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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. [ABSTRACT FROM AUTHOR]

Reduced capillary density is a feature of skeletal muscle (SkM) in type 2 diabetes (T2D), which is associated with multiple metabolic and functional abnormalities. SkM has been identified as a secretory tissue, releasing myokines that regulate multiple processes, including vascularization. We sought to determine how myokines secreted from T2D myotubes might influence SkM angiogenesis. Conditioned media (CM) were generated by myotubes from T2D and nondiabetic (ND) subjects. Primary human endothelial cells (HUVEC) and SkM explants were exposed to CM or recombinant myokines, and tube number or capillary outgrowth was determined as well as measurement of protein expression and phosphorylation. CM from ND myotubes stimulated tube formation of HUVEC to a greater extent than T2D myotubes (T2D-CM = 100%, ND-CM = 288 ± 90% after 48 h, P < 0.05). The effects of T2D myotube CM were mediated by IL-8, not IL-15 or GROα, and were due not to cell damage but rather through regulating tube production and maintenance (response to T2D-IL-8 = 100%, response to ND-IL-8 = 263 ± 46% after 48 h, P < 0.05). A similar effect was seen in SkM explants with exposure to IL-8. The dose-dependent effect of IL-8 on tube formation was also observable in the PI3K and FAK signaling pathways and mediated at least in part by PI3K, leading to regulation of Tie2 expression. These results suggest that elevated levels of IL-8 secreted from T2D myotubes create a muscle microenvironment that supports reduced capillarization in T2D. Impaired vascularization of SkM limits the availability of substrates, including glucose and contributes to the T2D phenotype.

Objectives: We designed this prospective observational study to clarify the associations between dialysate IL-6, a marker of ongoing peritoneal inflammation, Tie2, an important factor in angiogenesis in the peritoneum, and a high peritoneal solute transport rate (PSTR) in patients undergoing peritoneal dialysis (PD) and to investigate their outcome predictive roles. Methods: A total of 60 stable continuous ambulatory peritoneal dialysis (CAPD) patients from a single center in China were analyzed in this prospective study. We measured dialysate levels of IL-6 and Tie-2 using ELISAs. Our primary study endpoint was all-cause mortality with 10 years' follow-up. Results: For the evaluation of PSTR, we used the Dialysis/Plasma creatinine (D/Pcr) ratio. We subdivided the patients into two groups for statistical evaluation: low and low average D/Pcr (<0.64; L/A), and high and high average D/Pcr (=0.65; H/A) transporters. The mean levels of dialysates IL-6 (21.71 ± 8.88 pg/mL) and Tie-2 (1.23 ± 0.43 ng/mL) were significantly higher in the H/A (high and high average, group than those in the L/A group (13.94 ± 5.43 pg/mL, p<0.001 and 0.95 ± 0.43 ng/mL, p=0.019; respectively). Moreover, IL-6 and Tie-2 were positively correlated with D/Pcr (r=0.366, p=0.004 and r=0.402, p=0.001; respectively). Both dialysates IL-6 and Tie-2 were independent determinants of a high peritoneal solute transport rate. After follow-up for 42.65±18.08 months, 30 patients (50.0%) had died. An increased D/Pcr increased the risk of all-cause mortality in patients with CAPD (p=0.018), but the dialysates IL-6 and Tie2 were not independent predictors of all-cause mortality (p>0.05). Conclusion: Our results suggest that patients undergoing CAPD have a high peritoneal solute transport status with local peritoneal inflammation and angiogenesis. Increased D/Pcr is a relative risk factor for mortality and technique failure in patients undergoing CAPD. [ABSTRACT FROM AUTHOR]

Different housing conditions, including housing space and the physiological and social environment, may affect rodent behavior. Here, we examined the effects of different housing conditions on post-stroke angiogenesis and functional recovery to clarify the ambiguity about environmental enrichment and its components. Male rats in the model groups underwent right middle cerebral artery occlusion (MCAO) followed by reperfusion. The MCAO rats were divided into four groups: the physical enrichment (PE) group, the social enrichment (SE) group, the combined physical and social enrichment (PSE) group and the ischemia/reperfusion + standard conditioning (IS) group. The rats in the sham surgery (SS) group were housed under standard conditions. In a set of behavioral tests, including the modified Neurological Severity Score (mNSS), rotarod test, and adhesive removal test, we demonstrated that the animals in the enriched condition groups exhibited significantly improved neurological functions compared to those in the standard housing group. Smaller infarction volumes were observed in the animals of the PSE group by MRI detection. The enriched conditions increased the microvessel density (MVD) in the ischemic boundary zone, as revealed by CD31 immunofluorescent staining. The immunochemical and q-PCR results further showed that environmental enrichment increased the expression levels of angiogenic factors after ischemia/reperfusion injury. Our data suggest that all three enrichment conditions promoted enhanced angiogenesis and functional recovery after ischemia/reperfusion injury compared to the standard housing, while only exposure to the combination of both physical and social enrichment yielded optimal benefits.

Different housing conditions, including housing space and the physiological and social environment, may affect rodent behavior. Here, we examined the effects of different housing conditions on post-stroke angiogenesis and functional recovery to clarify the ambiguity about environmental enrichment and its components. Male rats in the model groups underwent right middle cerebral artery occlusion (MCAO) followed by reperfusion. The MCAO rats were divided into four groups: the physical enrichment (PE) group, the social enrichment (SE) group, the combined physical and social enrichment (PSE) group and the ischemia/reperfusion + standard conditioning (IS) group. The rats in the sham surgery (SS) group were housed under standard conditions. In a set of behavioral tests, including the modified Neurological Severity Score (mNSS), rotarod test, and adhesive removal test, we demonstrated that the animals in the enriched condition groups exhibited significantly improved neurological functions compared to those in the standard housing group. Smaller infarction volumes were observed in the animals of the PSE group by MRI detection. The enriched conditions increased the microvessel density (MVD) in the ischemic boundary zone, as revealed by CD31 immunofluorescent staining. The immunochemical and q-PCR results further showed that environmental enrichment increased the expression levels of angiogenic factors after ischemia/reperfusion injury. Our data suggest that all three enrichment conditions promoted enhanced angiogenesis and functional recovery after ischemia/reperfusion injury compared to the standard housing, while only exposure to the combination of both physical and social enrichment yielded optimal benefits. [ABSTRACT FROM AUTHOR]

In in vitro fertilization (IVF) the eggs are fertilized in the laboratory. The success of IVF depends on the angiogenesis. Angiopoetin-2 (ANGPT2) plays a critical role in angiogenesis and acts as a natural antagonist of the endothelial cell–specific receptor tyrosine kinase (Tie-2). A functional single-nucleotide polymorphism (SNP) in the ANGPT2 1087 G > A gene is known to influence in the gene expression in an allele-specific manner. The aim of this project was to study the effect of angiopoietin-2 gene polymorphism on in vitro fertilization and embryo transfer (IVF-ET) in Iran. The prevalence of G/A substitution in exon 4 was determined in 160 infertile patients enrolled who had unsuccessful IVF history and 160 control subjects who had successful IVF history. The genomic DNA was extracted from the whole blood using DNA extraction techniques. Genotype of ANGPT2 1087 G > A was performed using Allele-specific polymerase chain reaction (AS-PCR). The genotype frequencies of GG, AG and AA in the IVF negative were 7.5%, 67.5% and 25%, respectively and in the IVF positive were 15%, 77.5% and 7.5%, respectively. The allele frequencies of G, A in the IVF negative were 41.2%, 58.8%, respectively and in IVF positive were 53.8%, 46.2%, respectively. Statistical analysis showed that there is a significant correlation in the genotype between two groups (OR = 4.11, 95% CI = 2.065–8.18, P = 0.0001). It is concluded that ANGPT2 G/A polymorphism is associated with IVF-ET outcome in a population in the north of Iran.

We have previously characterized morphologic features of wounding-induced angiogenesis that occurs in response to acute and chronic gastric mucosal injury. As a means of investigating the molecular mechanisms underlying gastric angiogenesis, microvascular endothelial cells were isolated from stomachs of normal (non-injured) rats. The isolation procedure adapted and combined aspects of previous methods and employed positive selection using magnetic beads coated with monoclonal antibody specific for rat CD31 (PECAM-1), a cell surface marker restricted to platelets, monocytes, T lymphocytes and endothelial cells. The isolated microvascular endothelial cells expressed vascular endothelium-specific antigen and the endothelial-specific receptors, Tie2 and flt-1 (VEGFR1). When plated on growth factor-reduced matrigel, the isolated microvascular endothelial cells formed capillary-like structures reflecting in vitro angiogenesis. These cells were also responsive to vascular endothelial growth factor, VEGF, further verifying their endothelial nature. The rat microvascular endothelial cells isolated by this procedure should be useful in delineating molecular mechanisms and regulation of the angiogenesis that is essential for the healing of acute and chronic gastric injury.

The dysfunction of angiopoietin-1 (Ang-1)/Tie-2 signaling pathways has been implicated in diabetic complications. However, the underlying molecular mechanisms remain unclear. Fibronectin (FN) is thought to have an important role in regulating Ang-1/Tie-2 signaling activation. But no previous study has investigated the effects of FN glycation on Ang-1/Tie-2 signaling. In the present study, FN was glycated by methylglyoxal (MGO) to investigate whether the glycation of FN contributes to diabetes-induced Ang-1/Tie-2 signaling impairment and to understand the molecular mechanisms involved. The results demonstrated that MGO-glycated FN significantly impaired Ang-1-evoked phosphorylation of Tie-2 and Akt, Ang-1-induced endothelial cell migration and tube formation and Ang-1-mediated cell survival. The glycation of FN also inhibited the binding of α5β1 integrin to Tie-2. Moreover, FN was remarkably modified by AGEs in aortae derived from db/db mice, indicating the glycation of FN in vivo. Ang-1-induced aortic ring vessel outgrowth and Ang-1-mediated cell survival were also both significantly inhibited in aortae from db/db mice compared to that from the wild type littermates. Moreover, FN, rather than glycated FN partly restored aortic ring angiogenesis in db/db mice, indicating that the angiogenesis defect in the db/db mice are due to FN glycation. Collectively, the results in the present study suggest that the glycation of FN impairs Ang-1/Tie-2 signaling pathway by uncoupling Tie-2-α5β1 integrin crosstalk. This may provide a mechanism for Ang-1/Tie-2 signaling dysfunction and angiogenesis failure in diabetic ischaemic diseases. • MGO-glycated FN significantly impaired Ang-1/Tie-2 signaling activation and Ang-1-induced angiogenesis in vitro. • The glycation of FN inhibited the binding of α5β1 integrin to Tie-2. • FN was remarkably modified by AGEs in aortae derived from db/db mice, indicating the glycation of FN in vivo. • Ang-1-induced aortic ring vessel outgrowth was also significantly inhibited in db/db mice. [ABSTRACT FROM AUTHOR]

Angiopoietins (Ang-1, Ang-2) participate in vascular development and placental growth, both bind to Tie-2. This study aimed to determine the localization of angiopoietins in placental development of sows by immunohistochemistry and to validate the gene expression during gestation through a bioinformatic approach. Samples were collected from fifteen maternal-fetal interface from approximately 30 ( n = 5), 60 ( n = 5) and 114 ( n = 5) days of gestation for immunohistochemistry. A bioinformatic approach was performed by re-analysis of public datasets to determine the increase or decrease of genes involved in angiogenesis during pregnancy. There was no significant statistical difference of Ang-1 during gestation, although there was a tendency to increase from mid- to term-gestation ( P  = 0.7680). A notable decrease of Ang-2 was observed from early- to term-pregnancy ( P ≤ 0.05), consistent with the gene expression determined through bioinformatics. Furthermore, there were greater abundances of Tie-2 at both early and at term periods, but lesser abundances at mid-gestation ( P ≤ 0.05). The bioinformatics approach indicated that genes related to biological processes such as angiogenesis ( i.e. , development and morphogenesis of blood vessels) were expressed to a greater extent in early gestation as compared with later in gestation. The Ang-1 gene expression related to cell maturation, response to hypoxia and apoptosis, however, increased as gestation period advanced. In conclusion, angiopoietins may have an important role in the vascular development thus ensuring adequate placental growth in sows. The presence of angiopoietins in the trophoblast suggests a specific role for these pro-angiogenic factors in the tissue formation at the maternal-fetal interface. [ABSTRACT FROM AUTHOR]

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.

Background: The flowers of Carthamus tinctorius L. are widely used in traditional Chinese medicine to treat cerebrovascular and cardiovascular diseases. Hydroxysafflor yellow A (HSYA), the main constituent of C. tinctorius L. flowers, is known for its multiple biological activities. The present study investigated the effects of HSYA on angiogenesis in vitro and in a mouse hindlimb ischemia model. Methods: Using human umbilical vein endothelial cells (HUVEC) in vitro and a mouse hindlimb ischemia model in vivo, the angiogenic role of HSYA was evaluated. Results: HSYA significantly increased the capillary-like tube formation and migration of HUVEC. HSYA not only induced a rise in the expression of angiopoietin 1 and Tie-2 but it also increased phosphorylation of Tie- 2, Akt, and extracellular signal-regulated kinase 1/2. Furthermore, an anti-Tie-2 neutralizing antibody significantly inhibited HSYA-induced HUVEC tube formation and migration. In vivo, the recovery of perfusion of ischemic hindlimb tissue after femoral artery interruption was significantly increased in HSYA-treated mice compared to vehicle controls. Consistent with these results, the arteriole and capillary densities in ischemic gastrocnemius muscles were significantly increased in HSYA-treated mice. Conclusions: These results indicate the potential utility of HSYA for the treatment of ischemic diseases. [ABSTRACT FROM AUTHOR]

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 < 0.01). After TS-RDN, tripterygium and diosgenin treatment, VEGF and Ang-2 expression was lower than in the model group ( p < 0.01). However, Tie-2 expression showed no significant difference. The effects of TS-RDN on VEGF expression were more marked than those of tripterygium and diosgenin ( p < 0.01). Conclusion TS-RDN might reduce the expression of VEGF, Ang-2, and Tie-2 in the synovium, thus inhibiting synovial angiogenesis and playing a therapeutic role in RA. [ABSTRACT FROM AUTHOR]

Purpose: Angiogenesis plays a major role in tissue remodeling and repair after myocardial infarction (MI), and imaging it could provide information on the healing process. During angiogenesis, vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptors (PDGFRs), and Tie receptors are upregulated, and this study aimed to develop a C-11 positron emission tomography (PET) agent for imaging angiogenesis by targeting these receptors. Procedures: A VEGFR-2/Tie-2/PDGFRα inhibitor ( N-(6-{4-[3-(2-fluoro-5-trifluoromethyl-phenyl)-ureido]-phenoxy}-1 H-benzoimidazol-2-yl)-2-(4-methyl-piperazin-1-yl)-acetamide (ATV-1)) was synthesized and labeled with C-11. MicroPET imaging of a rat MI model was compared to proteins expression by immunohistochemistry. Results: [C]ATV-1 specifically accumulated in the infracted region of the left ventricular (LV) lateral wall more than in the interventricular septal wall, but not in sham-operated or healthy animals. Moreover, [C]ATV-1 uptake in the LV significantly correlated with Tie-2, VEGFR-2, and PDGFRα expression. Conclusion: Imaging angiogenesis in MI rats using [C]ATV-1 and PET has been demonstrated. These results merit further research and development of more hydrophilic modified [C]ATV-1 as a PET tracer. [ABSTRACT FROM AUTHOR]

Objective: To investigate the placental expression of angiopoietin (Ang)-1, Ang-2 and their receptor, Tie-2, in preeclampsia (PE) with or without intrauterine growth restriction (IUGR). Methods: Case-control study including placentas from 28 PE pregnancies, 30 PE-IUGR pregnancies and 40 controls. The expression status of the genes was evaluated by quantitative real-time PCR. Results: In both PE and PE-IUGR groups, compared to the control group, there was significantly higher expression of Ang-2 (p < 0.001) and Tie-2 (p = 0.008) and lower expression of Ang-1 (p = 0.001). The magnitude of the difference was similar for Ang-1 for both groups, whereas the magnitude of the differences was higher for Ang-2 and Tie-2 in PE-IUGR group compared to controls. Ang-2 and Tie-2 were correlated in both PE (r = 0.8602, p < 0.001) and PE-IUGR (r = 0.6342, p < 0.001) groups. In PE-IUGR group, Ang-1 was associated to Ang-2 (r = 0.3458, p = 0.0452) and Tie-2 (r = 0.4448, p = 0.0084). Log10Ang-1 but not Ang-2 was gestational age dependent (R2 = 0.40, p < 0.001). After conversion in Multiples of the Median (MoM) log10 MoM Ang-1 was reduced in the PE group (mean = -0.8181, p < 0.001) and the PE-IUGR group (mean = -1.2583, p < 0.001) compared to control group (mean = -0.0924). Discussion: We have demonstrated increased placental expression of Ang-2 and Tie-2 along with lower expression levels of Ang-1 in pregnancies with PE and PE-IUGR. Conclusion: The angiopoietin axis seems to be disrupted in PE pregnancies. Whether the results of this study represent the angiogenic imbalance observed in PE pregnancies or they are part of the pathophysiology of this condition has to be further investigated. [ABSTRACT FROM AUTHOR]

Abstract: The synthesis and biological evaluation of novel Tie-2 kinase inhibitors are presented. Based on the pyrrolopyrimidine chemotype, several new series are described, including the benzimidazole series by linking a benzimidazole to the C5-position of the 4-amino-pyrrolopyrimidine core and the ketophenyl series synthesized by incorporating a ketophenyl group to the C5-position. Medicinal chemistry efforts led to potent Tie-2 inhibitors. Compound 15, a ketophenyl pyrrolopyrimidine urea analog with improved physicochemical properties, demonstrated favorable in vitro attributes as well as dose responsive and robust oral tumor growth inhibition in animal models. [Copyright &y& Elsevier]

Abstract: Angiopoietins 1 and 2, ligands for the receptor kinase Tie-2, have been proposed to play critical but opposing roles in vascular development. Since signaling by Tie-2 is likely affected by other endothelial cell receptors such as Flk-1, the receptor for VEGF, and cell–cell adhesion receptors PECAM1 and VE-cad, we explored their interactions in a 3D model of vasculogenesis. When murine embryoid bodies (EBs) were treated with VEGF in Matrigel in the presence or absence of Ang-1 or Ang-2 for eight days, Ang-1 abrogated vascular sprouting for treatments started at days 0 or 3. In contrast, Ang-2 greatly accelerated vascular sprouting compared to untreated EBs. These results were confirmed in a second model system where VEGF treated HUVECs were grown in Matrigel in the presence or absence of Ang-1 or Ang-2. Since vascular sprouting must be precisely controlled in the developing embryo, it is likely that cell–cell adhesion molecules play a role in sensing the density of vascular sprouts. In this respect, we have shown that PECAM1 and CEACAM1 play essential roles in vascular sprouting. We now show that PECAM1 is associated with Tie-2, becomes phosphorylated on its ITIMs, and recruits the inhibitory phosphatases SHP-1 and SHP-2. In addition, PECAM1 is associated with VE-cad and may similarly regulate its signaling via recruitment of SHP-1/2. [Copyright &y& Elsevier]

Abstract: Angiogenesis consists in the growth of new blood vessels from pre-existing ones. Although anti-angiogenesis interventions have been shown to have therapeutic properties in human diseases such as cancer, their effect is only partial and the identification of novel modulators of angiogenesis is warranted. Recently, we reported the unexpected proteomic identification in endothelial cells (EC) of Myoferlin, a member of the Ferlin family of transmembrane proteins. Ferlins are well known to regulate the fusion of lipid vesicles at the plasma membrane in muscle cells, and we showed that Myoferlin gene knockdown not only decreases lipid vesicle fusion in EC but also attenuates Vascular Endothelial Growth Factor (VEGF) Receptor-2 (VEGFR-2) expression. Herein, we show that Myoferlin gene silencing in cultured EC also results in attenuated expression of a second tyrosine kinase receptor, Tie-2, which is another well-described angiogenic receptor. Most importantly, we provide evidence that delivery of a low-volume Myoferlin siRNA preparation in mouse tissues results in attenuated angiogenesis and edema formation. This provides the first evidence that acute Myoferlin knockdown has anti-angiogenic effects and validates Myoferlin as an anti-angiogenesis target. Furthermore, this supports the unexpected but increasingly accepted concept that proper tyrosine kinase receptors expression at the plasma membrane requires Myoferlin. [Copyright &y& Elsevier]

The angiopoietin (Ang)/Tie-2 ligand/receptor system is known to interact with the vascular endothelial growth factor (VEGF) pathway to determine the fate of blood vessels during angiogenesis. However, the precise contribution of this system to angiogenesis and the mechanisms of vascular maturation and remodelling in human tissue repair have yet to be elucidated. To examine the spatial and temporal expression of Ang-1, Ang-2, Tie-2 and VEGF in relation to angiogenesis in human surgical wounds. Punch biopsies were taken either from normal unwounded skin (controls) during surgery or from mastectomy scars between 3 days and 2 years postsurgery. Ang-1, Ang-2, Tie-2 and VEGF fibroblast/myofibroblast and endothelial expression were characterized by immunohistochemistry, analysed semiquantitatively and correlated with microvessel density (MVD) and scar age. The expression of VEGF, Ang-1, Ang-2 and Tie-2 in fibroblasts/myofibroblasts was increased significantly in early scars, decreased in older scars and was related to scar age ( P < 0·001) and MVD ( P < 0·0004), with strong correlations between all factors. In contrast, vascular expression of Ang-1 was decreased slightly in early scars, vascular Ang-2 remained constant and Tie-2 vascular expression increased, although there were no correlations with scar age or MVD. These data demonstrate that angiopoietins and their receptor, Tie-2, are expressed in both fibroblasts/myofibroblasts and endothelial cells in healing human wounds. Fibroblast/myofibroblast expression correlates with angiogenesis and VEGF expression, suggesting a role for the angiopoietin/Tie-2 system in normal wound repair and scarring. [ABSTRACT FROM AUTHOR]

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. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Objective: To investigate whether idiopathic menorrhagia (IM) is associated with alterations of the vascular expression of angiopoietin-1, angiopoietin-2, and tie-2 receptor. Design: Prospective clinical study. Setting: University Hospital, Department of Gynecology. Patient(s): Twenty-four patients with IM and 18 women with eumenorrhea. Intervention(s): Endometrial samples underwent immunohistochemical staining for CD34, angiopoetin-1, angiopoietin-2, tie-2, and smooth muscle actin-α. Previously published data on gap size and expression of vascular endothelial growth factor family members were used. Main Outcome Measure(s): Differences in immunostaining for these markers by computer-assisted stereological analysis. Result(s): There was significantly more angiopoetin-1 positive vessels in IM in the secretory phase, but not of angiopoetin-2 and tie-2, compared with controls. Densities of angiopoetin-1 positive vessels correlated significantly to those of angiopoetin-2 and vascular endothelial growth factor receptor 3. Smooth muscle actin-α positive pericytes covered the gaps. Double staining for CD34 and tie-2 receptor was partly identical, but gaps were covered by tie-2 stain. Conclusion(s): The discrete deregulation observed of the angiopoetin-1 expression before menstruation might affect vascular integrity, thereby contributing to the excessive blood loss in IM. [Copyright &y& Elsevier]

Abstract: A novel series of 8-(2-tetrahydropyranyl)-12,13-dihydroindazolo[5,4-a]pyrrolo[3,4-c]carbazoles (THP-DHI) was synthesized and evaluated as dual TIE-2 and VEGF-R2 receptor tyrosine kinase inhibitors. Development of the structure–activity relationships (SAR) with the support of X-ray crystallography led to identification of 7f and 7g as potent, selective dual TIE-2/VEGF-R2 inhibitors with excellent cellular potency and acceptable pharmacokinetic properties. Compounds 7f and 7g were orally active in tumor models with no observed toxicity. [Copyright &y& Elsevier]

Abstract: In recent years, many endogenous peptides have been identified by screening combinatory phage display peptide library, which play important roles in the process of angiogenesis. A heptapeptide, ATWLPPR, binds specifically to NRP-1 and selectively inhibits VEGF165 binding to VEGFR-2. Another heptapeptide, NLLMAAS, blocks both Ang-1 and Ang-2 binding to Tie-2 in a dose-dependent manner. In the present study, we aimed to connect ATWLPPR (V1) with NLLMAAS (V2) via a flexible linker, Ala-Ala, to reconstruct a novel peptide ATWLPPRAANLLMAAS (V3). We firstly investigated the anti-tumor and anti-angiogenic effects of peptide V3 on sarcoma S180 and hepatoma H22 bearing BALB/c nude mice. Mice were continuously subcutaneously administrated with normal saline, V1 (320μg/kg/d), V2 (320μg/kg/d), V1 + V2 (320μg/kg/d), and V3 (160, 320 and 480μg/kg/d), for 7 days. Treatment with peptide V3 could significantly reduce the tumor weight and volume. Pathological examination showed that the tumors treated with peptide V3 had a larger region of necrosis than that of peptide V1, V2, and V1 + V2 at the same dose. A significant decrease of microvessel density (MVD) in a dose-dependent manner was observed in each group of peptide V3. The results of pathological examination on normal tissue, lung, heart, liver, spleen, kidney and white blood cells showed that peptide V3 might have no significant toxicity. In conclusion, our results demonstrated that peptide V3 could be more effective on inhibiting tumor growth and angiogenesis than that of V1, V2, and V1 + V2. Peptide V3 could be considered as a novel chimeric peptide with potent anti-tumor activity. [Copyright &y& Elsevier]

Abstract: The SAR and improvement in potency against Tie2 of novel thienopyrimidine and thiazolopyrimidine kinase inhibitors are reported. The crystal structure of one of these compounds bound to the Tie-2 kinase domain is consistent with the SAR. These compounds have moderate potency in cellular assays of Tie-2 inhibition, good physical properties, DMPK, and show evidence of in vivo inhibition of Tie-2. [Copyright &y& Elsevier]

Objective: Recent observations have revealed an interaction between inflammation and angiogenesis, which may be mediated by angiopoietins and chemokines. Given the importance of inflammation in parturition, we sought to determine whether angiopoietin-2 (Ang-2) is present in amniotic fluid (AF) and if its concentration changes with gestational age, labor, and in intra-amniotic infection/inflammation (IAI) in patients with spontaneous preterm labor and intact membranes. Study design: This cross-sectional study included 486 patients in the following groups: 1) women in the mid-trimester of pregnancy (14–18 weeks) who underwent amniocentesis for genetic indications and delivered a normal neonate at term (n=52); 2) normal pregnant women at term with (n=48) and without (n=45) spontaneous labor; 3) patients with an episode of spontaneous preterm labor (PTL) and intact membranes who were classified into: a) PTL without IAI who delivered at term (n=152); b) PTL without IAI who delivered preterm (<37 weeks gestation; n=107); and c) PTL with IAI (n=82). Ang-2 concentration in AF was determined by enzyme-linked immunoassay. Non-parametric statistics were used for analysis. Results: 1) Ang-2 was detected in all AF samples; 2) the median AF Ang-2 concentration at term was significantly lower than that in the mid-trimester (1877.4 pg/mL vs. 3525.2 pg/mL; P<0.001); 3) among patients with PTL, the median AF Ang-2 concentration was significantly higher in patients with IAI than in those without IAI (4031.3 pg/mL vs. 2599.4 pg/mL; P<0.001) and those with PTL without IAI who delivered at term (4031.3 pg/mL vs. 2707.3 pg/mL; P<0.001); and 4) no significant differences were observed in the median AF Ang-2 concentration between patients with spontaneous labor at term and those at term not in labor (1722.9 pg/mL vs. 1877.4 pg/mL; P=0.6). Conclusions: 1) Ang-2, a protein involved in the process of vascular remodeling, is a physiologic constituent of the amniotic fluid and its concentration decreased with advancing gestation; 2) the median Ang-2 concentration in amniotic fluid is higher in patients with IAI than in those without; and 3) spontaneous parturition at term is not associated with changes in the AF concentration of Ang-2. These findings support the view of a link between angiopoietins and inflammation. [ABSTRACT FROM AUTHOR]

Abstract: Angiopoietins are integral to vasculogenesis and angiogenesis, which play crucial roles in the growth and development of the placenta. The current study assessed expression of angiopoietins (Ang-1 and Ang-2) and their receptors (Tie-1 and Tie-2) during development of the early human placenta. First-trimester placental tissues were obtained from women undergoing curettage during normal pregnancies. The use of immunohistochemistry (IHC) showed that Ang-1 was primarily localized to syncytiotrophoblasts where it displayed moderate immunoreactivity, whereas weak immunoreactivity for Ang-1 was observed in endothelial cells and angiogenic cell cords (ACC). Strong immunoreactivity for Ang-2 was also found predominantly in syncytiotrophoblasts with lower immunostaining levels evident in cytotrophoblasts. Moderate immunoreactivity for Ang-2 was observed in endothelial cells, ACC and Hofbauer cells. By contrast, the trophoblastic shell, as well as endothelial cells and ACC exhibited strong staining intensity for Tie-1 with the strongest immunoreactivity for Tie-2 observed in cytotrophoblasts, ACC and endothelial cells. Western blotting of tissue extracts confirmed the IHC results. Previous studies focused on VEGF and its receptors in controlling vasculogenesis and angiogenesis in human placenta. However, the specific localization patterns of angiopoietins and their receptors revealed by the current study emphasize the importance of these molecules in placental vascular development. Functional studies aimed at identifying the molecular mechanisms of actions of these factors and receptors may prove essential in elucidating the pathophysiology of placental disorders such as intrauterine growth restriction and pre-eclampsia. [Copyright &y& Elsevier]

Abstract: Tie-2 is a receptor tyrosine kinase which is involved in angiogenesis and thereby growth of human tumours. The discovery and SAR of a novel class of imidazole-vinyl-pyrimidine kinase inhibitors, which inhibit Tie-2 in vitro is reported. Their synthesis was carried out by condensation of imidazole aldehydes with methyl pyrimidines. These compounds are lead-like, with low molecular weight, good physical properties and oral bioavailability. [Copyright &y& Elsevier]

Objective. An anti-angiogenic state has been described in patients with preeclampsia, small-for-gestational age (SGA) fetuses and fetal death, and changes in the concentration of circulating angiogenic and anti-angiogenic factors can precede the clinical recognition of preeclampsia and SGA by several weeks. Gene deletion studies demonstrate that a selective group of endothelial growth factors are required for vascular development, including members of the vascular endothelial growth factor (VEGF) family, as well as angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), both ligands for the tyrosine kinase endothelial cell receptor Tie-2. These angiogenic factors have been proposed to promote angiogenesis in a coordinated and complementary fashion. Soluble Tie-2 (sTie-2) is the soluble form of the Tie-2 receptor, which is detectable in biological fluids. The purpose of this study was to determine whether patients with preeclampsia and mothers who deliver a SGA neonate have changes in the plasma concentrations of sTie-2. Study design. This cross-sectional study included patients in the following groups: (1) non-pregnant women (n = 40), (2) women with normal pregnancies (n = 135), (3) patients with preeclampsia (n = 112), and (4) patients who delivered an SGA neonate (n = 53). Maternal plasma concentrations of sTie-2 were measured by a sensitive immunoassay. Non-parametric statistics were used for analysis. Results. (1) The median maternal plasma concentration of sTie-2 was lower in normal pregnant women than in non-pregnant women [median 16.0 ng/mL (range 5.0-71.6) vs. median 20.7 ng/mL (range 10.8-52.4), respectively; p = 0.01)). (2) Plasma sTie-2 concentrations in normal pregnancy changed significantly as a function of gestational age. (3) Patients with preeclampsia and those who delivered SGA neonates had a lower median maternal plasma concentration of sTie-2 than those with a normal pregnancy [preeclampsia: median 14.9 ng/mL (range 4.9-67.3); SGA: median 10.9 ng/mL (range 5.1-29.1); normal pregnancy: median 16.0 ng/mL (range 5.0-71.6); p = 0.048 and p < 0.001, respectively]. (4) Patients with SGA neonates had a lower median plasma concentration of sTie-2 than that of those with preeclampsia [median 10.9 ng/mL (range 5.1-29.1) vs. median 14.9 ng/mL (range 4.9-67.3), respectively; p < 0.001]. (5) Patients with early-onset preeclampsia (≤34 weeks) had lower concentrations of sTie-2 than women with late-onset preeclampsia (>34 weeks) median of delta values: -0.13 ng/mL (range -0.47-0.58) vs. median of delta values: -0.09 ng/mL (range: -0.60-0.58), respectively; p = 0.043]. In contrast, there were no significant differences in the maternal plasma sTie-2 concentration between women with severe and mild preeclampsia (p = 0.6). Conclusion. Patients with preeclampsia and those with SGA fetuses have lower median plasma concentrations of soluble Tie-2 than women with normal pregnancies. [ABSTRACT FROM AUTHOR]

Summary: Background: The role of angiogenesis in the pathogenesis of pleural effusion (PE) has not been determined. The expression of angiogenic factors may represent useful markers for the diagnosis and prediction of disease outcome. To measure the pleural fluid (PF) and serum levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and Tie receptor tyrosine kinase (Tie-2) in order to investigate their role in the pathogenesis of PEs. Methods: Sixty-seven, 17 with transudative PEs due to heart failure and 50 with exudative PEs (malignant, 22; inflammatory, 15; undiagnosed, 13) were included in the study. PF and serum levels of the growth factors (VEGF, bFGF and Tie-2) were measured using enzyme-linked immunosorbent assays. Results: PF and serum VEGF levels but not bFGF and Tie-2 levels were higher (p<0.005) in exudates than in transudates. PF VEGF levels were significantly higher in malignant than inflammatory and undiagnosed PEs (p=0.03). In addition, PF Tie-2 levels were not found different in malignant or in parapneumonic PEs. Conclusion: Our results showed that VEGF is one of the main mediators in exudative PEs, but this effect is not mediated through the angiogenetic pathway Ang-1/Tie-2. However, the role of angiogenesis and its pathways in the pathogenesis of exudative PEs needs further exploration. [Copyright &y& Elsevier]

Abstract: Fused dihydroindazolopyrrolocarbazole oximes have been identified as low nanomolar, potent dual TIE-2 and VEGF-R2 receptor tyrosine kinase inhibitors with excellent cellular potency. Development of the structure–activity relationships (SAR) led to identification of compounds 35 and 40 as potent, selective dual TIE-2/VEGF-R2 inhibitors with favorable pharmacokinetic properties. Compound 35 was orally active in tumor models with no observed toxicity. [Copyright &y& Elsevier]

Abstract: Objective: To determine the effect of 17β-estradiol, medroxyprogesterone acetate, tibolone and tibolone metabolites on Angiopoietin-1, Tie-2, and tumor necrosis factor-α in Ishikawa cells, in vitro. We hypothesized that differential effects on angiogenic factors or inflammatory cytokines by individual hormones may be related to the endometrial bleeding in postmenopausal women using hormone therapy. Design: Ishikawa cells were cultured to 80% confluence, in vitro. After 24h incubation in serum-free media, 1.0, 0.1 and 0.01μM of 17β-estradiol, medroxyprogesterone acetate, tibolone, 3α-hydroxytibolone, 3β-hydroxytibolone, and Δ4-tibolone were added to the Ishikawa cells. The cells plus steroids were then incubated for a further 24h. Total RNA was extracted from control and treated Ishikawa cells. After reverse transcription, Angiopoietin-1, Tie-2, tumor necrosis factor-α, and β-actin cDNAs were amplified in a polymerase chain reaction spiked with 33p-dCTP. Relative abundance of Angiopoietin-1, Tie-2, and tumor necrosis factor-α mRNA was measured by scintillation spectroscopy. Results: 17β-Estradiol and medroxyprogesterone acetate increased Angiopoietin-1 mRNA significantly higher than control, tibolone and tibolone hydroxy metabolites. Δ4-Tibolone at all concentrations tested did not increase Angiopoietin-1. None of the steroids tested at any concentration altered Tie-2 mRNA expression compared to control. 17β-Estradiol at 1.0 and 0.1μM increased tumor necrosis factor-α mRNA significantly higher than control. Medroxyprogesterone acetate only at 1.0μM increased tumor necrosis factor-α mRNA above control levels. Tibolone, 3α-hydroxytibolone, 3β-hydroxytibolone, and Δ4-tibolone at every concentration had no effect on tumor necrosis factor-α mRNA abundance. Conclusions: Δ4-Tibolone did not stimulate Angiopoietin-1, while the other steroids had differential effects greater than control. None of the steroids changed the expression of Tie-2 mRNA. Tumor necrosis factor-α was increased by 17β-estradiol and by the highest concentration of medroxyprogesterone acetate. We interpret these results as supportive of our hypothesis that differential effects on angiogenic factors or inflammatory cytokines by individual steroids may be related to the clinical occurrence of endometrial bleeding in postmenopausal women using hormone therapy. [Copyright &y& Elsevier]

Abstract: During our effort to develop dual VEGFR2 and Tie-2 inhibitors as anti-angiogenic agents for cancer therapy, we discovered 4-amino-5-(4-((2-fluoro-5-(trifluoromethyl)phenyl)- aminocarbonylamino)phenyl)furo[2,3-d]pyrimidine (8a) possessing strong inhibitory activity at both the enzyme and cellular level against VEGFR2 and Tie-2. Compound 8a demonstrated high pharmacokinetic exposure through oral administration, and showed marked tumor growth inhibition and anti-angiogenic activity in mouse HT-29 xenograft model via once-daily oral administration. [Copyright &y& Elsevier]

Abstract: A novel class of 3,7-diphenyl-4-amino-thieno and furo[3,2-c]pyridine has been designed based on pharmacophore models of ATP competitive kinase inhibitors. Versatile synthetic methods via double Suzuki coupling to explore SAR have been established and potent inhibitors against angiogenetic targets, VEGFR2, Tie-2, and EphB4, have been successfully discovered. [Copyright &y& Elsevier]

Abstract: There are limited and conflicting studies investigating the role of the angiopoietin family in human thyroid cancer development and progression. We have investigated cytokines angiopoietin-1, -2 and their receptor (Tie-2), known to be involved in angiogenesis, in the serum of 52 thyroid cancer patients (21 cases of papillary cancers, PTC; 8 follicular cancers, FTC; 12 medullary cancers, MTC and 11 anaplastic cancers, ATC), using ELISA assays. The control consisted of 27 healthy volunteers. Statistically significant lower concentrations of Ang-1 were found in patients with thyroid cancers as compared with the control (p <0.003). The levels of Ang-2 and Tie-2 did not differ significantly between thyroid cancer patients and control. We have also compared the results of Ang-1, Ang-2, and Tie-2 determinations obtained in different histopathological subgroups of cancer patients. These results revealed lower Ang-1 concentrations in ATC (p <0.05), MTC (p <0.02), FTC (p <0.01) and in PTC patients (p <0.05) than control. We have also observed lower Ang-2 concentration in PTC patients (p <0.03) and Tie-2 in FTC patients (p <0.02 ) in comparison to controls. In conclusion, the Angs/Tie-2 system dysfunction may play an important role in thyroid cancerogenesis and decreased concentration of Ang-1 in serum can be a useful additional biomarker for the presence of thyroid cancers. [Copyright &y& Elsevier]

Vasculogenesis in the human placenta comprises differentiation and growth of newly forming blood vessels derived from hemangiogenic stem cells within the mesenchymal core of villi. In a second stage, angiogenesis leads to the expansion and remodeling of the already existing vessels. At present, relatively little is known about the regulatory mechanisms of vasculogenesis and angiogenesis during very early placentation. Using placental villous tissues from days 22 to 48 of pregnancy, we analyzed the spatial and temporal expression of Tie-1 and Tie-2 in parallel to vascular maturation in the human placenta. In immunohistochemistry both receptors, Tie-1 and Tie-2 show a cell and villous type specific expression during this early phase of placental development. Especially, cytotrophoblast and hemangiogenic cell cords in mesenchymal villi and Hofbauer cells in immature intermediate villi have the strongest immunoreactivities. Western blot analysis showed that no significant changes were detected for Tie-1 and Tie-2 as pregnancy advanced. Moreover, phospho-Tie-2 levels did not change significantly in parallel to pregnancy ages. We conclude that both receptors are involved in angiogenesis as well as vascular modulation of early vessels. Due to their spatial distribution we speculate on an additional role in regulation of villous and extravillous trophoblastic behavior. [Copyright &y& Elsevier]

Background Angiogenesis has been suggested to play an important role in inflammatory bowel disease (IBD). The aim of the study was to evaluate the serum markers of angiogenesis angiopoietin-2 (Ang-2) and soluble angiopoietin receptor Tie-2 in patients with ulcerative colitis (UC) and Crohn's disease (CD). Materials and methods Serum Ang-2 and Tie-2 serum levels were measured in 160 IBD patients (79 UC and 81 CD) and in 80 matched healthy controls using commercially available enzyme-linked immunosorbent assays. Serum Ang-2 and Tie-2 levels were correlated with the disease activity, as well as the type, localization and treatment of the disease. Results Median serum Ang-2 and Tie-2 levels were significantly higher in both the UC patients and the CD patients compared with the healthy controls ( P < 0·05 and P < 0·001, respectively). The IBD patients with early disease (diagnosis < 2 years) had significantly higher ( P = 0·04) median serum Ang-2 levels but significantly lower ( P = 0·02) median serum Tie-2 levels as compared with IBD patients with late disease (diagnosis > 2 years). The CD patients with active disease had significantly higher levels of Ang-2 compared with non-active disease ( P = 0·02). Serum levels of both Ang-2 and Tie-2 were not correlated with laboratory markers such as ESR, CRP, white blood cell count, platelet count and albumin. Conclusions Serum Ang-2 and Tie-2 levels are elevated in patients with IBD. These markers may mediate angiogenesis and vascular permeability in the mucosa of patients with IBD. [ABSTRACT FROM AUTHOR]

Background: Numerous recent reports demonstrated that changes in serum levels of angiogenesis-related factors were associated with preeclampsia. Here, we determined the serum concentration of angiopoietin-2 (Ang-2), a natural antagonist of angiopoietin-1 (Ang-1) involved in promoting angiogenesis in the presence of angiogenic stimuli such as vascular endothelial growth factor (VEGF), in women with preeclampsia. Methods: The levels of serum Ang-2 and Tie-2, a receptor for Ang-1 expressed on endothelial cells, were determined by enzyme-linked immunosorbent assay. Results: The concentrations of serum Ang-2 were significantly elevated in healthy pregnant women (18.9 ng/mL) as compared to nonpregnant women or women in postpartum period. Increase in the levels of serum Ang-2 was significantly suppressed in preeclamptic women (4.5 ng/mL). The serum Ang-2 concentrations inversely correlated with gestational age in healthy pregnant women, but not in preeclamptic women. The serum Ang-2 concentrations positively correlated with placental weight or mean blood pressure (BP) in healthy pregnant women, but not in preeclamptic women. The serum Ang-2 concentrations inversely correlated with proteinuria in preeclamptic women. The serum concentrations of Tie-2 were not significantly different between preeclamptic and nonpreeclamptic women. Conclusions: These results suggest the potential requirement of circulating Ang-2 in proper formation of placental vasculatures during pregnancy. Although we cannot exclude the possibility that suppression in the increase of serum Ang-2 levels during pregnancy in preeclampsia as a consequence rather than a cause, measurement of serum Ang-2 concentration in pregnant women may serve as a useful marker in the diagnosis and potentially in predicting subsequent development of preeclampsia. [Copyright &y& Elsevier]

Abstract: A novel class of furo[2,3-d]pyrimidines has been discovered as potent dual inhibitors of Tie-2 and VEGFR2 receptor tyrosine kinases (TK) and a diarylurea moiety at 5-position shows remarkably enhanced activity against both enzymes. One of the most active compounds, 4-amino-3-(4-((2-fluoro-5-(trifluoromethyl)phenyl)amino-carbonylamino)phenyl)-2-(4-methoxyphenyl)furo[2,3-d]pyrimidine (7k) is <3nM on both TK receptors and the activity is rationalized based on the X-ray crystal structure. [Copyright &y& Elsevier]

Angiogenesis is a feature of hematological malignancies which may provide prognostic information. However, there is, as yet, no established marker for leukemia-associated vessels in bone marrow. In this study, immunohistochemical stainings for von Willebrand factor (vWf), CD34, Tie-2, angiomodulin, glycodelin, cycloxygenase-2 (Cox-2) and endoglin were compared in order to identify the bone marrow vasculature. Chronic myeloid leukemia (CML), a disease displaying intense angiogenesis, and polycythemia vera (PV), a disease with a low microvascular density (MVD), were studied, as well as normal bone marrow. Only vWf, CD34 and Tie-2 stained the bone marrow endothelium. Although more vessels were stained for vWf than for CD34, there was no evidence that vWf stained more disease-associated vessels. In double staining, Tie-2 co-localized with CD34, but vessels staining only for Tie-2 were also found. However, the number of Tie-2-positive vessels did not correlate to either the MVD or the disease. Angiomodulin, glycodelin, Cox-2 and endoglin did not stain vessel-like structures. In conclusion, estimating the MVD by means of CD34 staining appears to be the most reliable method, but none of the tested molecules qualified as a specific marker for leukemia-associated vessels in the bone marrow. [ABSTRACT FROM AUTHOR]

To study a potential feedback system in the angiopoietin (Ang)-Tie2 system, the authors examined effects of Ang1 and Ang2 on Tie2 expression on human umbilical vein endothelial cells (HUVECs) with or without stimulation by a potent inflammatory cytokine, tumor necrosis factor-alpha (TNF-α). Ang1, but not Ang2, down-regulated Tie2 expression on HUVECs without TNF-α stimulation. Both Ang1 and Ang2 attenuated TNF-α–induced Tie2 up-regulation. Regulation of Tie2 expression by Ang1 or Ang2 was not dependent on phosphatidylinositol 3-kinase. The Ang-Tie2 system appears to have an autoregulatory feedback system that may be regulating the overall activity of the Tie2 system in both physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Human CD34+ cells represent a heterogeneous population of immature cells which may differentiate to various cell types. The aim of the study was to determine angiogenesis regulating genes expression in CD34+ cells, their subpopulations, and during their differentiation induced by hematopoietic growth factors. Material and methods: We have measured the expression of angiogenesis regulating genes angiopoietin-1 (Ang-1), angiopoietin-1 (Ang-2) and their receptor Tie-2, vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 in sorted population of CD34+ and CD34+/CD133+ cells from human cord blood and bone marrow, and in their differentiating progeny, using real time reverse transcriptase polymerase chain reaction. The hematopoietic differentiation of CD34+ cells was induced in semisolid or liquid differentiation supporting media containing appropriate hematopoietic growth factors. Results: A higher expression of Ang-1, Ang-2, and Tie-2 mRNAs was detected in CD34+/CD133+ cord blood cells as compared with CD34-/CD133- fraction, but no expression of these genes was detected in burst-forming unit erythroid (BFU-E) nor colony-forming unit granulocyte macrophage (CFU-GM) colonies. The level of Ang-1 and Tie-2 mRNAs, but not that of Ang-2 mRNA gradually decreased during a 14-d incubation of cord blood CD34+ cells in a liquid culture. A significantly higher expression of VEGF mRNA was in BFU-E as compared with CFU-GM cell colonies and CD34+/CD133+ cells. VEGFR-1 mRNA was equally expressed in CD34+/CD133+ and CD34-/CD133- cells as well as in BFU-E and CFU-GM colonies. Expression of VEGFR-2 mRNA was detected at the borderline of method sensitivity only in CD34+/CD133+ cells. Conclusion: CD34+/CD133+ cord blood cells express Ang-1, Ang-2 and VEGF as well as their receptor mRNAs, suggesting a role of these cells in regulation both angiopoiesis and hematopoiesis. [ABSTRACT FROM AUTHOR]

Estrogen promotes endothelial cell proliferation and survival in the vasculture of non-reproductive organs. The main mechanisms through which estrogen exerts its effects on endothelial cells remain unknown. Angiopoietins are newly described modulators of endothelial cell survival and they exert their effects through the activation of endothelial cell-specific Tie-2 receptors. In this study, we evaluated whether estrogen modulates the activity and expression of Tie-2 receptors, Ang-1 and its endogenous antagonist; angiopoietins-2 (Ang-2) in non-reproductive organs. Using RT-PCR, we found that daily administration of 17-β-estradiol for 8 days in ovariectomized rats results in a significant reduction in tissue Ang-1 mRNA expression. By comparison, estrogen therapy produced a significant increase in Ang-2 mRNA in estrogen-treated rats with heart, kidney and lung Ang-2 mRNA levels reaching 169%, 152% and 224% of those of oil-treated animals, respectively. We also observed that tyrosine phosphorylation of Tie-2 receptors is significantly attenuated in ovariectomized rats treated with 17-β-estradiol. Our results suggest that the effects of estrogen on the vasculature of non-reproductive organs require the inhibition of angiopoietin-1-Tie-2 receptor pathway and that this inhibition is achieved through simultaneous down-regulation of Ang-1 and Tie-2 expression and elevation in Ang-2 expression. [Copyright &y& Elsevier]

Tissue hypoxia has been identified as being a particularly important stimulus for triggering angiogenesis. Here we report early effects of hypoxia/reoxygenation (H/R) on the protein expression profiles and localization patterns of the VEGF and Angiopoietin-Tie systems in adult rat myocardium. Western blot as well as immunohistochemical analyses were performed on hearts obtained from rats exposed to various durations of in vivo systemic hypoxemic hypoxia followed by 24 h reoxygenation. The relative time course of protein expression in response to increasing durations of hypoxia, as indicated from our experiments, seems to suggest the involvement of the VEGF system and the Ang-Tie system in early angiogenesis. An apparent relationship between the expression profiles of Flk-1 and Ang-2 was observed. The most significant and interesting relationship which came to light was the surprisingly coincident yet opposite temporal trends between Ang-1 and Ang-2 protein levels. In the 1 h hypoxia group, there was significant induction of Ang-2 expression (31.3% compared to its baseline control) in contrast to relatively mild Ang-1 expression (23.8% compared to its baseline control). Thereafter Ang-1 displayed a progressive increase in expression, parallel to a progressive decrease in Ang-2 expression, becoming most pronounced in the 4 h hypoxia group (Ang-1, 50% and Ang-2, 12.6% compared to respective baseline control values). This suggests that despite their being antagonists at the receptor level, regulation of Ang-1 and Ang-2 protein levels in response to hypoxia runs much deeper and seems to indicate modulatory control at the transcriptional and/or translational level. Two additional groups of rats were sacrificed 7 days after 4 h hypoxia + 24 h reoxygenation, or after a 28 h period of time-matched normoxia. Left ventricular tissue sections were used to determine capillary density (CD) by using anti-CD31 immunohistochemistry and computer-assisted morphometry. CD was significantly increased in the 4 h hypoxia group compared to control (1814 ± 56 vs. 1642 ± 43 counts/mm2) confirming that modulation of angiogenic factors and their receptors by H/R is capable of stimulating capillary proliferation in the myocardium. Our study presents the first evidence for the Ang-Tie system's involvement in early stages of myocardial angiogenesis along with the VEGF-Flk-1-Flt-1 system. The stimulation of myocardial angiogenesis by H/R may constitute a potential basis for a possible more long-lived adaptive response to stress afforded by preconditioning stimuli. [ABSTRACT FROM AUTHOR]