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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.

Background: Delayed wound healing is a diverse, multifactorial, complex and inter-related complication of diabetes resulting in significant clinical morbidity. Hesperidin possesses potent antidiabetic and wound healing activity. Aim: To evaluate the potential of hesperidin against experimentally induced diabetes foot ulcers. Methods: Diabetes was induced experimentally by streptozotocin (STZ, 55 mg/kg, i.p.) in Sprague Dawley rats (180-220 g) and wounds were created on the dorsal surface of the hind paw of rats. Hesperidin (25, 50 and 100 mg/kg, p.o.) was administered for 21 days after wound stabilization. Various biochemical, molecular and histopathological parameters were evaluated in wound tissue. Results: STZ-induced decrease in body weight and increase in blood glucose, food, and water intake was significantly (p < 0.05) inhibited by hesperidin (50 and 100 mg/kg) treatment. It showed a significant increase (p < 0.05) in percent wound closure and serum insulin level. The STZ-induced decrease in SOD and GSH level, as well as elevated MDA and NO levels, were significantly (p < 0.05) attenuated by hesperidin (50 and 100 mg/kg) treatment. Intraperitoneal administration of STZ caused significant down-regulation in VEGF-c, Ang-1, Tie-2, TGF-β and Smad 2/3 mRNA expression in wound tissues whereas hesperidin (50 and 100 mg/kg) treatment showed significant up-regulation in these mRNA expressions. STZ-induced alteration in would architecture was also attenuated by hesperidin (50 and 100 mg/kg) treatment. Conclusion: Together, treatment with hesperidin accelerate angiogenesis and vasculogenesis via up-regulation of VEGF-c, Ang-1/Tie-2, TGF-β and Smad-2/3 mRNA expression to enhance wound healing in chronic diabetic foot ulcers., EXCLI Journal; 17:Doc399; ISSN 1611-2156

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.

Angiopoietin (Ang)-1 and Ang-2 interact in angiogenesis to activate the Tie-2 receptor, which may be involved in new vessel maturation and regression. Mast cells (MCs) are also involved in formation of new blood vessels and angiogenesis. The present study was designed to test whether MCs can mediate angiogenesis in myocardial microvascular endothelial cells (MMVECs). Using a rat MMVEC and MC co-culture system, we observed that Ang-1 protein levels were very low even though its mRNA levels were increased by MCs. Interestingly, MCs were able to enhance migration, proliferation, and capillary-like tube formation, which were associated with suppressed Ang-2 protein expression, but not Tie-2 expression levels. These MCs induced effects that could be reversed by either tryptase inhibitor [N-tosyl-L-lysine chloromethyl ketone (TLCK)] or chymase inhibitor (N-tosyl-L-phenylalanyl chloromethyl ketone), with TLCK showing greater effects. In conclusion, our data indicated that MCs can interrupt neovessel maturation via suppression of the Ang-2/Tie-2 signaling pathway.

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.

Angiopoietin-2 (ANG2) regulates blood vessel remodeling in many pathological conditions through differential effects on Tie2 signaling. While ANG2 competes with ANG1 to inhibit Tie2, it can paradoxically also promote Tie2 phosphorylation (p-Tie2). A related paradox is that both inactivation and overactivation of Tie2 can result in vascular remodeling. Here, we reconciled these opposing actions of ANG2 by manipulating conditions that govern its actions in the vasculature. ANG2 drove vascular remodeling during Mycoplasma pulmonis infection by acting as a Tie2 antagonist, which led to p-Tie2 suppression, forkhead box O1 (FOXO1) activation, increased ANG2 expression, and vessel leakiness. These changes were exaggerated by anti-Tie2 antibody, inhibition of PI3K signaling, or ANG2 overexpression and were reduced by anti-ANG2 antibody or exogenous ANG1. In contrast, under pathogen-free conditions, ANG2 drove vascular remodeling by acting as an agonist, promoting high p-Tie2, low FOXO1 activation, and no leakage. Tie1 activation was strong under pathogen-free conditions, but infection or TNF-α led to Tie1 inactivation by ectodomain cleavage and promoted the Tie2 antagonist action of ANG2. Together, these data indicate that ANG2 activation of Tie2 supports stable enlargement of normal nonleaky vessels, but reduction of Tie1 in inflammation leads to ANG2 antagonism of Tie2 and initiates a positive feedback loop wherein FOXO1-driven ANG2 expression promotes vascular remodeling and leakage.

The angiopoietin/Tie (ANG/Tie) receptor system controls developmental and tumor angiogenesis, inflammatory vascular remodeling, and vessel leakage. ANG1 is a Tie2 agonist that promotes vascular stabilization in inflammation and sepsis, whereas ANG2 is a context-dependent Tie2 agonist or antagonist. A limited understanding of ANG signaling mechanisms and the orphan receptor Tie1 has hindered development of ANG/Tie-targeted therapeutics. Here, we determined that both ANG1 and ANG2 binding to Tie2 increases Tie1-Tie2 interactions in a β1 integrin-dependent manner and that Tie1 regulates ANG-induced Tie2 trafficking in endothelial cells. Endothelial Tie1 was essential for the agonist activity of ANG1 and autocrine ANG2. Deletion of endothelial Tie1 in mice reduced Tie2 phosphorylation and downstream Akt activation, increased FOXO1 nuclear localization and transcriptional activation, and prevented ANG1- and ANG2-induced capillary-to-venous remodeling. However, in acute endotoxemia, the Tie1 ectodomain that is responsible for interaction with Tie2 was rapidly cleaved, ANG1 agonist activity was decreased, and autocrine ANG2 agonist activity was lost, which led to suppression of Tie2 signaling. Tie1 cleavage also occurred in patients with hantavirus infection. These results support a model in which Tie1 directly interacts with Tie2 to promote ANG-induced vascular responses under noninflammatory conditions, whereas in inflammation, Tie1 cleavage contributes to loss of ANG2 agonist activity and vascular stability.

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.

Angiogenesis plays a critical role in the development and growth of solid tumors and hematologic malignancies. The system involving angiopoietin-2 [Ang-2] and its receptor Tie-2 appears to play an important role in tumor angiogenesis and in the biology of hematological and non-hematological malignancies. We evaluated the levels of soluble (s)Ang-2 and sTie-2 in acute myeloid leukemia (AML) patients and investigated the impact of their circulating levels on the overall survival in those patients.Ang-2 and Tie-2 were measured in plasma samples from AML patients and controls using enzyme-linked immunosorbent assay (ELISA).The levels of sAng-2 and sTie-2 were significantly higher in AML patients (2382.1±1586.1 pg/ml and 6.74±3.47 ng/ml, respectively) than in controls (649.5±402.6 pg/ml and 2.63±0.57 ng/ml, respectively; p0.01). AML patients with high levels of sAng-2 and sTie-2 (≥2500 pg/ml and ≥8 ng/ml, respectively) had significantly shorter overall survival than those patients with low levels (2500 pg/ml and8 ng/ml, respectively).The results of our study demonstrated the prognostic significance of circulating sAng-2 and sTie-2 in AML patients. Modulation of the angiopoietin / Tie-2 axis may be a promising approach to improve the outcome in those patients.AMAÇ: Anjiyojenez, katı tümörler ve hematolojik hastalıkların gelişimi ve büyümesinde kritik bir rol oynamaktadır. Anjiyopoietin-2 [Ang-2] ve onun reseptörü Tie-2’yi kapsayan sistemin, tümör anjiyojenezinde ve hematolojik ve hematoloji dışı hastalıkların biyolojisinde önemli bir rol oynadığı görülmektedir. AML hastalarında çözünebilen sAng-2 ve sTie-2 düzeyleri değerlendirilmiş olup, bu hastalarda dolaşımdaki düzeylerinin toplam sağkalım üzerindeki etkileri araştırılmıştır. YÖNTEMLER: AMLli hastalar ve kontrollerden alınan plazma örneklerinde, Enzim bağlı immünassay (ELISA) ile Ang-2 ve Tie -2 ölçülmüştür.AML hastalarında, sAng-2 ve sTie-2 değerleri [sırasıyla 2382.1±1586.1 pg/ml ve 6.74±3.47ng/ml], kontrollere [sırasıyla 649.5±402.6 pg/ml ve 2.63±0.57 ng/ml] kıyasla anlamlı derecede daha yüksekti (p0.01). Toplam sağkalım değeri,yüksek sAng-2 ve sTie-2 [sırasıyla ≥2500pg/ml ve ≥8ng/ml] değerli AML hastalarında düşük düzeylere [SONUÇ: Çalışmada elde edilen sonuçlarda, AML hastalarının dolaşımdaki Ang-2 ve sTie-2 düzeylerinin prognostik anlamlılığı gösterilmiştir. Anjiyopoietin / Tie2 aksisinin modülasyonu, söz konusu hastalarda sonucu geliştirmeye yönelik ümit verici bir yaklaşım olabilir.

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.

Extrahepatic bile duct cancer is a high mortal malignancy. Angiopoietin (Ang) and its receptor Tie, which are known to contribute to angiogenesis, have recently been reported to participate in the proliferation and differentiation of malignant tumor cells. The aim of this study is to investigate the expression and the significance of Ang-1, 2 and Tie-2 in extrahepatic bile duct carcinoma cells. We used immunohistochemistry to study 119 cases of surgically resected human extrahepatic bile duct carcinoma, and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) to confirm the expression of Ang-1, 2 and Tie-2 mRNA. Among these 119 cases, 52 (43.7%), 50 (42.0%) and 89 (74.8%) cases showed positive staining for Ang-1, 2 and Tie-2, respectively, in bile duct carcinoma cells. In 38 cases of normal mucosa, 6 (15.8%), 10 (26.3%) and 9 (23.7%) cases were positive for Ang-1, 2 and Tie-2, respectively. The positivity for Ang-1 and Tie-2 in normal mucosa was significantly different from all carcinomas (p, Acta medica Nagasakiensia, 53(4), pp.89-95 ; 2009

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.

The aim of the study was to evaluate the plasma concentrations of vascular endothelial growth factor (VEGF-A), its soluble receptors VEGFR-1 and VEGFR-2, insulin like growth factor (IGF-1) and soluble Tie-2 in twin infants with and without retinopathy of prematurity (ROP). Eleven pairs of twin infants (total 22 infants) were included in the study: There were 11 infants with advanced stage ROP in the study group and 11 infants with no ROP in the control group. Before time-point measurements, the peripheral venous blood samples were first centrifuged, and the plasma was then stored at -70°C. The plasma concentrations of the angiogenic factors were measured by using high sensitivity enzyme-linked immunosorbent assay (ELISA) kits. The average gestational age of the infants at the time of birth and the time at which the serum samples were collected were 30.5±2.3 and 37.5±2.5 weeks, respectively. The average serum levels of all the angiogenic factors were higher in infants with ROP, but the differences were not statistically significant (p>0.05). In this study, serum samples of twins with and without ROP were evaluated simultaneously. The time of serum sampling in the study group corresponded to the period of phase 2 ROP. Therefore, there appeared to be no difference between the ROP group and the control group. J. Exp. Clin. Med., 2013; 30:39-43

„Angioadaptation“ bezeichnet sowohl die Änderung der Anzahl von Blutgefäßen als auch den kontinuierlichen Umbau bestehender Gefäßnetzwerke an die Bedürfnisse des versorgten Gewebes. Die erfolgreiche Adaptation eines Gefäßnetzes äußert sich in der funktionsgerechten Sauerstoffversorgung eines Gewebes und durch die Optimierung der Strömungsmechanik zur Minimierung der Perfusionsenergie. Tatsächlich kann die Expression verschiedener vaskulärer Gene durch Sauerstoffpartialdruck, Dehnung (Stretch) und Schubspannung (Shear Stress) reguliert werden, wodurch auf zellulärer Ebene Rückkopplungsmechanismen im Sinne einer „Angioadaptation“ bestehen könnten. Unsere Publikationen konzentrierten sich auf die Identifikation von Mechanismen, welche eine Regulation der Angiogenese überwiegend durch Schubspannung ermöglichen. Das von Douglas Hanahan (Science. 1997 Jul 4;277(5322):48-50) etablierte Modell der Kontrolle von Vaskulogenese, Angiogenese, Maturation, Stabilisierung und Regression von Blutgefäßen durch ein Sauerstoffpartialdruck reguliertes Zusammenspiel von Ang1/Tie2 sowie VEGF konnte so unter Einbeziehung von ADAMTS1, Foxo-1 und der Schubspannung erweitert werden. Die Untersuchung endothelialer Gene, deren Expression durch Wandschubspannung geändert wird, führte uns darüber hinaus zu dem Transkriptionsfaktor ZNF580, welchem durch Beeinflussung von IL-8 eine Rolle sowohl bei der Angiogenese als auch bei der Atherogenese zuzuweisen ist., Angiogenesis is correlated with changes in hemodynamic forces, especially changes in wall shear stress. Our studies show that Foxo-1, Ang-2, Tie2 and ADAMTS1 are regulated by shear stress acting on endothelial cells. This will have implications for the regulation of different types of angiogenesis. In the last study we discovered ZNF580 as a novel factor in the regulation of IL-8 and monocyte arrest. We showed that high oxLDL/LDL-ratios are correlated with increased IL-8 serum levels in humans. Therefore it may be a potential target for intervention in atherosclerosis.

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.

Molecular mechanisms regulating the remodeling of the lymphatic vasculature from an immature plexus of vessels to a hierarchal network of initial and collecting lymphatics are not well understood. One gene thought to be important for this process is Angiopoietin-2 (Ang-2). Ang2−/− mice have previously been reported to exhibit an abnormal lymphatic phenotype but the precise nature of the lymphatic defects and the underlying mechanisms have yet to be defined. Here we demonstrate by whole-mount immunofluorescence staining of ear skin and mesentery that lymphatic vessels in Ang2−/− mice fail to mature and do not exhibit a collecting vessel phenotype. Furthermore, dermal lymphatic vessels in Ang2−/− pups prematurely recruit smooth muscle cells and do not undergo proper postnatal remodeling. In contrast, Ang2 knock-out Ang1 knock-in mice do develop a hierarchal lymphatic vasculature, suggesting that activation of Tie-2 is required for normal lymphatic development. Taken together, this work pinpoints a specific lymphatic defect of Ang2−/− mice and further defines the sequential steps in lymphatic vessel remodeling.

Hemoragijsku vrućicu s bubrežnim sindromom (HVBS) uzrokuju virusi roda Hantavirus, familije Bunyaviridae, dok spirohete roda Leptospira izazivaju leptospirozu. Ove dvije zoonoze imaju slične kliničke slike i u teškim slučajevima mogu uzrokovati hemoragije, zatajenje bubrega, a mortalitet je oko 1 – 10%. Poznato je da aktivacija Tie-2 putem angiopoietina-1 (Ang-1) sprječava apoptozu u endotelnim stanicama, ali objavljeni su i proturječni podaci o djelovanju angiopoietina-2 (Ang-2). Cilj ove studije je definirati moguće interakcije Ang-1/Ang-2 s njihovim receptorom Tie-2 kod bolesnika inficiranih hantavirusima ili leptospirama. U ovoj studiji smo testirali serume 35 HVBS bolesnika, 11 bolesnika s leptospirozom (uključujući osam parnih seruma) i 14 kontrola (opća populacija). Serološko testiranje je obavljeno ELISA testom (HVBS) ili testom mikroskopske aglutinacije (MAT) - korišteno je 12 serovara Leptospira interrogans. Bolesnici su bili hospitalizirani u Klinici za infektivne bolesti "Dr Fran Mihaljević" i u drugim odjelima za zarazne bolesti u Hrvatskoj. Prikupljeni su i klinički, te laboratorijski nalazi. Svi serumi su testirani ELISA-testom na Ang-1, Ang-2 i Tie-2 prema protokolu proizvođača (R&D Systems, Minneapolis, MN, USA). U statističkoj obradi podataka korišteni su neparametrijski testovi. HVBS bolesnici su bili većinom zaraženi virusom Puumala. Titar antitijela na leptospire se kretao u rasponu od 1:100 do 1:16000 za svih 12 serovara. Najčešći serovari bili su Australis, Saxkoebing i Hardjo. Značajno snižene koncentracije Tie-2 nađene su u serumima bolesnika s leptospirozom, kako u akutnoj (p=0, 00017), tako i u fazi regresije (p=0, 0022) bolesti. Nikakve razlike nisu pronađene u HVBS bolesnika u odnosu na kontrolnu skupinu. Nasuprot tome, koncentracije Ang-1 su više kod HVBS bolesnika nego u zdravih ispitanika, dok nisu pronađeni nikakve razlike između kontrolne skupine i bolesnika s leptospirozom. Koncentracije Ang-2 nisu pokazale nikakve značajnije razlike u odnosu na kontrolnu skupinu. Značajna korelacija je nađena između testiranih imunoloških parametara i nekih biokemijskih parametara u bolesnika oboljelih od leptospiroze. Snižene koncentracije solubilnog Tie-2, koji djeluje kao inhibitor angiopoietina, u bolesnika s leptospirozom mogle bi imati ulogu u preživljavanju endotelnih stanica koje su važan čimbenik u imunopatogenezi (generalno oštećenje i aktivacija endotela) kako leptospiroze, tako i HVBS-a. Promjene u imunološkim parametrima bila su najizraženije u bolesnika s teškim oblicima leptospiroze (Weilova bolest).

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.

Morphogenesis of a vascular network requires dynamic vessel growth and regression. To investigate the cellular mechanism underlying this process, we deleted focal adhesion kinase (FAK), a key signaling mediator, in endothelial cells (ECs) using Tie2-Cre mice. Targeted FAK depletion occurred efficiently early in development, where mutants exhibited a distinctive and irregular vasculature, resulting in hemorrhage and lethality between embryonic day (e) 10.5 and 11.5. Capillaries and intercapillary spaces in yolk sacs were dilated before any other detectable abnormalities at e9.5, and explants demonstrate that the defects resulted from the loss of FAK and not from organ failure. Time-lapse microscopy monitoring EC behavior during vascular formation in explants revealed no apparent decrease in proliferation or migration but revealed increases in cell retraction and death leading to reduced vessel growth and increased vessel regression. Consistent with this phenotype, ECs derived from mutant embryos exhibited aberrant lamellipodial extensions, altered actin cytoskeleton, and nonpolarized cell movement. This study reveals that FAK is crucial for vascular morphogenesis and the regulation of EC survival and morphology.