes downstream TGFβ activation. Ultimately, this manifests in the upregulation of a poorly studied effector molecule, TGFβ1-induced-1, which is a TGFβ-rheostat known to have antagonistic effects on the endothelial cell and smooth muscle cell. Dual knockdown studies confirmed the reversibility of the proposed mechanism, in vitro. These results suggest that loss of CDKN2B may not only promote cardiovascular disease through the development of atherosclerosis but may also impair TGFβ signaling and hypoxic neovessel maturation.

Downregulation of ERRα inhibits angiogenesis in human umbilical vein endothelial cells through regulating VEGF production and PI3K/Akt/STAT3 signaling pathway

The human estrogen related receptor α (ERRα) is a pivotal regulator involved in energy homeostasis and mitochondrial biogenesis. It has been demonstrated that activation of ERRα in various breast cancer cells results in a significant increase of vascular endothelial growth factor (VEGF) mRNA and protein secretion. However, little is known about the relationship between ERRα and angiogenesis. Thus, the present study is aimed to investigate the effects and mechanism of ERRα suppression on the angiogenesis in human umbilical vein endothelial cells (HUVECs). Here we show that ERRα suppression powerfully inhibits proliferation, migration and capillary-like structures formation of HUVECs. Importantly, we demonstrate that these inhibitory effects are associated with the significantly reduced expression and production of VEGF. Results from further experiments using western blot and luciferase reporter assay exhibit that ERRα suppression inhibits hypoxia-inducible factor 1α (HIF-1α) expression, and phosphorylation of protein kinase B (Akt) and signal transducer and activator of transcription (STAT3) which up-regulated VEGF expression. In summary, we show that ERRα suppression inhibits angiogenesis in HUVECs and deserves further studies for application of rationale therapeutic target for patient with diseases related with aberrant angiogenesis.

Abstract 12334: A Flavor of Metabolic Disease in Pulmonary Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a severe occlusive vascular disease of the lungs. One of the primary origin of PAH is pulmonary endothelial dysfunction driving vasoconstriction, aberrant angiogenesis and smooth muscle cell proliferation, endothelial-to-mesenchymal transition, thrombosis and inflammation. Interestingly, endothelial dysfunction is maintained in culture, out of fluid and hemodynamic stress, humoral/hormonal, and inflammatory environment. Hypothesis: This aberrant phenotype may be imprinted in pulmonary endothelial cells (PEC) DNA though a specific pattern of DNA methylations. Methods: Genomic DNA was extracted from cultured PEC (passage 3): idiopathic PAH (n=11), heritable PAH (BMPR2 mutation carriers, n=10), controls (n=18). DNA methylation was assessed at over 485 000 CpG sites using the Illumina Infinium HumanMethylation450 Bead Chip. We normalized all arrays against each other using functional normalization. Differentially methylated sites were clustered with Cluster3.0 and heatmap were obtained with Treeview. Results: We discriminated controls vs PAH into 2 clusters of hypermethylated loci (119 probes= 31 promoters) and hypomethylated loci (331 probes= 116 promoters). Interestingly, 46 promoters/147 (clusters 1+2) (31%) were related to metabolic diseases (Ingenuity pathway analysis), and top molecules (fold changes up- and down regulated) includes molecules highly involved in cellular lipid metabolic process (ABCA1, Q=0.002 and ABCB4, Q=0.003), regulation of glucogenesis (ACN9, Q=2.78.10-5), lipid and glucose metabolism (ADIPOQ, Q=10-4), and insulin sensitivity and metabolism of glucose and lipids (miR-26a, Q=0.005), among others. Conclusions: the methylation fingerprint of PAH highlighted a set of molecules involved in metabolic disease and metabolism regulation. This may have fundamental and clinical implications in PAH.

Astrocytes Regulate Angiogenesis Through the Jagged1-Mediated Notch1 Pathway After Status Epilepticus.

Vascular disruptions including blood-brain barrier breakdown and pathologic angiogenesis contribute to the development of epilepsy in normal brains. The Notch signaling pathway is activated in response to seizure activity, and its activation promotes seizures, although its exact role in angiogenesis is poorly understood. Here, we have examined the role of Notch signaling in angiogenesis in a kainic acid-induced mouse model of epilepsy. We show that following seizures, expression of the Notch ligand Jagged1 in the hippocampus is upregulated in astrocytes and levels of activated Notch1 are increased in endothelial cells. Using an in vitro model of angiogenesis, we provide evidence that brain endothelial tube formation is promoted in the presence of astrocytes. Isolated primary brain endothelial cells develop significantly longer vascular sprouts when cultured in the presence of astrocytes. Notch1 signaling is activated in brain endothelial cells cocultured with astrocytes, and astrocytic Jagged1 expression is required for angiogenic enhancement, as shown by the inhibitory effect of Jagged1 small interfering RNA (siRNA) expression in astrocytes on endothelial cell vascular sprouting in vitro. Therapies targeting the Jagged1/Notch1 signaling pathway may therefore be effective in limiting aberrant angiogenesis following status epilepticus.

Halofuginone attenuates osteoarthritis by inhibition of TGF-β activity and H-type vessel formation in subchondral bone

ObjectivesExamine whether osteoarthritis (OA) progression can be delayed by halofuginone in anterior cruciate ligament transection (ACLT) rodent models.Methods3-month-old male C57BL/6J (wild type; WT) mice and Lewis rats were randomised to...