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Background Individuals infected with HIV have an increased risk of developing cardiovascular disease; yet, the underlying mechanisms remain unknown. Recent evidence has implicated the Tie‐2 tyrosine kinase receptor system and its associated ligands ANG1 (angiopoietin 1) and ANG2 (angiopoietin 2) in maintaining vascular homeostasis. In the general population, lower ANG1 levels and higher ANG2 levels are strongly correlated with the development of cardiovascular disease. In this study, we aim to investigate the associations of HIV infection with angiopoietin levels and endothelial dysfunction. Methods and Results In this cross‐sectional study, we compared measures of ANG1, ANG2, and endothelial dysfunction using flow‐mediated vasodilation of the brachial artery in 39 untreated subjects infected with HIV, 47 treated subjects infected with HIV, and 46 uninfected subjects from the SCOPE (Observational Study of the Consequences of the Protease Inhibitor Era) cohort. Compared with uninfected controls, treated individuals infected with HIV had 53.1% lower mean ANG1 levels ( P P P P P P =0.05). In contrast, HIV status, ANG1 levels, and ANG2 levels were not associated with macrovascular dysfunction as measured by flow‐mediated dilatation and brachial artery diameter, 2 other measures of vascular homeostasis. Conclusions HIV infection affects the balance between levels of ANG1 and ANG2 and may disturb endothelial homeostasis through disruption of vascular homeostasis. Individuals with treated HIV had decreased ANG1 levels and similar ANG2 levels, whereas individuals with untreated HIV had similar ANG1 levels and increased ANG2 levels, suggesting that treatment status may alter the balance between ANG1 and ANG2. HIV also promotes endothelial dysfunction via impairment of microvascular dysfunction, independent of the Tie‐2 receptor system; the finding of worse microvascular dysfunction in the setting of treated HIV infection may reflect the impact of viral persistence on the microvasculature or toxicities of specific antiretroviral regimens. Further research to clarify the mechanism of HIV‐mediated endothelial dysfunction is necessary to advance treatment of cardiovascular complications of HIV infection.

The impairment of the angiopoietin-1 (Ang-1)/Tie-2 signaling pathway has been thought to play a critical role in diabetic complications. However, the underlying mechanisms remain unclear. The present study aims to investigate the effects of Tie-2 glycation on Ang-1 signaling activation and Ang-1-induced angiogenesis. We identified that Tie-2 was modified by advanced glycation end products (AGEs) in aortae derived from high fat diet (HFD)-fed mice and in methylglyoxal (MGO)-treated human umbilical vein endothelial cells (HUVECs). MGO-induced Tie-2 glycation significantly inhibited Ang-1-evoked Tie-2 and Akt phosphorylation and Ang-1-regulated endothelial cell migration and tube formation, whereas the blockade of AGE formation by aminoguanidine remarkably rescued Ang-1 signaling activation and Ang-1-induced angiogenesis in vitro. Furthermore, MGO treatment markedly increased AGE cross-linking of Tie-2 in cultured aortae ex vivo and MGO-induced Tie-2 glycation also significantly decreased Ang-1-induced vessel outgrow from aortic rings. Collectively, these data suggest that Tie-2 may be modified by AGEs in diabetes mellitus and that Tie-2 glycation inhibits Ang-1 signaling activation and Ang-1-induced angiogenesis. This may provide a novel mechanism for Ang-1/Tie-2 signal dysfunction and angiogenesis failure in diabetic ischaemic diseases.

Intravitreal antivascular endothelial growth factor (VEGF) drugs represent the first-line treatment option for wet age-related macular degeneration (w-AMD) and diabetic macular edema (DME); however, the frequent injection intervals have illuminated to the necessity for new molecules allowing a more prolonged treatment regimen. Faricimab is a promising bispecific drug targeting VEGF-A and the Ang-Tie/pathway. Phase II STAIRWAY and AVENUE Trials showed its clinical efficacy for the treatment of w-AMD, while the phase II BOULEVARD Trial revealed its superiority to monthly ranibizumab in the management of DME with a monthly treatment regimen. The agents are awaiting approval for the treatment of w-AMD and DME.This article presents an overview of w-AMD and diabetic retinopathy and examines the progress of Faricimab through clinical trials. It offers insights on where Faricimab may be placed in the future market of anti-VEGF treatments and discusses the role of Ang/Tie pathway as a potential additive weapon for the treatment of w-AMD, DME, and retinal vein occlusion (RVO).The possibility of administering faricimab with more prolonged treatment intervals represents an important advantage to decrease the treatment burden and improve patient compliance. Further phase III trials should provide more evidence on clinical efficacy.

Purpose Tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Tie2) activation in Schlemm's canal (SC) endothelium is required for the maintenance of IOP, making the angiopoietin/Tie2 pathway a target for new and potentially disease modifying glaucoma therapies. The goal of the present study was to examine the effects of a Tie2 activator, AKB-9778, on IOP and outflow function. Methods AKB-9778 effects on IOP was evaluated in humans, rabbits, and mice. Localization studies of vascular endothelial protein tyrosine phosphatase (VE-PTP), the target of AKB-9778 and a negative regulator of Tie2, were performed in human and mouse eyes. Mechanistic studies were carried out in mice, monitoring AKB-9778 effects on outflow facility, Tie2 phosphorylation, and filtration area of SC. Results AKB-9778 lowered IOP in patients treated subcutaneously for diabetic eye disease. In addition to efficacious, dose-dependent IOP lowering in rabbit eyes, topical ocular AKB-9778 increased Tie2 activation in SC endothelium, reduced IOP, and increased outflow facility in mouse eyes. VE-PTP was localized to SC endothelial cells in human and mouse eyes. Mechanistically, AKB-9778 increased the filtration area of SC for aqueous humor efflux in both wild type and in Tie2+/- mice. Conclusions This is the first report of IOP lowering in humans with a Tie2 activator and functional demonstration of its action in remodeling SC to increase outflow facility and lower IOP in fully developed mice. Based on these studies, a phase II clinical trial is in progress to advance topical ocular AKB-9778 as a first in class, Tie2 activator for treatment for ocular hypertension and glaucoma.

Emerging evidence suggests that gamma-tocotrienol (&gamma, T3), a vitamin E isomer, has potent anti-cancer properties against a wide-range of cancers. &gamma, T3 not only inhibited the growth and survival of cancer cells in vitro, but also suppressed angiogenesis and tumour metastasis under in vivo conditions. Recently, &gamma, T3 was found to target cancer stem cells (CSCs), leading to suppression of tumour formation and chemosensitisation. Despite its promising anti-cancer potential, the exact mechanisms responsible for the effects of &gamma, T3 are still largely unknown. Here, we report the identification of Ang-1 (Angiopoietin-1)/Tie-2 as a novel &gamma, T3 downstream target. In prostate cancer cells, &gamma, T3 treatment leads to the suppression of Ang-1 at both the mRNA transcript and protein levels. Supplementing the cells with Ang-1 was found to protect them against the anti-CSC effect of &gamma, T3. Intriguingly, inactivation of Tie-2, a member receptor that mediates the effect of Ang-1, was found to significantly enhance the cytotoxic effect of &gamma, T3 through activation of AMP-activated protein kinase (AMPK) and subsequent interruption of autophagy. Our results highlighted the therapeutic potential of using &gamma, T3 in combination with a Tie-2 inhibitor to treat advanced prostate cancer.

Background: This study aimed to determine the effects of total saponins from Rhizoma Dioscoreae Nipponicae (TS-RDN) on the expression of vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-2 and Tie-2 (endothelial tyrosine kinase receptor) receptors in the synovium of rats with rheumatoid arthritis (RA) (collagen-induced arthritis; CIA), and to examine the mechanisms of TS-RDN in alleviating RA. Methods: The CIA rat model was established and the animals were randomly divided into control, CIA model, TS-RDN, diosgenin, and tripterygium groups. Fluorescent polymerase chain reaction was performed to detect VEGF expression in the rat knee joint synovium. Additionally, immunohistochemical assay was used to detect protein expression of Ang-2 and Tie-2 in the rat knee joint synovium. Results: Expression of VEGF, Ang-2, and Tie-2 in the model group was significantly higher than in the control group (p

Ample evidence supports that prostate tumor metastasis originates from a rare population of cancer cells, known as cancer stem cells (CSCs). Unfortunately, little is known about the identity of these cells, making it difficult to target the metastatic prostate tumor. Here, for the first time, we report the identification of a rare population of prostate cancer cells that express the Tie-2 protein. We found that this Tie-2High population exists mainly in prostate cancer cell lines that are capable of metastasizing to the bone. These cells not only express a higher level of CSC markers but also demonstrate enhanced resistance to the chemotherapeutic drug Cabazitaxel. In addition, knockdown of the expression of the Tie-2 ligand angiopoietin (Ang-1) led to suppression of CSC markers, suggesting that the Ang-1/Tie-2 signaling pathway functions as an autocrine loop for the maintenance of prostate CSCs. More importantly, we found that Tie-2High prostate cancer cells are more adhesive than the Tie-2Low population to both osteoblasts and endothelial cells. Moreover, only the Tie-2High, but not the Tie-2Low cells developed tumor metastasis in vivo when injected at a low number. Taken together, our data suggest that Tie-2 may play an important role during the development of prostate tumor metastasis.

Foretinib, a multiple kinase inhibitor undergoing clinical trials, could suppress the activity of hepatocyte growth factor (HGF) receptor c-MET and vascular endothelial growth factor receptor-2 (VEGFR-2). In addition, Foretinib may inhibit two critical lymphangiogenic signaling receptors VEGFR-3 and TIE-2. However, the effect of Foretinib on lymphatic endothelial cells (LECs) in vitro and lymphangiogenesis in vivo is still unknown. We found Foretinib decreased basal- and HGF-induced c-MET activity at low concentrations. However, Foretinib only reduced the proliferation of pancreatic cancer cells at high concentration reflecting the intrinsic chemoresistance of pancreatic cancer cells. Foretinib inhibited VEGF-A, VEGF-C and Angiopoetin-2 (ANG-2)-stimulated tube formation and sprouting of LECs by reducing VEGFR-2, VEGFR-3 and TIE-2 activation and increased apoptosis of LECs. In xenograft animal study, Foretinib suppressed tumor growth by inhibiting proliferation, angiogenesis and lymphangiogenesis. Additionally, Foretinib inhibited angiogenesis and lymphangiogenesis more significantly and exhibited low detrimental effect in orthotopic animal study. Collectively, we suggested that Foretinib simultaneously inhibits cancer cells and LECs to reduce pancreatic tumor growth in vivo and demonstrated for the first time that Foretinib suppresses angiogenesis and lymphangiogenesis by blocking VEGFR-2/3 and TIE-2 signaling.

The lymphatic vasculature is a blind-ended network crucial for tissue-fluid homeostasis, immune surveillance and lipid absorption from the gut. Recent evidence has proposed an entirely venous-derived mammalian lymphatic system. By contrast, here we show that cardiac lymphatic vessels in mice have a heterogeneous cellular origin, whereby formation of at least part of the cardiac lymphatic network is independent of sprouting from veins. Multiple Cre–lox-based lineage tracing revealed a potential contribution from the putative haemogenic endothelium during development, and discrete lymphatic endothelial progenitor populations were confirmed by conditional knockout of Prox1 in Tie2+ and Vav1+ compartments. In the adult heart, myocardial infarction promoted a significant lymphangiogenic response, which was augmented by treatment with VEGF-C, resulting in improved cardiac function. These data prompt the re-evaluation of a century-long debate on the origin of lymphatic vessels and suggest that lymphangiogenesis may represent a therapeutic target to promote cardiac repair following injury.

While tumor blood vessels share many characteristics with normal vasculature, they also exhibit morphological and functional aberrancies. For example, the neural adhesion molecule L1, which mediates neurite outgrowth, fasciculation, and pathfinding, is expressed on tumor vasculature. Here, using an orthotopic mouse model of pancreatic carcinoma, we evaluated L1 functionality in cancer vessels. Tumor-bearing mice specifically lacking L1 in endothelial cells or treated with anti-L1 antibodies exhibited decreased angiogenesis and improved vascular stabilization, leading to reduced tumor growth and metastasis. In line with these dramatic effects of L1 on tumor vasculature, the ectopic expression of L1 in cultured endothelial cells (ECs) promoted phenotypical and functional alterations, including proliferation, migration, tubulogenesis, enhanced vascular permeability, and endothelial-to-mesenchymal transition. L1 induced global changes in the EC transcriptome, altering several regulatory networks that underlie endothelial pathophysiology, including JAK/STAT-mediated pathways. In particular, L1 induced IL-6–mediated STAT3 phosphorylation, and inhibition of the IL-6/JAK/STAT signaling axis prevented L1-induced EC proliferation and migration. Evaluation of patient samples revealed that, compared with that in noncancerous tissue, L1 expression is specifically enhanced in blood vessels of human pancreatic carcinomas and in vessels of other tumor types. Together, these data indicate that endothelial L1 orchestrates multiple cancer vessel functions and represents a potential target for tumor vascular-specific therapies.

Background and objectives: Ischemic and idiopathic heart failure are characterized by reactive cardiac fibrosis and impaired vasculogenesis involving pro-angiogenic factors such as angiogenin, angiopoietin-1 (Ang-1), and angiopoietin-2 (Ang-2), as demonstrated in experimental models of heart failure. However, differences in the molecular pathways between these cardiomyopathies are still unclear. In this short communication, we evaluate and compare the expression of pro-angiogenic molecules in the heart tissue of patients with advanced chronic heart failure (CHF) of ischemic vs. nonischemic etiology. Materials and Methods: We obtained heart tissue at transplantation from left ventricular walls of 16 explanted native hearts affected by either ischemic (ICM) or nonischemic dilated cardiomyopathy (NIDCM). Tissue samples were examined using immunohistochemistry for angiogenic molecules. Results: We found immunopositivity (I-pos) for angiopoietin-1 mainly in the cardiomyocytes, while we observed I-pos for Ang-2 and Tie-2 receptor mainly in endothelial cells. Expression of Procollagen-I (PICP), angiogenin, Ang-1, and Tie-2 receptor was similar in ICM and NIDCM. In contrast, endothelial immunopositivity for Ang-2 was higher in ICM samples than NIDCM (p = 0.03). Conclusions: In our series of CHF heart samples, distribution of Ang-1 and angiogenin was higher in cardiomyocytes while that of Ang-2 was higher in endothelial cells; moreover, Ang-2 expression was higher in ICS than NIDCM. Despite the small series examined, these findings suggest different patterns of angiogenic stimulation in ICM and NIDCM, or at least a more altered endothelial integrity in ICD. Our data may contribute to a better understanding of the angiogenesis signaling pathways in CHF. Further studies should investigate differences in the biochemical processes leading to heart failure.

Dynamic changes in microvascular endothelial structure and function are pivotal in the acute inflammatory response, the body's rapid, coordinated effort to localize, sequester, and eliminate microbial invaders at their portal of entry. To achieve this, the endothelium becomes leaky and inflamed, providing innate immune cells and humoral effector molecules access to the site of infection. During sepsis this locally adaptive response becomes manifest throughout the body, leading to dangerous host consequences. Increased leakiness in the pulmonary circulation contributes to acute respiratory distress syndrome (ARDS), a complication of sepsis associated with 40% mortality. Understanding the molecular governance of vascular leak and inflammation has major diagnostic, prognostic, and potentially therapeutic implications for this common and pernicious disease. This review summarizes results from cell-based experiments, animal models, and observational human studies; together, these studies suggest that an endothelial receptor called Tie2 and its ligands, called angiopoietins, form a signaling axis key to the vascular dyshomeostasis that underlies sepsis.

Background: Gastro-entero-pancreatic/neuroendocrine (NET) tumors are highly vascularized neoplasms. However, our knowledge concerning circulating levels of the angiogenic factors in NET patients still remains insufficient. Methods: The aim of this study was to measure plasma concentrations of VEGF, angiopoietin 1 (Ang-1), angiopoietin 2 (Ang-2), soluble Tie-2, endostatin, osteopontin (OPN) and chromogranin A (CgA) in 36 NET patients and 16 controls. Results: Only the plasma concentrations of Tie-2 and CgA were higher in NET patients as compared to controls. These levels were within the reference range in controls; however one control demonstrated slightly elevated Tie-2 and 4 elevated CgA. Similarly, in the subgroup of patients with carcinoid syndrome, only Tie-2 and CgA concentrations were higher than those in patients with non-functioning NETs. In turn, in the subgroup of metastatic patients, only Ang-2 levels were higher than in those with localized disease. A positive correlation was found between Ang-2 and Tie-2 levels in metastatic patients and between Ang-1 and Tie-2 in localized NETs. Conclusions: The plasma concentration of Tie-2 is proposed as an additional marker for NET patients and seems to be similarly effective as the currently used CgA level. Moreover, higher plasma levels of Ang-2 together with the positive correlation between Ang-2 and Tie-2 levels in metastatic subjects, implies that cases with a Tie-2 level above the upper limits, together with higher level of Ang-2 seem to be highly predictive of metastases.

Introduction: Endothelial activation leading to vascular barrier breakdown plays an essential role in the pathophysiology of multiple-organ dysfunction syndrome (MODS) in sepsis. Increasing evidence suggests that the function of the vessel-protective factor Angiopoietin-1 (Ang-1), a ligand of the endothelial-specific Tie2 receptor, is inhibited by its antagonist Angiopoietin-2 (Ang-2) during sepsis. In order to reverse the effects of the sepsis-induced suppression of Ang-1 and elevation of Ang-2 we aimed to investigate whether an intravenous injection of recombinant human (rh) Ang-1 protects against MODS in murine sepsis.Methods: Polymicrobiological abdominal sepsis was induced by cecal ligation and puncture (CLP). Mice were treated with either 1 mu g of intravenous rhAng-1 or control buffer immediately after CLP induction and every 8 h thereafter. Sham-operated animals served as time-matched controls.Results: Compared to buffer-treated controls, rhAng-1 treated septic mice showed significant improvements in several hematologic and biochemical indicators of MODS. Moreover, rhAng-1 stabilized endothelial barrier function, as evidenced by inhibition of protein leakage from lung capillaries into the alveolar compartment. Histological analysis revealed that rhAng-1 treatment attenuated leukocyte infiltration in lungs and kidneys of septic mice, probably due to reduced endothelial adhesion molecule expression in rhAng-1 treated mice. Finally, the protective effects of rhAng-1 treatment were reflected by an improved survival time in a lethal CLP model.Conclusions: In a clinically relevant murine sepsis model, intravenous rhAng-1 treatment alone is sufficient to significantly improve a variety of sepsis-associated organ dysfunctions and survival time, most likely by preserving endothelial barrier function. Further studies are needed to pave the road for clinical application of this therapy concept. (C) 2011 Elsevier Ltd. All rights reserved.

Objective: To evaluate whether fibroblasts derived from periodontal ligament retain the ability to differentiate into putative vascular cells and construct vascular cell-specific marker-positive blood vessel structures. We also evaluated the morphological features of the structure and investigated the intracellular molecular mechanism underlying the angiogenic activity of these cells. Methods: Single cell-derived cultures (SCDCs) were established from primary rat ligament fibroblast cultures, and their expression of ligament cell-, mesenchymal stem cell- and vascular cell-specific markers was evaluated by RT-PCR and immunocytochemistry. The ability of the cells to construct a blood vessel structure was evaluated in a three-dimensional type I collagen scaffold. The morphological and immunohistological characteristics of the structure were then evaluated. Results: Each SCDC expressed endothelial cell (EC)-specific and smooth muscle cell-specific markers, in addition to mesenchymal stem cell- and ligament cell-specific markers. SCDC2 cells, which abundantly expressed the EC markers Flk-1 and Tie-2, vigorously constructed a blood vessel structure in a phosphoinositide 3-kinase activation-dependent manner. Conclusion: Periodontal ligament fibroblasts have the potential to construct an EC marker-positive blood vessel-like structure. Consequently, the fibroblastic lineage in ligament tissue could be a candidate precursor for construction of a vascular system around damaged ligament tissue to facilitate its regeneration.

WOS: 000375821000021, PubMed ID: 26851415, Study Objective: To evaluate serum values of cluster of differentiation 95 (CD95/FAS), hypoxia-inducible factor 1-alpha (HIF-1 alpha), and tyrosine kinase receptor 2 (Tie-2) as possible biomarkers of disease presence and severity in women with endometriosis, and to characterize the changes in these values in women with stage I/II and stage III/IV endometriosis. Design: Prospective study (Canadian Task Force classification I). Setting: University hospital. Patients: Thirty women with endometriosis and 30 healthy women without endometriosis. Intervention: For the diagnosis of endometriosis and prediction of its severity, we measured the serum levels of CD95/FAS, which assess apoptotic conditions, and of HIF-1 alpha and Tie-2, which assess angiogenesis. Endometriosis was diagnosed and staged through surgical laparoscopy and later confirmed histologically. During the surgery, the patients with endometriosis were divided into 2 groups based on disease stage. Eleven patients had stage I/II endometriosis, and 19 had stage III/IV endometriosis. Measurements and Main Results: Endometriosis was associated with increased serum CD95/FAS and HIF-1 alpha levels, but not Tie-2 levels. We also determined that stage III/IV endometriosis was associated with higher serum CD95/FAS and HIF-1 alpha levels, but not Tie-2 levels, compared with stage I/II endometriosis. Conclusion: Endometriosis, in accordance with its severity, increases serum CD95/FAS and HIF-1 alpha levels, but not Tie-2 levels. These biomarkers may be useful for reproductive surgeons to improve the quality of counseling women about the presence and severity of endometriosis. (C) 2016 AAGL. All rights reserved.

WOS: 000358411800003, PubMed ID: 25831422, Background: Crimean-Congo hemorrhagic fever (CCHF) causes endothelial activation and dysfunction by affecting the endothelium directly or indirectly. In maintaining the vascular integrity, vascular endothelial growth factor (VEGF-A) and its receptor (VEGFR1) and angiopoietin-2 (Ang-2) and its receptor (Tie-2) are very important mediators. For this reason, we aimed at studying the association of Ang-2 and VEGF and their receptors Tie-2 and VEGFR1 with CCHF infection. Methods: Thirty one CCHF patients and 31 healthy controls (HC) were included in the study. CCHF patients were classified into 2 groups in terms of disease severity (severe and nonsevere). VEGF-A, VEGFR1, Ang-2 and Tie-2 levels were measured in all groups. Result: Serum levels of Tie-2, Ang-2, VEGF-A and VEGFR1 were significantly increased in CCHF patients compared with the HC. Furthermore, serum Tie-2, Ang-2, VEGF and VEGFR1 levels were found to be significantly higher in the severe group than in the nonsevere and HC groups (P < 0.05 and P < 0.001, respectively). Also, Tie-2, Ang-2, VEGF-A and VEGFR1 levels were significantly higher in the nonsevere group than in the HC group (P < 0.05). Conclusion: Having statistically significant higher Ang-2, Tie-2, VEGF-A and VEGFR1 levels in the severe group when compared with the other groups suggested that VEGF-related Ang-2/Tie-2 system played a critical role in the pathogenesis of the disease, and these markers could be used as the severity criteria.

Fetal growth restriction (FGR) is associated with increased perinatal morbidity and mortality, and often results from functional placental insufficiency. Placentation requires extensive vasculogenesis and subsequent angiogenesis, in both maternal and fetal tissues. Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2) are angiogenic growth factors expressed in the placenta, and compete for binding to a common receptor, Tunica interna endothelial cell kinase-2 (Tie-2). Our objective was to examine Ang-1, Ang-2 and Tie-2 expression in ovine placental tissue obtained from normal and FGR pregnancies throughout gestation. Fetal cotyledon and maternal caruncle tissue concentrations of Ang-1, Ang-2 and Tie-2 mRNA were assessed by real-time reverse transcriptase-polymerase chain reaction and protein concentrations were assessed by Western immunoblot analysis, at 55, 90 and 135 d gestational age (dGA). Concentrations of Ang-1, Ang-2 and Tie-2 mRNA in FGR fetal cotyledons were increased at 55 dGA, and Tie-2 mRNA concentrations were decreased in FGR fetal cotyledons and maternal caruncles at 135 dGA. Immunoblot analysis demonstrated increased concentrations of Ang-2 in the fetal cotyledon at 55 dGA, and lower concentrations at 135 dGA. In contrast, concentrations of Tie-2 were increased at 90 dGA, but tended to decrease at 135 dGA in FGR maternal caruncles. The changes observed during early- to mid-gestation may result in increased branching angiogenesis, but may also set the stage for increased nonbranching angiogenesis during late gestation, altered placental architecture and placental insufficiency that result in FGR.

Diabetes mellitus is characterized by changes in endothelial cells that alter monocyte recruitment, increase classic (M1-type) tissue macrophage infiltration and lead to self-sustained inflammation. Our and other groups recently showed that chronic inhibition of phosphodiesterase-5 (PDE5i) affects circulating cytokine levels in patients with diabetes; whether PDE5i also affects circulating monocytes and tissue inflammatory cell infiltration remains to be established. Using murine streptozotocin (STZ)-induced diabetes and in human vitro cell-cell adhesion models we show that chronic hyperglycemia induces changes in myeloid and endothelial cells that alter monocyte recruitment and lead to self-sustained inflammation. Continuous PDE5i with sildenafil (SILD) expanded tissue anti-inflammatory TIE2-expressing monocytes (TEMs), which are known to limit inflammation and promote tissue repair. Specifically, SILD: 1) normalizes the frequency of circulating pro-inflammatory monocytes triggered by hyperglycemia (53.7 ± 7.9% of CD11b+Gr-1+ cells in STZ vs. 30.4 ± 8.3% in STZ+SILD and 27.1 ± 1.6% in CTRL, P

Epoxyeicosatrienoic acids (EETs), lipid mediators produced by cytochrome P450 epoxygenases, regulate inflammation, angiogenesis, and vascular tone. Despite pleiotropic effects on cells, the role of these epoxyeicosanoids in normal organ and tissue regeneration remains unknown. EETs are produced predominantly in the endothelium. Normal organ and tissue regeneration require an active paracrine role of the microvascular endothelium, which in turn depends on angiogenic growth factors. Thus, we hypothesize that endothelial cells stimulate organ and tissue regeneration via production of bioactive EETs. To determine whether endothelial-derived EETs affect physiologic tissue growth in vivo, we used genetic and pharmacological tools to manipulate endogenous EET levels. We show that endothelial-derived EETs play a critical role in accelerating tissue growth in vivo, including liver regeneration, kidney compensatory growth, lung compensatory growth, wound healing, corneal neovascularization, and retinal vascularization. Administration of synthetic EETs recapitulated these results, whereas lowering EET levels, either genetically or pharmacologically, delayed tissue regeneration, demonstrating that pharmacological modulation of EETs can affect normal organ and tissue growth. We also show that soluble epoxide hydrolase inhibitors, which elevate endogenous EET levels, promote liver and lung regeneration. Thus, our observations indicate a central role for EETs in organ and tissue regeneration and their contribution to tissue homeostasis.

Occlusion of the main arterial route redirects blood flow to the collateral circulation. We previously reported that macrophages genetically modified to express low levels of prolyl hydroxylase domain protein 2 (PHD2) display an arteriogenic phenotype, which promotes the formation of collateral vessels and protects the skeletal muscle from ischaemic necrosis. However, the molecular mechanisms underlying this process are unknown. Here, we demonstrate that femoral artery occlusion induces a switch in macrophage phenotype through angiopoietin-1 (ANG1)-mediated Phd2 repression. ANG blockade by a soluble trap prevented the downregulation of Phd2 expression in macrophages and their phenotypic switch, thus inhibiting collateral growth. ANG1-dependent Phd2 repression initiated a feed-forward loop mediated by the induction of the ANG receptor TIE2 in macrophages. Gene silencing and cell depletion strategies demonstrate that TIE2 induction in macrophages is required to promote their proarteriogenic functions, enabling collateral vessel formation following arterial obstruction. These results indicate an indispensable role for TIE2 in sustaining in situ programming of macrophages to a proarteriogenic, M2-like phenotype, suggesting possible new venues for the treatment of ischaemic disorders., The authors thank Y. Jonsson for technical assistance. The authors are thankful to P. Carmeliet for scientific discussion and support. AH was granted by Deutsche Forschungsgemeinschaft (DFG), LV by the European Society of Cardiology (ESC), SC by Fundacao para a Ciencia e a Tecnologia (FCT), ED by Fondation ARC pour la recherche sur le cancer, A-T.H by Fonds Wetenschappelijk Onderzoek (FWO). This work was supported by grants from the Fonds Wetenschappelijk Onderzoek (FWO G.0726.10 to MM) and the European Research Council (OxyMO to MM and Tie2+ Monocytes to MDP).

Primary myelofibrosis (PMF) is a Philadelphia-negative (Ph-) myeloproliferative disorder, showing abnormal CD34+ progenitor cell trafficking, splenomegaly, marrow fibrosis leading to extensive extramedullary haematopoiesis, and abnormal neoangiogenesis in either the bone marrow or the spleen. Monocytes expressing the angiopoietin-2 receptor (Tie2) have been shown to support abnormal angiogenic processes in solid tumors through a paracrine action that takes place in proximity to the vessels. In this study we investigated the frequency of Tie2 expressing monocytes in the spleen tissue samples of patients with PMF, and healthy subjects (CTRLs), and evaluated their possible role in favouring spleen angiogenesis. We show by confocal microscopy that in the spleen tissue of patients with PMF, but not of CTRLs, the most of the CD14+ cells are Tie2+ and are close to vessels; by flow cytometry, we found that Tie2 expressing monocytes were Tie2+CD14lowCD16brightCDL62-CCR2- (TEMs) and their frequency was higher (p = 0.008) in spleen tissue-derived mononuclear cells (MNCs) of patients with PMF than in spleen tissue-derived MNCs from CTRLs undergoing splenectomy for abdominal trauma. By in vitro angiogenesis assay we evidenced that conditioned medium of immunomagnetically selected spleen tissue derived CD14+ cells of patients with PMF induced a denser tube like net than that of CTRLs; in addition, CD14+Tie2+ cells sorted from spleen tissue derived single cell suspension of patients with PMF show a higher expression of genes involved in angiogenesis than that found in CTRLs. Our results document the enrichment of Tie2+ monocytes expressing angiogenic genes in the spleen of patients with PMF, suggesting a role for these cells in starting/maintaining the pathological angiogenesis in this organ.

Despite the high prevalence of intervertebral disc disease, little is known about changes in intervertebral disc cells and their regenerative potential with ageing and intervertebral disc degeneration. Here we identify populations of progenitor cells that are Tie2 positive (Tie2+) and disialoganglioside 2 positive (GD2+), in the nucleus pulposus from mice and humans. These cells form spheroid colonies that express type II collagen and aggrecan. They are clonally multipotent and differentiated into mesenchymal lineages and induced reorganization of nucleus pulposus tissue when transplanted into non-obese diabetic/severe combined immunodeficient mice. The frequency of Tie2+ cells in tissues from patients decreases markedly with age and degeneration of the intervertebral disc, suggesting exhaustion of their capacity for regeneration. However, progenitor cells (Tie2+GD2+) can be induced from their precursor cells (Tie2+GD2−) under simple culture conditions. Moreover, angiopoietin-1, a ligand of Tie2, is crucial for the survival of nucleus pulposus cells. Our results offer insights for regenerative therapy and a new diagnostic standard., Back pain and sciatica are often caused by intervertebral disc degeneration. Sakai and colleagues identify a subset of nucleus pulposus progenitor cells from the intervertebral disc and show that loss of these progenitor cells correlates with ageing and intervertebral disc degeneration.

FMS-related tyrosine kinase 3 (FLT3) mutations are found in 30% of cases of acute myeloid leukaemia (AML). In addition, recent studies have lead to the identification of about 10-15% of AML patients displaying high expression of FLT3, not associated with mutations of the receptor (FLT3 Wild-type High, FLT3WTH). These AMLs, as well as those displaying internal tandem duplication (ITD) are associated with an unfavourable prognosis. However, the biological features of these AMLs are poorly characterized. The present study explored the immunophenotypic features of FLT3WTH AMLs in 94 de novo cases of AML. The levels of FLT3 expression, as assessed by flow cytometry and FLT3 mutational status, was used to identify four AML subgroups: FLT3WTH (14/94); FLT3 Wild-type low (FLT3WTL, 48/94); FLT3 internal tandem duplication (FLT3ITD 26/94); FLT3 aspartic acid 835 (FLT3D835, 6/94). FLT3WTH and FLT3ITD were characterized by: high white blast cell counts; predominance of M4 and M5 French-American-British classification subtypes and associated expression of myelo-monocytic markers; high expression of CD123 and TRAIL-Rs; high expression of receptors for angiogenic growth factors. Addition of FLT3 Ligand to human CD34(+) or monocytic cells stimulated CD123 and TRAIL-R expression. These findings are of potential value for the development of new therapeutic strategies.

Haznedaroglu, Ibrahim C./0000-0001-8028-9462; Haznedaroglu, Ibrahim C./0000-0001-8028-9462 WOS: 000266260300010 PubMed: 19082594 The roles of vascular endothelial growth factor (VEGF), Tie-2, and angiopoietins in the mobilization of the hematopoietic stem cells (HSCs) in humans are not yet clearly understood. In order to elucidate mechanisms of HSC mobilization from their niches, we aimed to investigate the effects of mobilization with granulocyte colony-stimulating factor to the levels of VEGF, Tie-2, and angiopoietins 1 and 2 in the allogeneic HSC transplantation donors. Soluble VEGF, Tie-2, angiopoietin 1 and angiopoietin 2 levels were studied in 20 healthy allogeneic HSC transplantation donors before (from peripheral blood) and 5 days after mobilization (from apheresis material). Mean VEGF level in the postmobilization apheresis sample was significantly higher compared to baseline premobilization peripheral blood (t test, p < 0.001). In contrast, mean Tie-2 level in the postmobilization aphaeresis sample was significantly lower compared to baseline premobilization peripheral blood (t test, p = 0.01). Angiopoietin 1 and angiopoietin 2 levels did not differ between baseline and postmobilization samples. A significant rise in VEGF level after mobilization suggests stimulation of the angiogenesis. A significant fall in Tie-2 level suggests suppression of the angiopoietin 1/Tie-2 signaling, leading to release of HSC from the hematopoietic niches and mobilization to the peripheral blood.

Compared to lower metazoans, vertebrates built up an exclusively new set of adhesion-related genes involved in the tissue development and in their functions. They include a large variety of extracellular matrix proteins and their heterodimeric integrin adhesive receptors. Integrins control the adhesive state of the cell through complex molecular mechanisms. Outside-in signalling informs the cell about the extracellular matrix environment, while Inside-out signalling results in changes in integrin functional activity. In the last 10 years it has well established a reciprocal integration of signals originating from integrins and receptors for soluble growth factors. This review summarizes the current understanding of this connection in vascular endothelial cells and highlights how integrins regulate a genetic program triggered by angiogenic inducers during embryo development and in adult life.

During angiogenic remodeling, Ang-1, the ligand of Tie2 tyrosine kinase, is involved in vessel sprouting and stabilization through unclear effects on nascent capillaries and mural cells. In our study, we hypothesized that the Ang-1/Tie2 system could cross-talk with integrins, and be influenced by the dynamic interactions between extracellular matrix and endothelial cells (ECs). Here, we show that alpha5beta1 specifically sensitizes and modulates Tie2 receptor activation and signaling, allowing EC survival at low concentrations of Ang-1 and inducing persistent EC motility. Tie2 and alpha5beta1 interact constitutively; alpha5beta1 binding to fibronectin increases this association, whereas Ang-1 stimulation recruits p85 and FAK to this complex. Furthermore, we demonstrate that Ang-1 is able to mediate selectively alpha5beta1 outside-in FAK phosphorylation. Thus, Ang-1 triggers signaling pathways through Tie2 and alpha5beta1 receptors that could cross-talk when Tie2/alpha5beta1 interaction occurs in ECs plated on fibronectin. By using blocking antibodies, we consistently found that alpha5beta1, but not alphavbeta3 activation, is essential to Ang-1-dependent angiogenesis in vivo.

Angiopoietin-1 is implicated in the maturation and remodeling of the vascular network during embryo development and in adult life. Through its tyrosine kinase receptor Tie-2 it stimulates endothelial cells to migrate and change shape. Here we show that angiopoietin-1 elicits chemokinesis of endothelial cells by a phosphoinositide 3-OH kinase/son of sevenless-dependent modulation of Rac1 and RhoA. The resulting temporal events are associated with cytoskeletal rearrangements and occur in discrete zones of the cell. Endothelial cells carrying dominant-negative mutants of RhoA and Rac1 or treated with LY294002, an inhibitor of phosphoinositide 3-OH kinase, dramatically decrease their chemokinetic velocity. Taken together, these results further expand our understanding of angiopoietin-1-mediated endothelial cell motility during vascular network assembly and angiogenesis. (Blood. 2003;102:2482-2490)

Background/aim: Crimean-Congo hemorrhagic fever (CCHF) is a serious illness characterized by fever and hemorrhage. Endothelin-1 (ET-1), angiopoietin-2 (Ang-2), and endothelial cell-specific receptor tyrosine kinase (Tie-2) are believed to be important markers of the pathogenesis, clinical course, and prognosis of the disease. The aim of this study was to determine ET-1, Ang-2, and Tie-2 levels in adults with CCHF and investigate the associations between these markers and pathogenesis and disease course.Materials and methods: Sixty CCHF patients were included in the study. The patients were classified according to disease severity criteria and Ang-2, Tie-2, and ET-1 levels were compared.Results: Mean serum ET-1 level was 36.62 ± 27.99 pg/mL in the patient group and 3.70 ± 4.71 pg/mL in the control group (P = 0.001). Mean serum Ang-2 levels were 2511.18 ± 1018.64 pg/mL in the patient group and 3570.76 ± 209.52 pg/mL in the control group (P = 0.001). Mean serum Tie-2 levels were 7.35 ± 7.75 ng/mL in the patient group and 0.67 ± 1.26 ng/mL in the control group (P = 0.001). Conclusion: Elevated ET-1 and Tie-2 levels were associated with more severe disease course, while Ang-2 level was negatively correlated with severity in adult CCHF patients. ET-1, Tie-2, and Ang-2 levels are important prognostic parameters in CCHF and may contribute significantly to treatment and follow-up.

Background The lymphatic system complements the blood circulatory system in absorption and transport of nutrients, and in the maintenance of homeostasis. Angiopoietins 1 and 2 (Ang1 and Ang2) are regulators of both angiogenesis and lymphangiogenesis through the Tek/Tie-2 receptor tyrosine kinase. The response of endothelial cells to stimulation with either Ang1 or Ang2 is thought to be dependent upon the origin of the endothelial cells. In this study, we examined the effects of the angiopoietins on lymphatic, venous and arterial primary endothelial cells (bmLEC, bmVEC and bmAEC, respectively), which were isolated and cultured from bovine mesenteric vessels. Results BmLEC, bmVEC and bmAEC cell populations all express Tie-2 and were shown to express the appropriate cellular markers Prox-1, VEGFR3, and Neuropilin-1 that define the particular origin of each preparation. We showed that while bmLECs responded slightly more readily to angiopoietin-2 (Ang2) stimulation, bmVECs and bmAECs were more sensitive to Ang1 stimulation. Furthermore, exposure of bmLECs to Ang2 induced marginally higher levels of proliferation and survival than did exposure to Ang1. However, exposure to Ang1 resulted in higher levels of migration in bmLECs than did to Ang2. Conclusion Our results suggest that although both Ang1 and Ang2 can activate the Tie-2 receptor in bmLECs, Ang1 and Ang2 may have distinct roles in mesenteric lymphatic endothelial cells.