Ang-2 mRNA Research Articles

Modulation of Angiogenesis during Adipose Tissue Development in Murine Models of Obesity

Development of vasculature and mRNA expression of 17 pro- or antiangiogenic factors were studied during adipose tissue development in nutritionally induced or genetically determined murine obesity models. Subcutaneous (SC) and gonadal (GON) fat pads were harvested from male C57Bl/6 mice kept on standard chow [standard fat diet (SFD)] or on high-fat diet for 0-15 wk and from male ob/ob mice kept on SFD. Ob/ob mice and C57Bl/6 mice on high-fat diet had significantly larger SC and GON fat pads, accompanied by significantly higher blood content, increased total blood vessel volume, and higher number of proliferating cells. mRNA and protein levels of angiopoietin (Ang)-1 were down-regulated, whereas those of thrombospondin-1 were up-regulated in developing adipose tissue in both obesity models. Ang-1 mRNA levels correlated negatively with adipose tissue weight in the early phase of nutritionally induced obesity as well as in genetically determined obesity. Placental growth factor and Ang-2 expression were increased in SC adipose tissue of ob/ob mice, and thrombospondin-2 was increased in both their SC and GON fat pads. mRNA levels of vascular endothelial growth factor (VEGF)-A isoforms VEGF-B, VEGF-C, VEGF receptor-1, -2, and -3, and neuropilin-1 were not markedly modulated by obesity. This modulation of angiogenic factors during development of adipose tissue supports their important functional role in obesity.

Differential expression of pro- and antiangiogenic factors in mouse strain-dependent hypoxia-induced retinal neovascularization

Clinical observations suggest that genetic factors may influence heterogeneity of angiogenic responses in cardiovascular disease, proliferative diabetic retinopathy, and neoplasia. Experiments among mouse strains using a corneal micropocket assay indicate that extent of angiogenesis may be genetically determined. Here, we established the strain-dependence of hypoxia-induced retinal angiogenesis in multiple mouse strains which paralleled the rank order found for bFGF-induced corneal angiogenesis. Using quantitative real-time RT-PCR, strain-related gene expression differences in retina/choroid between C57BL/6J and 129S3/SvIM, inbred strains with relatively low and high levels of angiogenesis, respectively, after 0, 6, 12, 24, 48, and 96 h hypoxia were determined for vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2), angiogenic ligands potently induced by hypoxia, and for pigment epithelium-derived factor (PEDF) and thrombospondin-1 (TSP-1), endogenous broad-spectrum antiangiogenic factors. Indirect ELISA was used to correlate VEGF and PEDF protein levels with mRNA expression. At the onset of hypoxia, both PEDF and TSP-1 levels were increased over 15-fold and VEGF was increased over 10-fold compared to Ang-2 in both strains. At the onset of neovascularization (48 h), both VEGF and Ang-2 mRNA levels were increased in the more angiogenic 129S3/SvIM strain (P<0.02), which was not observed among developmental control animals. PEDF expression was higher in the less angiogenic C57BL/6J strain at 6, 12, 24, and 96 h hypoxia (P<0.03), while TSP-1 expression was higher in C57BL/6J throughout the entire time course of hypoxia (4 days) compared to 129S3/SvIM (P<0.02). Among developmental control animals, PEDF and TSP-1 expression was also increased at P14 and P16 in C57BL/6J strain compared to 129S3/SvIM (P<0.02). Strain-dependent expression of both pro- and antiangiogenic growth factors may determine heterogeneity in the angiogenic response and potentially, susceptibility to angiogenesis-dependent diseases.

Increased mRNA Expression of VEGF within the Hematoma and Imbalance of Angiopoietin-1 and -2 mRNA within the Neomembranes of Chronic Subdural Hematoma

The aim of the study was to determine the source of vascular endothelial growth factor (VEGF) in hematoma fluid of patients suffering from chronic subdural hematoma (CSH) and to identify the level of gene expression of the pro-angiogenic factors angiopoietin 1 (ANG-1) and ANG-2 in hematoma membranes. Samples of venous blood, hematoma fluid, and outer membrane were obtained during surgery for CSH. The numbers of mononuclear cells were determined in hematoma fluid and in venous blood samples taken from 11 patients. The concentration of VEGF was measured by ELISA technique in hematoma fluid and in plasma. RT-PCR methodology was used to study the expression of different mRNA species in 11 patients. The mRNA species analyzed include VEGF and its receptors, VEGFR-1 and VEGFR-2, and ANG-1, ANG-2 and their receptor, Tie-2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as housekeeping gene and was used for semiquantitative analysis. The VEGF concentration was several hundred fold higher in the hematoma fluid than in corresponding plasma samples. A significant correlation was found between the number of neutrophils and the VEGF content in the hematoma fluid. The expression levels of VEGF, mainly VEGF165 and VEGF121 mRNA were highest in cells obtained from the hematoma fluid. In membrane samples, mRNA encoding for VEGF and its receptors was only inconsistently detected while the mRNA species encoding for ANG-1, ANG-2, and Tie-2 were found throughout all samples. The mean ratio of ANG-1/ANG-2 mRNA expression was 0.48 as opposed to 1.9 in a normal human brain tissue sample. The results suggest that the hematoma cells are the primary source of VEGF. A marked increase in the expression of ANG-2 mRNA over ANG-1 mRNA demonstrates a pro-angiogenic pattern in the hematoma membranes. Persistent activation of the ANG/Tie-2 system in addition to high levels of VEGF may keep the vasculature in a destabilized condition and may account for the continuous formation of new and immature blood vessels resulting in massive plasma extravasation and repeated bleeding episodes. Thus, the present study provides new evidence in favor of pro-angiogenic mechanisms playing an important role in the pathophysiology of CSH.

Analysis of Concerted Expression of Angiogenic Growth Factors in Acute Myeloid Leukemia: Expression of Angiopoietin-2 Represents an Independent Prognostic Factor for Overall Survival

Bone marrow neoangiogenesis plays an important pathogenetic and possible prognostic role in acute myeloid leukemia (AML). Members of the vascular endothelial growth factor (VEGF) and angiopoietin family represent the most specific inducers of angiogenesis secreted by AML blasts. We therefore correlated expression of angiogenic factors with clinical variables. We investigated the expression of VEGF-A, VEGF-C, angiopoietin-1 (Ang1), angiopoietin-2 (Ang2), and the receptor Tie2 by quantitative polymerase chain reaction in a cohort of 90 patients younger than 61 years with de novo AML entered into the German AML Süddeutsche Hämoblastose Gruppe Hannover 95 trial. Uni- and multivariate analyses were performed using clinical and gene expression variables. Univariate analysis of overall survival indicated the following variables as prognostic factors: good response on a day-15 bone marrow examination after initiation of induction chemotherapy, karyotype, and high Ang2 expression. In multivariate analysis, only bad response and log Ang2 expression remained of statistical significance, with a hazard ratio of 3.51 (95% CI, 1.91 to 6.47) and 0.75 (95% CI, 0.61 to 0.91), respectively. Subgroup analysis suggested that the prognostic impact of Ang2 expression was especially evident in cohorts with low VEGF-C and Ang1 mRNA levels. These results show that expression of Ang2 represents an independent prognostic factor in AML. Additional research into interactions of angiogenic cytokines in the pathogenesis of bone marrow angiogenesis in AML is warranted.

TNF-α modulates angiopoietin-1 expression in rheumatoid synovial fibroblasts via the NF-κB signalling pathway

Angiopoietin-1 (Ang-1) is one of a family of ligands for the Tie-2 receptor which has been demonstrated to be involved in angiogenesis. Little is known about the regulation of Ang-1 gene expression. We have previously demonstrated that TNF-α is able to up-regulate the expression of Ang-1 mRNA in synovial fibroblasts. This present study investigated the signal transduction pathways involved in the TNF-α induced expression of Ang-1. TNF-α signals primarily through the p38, JNK, MAP kinase, and IKK pathways resulting in the activation of the transcription factors AP-1 and NF-κB. Experiments with inhibitors and siRNA for these various signal transduction pathways revealed that TNF-α stimulation of Ang-1 expression occurs via the NF-κB signal transduction pathway.

Estradiol Regulates Angiopoietin-1 mRNA Expression Through Estrogen Receptor-α in a Rodent Experimental Stroke Model

Female, compared with male, animals are protected from cerebral ischemic injury. Physiological concentrations of 17beta-estradiol (E2) reduce damage in experimental stroke. E2 augments angiogenesis in reproductive organs and noncerebral vascular beds. We hypothesized that E2 protects brain in stroke through modulation of angiogenesis. We quantified molecular markers of angiogenesis and capillary density before and after unilateral middle cerebral artery occlusion (MCAO). Female animals were ovariectomized, treated with 25 microg E2 or placebo implants, and subjected to 2-hour MCAO and 22 hours of reperfusion. Brain angiopoietin-1 (Ang-1), Ang-2, Tie-1, Tie-2, vascular endothelial growth factor (VEGF), VEGF R1, and VEGF R2 mRNA levels were determined by RNAse protection assays, and CD31-positive vessels were counted. E2, but not ischemia, upregulated cerebral Ang-1 mRNA by 49%. Capillary density was higher in the brains of E2-treated animals. In estrogen receptor-alpha knockout (ERKO) mice, E2-mediated induction of Ang-1 mRNA was absent relative to wild-type littermates. These results suggest that E2 increases Ang-1 and enhances capillary density in brain under basal conditions, priming the MCA territory for survival after experimental focal ischemia.

Vascular endothelial growth factor and angiopoietins regulate sinusoidal regeneration and remodeling after partial hepatectomy in rats

To study the regulatory mechanisms of sinusoidal regeneration after partial hepatectomy. We investigated the expression of angiopoietin (Ang)-1, Ang-2, Tie-2, and vascular endothelial growth factor (VEGF) in regenerating liver tissue by quantitative reverse-transcription polymerase chain reaction (RT-PCR) using a LightCycler (Roche Diagnostics) and also immunohistochemical staining after 70% hepatectomy in rats. In the next step, we isolated liver cells (hepatocytes, sinusoidal endothelial cell (SEC), Kupffer cell, and hepatic stellate cells (HSC)) from regenerating liver tissue by in situ collagenase perfusion and counterflow elutriation, to determine potential cellular sources of these angiogenic factors after hepatectomy. Proliferation and apoptosis of SECs were also evaluated by proliferating cell nuclear antigen (PCNA) staining and the terminal deoxynucleotidyl transferase d-uridine triphosphate nick end labeling (TUNEL) assay, respectively. VEGF mRNA expression increased with a peak at 72 h after hepatectomy, decreasing thereafter. The expression of Ang-1 mRNA was present at detectable levels before hepatectomy and increased slowly with a peak at 96 h. Meanwhile, Ang-2 mRNA was hardly detected before hepatectomy, but was remarkably induced at 120 and 144 h. In isolated cells, VEGF mRNA expression was found mainly in the hepatocyte fraction. Meanwhile, mRNA for Ang-1 and Ang-2 was found in the SEC and HSC fractions, but was more prominent in the latter. The PCNA labeling index of SECs increased slowly, reaching a peak at 72 h, whereas apoptotic SECs were detected between 120 h and 144 h. Ang-Tie system, together with VEGF, plays a critical role in regulating balance between SEC proliferation and apoptosis during sinusoidal regeneration after hepatectomy. However, the VEGF system plays a more important role in the early phase of sinusoidal regeneration than angiopoietin/Tie system.

Changes of mRNA expression of vascular endothelial growth factor, angiopoietins and their receptors during the periovulatory period in eCG/hCG-treated immature female rats

Angiogenic factors can induce the perifollicular capillary network in the theca interna that shows marked changes in and around the preovulatory luteinizing hormone (LH) surge. To get more information on their functional crosstalk, the aim of the present study was to investigate the manner of mRNA expression of vascular endothelial growth factors (VEGFs) 120, 164, angiopoietin (Ang)-1, Ang-2 and their specific receptors during the periovulatory phase. We used an established equine and human chorionic gonadotropins (eCG/hCG)-derived experimental model capable of stimulating naturally occurring follicular maturation, ovulation and corpus luteum (CL) formation. On day 28 postpartum, immature female rats were administrated s.c. with 10 IU of eCG to promote follicular development, followed 48 hr later by i.p. administration of 20 IU of hCG. Ovaries were dissected at 0, 6, 12, 18 and 24 hr after hCG treatment, and were obtained on day 30 in the untreated control. After induction of follicular growth by the eCG treatment, each mRNA expression of VEGF 120, VEGF 164, Neuropilin-1 and Flt-1 significantly increased. The peaks in mRNA expressions of VEGF120 and VEGF164 were both found at 18 hr after hCG treatment. Flk-1 mRNA expression maintained up to 6 hr after hCG treatment, and then decreased at 12, 18 and 24 hr after hCG treatment. Ang-2 mRNA expression increased in the ovaries at 6 and 12 hr after hCG treatment. Tie-2 mRNA expression decreased at 24 hr after the treatment of gonadotropins. Our findings suggest that ovarian vascular formation during the periovulatory period including preovulatory follicles, ovulation and CL formation may develop via crosstalk of the VEGF-Flt-1 and Ang-Tie2 systems.

Localization of the VEGF and angiopoietin genes in uterine carcinosarcoma

Objective Carcinosarcoma of the uterus is a highly aggressive tumor. However, the angiogenesis in this tumor remains unclear. This is the first study to examine the characteristics of angiogenesis in this tumor at the molecular level while also comparing the findings with those of high-grade endometrial carcinoma. Methods The expression of vascular endothelial growth factors (VEGF) and angiopoietins (Ang) genes were examined in 35 primary uterine carcinosarcomas as well as in 12 high-grade endometrial carcinomas by in situ hybridization. Results A strong expression of VEGF-A mRNA was significantly seen in carcinosarcomas compared to high-grade endometrial carcinomas. Interestingly, in uterine carcinosarcoma, VEGF-A mRNA was more strongly expressed in the carcinoma cells than in the sarcoma cells. In addition, a decrease in the VEGF-A mRNA expression was found in the transitional areas between carcinomatous and sarcomatous elements in most carcinosarcomas evaluated. Moreover, the Ang-2 mRNA expression was significantly seen in the vasculature adjacent to the periphery of the carcinoma cells in most carcinosarcomas, in comparison to that of endometrial carcinomas. Conclusions A high angiogenic activity in uterine carcinosarcoma was shown, in comparison to that of endometrial carcinoma. Tumor angiogenesis in uterine carcinosarcoma might be chiefly influenced by VEGF-A in the carcinoma cells, in cooperation with Ang-2 in the surrounding microvessels, however, this is not fully usually the case in sarcoma cells.

Overexpression of Ang-2 mRNA in non-small cell lung cancer: Association with angiogenesis and poor prognosis

Overexpressed Angiopoietin-2 (Ang-2) derived mainly from cancer cells was reported to promote tumor angiogenesis. The aim of this study was to evaluate the expression of Ang-2 in non-small cell lung cancers (NSCLCs). We investigated Ang-2 expression in 77 patients with NSCLC who underwent curative tumor resection by means of reverse transcription-polymerase chain reaction (RT-PCR). We also determined whether or not expression of Ang-2 mRNA correlates with immunohistochemical assays of Ang-2 protein and microvessel density (MVD) level. The level of Ang-2 mRNA expression was presented by the relative yield of each gene to the S14 mRNA, respectively. Ang-2 mRNA expression in NSCLC was significantly greater than that in non-cancerous normal lung (p=0.0178). The Ang-2 mRNA expression was significantly associated with lymph node metastasis, stage, Ang-2 protein, and microvessel density (MVD) level (p=0.0009 for lymph node metastasis; p=0.0002 for stage; p<0.0001 for Ang-2 protein; p<0.0001 for MVD). With regard to prognosis, the overall and stage I survival rates for patients in the high Ang-2 mRNA expression group were significantly poorer when compared with the low Ang-2 mRNA expression group (p<0.0001 for overall; p=0.0201 for stage I). Furthermore, expression of Ang-2 mRNA was an independent predictor of prognosis by multivariate analysis (p=0.0028). These data indicate that Ang-2 may contribute to tumor angiogenesis and progression and that Ang-2 gene expression can serve as a useful prognostic marker in NSCLC.

Reciprocal regulation of angiopoietin-1 and angiopoietin-2 following myocardial infarction in the rat.

This study sought to characterize changes in the angiopoietin system in a rat model of myocardial infarction (MI). Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) bind to the endothelial-specific receptor tyrosine kinase, TIE-2. Ang-2 has been suggested to be an antagonist of TIE-2, possibly acting to release endothelial cells from the tonic stabilizing influence of Ang-1. However, on prolonged exposure, Ang-2 has been shown to acquire agonistic activity at TIE-2, raising the possibility that this isoform may play a direct role in neovascularization. Sprague-Dawley rats were subjected to left coronary ligation and myocardial tissues were harvested from the infarct and peri-infarct regions, or from non-infarcted myocardium. Changes in gene expression were determined by RT-PCR and confirmed by Northern analysis. Changes in protein expression were confirmed by Western analysis and immunocytochemistry, and TIE-2 activity was determined by immunoprecipitation with anti-TIE-2 and antiphosphotyrosine immunoblotting. At 24 h, Ang-1 mRNA and protein expression within the infarct and peri-infarct regions were decreased compared to non-infarcted myocardium, whereas Ang-2 mRNA levels were markedly increased and TIE-2 expression was unchanged. Immunohistochemical staining revealed Ang-1 and TIE-2 immunoreactivity localized to vascular endothelium. In the infarct territory, Ang-2 immunostaining was localized primarily to invading leukocytes at 24 h. At 1 week, Ang-1 expression was partially restored, whereas Ang-2 expression remained elevated. At the time of peak elevation in Ang-2, Tie2 phosphorylation was found to be markedly increased, consistent with receptor activation. Thus, myocardial ischemia induced by left coronary artery ligation resulted in a sustained increase in Ang-2 expression and a reciprocal decrease in Ang-1, consistent with a predominant role for Ang-2 in the angiogenic response to MI.

Endometrial Angiopoietin Expression and Modulation by Thrombin and Steroid Hormones: A Mechanism for Abnormal Angiogenesis Following Long-Term Progestin-Only Contraception

The angiopoietins (Ang) are endothelial cell-related factors necessary for the development and maintenance of all vessels. Altering the expression of these proteins would be expected to result in aberrant angiogenesis. Indeed the fragile endometrial vasculature and bleeding observed in women treated with long-term progestin-only contraceptives has been associated with changes in the expression of Ang-1 and Ang-2. Since bleeding would result in thrombin formation, we have assessed the effects of thrombin on the expression of the Angs in human endometrial cells. This study shows that thrombin significantly reduces the expression of Ang-1 protein and mRNA expression in human endometrial stromal cells (HESCs) and minimally decreases the production of Ang-2 protein in human endometrial endothelial cells (HEECs). Hence the presence of thrombin due to aberrant bleeding could affect the angiogenic potential of the endometrium, creating a feed forward loop resulting in more thrombin, weak vasculature, and more bleeding. In addition, since the exact localization of Ang in the human endometrium remains a subject of controversy, we have addressed this issue in an in vivo system by analyzing the expression of Angs by microdissection of HESCs, HEECs, and human endometrial glandular epithelial cells followed by real time, quantitative RT-PCR.

Morphologic changes of peritoneum and expression of VEGF in encapsulated peritoneal sclerosis rat models

Simple sclerosis consisted of a thin layer of submesothelial sclerotic tissue in peritoneal dialysis patients. Encapsulated peritoneal sclerosis (EPS) was characterized by thick sclerotic tissue involving vascular alterations in peritoneal dialysis patients. The objective of the present study is to evaluate serial morphologic changes and expressions of angiogenic factors [i.e., vascular endothelial growth factor (VEGF), angiopietin-1 (Ang-1), and angiopoietin-2 (Ang-2)] in EPS rat models. Twenty-four rats were given a daily intraperitoneal injection of chlorhexidine gluconate and ethanol dissolved in saline (CH). Nine rats were injected with CH and anti-VEGF neutralizing antibody simultaneously. Quantitative blood vessel evaluation was performed by staining for GS1-lectin. The mRNA expression of VEGF, Ang-1, Ang-2, and their receptors was evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemical staining was performed in peritoneal vessels using anti-VEGF, Ang-1, and Ang-2 antibodies. Hematopoietic stem cells were detected using anti-CD34 antibody. The vessel area, diameter, and length gradually increased until day 21, and then decreased. VEGF and Ang-2 mRNA expressions gradually increased until day 35. In contrast, Ang-1 peaked at day 21 and then decreased significantly. VEGF blockade improved the experimental EPS. In immunohistochemistry, the vessels stained by VEGF and Ang-2 were detected in subfibrous layer. CD34-positive cells were markedly stained at day 21. Neoangiogenesis was observed in a rat model of experimental EPS. VEGF and angiopoietin/Tie system play an important role in the neoangiogenesis in this model. An analysis using this experimental rat model may elucidate the development of EPS in peritoneal dialysis patients.

Interleukin-1beta regulates angiopoietin-1 expression in human endothelial cells.

Angiopoietin (Ang)-1 is an important regulator of endothelial cell (EC) survival and stabilization. Ang-1 exerts its biological effects by binding to the EC-specific tyrosine kinase receptor Tie-2, and initiates intracellular signaling in ECs. However, regulatory mechanisms for endothelial Ang-1 expression have not been completely elucidated. In this study, we investigated the effects of angiogenic cytokines and growth factors on Ang-1 expression in human umbilical vein ECs (HUVECs). Northern blot analysis was performed after HUVECs were exposed to interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, platelet-derived growth factor-BB, insulin-like growth factor-1, or vascular endothelial growth factor (VEGF). Both IL-1beta and tumor necrosis factor-alpha caused marked down-regulation of Ang-1 mRNA levels at 4 h with a further decrease observed at 24 h. Using signaling inhibitors, we identified the P38 pathway as the pathway that mediates IL-1beta down-regulation of Ang-1. Furthermore, treatment of cells with IL-1beta indirectly (via down-regulation of Ang-1) led to a decrease in Tie-2 autophosphorylation levels in HUVECs. We previously demonstrated that IL-1beta regulates VEGF expression in tumor cells. This observation was confirmed in ECs in the present study. Because pericytes play a role in regulating EC function, we also determined whether IL-1beta would also down-regulate Ang-1 in human vascular smooth muscle cells. Similar to our findings in HUVECs, we found that IL-1beta decreased Ang-1 expression in human vascular smooth muscle cells. Direct effects of IL-1beta on angiogenesis were investigated by use of an in vivo Gelfoam angiogenesis assay in which IL-1beta produced a significant increase in vessel counts (P = 0.0189). These results suggest that IL-1beta indirectly regulates angiogenesis by modulating the expression of Ang-1. IL-1beta may trigger a proangiogenic response by decreasing Ang-1 levels in ECs and pericytes and up-regulating VEGF in ECs and tumor cells.

Hypoxic Regulation of Angiopoietin-2 Expression in Endothelial Cells

Exposure of endothelial cells to hypoxia-induced angiopoietin-2 (Ang2) expression. The increase in Ang2 mRNA levels occurred by transcriptional regulation and by post-transcriptional increase in mRNA stability. Induction of Ang2 mRNA resulted in an increase of intracellular and secreted Ang2 protein levels. Since the transcriptional regulation of several genes involved in angiogenesis during hypoxia is mediated by hypoxia-inducible factor-1 (HIF-1), it was conceivable that Ang2 expression might be regulated by the same oxygen-dependent mechanism. However, our data showed that pharmacological HIF inducers, CoCl(2) and DFO, did not affect Ang2 expression. Moreover, HIF-1-deficient hepatoma cell (Hepa1 c4) and its wild-type counterpart (Hepa1 c1c4) up-regulates Ang2 during hypoxia. These results indicated that hypoxia-driven Ang2 expression may be independent of the HIF pathway. Using neutralizing VEGF antibody or pharmacological inhibitors of VEGF receptors, we showed that hypoxia-induced VEGF participates but could not account completely for Ang2 expression during hypoxia. In addition, hypoxia elicited an increase of cyclooxygenase-2 (COX-2) expression and a parallel increase in prostanglandin E(2) (PGE(2)) and prostacyclin (PGI(2)) production. COX-2 inhibitors decreased the hypoxic induction of Ang2 and the hypoxic induction of PGE(2) and PGI(2) in a dose-dependent manner. Similarly, COX-2 but not COX-1 antisense treatment decreased hypoxic induction of Ang2 expression, and this effect was reversed by exogenous PGE(2). Finally, exogenous PGE(2) and PGI(2) were able to stimulate Ang2 under normoxic conditions. These findings suggest that COX-2-dependent prostanoids may play an important role in the regulation of hypoxia-induced Ang2 expression.

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.

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.

Endoluminal aortic stent grafts — morphology of complications

Infantile hemangiomas are endothelial tumors that grow rapidly in the first year of life and regress slowly during early childhood. Although hemangiomas are well-known vascular lesions, little is known about the mechanisms that cause the excessive endothelial cell proliferation in these most common tumors of infancy. To investigate the molecular basis of hemangioma, we isolated endothelial cells from several proliferative-phase lesions and showed that these cells are clonal and exhibit abnormal properties in vitro (E. Boye, Y. Yu, G. Paranya, J. B. Mulliken, B. R. Olsen, J. Bischoff: Clonality and altered behavior of endothelial cells from hemangiomas. J Clin Invest 2001, 107:745–752). Here, we analyzed mRNA expression patterns of genes required for angiogenesis, including members of the vascular endothelial growth factor (VEGF)/VEGF receptor family and the angiopoietin/Tie family, in hemangioma-derived and normal endothelial cells. KDR, Flt-1, Tie1, Tie2, and angiopoietin-2 (Ang2) were strongly expressed in cultured hemangioma-derived endothelial cells and in hemangioma tissue. In contrast, there was little expression of angiopoietin-1 (Ang1) or VEGF. We found Tie2 mRNA and protein up-regulated with a concomitant increase in cellular responsiveness to Ang1 in most hemangioma-derived endothelial cells. Ang2 mRNA was down-regulated in response to serum in hemangioma-derived endothelial cells, but not in normal endothelial cells, suggesting altered regulation. These findings implicate Tie2 and its ligands Ang1 and Ang2 in the pathogenesis of hemangioma.

Expression of angiopoietin-2 gene in non-small cell lung cancer.

To study the expression of Angiopoietin 2 (Ang-2) gene and its relationship with clinical pathological characteristics of non-small cell lung cancer (NSCLC), expression of the Ang-2 mRNA was evaluated by employing reverse transcription polymerase chain reaction (RT-PCR) in cancerous tissues and paired adjacent noncancerous tissues from 52 patients. The expression of Ang-2 gene was detected in a significantly greater proportion of cancerous tissues (80.8%) than paired adjacent noncancerous tissues (53.8%, P<0.01). No significant relationship was found between Ang-2 gene expression and patients' age, sex, histology of tumors, differentiation and TNM stages (P>0.05). It is concluded that the up-regulation of Ang-2 gene may play a role in the pathway of NSCLC carcinogenesis and Ang-2 may be used as a potential therapeutic agent for lung cancer.

Differential expression of angiopoietins 1 and 2 and their receptor Tie-2 in human endometrium

Angiogenesis, the growth of new capillaries from pre-existing blood vessels, is a physiological process involved in both normal menstrual cycling and implantation of the embryo. So far, very little is known about the expression of angiopoietins, growth factors involved in angiogenesis, in human endometrium. Both angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are ligands for the endothelial cell-specific receptor tyrosine kinase Tie-2. In this study we determined the mRNA expression of Ang-1, Ang-2 and Tie-2 by quantitative competitive RT/(QC)-PCR (including specifically designed competitor cDNA) in biopsied human endometrium throughout the menstrual cycle. We detected the mRNA for the angiopoietins in 30 out of 32 endometrial biopsies (94%), covering early proliferative (n = 4), mid proliferative (n = 12), late proliferative (n = 3), early secretory (n = 3), mid secretory (n = 5) and late secretory (n = 3) phases. Analysis of the target/competitor ratios (QC-PCR) revealed that Ang-1 mRNA expression was significantly up-regulated (P = 0.027) during the secretory phase of the menstrual cycle. In contrast, the expression levels of both Ang-2 mRNA and Tie-2 mRNA showed only minor variations at different cycle stages. These findings were confirmed by the relative expression ratio of Ang-1 versus Ang-2 in a multiplex PCR. The expression of Ang-1, Ang-2 and Tie-2 mRNA was detected in both isolated endometrial epithelial and stromal cell fractions. Immunohistochemical localization of the proteins revealed qualitative differences in both cell type and cycle stage expression. In conclusion, the enhanced Ang-1 expression during the secretory phase might serve to stabilize the newly developed blood vessels.