Dynamic changes in serum angiopoietin-1, angiopoietin-2, and angiopoietin-2/angiopoietin-1 ratio in acute myocardial infarction patients treated with primary percutaneous coronary intervention

Context: Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) play divergent roles in myocardial ischemia and reperfusion injury.

Objective: To investigate serum Ang-1 and Ang-2 levels in ST-segment elevation myocardial infarction (STEMI) patients treated with primary percutaneous coronary intervention (PCI).

Methods: Serum Ang-1 and Ang-2 were measured in 85 STEMI patients in the first week after PCI.

Results: Ang-1, Ang-2 and Ang-2/Ang-1 ratio (Ang-2/1) were all increased at admission, and had dynamic changes after PCI. Ang-2 and Ang-2/1 at admission and 2 h after PCI were positively correlated with peak cardiac troponin T levels.

Conclusion: The extent of myocardial damage may be linked to circulating Ang-2 and Ang-2/1.     

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.     

Regulation of the expression balance of angiopoietin-1 and angiopoietin-2 by Shh and FGF-2

Sonic hedgehog (Shh) is a typical morphogen to regulate epithelial–mesenchymal interactions during embryonic development. Shh is also an indirect angiogenic agent upregulating other angiogenic factors, including angiopoietin-1 (Ang-1). Recent studies revealed that angiogenesis induced by Shh is characterized by distinct large-diameter vessels with less branching. Ang-1 promotes blood vessel maturation, and angiopoietin-2 (Ang-2) counteracts Ang-1 activity and regulates vascular branching. Thus, we hypothesized that Shh-induced angiogenesis is affected by expression of Ang-1 and Ang-2, and we investigated the regulatory system of angiopoietins by Shh in vitro. Shh enhanced Ang-1 expression but did not enhance vascular endothelial growth factor in fibroblasts. The upregulation of Ang-1 expression by Shh was significantly decreased by fibroblast growth factor-2 (FGF-2), a potent angiogenic factor. Furthermore, FGF-2 increased the expression of Ang-2 in endothelial cells. These findings suggest that Shh and FGF-2 regulate the expression balance of vascular morphogens Ang-1 and Ang-2 and are involved in angiogenesis.     

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.     

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.     

VEGF, Bcl-2 and Bad regulated by angiopoietin-1 in oleic acid induced acute lung injury

Molecular mechanisms of acute lung injury (ALI) are poorly defined. Our previous study demonstrated that recombinant angiopoietin-1 (Ang1) can protect against oleic acid (OA) induced ALI at an early stage. The purpose of this study was to elucidate whether vascular endothelial growth factor (VEGF), Bcl-2, and Bad, phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) play any role in the protective mechanism of recombinant Ang1 in OA-induced ALI. All BALB/C mice were administered a single dose of OA to induce lung injury. Lungs, bronchoalveolar lavage fluid (BALF), and serum were harvested at certain time points. The expression of VEGF, Bcl-2, Bad, PI3K/Akt, and the histological changes in the lung, and the levels of VEGF, IL-6, and IL-10 in serum and BALF were examined. A second cohort of mice was followed for survival for 7 days. We observed increased expression of VEGF in BALF and serum and reduced expression of VEGF in lung tissue. Recombinant Ang1 treatment, however, up-regulated VEGF expression and p-Akt/Akt in lung tissue but down-regulated VEGF expression in BALF and serum. OA led to a decrease of anti-apoptotic marker Bcl-2 and a marked increase of pro-apoptotic marker Bad. Compared with the ALI group, in the recombinant Ang1 treated group, Bcl-2 expression was restored, and Bad expression was clearly attenuated. In addition, recombinant Ang1 attenuated the lung pathological changes and improved the survival of mice. These findings suggest that recombinant Ang1 may be a promising potential treatment for ALI. It seems that VEGF is mediated by PI3K/Akt pathway which is required for Ang1-mediated protection of lung injury. Activation of Akt stimulates expression of Bcl-2 and inhibits the expression of Bad, thus inhibiting the execution of apoptosis.     

Tie1 regulates the Tie2 agonistic role of angiopoietin-2 in human lymphatic endothelial cells

Although Angiopoietin (Ang) 2 has been shown to function as a Tie2 antagonist in vascular endothelial cells, several recent studies on Ang2-deficient mice have reported that, like Ang1, Ang2 acts as a Tie2 agonist during in vivo lymphangiogenesis. However, the mechanism governing the Tie2 agonistic activity of Ang2 in lymphatic endothelial cells has not been investigated. We found that both Ang1 and Ang2 enhanced the in vitro angiogenic and anti-apoptotic activities of human lymphatic endothelial cells (HLECs) through the Tie2/Akt signaling pathway, while only Ang1 elicited such effects in human umbilical vein vascular endothelial cells (HUVECs). This Tie2-agonistic effect of Ang2 in HLECs resulted from low levels of physical association between Tie2 and Tie1 receptors due to a reduced level of Tie1 expression in HLECs compared to HUVECs. Overexpression of Tie1 and the resulting increase in formation of Tie1/Tie2 heterocomplexes in HLECs completely abolished Ang2-mediated Tie2 activation and the subsequent cellular responses, but did not alter the Ang1 function. This inhibitory role of Tie1 in Ang2-induced Tie2 activation was also confirmed in non-endothelial cells with adenovirus-mediated ectopic expression of Tie1 and/or Tie2. To our knowledge, this study is the first to describe how Ang2 acts as a Tie2 agonist in HLECs. Our results suggest that the expression level of Tie1 and its physical interaction with Tie2 defines whether Ang2 functions as a Tie2 agonist or antagonist, thereby determining the context-dependent differential endothelial sensitivity to Ang2.     

Expression, purification and characterization of rat angiopoietin-2 in Pichia pastoris

Angiopoietin-2 (Ang2) is a member of the Ang family. Its potential in clinical use has been highlighted for its important roles in angiogenesis during the individual development and the growth of tumors. Ang2 is difficult to be expressed in E. coli for its unique structure. The expressions of Ang2 in insect cells (Sf9) and Chinese hamster ovary (CHO) cell line have been reported, however, the large-scale production of Ang2 for application is still pendent. In this study, the expression of Ang2 in Pichia pastoris expression system was described for the first time. The cDNA encoding Ang2 was cloned from the rat vascular tissue by RT-PCR, and inserted in the eukaryotic expression vector pPIZαA, and then transformed into P. pastoris KM71H cells. The expression of recombinant rat Ang2 (rrAng2) was induced by methanol and accounted for about 75% of the total secreted proteins. The recombinant protein was subsequently purified by HisTrap FF crude with a purity of 90%. Functional analysis of the purified rrAng2 demonstrated a specific activity in promoting the survival of ECV304 cells and binding to the Tie2 receptor. Preparation of bioactive rrAng2 not only lays the basis for further functional study but also provides a new strategy for soluble and large-scale production of human Ang2.     

Leptin induces angiopoietin-2 expression in adipose tissues

Adipose tissues consisting of adipocytes, microvasculature, and stroma are completely ablated upon over-expression of leptin in rats. This tissue regression is mediated by enhanced lipid beta-oxidation, adipocyte dedifferentiation, and apoptosis. To further characterize this phenomenon, we studied the possible effect of leptin on the adipose microvasculature. Tissue microvasculature is maintained by the interplay between positive and negative signals mediated by factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor, angiopoietin-1 (Ang-1), and Ang-2. Expression of the negative signal Ang-2 was reported in fetal tissues and in the adult ovary, which undergoes vascular remodeling or regression. We demonstrate that leptin induces the expression of Ang-2 in adipose tissue without a concomitant increase in VEGF. Induction of Ang-2 occurred in an autocrine manner, as demonstrated in cultured adipocytes but not in several other cell types. This tissue-specific induction of Ang-2 coincided with initiation of apoptosis in adipose endothelial cells. We propose that induction of Ang-2 by leptin in adipose cells is one of the events leading to adipose tissue regression.     

Angiopoietin-1, unlike angiopoietin-2, is incorporated into the extracellular matrix via its linker peptide region.

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) affect angiogenesis differently during embryogenesis and tumorigenesis. In an attempt to understand the molecular basis underlying the distinct roles of those two homologous molecules, we investigated the association of Ang-1 and Ang-2 with the extracellular matrix (ECM). TA3 murine mammary carcinoma (TA3) and Lewis lung carcinoma cells expressing v5 epitope-tagged Ang-1 and Ang-2 were used in our studies. The results indicated that Ang-1 is secreted and incorporated into the ECM of the tumor cells, whereas Ang-2 is not associated with the ECM. The mutagenesis study indicated the domain that is responsible for the ECM association of Ang-1 is the linker peptide region between the coiled-coil and the fibrinogen-like domains. A weak binding between the coiled-coil domain of Ang-1 and the ECM was observed. Immunocytochemistry study revealed a distinct ECM distribution pattern of Ang-1, which is quite different from that of fibronectin, laminin, and collagen types I and IV. The ECM-associated Ang-1 proteins are released, and Tie-2 receptors are phosphorylated upon the adhesion of human umbilical vein endothelial cells. Implications of the difference in the ECM association of Ang-1 and Ang-2, which are related to the regulation of angiopoietin activity and their roles in local versus distant angiogenesis during tumor metastasis, are discussed.     

Intra and extravascular transmembrane signalling of angiopoietin-1-Tie2 receptor in health and disease

Angiopoietin-1 (Ang-1) is the primary agonist for Tie2 tyrosine kinase receptor (Tie2), and the effect of Ang-1-Tie2 signalling is context-dependent. Deficiency in either Ang-1 or Tie2 protein leads to severe microvascular defects and subsequent embryonic lethality in murine model. Tie2 receptors are expressed in several cell types, including endothelial cells, smooth muscle cells, fibroblasts, epithelial cells, monocytes, neutrophils, eosinophils and glial cells. Ang-1-Tie2 signalling induces a chemotactic effect in smooth muscle cells, neutrophils and eosinophils, and induces differentiation of mesenchymal cells to smooth muscle cells. Additionally, this signalling pathway induces the secretion of serotonin, matrix metalloproteinases (MMPs) and plasmin. Ang-1 inhibits the secretion of tissue inhibitor of matrix metalloproteinase (TIMPs). Aberrant expression and activity of Tie2 in vascular and non-vascular cells may result in the development of rheumatoid arthritis, cancer, hypertension and psoriasis. Ang-1 has an anti-inflammatory effect, when co-localized with vascular endothelial growth factor (VEGF) in the vasculature. Thus, Ang-1 could be potentially important in the therapy of various pathological conditions such as pulmonary hypertension, arteriosclerosis and diabetic retinopathy. In this article, we have summarized and critically reviewed the pathophysiological role of Ang-1-Tie2 signalling pathway.     

The enigmatic role of angiopoietin-1 in tumor angiogenesis

A tumor vasculature is highly unstable and immature, characterized by a high proliferation rate of endothelial cells, hyper-permeability, and chaotic blood flow. The dysfunctional vasculature gives rise to continual plasma leakage and hypoxia in the tumor, resulting in constant on-sets of inflammation and angiogenesis. Tumors are thus likened to wounds that will not heal. The lack of functional mural cells, including pericytes and vascular smooth muscle cells, in tumor vascular structure contributes significantly to the abnormality of tumor vessels. Angiopoietin-1 (Ang 1) is aphysiological angiogenesis promoter during embryonic development. The function of Angl is essential to endothelial cell survival, vascular branching, and pericyte recruitment. However, an increasing amount of experimental data suggest that Angl-stimulated association of mural cells with endothelial cells lead to stabilization of newly formed blood vessels. This in turn may limit the otherwise continuous angiogenesis in the tumor, and consequently give riseto inhibition of tumor growth. We discuss the enigmatic role of Angl in tumor angiogenesis in this review.     

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.