Enhanced expression of angiopoietin-2 and the Tie2 receptor but not angiopoietin-1 or the Tie1 receptor in a rat model of myocardial infarction

Modulation of Tie2 receptor activity by angiopoietin ligands is crucial for angiogenesis, blood vessel maturation, and vascular endothelium integrity. The role of the angiopoietin (Ang) and Tie system in myocardial infarction is not well understood. To investigate the participation of the Ang/Tie in myocardial infarction, adult Sprague-Dawley rats with ligation of the left anterior descending coronary artery to induce myocardial infarction were studied. Ang1, Ang2, Tie1, and Tie2 were measured immediately after ligation of the coronary artery, and at 6 h, 1 and 3 days, and 1, 2, 3 and 4 weeks after ligation by Northern blotting, Western blotting, and immunohistochemical staining. Ang2 mRNA significantly increased from 2 weeks (2.1-fold) to 4 weeks (2.9-fold) after the infarction in the left ventricular free wall. Tie2 mRNA increased significantly from 1 week (2.1-fold) to 4 weeks (3.8-fold) after the infarction. Ang2 protein also significantly increased from 3 days (1.9-fold) to 4 weeks (3-fold) after the infarction in the left ventricular free wall. Tie2 protein increased 2.4-fold at 3 weeks and 2.8-fold at 4 weeks after the infarction. Neither Ang1 nor Tie1 mRNA or protein showed any significant change at any time point after the infarction. The ratio of Ang2/Ang1 mRNA and protein in the study group was higher than that in the control group. Ang2 and Tie2 expression in nonischemic myocardium showed no significant change. Immunohistochemical study also showed increased immunoreactivity of Ang2 and Tie2 at the infarct border. In conclusion, Ang2 and Tie2 expressions significantly increased both spatial and temporal patterns after myocardial infarction in the rat ventricular myocardium, while Ang1 and Tie1 receptor expression did not.     

2-Methoxycinnamaldehyde inhibits tumor angiogenesis by suppressing Tie2 activation

Blood vessels are mainly composed of intraluminal endothelial cells (ECs) and mural cells adhering to the ECs on their basal side. Immature blood vessels lacking mural cells are leaky; thus, the process of mural cell adhesion to ECs is indispensable for stability of the vessels during physiological angiogenesis. However, in the tumor microenvironment, although some blood vessels are well-matured, the majority is immature. Because mural cell adhesion to ECs also has a marked anti-apoptotic effect, angiogenesis inhibitors that destroy immature blood vessels may not affect mature vessels showing more resistance to apoptosis. Activation of Tie2 receptor tyrosine kinase expressed in ECs mediates pro-angiogenic effects via the induction of EC migration but also facilitates vessel maturation via the promotion of cell adhesion between mural cells and ECs. Therefore, inhibition of Tie2 has the advantage of completely inhibiting angiogenesis. Here, we isolated a novel small molecule Tie2 kinase inhibitor, identified as 2-methoxycinnamaldehyde (2-MCA). We found that 2-MCA inhibits both sprouting angiogenesis and maturation of blood vessels, resulting in inhibition of tumor growth. Our results suggest a potent clinical benefit of disrupting these two using Tie2 inhibitors.     

Combination of angiopoietin-1 and vascular endothelial growth factor gene therapy enhances arteriogenesis in the ischemic myocardium

We hypothesised that angiopoietin-1 (Ang-1), in conjunction with vascular endothelial growth factor (VEGF) gene therapy, can enhance arteriogenesis and angiogenesis during myocardial ischemia. Mice were given a single intramyocardial injection of saline, phVEGF-A(165) and phAng-1 or a combination thereof into the non-ischemic normal heart or into the ischemic border zone of the infarcted heart. In the normal and the ischemic myocardium, gene transfer of phVEGF-A(165) alone increased the myocardial capillary density by 16% and 36%, respectively, and phAng-1 had a similar effect. In the normal heart, the ratio of arteriolar to capillary densities increased with phVEGF-A(165) and more so in the ischemic myocardium where phAng-1 also had an effect. Furthermore, the combination of plasmids induced an up to 7.5-fold increase. Transient overexpression of VEGF-A(165) boosts endogenous arteriogenesis in addition to capillary angiogenesis. Ang-1 further boosts this effect at the arteriolar level.     

Oligomerized Tie2 localizes to clathrin-coated pits in response to angiopoietin-1

The tyrosine kinase receptor Tie2 is expressed on endothelial cells, and together with its ligand angiopoietin-1 (Ang1), is important for angiogenesis and vascular stability. Upon activation by Ang1, Tie2 is rapidly internalized and degraded, a mechanism most likely necessary to attenuate receptor activity. Using immunogold electron microscopy, we show that on the surface of endothelial cells, Tie2 is arranged in variably sized clusters containing dimers and higher order oligomers. Clusters of Tie2 were expressed on the apical and basolateral plasma membranes, and on the tips of microvilli. Upon activation by Ang1, Tie2 co-localized with the clathrin heavy chain at the apical and basolateral plasma membranes and within endothelial cells indicating that Tie2 internalizes through clathrin-coated pits. Inhibiting cellular endocytosis by depleting cellular potassium or by acidifying the cytosol blocked the internalization of Tie2 in response to Ang1. Our results suggest that one pathway mediating the internalization of Tie2 in response to Ang1 is through clathrin-coated pits. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Adipose tissue is a regulated source of interleukin-10

White adipose tissue (WAT) is the source of pro- and anti-inflammatory cytokines and we have recently shown that this tissue is a major source of the anti-inflammatory interleukin (IL)-1 receptor antagonist (IL-1Ra). We now aimed at identifying additional adipose-derived cytokines, which might serve as regulators of IL-1Ra. We demonstrate here for the first time that the antiinflammatory cytokine IL-10 is secreted by human WAT explants and that it is up-regulated by LPS and TNF-alpha in vitro, as well as in obesity in humans (2- and 6-fold increase in subcutaneous and visceral WAT, respectively) and rodents (4-fold increase).     

Enhancement of bone formation by genetically-engineered bone marrow stromal cells expressing BMP-2, VEGF and angiopoietin-1

To explore the potential of combined delivery of osteogenic and angiogenic factors to bone marrow stromal cells (BMSCs) for repair of critical-size bone defects, we followed the formation of bone and vessels in tissue-engineered constructs in nude mice and rabbit bone defects upon introducing different combinations of BMP-2, vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) to BMSCs with adenoviral vectors. Better osteogenesis and angiogenesis were found in co-delivery group of BMP-2, VEGF and angiopoietin-1 than any other combination of these factors in both animal models, indicating combined gene delivery of angiopoietin-1 and VEGF165 into a tissue-engineered construct produces an additive effect on BMP-2-induced osteogenesis.     

Effects of intraluteal implants of prostaglandin E1 or E2 on angiogenic growth factors in luteal tissue of Angus and Brahman cows

Previously, it was reported that intraluteal implants containing prostaglandin E₁ or E₂ (PGE₁ and PGE₂) in Angus or Brahman cows prevented luteolysis by preventing loss of mRNA expression for luteal LH receptors and luteal unoccupied and occupied LH receptors. In addition, intraluteal implants containing PGE₁ or PGE₂ upregulated mRNA expression for FP prostanoid receptors and downregulated mRNA expression for EP₂ and EP₄ prostanoid receptors. Luteal weight during the estrous cycle of Brahman cows was reported to be lesser than that of Angus cows but not during pregnancy. The objective of this experiment was to determine whether intraluteal implants containing PGE₁ or PGE₂ alter vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), angiopoietin-1 (ANG-1), and angiopoietin-2 (ANG-2) protein in Brahman or Angus cows. On Day 13 of the estrous cycle, Angus cows received no intraluteal implant and corpora lutea were retrieved, or Angus and Brahman cows received intraluteal silastic implants containing vehicle, PGE₁, or PGE₂ on Day 13 and corpora lutea were retrieved on Day 19. Corpora lutea slices were analyzed for VEGF, FGF-2, ANG-1, and ANG-2 angiogenic proteins via Western blot. Day-13 Angus cow luteal tissue served as preluteolytic controls. Data for VEGF were not affected (P > 0.05) by day, breed, or treatment. PGE₁ or PGE₂ increased (P < 0.05) FGF-2 in luteal tissue of Angus cows compared with Day-13 and Day-19 Angus controls but decreased (P < 0.05) FGF-2 in luteal tissue of Brahman cows when compared w Day-13 or Day-19 Angus controls. There was no effect (P > 0.05) of PGE₁ or PGE₂ on ANG-1 in Angus luteal tissue when compared with Day-13 or Day-19 controls, but ANG-1 was decreased (P < 0.05) by PGE₁ or PGE₂ in Brahman cows when compared with Day-19 Brahman controls. ANG-2 was increased (P < 0.05) on Day 19 in Angus Vehicle controls when compared with Day-13 Angus controls, which was prevented (P < 0.05) by PGE₁ but not by PGE₂ in Angus cows. There was no effect (P > 0.05) of PGE₁ or PGE₂ on ANG-2 in Brahman cows. PGE₁ or PGE₂ may alter cow luteal FGF-2, ANG-1, or ANG-2 but not VEGF to prevent luteolysis; however, species or breed differences may exist.     

Angiogenic activity of endothelial progenitor cells through angiopoietin-1 and angiopoietin-2

Angiogenesis is a regulated process involving the proliferation, migration, and remodeling of different cell types particularly mature endothelial cells and recently discovered progenitor cells, named as endothelial progenitor cells (EPCs). Up to now, many attempts have been made to understand the dynamic balance of pro- and anti-angiogenic factors on EPCs on different milieu. It has been accepted that Ang-1, -2 and Tie-1, -2 signaling play a key role on angiogenesis pathways in endothelial lineage cells. In the current experiment, the angiogenic/angio-modulatory potency of Ang-1 and -2 was investigated on isolated EPCs. Freshly isolated EPCs were exposed to different concentrations of Ang-1 and -2 (25 and 50?ng/ml) over a course of 7 and 14 days. Corroborating to our results, a superior effect of Ang-1 on angiogenic properties, including an increased concentration of vascular endothelial growth factor, in vitro tubulogenesis, EPC migratory, Tie-2 expression and clonogenicity, was determined. A large amount of positive mature endothelium markers was achieved in EPCs being-exposed to Ang-1 peptide. Nonetheless, the number of CD133 positive cells increased in the presence of Ang-2. Collectively, we conclude that Ang-1 potentially induces functional and mature vascular-like behavior in EPCs more than Ang-2.     

Single chain Fv antibody against angiopoietin-2 inhibits VEGF-induced endothelial cell proliferation and migration in vitro

Angiopoietin-2 (Ang2) promotes tumor growth and metastasis by specifically priming endothelial cells for angiogenesis. Multiple angiogenic factors up-regulate expression of Ang2, suggesting that Ang2 may be the common pathway in growth factor initiated-angiogenesis. Using phage display technology, we generated single chain Fv molecule against human Ang2 (scFv-Ang2) with high affinity (K(d)=0.01 microM) from a mouse phage antibody library. Compared with control scFv, the mouse scFv-Ang2 completely inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) treated with vascular endothelial growth factor (VEGF, 10 ng/ml), but not that of the cells treated with either basic fibroblast growth factor, or angiotensin II, or Ang2. Chemotaxis assay showed that scFv-Ang2 could block completely Ang2-induced (100%) and partially VEGF-induced (49%) migration of HUVECs. The results indicate that Ang2 takes part in the VEGF-induced angiogenesis and scFv-Ang2 might be a promising compound in blocking both VEGF and Ang2 induced angiogenesis.     

Adipose tissue,angiogenesis and angio-MIR under physiological and pathological conditions

Angiogenesis is a crucial process for the maintenance of normal tissue physiology and it is involved in tissue remodeling and regeneration. This process is essential for adipose tissue maintenance. The adipose tissue is composed by different cell types including stromal vascular cells as well as adipose stem cells (ASCs). In particular, ASCs are multipotent somatic stem cells that are able to differentiate and secrete several growth factors; they are recently emerging as a new cell reservoir for novel therapies and strategies in many diseases. Several studies suggest that ASCs have peculiar properties and participate in different disease-related processes such as angiogenesis. Furthermore, pathological expansion of adipose tissue brings to hypoxia, a major condition of unhealthy angiogenesis.Recent evidences have shown that microRNAs (miRNAs) play a crucial role also on ASCs as they take part in stemness maintenance, proliferation, and differentiation. It has been suggested that some miRNAs (MIR126, MIR31, MIR221 MIR222, MIR17-92 cluster, MIR30, MIR100 and MIR486) are directly involved in the angiogenic process by controlling multiple genes involved in this pathway. With the present review, we aim at providing an updated summary of the importance of adipose tissue under physiological and pathological conditions and of its relationship with neovascularization process. In particular, we report an overview of the most important miRNAs involved in angiogenesis focusing on ASCs. Hopefully the data presented will bring benefit in developing new therapeutic strategies.     

Angiopoietin 1 (Ang-1), angiopoietin 2 (Ang-2) and Tie-2 (a receptor tyrosine kinase) concentrations in peripheral blood of patients with thyroid cancers

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.     

Adipose tissue in Walker 256 tumour-induced cachexia: possible association between decreased leptin concentration and mononuclear cell infiltration

The adipose tissue (AT) is severely affected by cachexia, a paraneoplastic syndrome, which increases the morbidity and mortality of cancer. There is, however, a heterogeneous response to the condition, according to the AT depot. As plasma leptin concentration has been often reported to vary in cachexia, we have measured (species specific radioimmunoassay) the local concentration of leptin in three AT depots: retroperitoneal (RPAT), epididymal (EAT) and mesenteric (MES) of Walker 256 tumour-bearing rats. A reduced concentration of leptin (P<0.0001) was found in all the depots and in the plasma of the cachectic rats, compared with controls already from day 4 after tumour cell injection. The presence of a cell infiltrate was observed in all AT obtained from the tumour-bearing animals. Ultrastructural analysis, along with immunocytochemistry for RT1B (indicating the presence of MHCII) and using antibody against mononuclear phagocytes, showed the cells to be macrophages. The profile of TNF and PGE₂ secretion by the infiltrate was investigated (commercial kits). There was increased production of both factors by the cells of all AT (P<0.05) compared with peritoneal macrophages obtained from the cachectic rats, while the cells isolated from MES showed the highest synthesis of TNF. The results suggest a possible modulation of the chronic locally produced TNF and PGE₂ upon leptin synthesis by the AT of the cachectic rats.Financial support by FAPESP     

驱动蛋白-1在小鼠脂肪组织糖脂代谢中的作用研究

目的:本文旨在探讨动物体内水平驱动蛋白-1在脂肪组织糖、脂代谢中的作用。方法:通过Cre/Loxp重组系统构建脂肪组织特异性敲除驱动蛋白-1的小鼠模型,在生理水平观察驱动蛋白-1表达缺陷对小鼠糖代谢、脂代谢和脂肪因子分泌的影响。结果:与六月龄对照组小鼠相比,同月龄驱动蛋白-1敲除小鼠的体重、脂肪组织重量和空腹血糖水平没有显著差异,但是其血清胰岛素水平显著升高;使用葡萄糖耐量试验(GTT)和胰岛素耐量实验(ITT)对小鼠的糖代谢水平进行评估,结果显示驱动蛋白-1敲除小鼠表现为葡萄糖不耐受、胰岛素不耐受;进一步血清检测显示驱动蛋白-1敲除小鼠表现为高甘油三酯血症和血清脂联素水平降低。结论:驱动蛋白-1在脂肪组织中参与调节糖、脂代谢过程,其表达或功能障碍是2型糖尿病等代谢性疾病的一个重要的发病因素。     

Tie receptors and their angiopoietin ligands are context-dependent regulators of vascular remodeling

Angiopoietins are ligands of the Tie2 receptor that control angiogenic remodeling in a context-dependent manner. Tie signaling is involved in multiple steps of the angiogenic remodeling process during development, including destabilization of existing vessels, endothelial cell migration, tube formation and the subsequent stabilization of newly formed tubes by mesenchymal cells. Beyond this critical role in blood vessel development, recent studies suggest a wider role for Tie2 and angiopoietins in lymphangiogenesis and the development of the hematopoietic system, as well as a possible role in the regulation of certain non-endothelial cells. The outcome of Tie signaling depends on which vascular bed is involved, and crosstalk between different VEGFs has an important modulating effect on the properties of the angiopoietins. Signaling through the Tie1 receptor is not well understood, but Tie1 may have both angiopoietin-dependent and ligand-independent functions. Changes in the expression of Tie receptors and angiopoietins occur in many pathological conditions, and mutations in the Tie2 gene are found in familial cases of vascular disease.     

Expression and purification of recombinant human Angiopoietin-1 produced in Chinese hamster ovary cells

Angiopoietin-1 (Ang1) is an essential molecule for blood vessel formation. In an effort to produce large quantities of Ang1, recombinant Chinese hamster ovary (rCHO) cells expressing a high level of recombinant human Ang1 protein (rhAng1) with an amino terminal FLAG-tag were constructed by transfecting the expression vector into dihydrofolate reductase-deficient CHO cells and subsequent gene amplification in a medium containing step-wise increments of methotrexate, such as 0.02, 0.08, and 0.32 μM. The rhAng1 secreted from rCHO cells was purified at a purification yield of 18.4% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng1 as heterogeneous multimers. Moreover, rhAng1 expressed in rCHO cells is biologically active in vitro as demonstrated by its ability to bind to the Tie2 receptor and to phosphorylate Tie2. Therefore, the rhAng1 produced from CHO cells could be useful for clarifying the biological effects of exogenous rhAng1 in the future.     

Transplantation of wild-type white adipose tissue normalizes metabolic, immune and inflammatory alterations in leptin-deficient ob/ob mice

Leptin-deficient ob/ob mice exhibit several metabolic and immune abnormalities, including thymus atrophy and markedly reduced inflammatory responses. We evaluated whether transplantation of wild-type (WT) white adipose tissue (WAT) into ob/ob mice could mimic the effect of recombinant leptin administration in normalizing metabolic, immune and inflammatory abnormalities. Female ob/ob mice received a subcutaneous transplantation of WAT obtained from WT littermates. A separate group of ob/ob mice was sham-operated. Despite raising leptin levels to only 15% of those observed in WT mice, WAT transplantation normalized metabolic abnormalities (glycemia, ALT, liver weight) in ob/ob mice and prevented further body weight gain. The transplanted group demonstrated normalization of thymus and spleen cellularity, thymocyte subpopulations and rates of thymocyte apoptosis. In the model of dextran sulfate sodium-induced colitis, WAT transplantation restored inflammation to levels equivalent to those of WT mice. Colonic production of IL-6 and MIP-2 was markedly reduced in the non-transplanted ob/ob group compared to transplanted ob/ob and WT mice. Our data indicate that WAT transplantation is an effective way to normalize metabolic as well as immune and inflammatory parameters in ob/ob mice. The threshold of leptin sufficient to normalize metabolic, immune and inflammatory function is significantly lower than levels present in lean WT mice. Finally, leptin derived exclusively from WAT is sufficient to normalize metabolic, immune and inflammatory parameters in ob/ob mice.     

White adipose tissue genome wide-expression profiling and adipocyte metabolic functions after soy protein consumption in rats

Obesity is associated with an increase in adipose tissue mass due to an imbalance between high dietary energy intake and low physical activity; however, the type of dietary protein may contribute to its development. The aim of the present work was to study the effect of soy protein versus casein on white adipose tissue genome profiling, and the metabolic functions of adipocytes in rats with diet-induced obesity. The results showed that rats fed a Soy Protein High-Fat (Soy HF) diet gained less weight and had lower serum leptin concentration than rats fed a Casein High-Fat (Cas HF) diet, despite similar energy intake. Histological studies indicated that rats fed the Soy HF diet had significantly smaller adipocytes than those fed the Cas HF diet, and this was associated with a lower triglyceride/DNA content. Fatty acid synthesis in isolated adipocytes was reduced by the amount of fat consumed but not by the type of protein ingested. Expression of genes of fatty acid oxidation increased in adipose tissue of rats fed Soy diets; microarray analysis revealed that Soy protein consumption modified the expression of 90 genes involved in metabolic functions and inflammatory response in adipose tissue. Network analysis showed that the expression of leptin was regulated by the type of dietary protein and it was identified as a central regulator of the expression of lipid metabolism genes in adipose tissue. Thus, soy maintains the size and metabolic functions of adipose tissue through biochemical adaptations, adipokine secretion, and global changes in gene expression.