Ang-2 Gene Expression Research Articles

Angiopoietin-1 targeted RNA interference suppresses angiogenesis and tumor growth of esophageal cancer

To determine the inhibitory effect of the adenovirus-based angiopoietin-1 (Ang-1) targeted small interfering RNA expression system (Ad/Ang-1si) on the expression of the Ang-1 gene, cell growth and apoptosis in human esophageal cancer cell line Eca109. siRNA-expressing adenovirus targeting Ang-1 gene was constructed using the Ad Easy System. Cultured Eca109 cells were transfected with Ad/Ang-1si (Eca109/Ang-1si), and Ad/si was used to infect Eca109 cells as control (Eca109/si). Ang-1 gene expression and concentration was determined with RT-PCR and ELISA, respectively. Human umbilical vein endothelial cell (HUVEC) migration and proliferation were analyzed. After s.c. injection into athymic nu/nu mice, the tumor growth, vessel density and apoptosis of each group was also determined. HUVEC migration induced by conditioned medium from Ang-1si-transfected Eca109 cells was significantly less than that induced by conditioned medium from Eca109 cells and control adenovirus-transfected Eca109 cells. Furthermore, after s.c. injection into athymic nu/nu mice, the tumor growth and cell apoptosis of Ad/Ang-1si -expressing Eca109 cells was significantly lower than that of parental or control adenovirus-transfected cells. Vessel density assessed by CD31 immunohistochemical analysis and Ang-1 expression by RT-PCR were also decreased. The targeting Ang-1 may provide a therapeutic option for esophageal cancer.

ANGIOTENSIN II DEPENDENT GENE REGULATION IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS INVOLVES VEGF-SIGNALING AND ANGIOGENESIS

The present study examined the effect of angiotensin II on the regulation of different angiogenesis associated genes in human endothelial umbilical vein cells (HUVEC) and investigated whether administration of At1R blocker candesartan could suppress this angiotensin II dependent proangiogenic effect. Angiotensin II dependent expression of different angiogenesis-associated genes was analysed in HUVECs 1) after stimulation with angiotensin II, 2) after blocking of At1R by candesartan and 3) after neutralizing of vascular endothelial growth factor (VEGF) by VEGF trap FLT-Fc. Gene expression of VEGF, Angiopoeitin 1 (Ang1), Angiopoeitin 2 (Ang2), Von-Hippel-Lindau-Tumorsuppressorgene (VHL), tissue inhibitor of matrix metalloproteinases 1 (TIMP-1), and hypoxia inducible transcription factor 2alpha (HIF-2alpha) was investigated using TaqMan Real-Time-PCR. A possible proangiogenic effect of angiotensin II on HUVEC was investigated using ki-67 immunocytofluorescence. VEGF, Ang1, VHL, TIMP-1, HIF-2alpha and the At1R were expressed in HUVEC. Angiotensin II significantly increased gene expression of VEGF and VHL and decreased gene expression of Ang1. Expression of Ang2, TIMP-1, and HIF-2alpha were not affected significantly. In addition significantly increased endothelial cell proliferation was observed after stimulation with angiotensin II. This was suppressed by simultaneous administration of candesartan or VEGF trap FLT-Fc. It is hypothesized that angiotensin II influences angiogenesis by regulation of angiogenesis associated genes via At1R. Since VEGF inhibition opposed the effect of angiotensin II on cell proliferation, we suggest VEGF acts as a mediator of this angiotensin II-dependent effect. In addition, we hypothesize, that targeting angiogenesis by inhibition of At1R may lead to new therapeutical anti-angiogenic strategies. (poster)

Expression of heparanase and angiopoietin-2 in patients with endometriosis

Objective The objective was to investigate the expression of heparanase (Hpa) and angiopoietin-2 (Ang-2) in endometriosis. Study design In ectopic and eutopic endometrium of patients undergoing laparoscopy for endometriosis ( n = 86) and in normal endometrium of patients undergoing laparoscopic tubal ligation or hysteroscopic resection because of uterus septus ( n = 30), we determined Hpa and Ang-2 gene expression by RT-PCR. To support the mRNA data, the expression of Hpa and Ang-2 protein was measured by Western blot analysis. Finally, Hpa and Ang-2 in these tissues was localized by immunohistochemical staining. Result(s) The positive rate of Hpa and Ang-2 mRNA in ectopic and eutopic endometrium in the study group was significantly higher than that in normal endometrium in the control group. In the study group, ectopic and eutopic endometrium expressed a higher positive rate of Hpa and Ang-2 protein, whereas in the control group, normal endometrium expressed a lower positive rate of Hpa and Ang-2 protein. In eutopic and ectopic endometrium, there was balanced expression between Hpa and Ang-2. Both Hpa and Ang-2 showed a balanced expression between eutopic and ectopic endometrium. In ectopic endometrium, strong staining for Hpa and Ang-2 was observed both in epithelial cells and in stromal cells, but in eutopic endometrium, Hpa and Ang-2 were mainly expressed in epithelial cells. Conclusion The higher expression of Hpa and Ang-2 in ectopic and eutopic endometrium may play an important role in the pathogenesis and development of endometriosis.

Angiopoietin-2 Expression in B-Cell Chronic Lymphocytic Leukemia: Association with Clinical Outcome and Immunoglobulin Heavy-Chain Mutational Status.

B-cell Chronic Lymphocytic Leukemia (B-CLL) follows an extremely variable clinical course. For some patients CLL is an indolent disease that never progresses to the point of requiring therapy and these patients have a survival time similar to age-matched controls. On the contrary, other patients experience rapidly deteriorating blood count and organomegaly which requires prompt treatment. The overall survival (OS) times range from months to decades. B-CLL patients can be divided into two subgroups on the basis of the presence or absence of somatic mutations in the specific immunoglobulin heavy-chain variable region (IgVH) genes used by leukemic cells. Patients with unmutated IgVH genes usually have an advanced stage and an unfavourable cytogenetic features, require therapy and have a short survival. The biological reasons of the different behaviour of Ig-mutated and Ig-unmutated leukemic clones have not been fully elucidated yet. Angiogenesis is a very complex network which is closely regulated by the orchestrating functions of many angiogenic factors. The balance of cellular expression of all these angiogenic factors determines vascular stabilization or angiogenic remodelling and sprouting or vessel regression. Increasing evidence shows that neovascularization plays a role in the biology of chronic lymphocytic leukemia. The goal of this study was to evaluate the angiogenic status of Ig-mutated and Ig-unmutated CLL in the attempt to identify a possible role of angiogenesis in the adverse clinical outcome of Ig-unmutated CLL patients. So, we first performed a large scale gene-expression analysis on 29 B-CLL patients using microarrays comprising about 20,000 probes and 208 angiogenesis-related genes. We identified 64 up-regulated genes in Ig-unmutated CLL relative to Ig-mutated CLL. Among them, we found angiopoietin-2 (Ang-2) as one of the highest differentially expressed gene (p=3.02x10−6). Then, we evaluated the Ang-2 expression both at transcript and protein level in a wide cohort of CLL, in normal controls and in other haematological malignancies. The data showed an extremely wide range of Ang-2 expression in B-CLL: Ang-2 high-expressing cases were characterized by advanced Binet stage (p=0.032) and significantly shorter progression-free survival than Ang-2 low-expressing subset (median, 16 vs. 146 months) (p=0.006). Moreover, there was a strong correlation between the IgVH mutational status and the Ang-2 gene expression level (p<0.0001). Ig-mutated CLL exhibited very low Ang-2 expression, absolutely similar to the levels measured in healthy controls (median, 0.27 vs. 0.32, p=0.959). On the contrary, Ig-unmutated CLL expressed up to one hundred-fold higher levels of Ang-2 than normal controls(median, 38.61 vs. 0.32, p=0.002). Moreover, Ang-2 was up-regulated in all investigated CML, ALL and AML samples. We observed extremely high levels of expression in CML and ALL (median Ang-2 mRNA, 3948.96 and 190.00, respectively), whereas moderately high levels of Ang-2 were found in AML patients (median, 16.21). These data suggest that increased angiogenesis due to high Ang-2 expression may be involved in adverse clinical outcome of Ig-unmutated CLL patients.

Effects of angiogenic factor overexpression by human and rodent cholangiocytes in polycystic liver diseases

Liver involvement in autosomal dominant polycystic kidney disease (ADPKD) is characterized by altered remodeling of the embryonic ductal plate (DP) with presence of biliary cysts and aberrant portal vasculature. The genetic defect causing ADPKD has been identified, but mechanisms of liver cyst growth remain uncertain. To investigate the possible role of angiogenic mechanisms, we have studied the immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and their receptors (VEGFR-1, VEGFR-2, Tie-2) in ADPKD, Caroli's disease, normal and fetal livers. In ADPKD and control livers Ang-1 and Ang-2 gene expression was studied by real-time-PCR. Effects of VEGF on cholangiocyte proliferation were studied by PCNA Western Blot in isolated rat cholangiocytes and by MTS assay in cultured cholangiocytes isolated from ADPKD patients and from an ADPKD mouse model (Pkd2(WS25/-)). Cholangiocytes were strongly positive for VEGF, VEGFR-1, VEGFR-2 and Ang-2 in ADPKD and Caroli, and also for Ang-1 and Tie-2 in ADPKD, similar to fetal ductal plate cells. VEGF stimulated proliferation in both normal and ADPKD cholangiocytes, but the effect was particularly evident in the latter. Ang-1 alone had no effect, but was synergic to VEGF. VEGF expression on cholangiocytes positively correlated with microvascular density. In conclusion, consistent with the immature phenotype of the cystic epithelium, expression of VEGF, VEGFRs, Ang-1 and Tie-2 is strongly upregulated in cholangiocytes from polycystic liver diseases. VEGF and Ang-1 have autocrine proliferative effect on cholangiocyte growth and paracrine effect on portal vasculature, thus promoting the growth of the cysts and their vascular supply. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

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.

Temporal renal expression of angiogenic growth factors and their receptors in experimental diabetes

It has been postulated that vascular endothelial growth factor (VEGF) plays a role in the progression of renal injury. However, the role of other angiogenic factors and their receptors, such as the angiopoietins and Tie2, and in particular their relation to renoprotective therapies, such as agents that interrupt the renin-angiotensin system, have not been studied in the context of diabetes-related renal injury. Renal expression of VEGF, angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and their receptors, VEGF-R2 and Tie-2, were assessed using reverse transcription-polymerase chain reaction, immunohistochemistry and Western blotting, in control and streptozotocin diabetic rats, untreated or receiving the AT1 receptor antagonist, valsartan, or the AT2 receptor antagonist, PD123319. Diabetes was associated with increased gene and protein expression of VEGF, VEGF-R2, Ang-1, Ang-2 and Tie-2. AT1 receptor antagonism attenuated gene expression of these cytokines and receptors, yet PD123319, which had no effect on blood pressure, reduced VEGF-R2 and Ang-1 gene expression and decreased VEGF, Ang-1 and Ang-2 protein levels. In experimental diabetes, there is significant upregulation within the kidney of various angiogenic cytokines and their receptors. Furthermore, the effects of angiotensin II receptor blockade on these parameters is consistent with the VEGF-VEGF-R2 and angiopoietin-Tie-2 axes being modulated in the kidney by haemodynamic factors in the diabetic context.

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.

174 Expression of Angiopoietin-2 and Endostatin in Intrauterine Growth Restriction During The Perinatal Period

Background: Angiopoietin-2 (Ang2) is an endothelial cell-specific growth factor, which acts by blocking stabilization and maturation of vessels allowing them to remain in a more plastic state and respond to sprouting signals provided by other angiogenic factors. Ang2 gene expression is up-regulated by hypoxia, present in intrauterine growth restriction (IUGR) pregnancies. On the contrary, hypoxia down-regulates production of endostatin (End), a potent angiostatic factor derived from collagen XVIII. This study aimed at investigating circulating Ang2 and End levels in maternal blood (MS) during the first stage of labor, in the doubly clamped umbilical cord (UC) at delivery, representing fetal state and in the neonate in the first (N1) and fourth (N4) day of life, reflecting transition and stabilization to extrauterine life, respectivelyMethods: Ang2 and End were determined by enzyme immunoassay methods in serum, deriving from 20 fullterm appropriate for gestational age (AGA) and 40 fullterm IUGR infants, as well as from their mothers.Results: Statistical significant difference was noted in N4 Ang2, being higher in IUGR (p=0.030), (while in N1 Ang2 was only indicative-p=0.057) and in UC End, being lower in IUGR (p=0.002), as compared to AGA cases. Statistical significant correlations existed for Ang2 between: MS and UC, N1, N4 (p=0.000, p=0.001, p=0.017 respectively), UC and N1, N4 (p=0.000, p=0.000 respectively), N1 and N4 (p=0.000) and for End between N1 and N4 (p=0.006). In the IUGR group variables presenting a statistically significant association with: a) MS Ang2, were gestational age (p=0.011), placental weight (p=0.013), gender (p=0.002) and birth length (p=0.023), b) UC Ang2, was gestational age (p=0.042), c) N1 Ang2, were gestational age (p=0.000) and birth weight (p=0.003) d) N4 Ang2, were gestational age (p=0.000) and birth weight (p=0.023), e) MS End, were gender (p=0.003), gestational age (p=0.007) and birth length (p=0.006), f) UC End, were placental weight (p=0.038) and gender (p=0.031), g) N1 End, was gender (p=0.050).Conclusion: High neonatal Ang2 and low fetal End is documented in IUGR infants, possibly due to intrauterine action of hypoxia on these factors. In IUGR fetuses End is associated with placental weight. Similarly, Ang2 in IUGR fetuses is associated with gestational age and in IUGR neonates with gestational age and birth weight, in general indicating the impact of these factors on angiogenesis and tissue growth.

ESE-1 Is a Novel Transcriptional Mediator of Angiopoietin-1 Expression in the Setting of Inflammation

Angiogenesis is a critical component of the inflammatory response associated with a number of conditions. Angiopoietin-1 (Ang-1) is an angiogenic growth factor that promotes the chemotaxis of endothelial cells and facilitates the maturation of new blood vessels. Ang-1 expression is up-regulated in response to tumor necrosis factor-alpha (TNF-alpha). To begin to elucidate the underlying molecular mechanisms by which Ang-1 gene expression is regulated during inflammation, we isolated 3.2 kb of the Ang-1 promoter that contain regulatory elements sufficient to mediate induction of the promoter in response to TNF-alpha, interleukin-1beta, and endotoxin. Surprisingly, sequence analysis of this promoter failed to reveal binding sites for transcription factors that are frequently associated with mediating inflammatory responses, such as NF-kappaB, STAT, NFAT, or C/EBP. However, putative binding sites for ETS and AP-1 transcription factor family members were identified. Interestingly, among a panel of ETS factors tested in a transient transfection assay, only the ETS factor ESE-1 was capable of transactivating the Ang-1 promoter. ESE-1 binds to specific ETS sites within the Ang-1 promoter that are functionally important for transactivation by ESE-1. ESE-1 and Ang-1 are induced in synovial fibroblasts in response to inflammatory cytokines, with ESE-1 induction slightly preceding that of Ang-1. Mutation of a high-affinity ESE-1 binding site leads to a marked reduction in Ang-1 transactivation by ESE-1, inducibility by inflammatory cytokines, and DNA binding to the ESE-1 protein. Transcriptional profiling of cells transiently transfected with an ESE-1 expression vector demonstrates that the endogenous Ang-1 gene is directly inducible by ESE-1. Finally, Ang-1 and ESE-1 exhibit a similar and strong expression pattern in the synovium of patients with rheumatoid arthritis. Our results support a novel paradigm for the ETS factor ESE-1 as a transcriptional mediator of angiogenesis in the setting of inflammation.

Expression of the angopoietin-1, angopoietin-2, Tie2, and vascular endothelial growth factor gene in epithelial ovarian cancer

Objective. Angiopoietin/Tie2 system with vascular endothelial growth factor (VEFG) is known to be important for the initiation of angiogenesis in tumors. The aim was to evaluate whether angiopoietin/Tie2 system with VEFG affects prognosis in patients of epithelial ovarian cancer. Methods. Angiopoietin-1 ( Ang-1), Angiopoietin-2 ( Ang-2), Tie2, and VEGF gene expression were analyzed by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) in 85 epithelial ovarian cancer surgical specimens. These gene expressions were correlated with clinical–pathological parameters, microvessel density (MVD), and patients' survival. Results. Ang-1/Ang-2 gene expression ratio, VEGF, and Tie2 gene expression significantly associated with MVD, respectively ( P < 0.0001, P = 0.024, P = 0.005). The patients with low Ang-1/Ang-2 gene expression ratio and high VEGF gene expression were found to have a significantly higher MVD when compared to others ( P = 0.0003). Moreover, there was a significant difference between the values of MVD in patients with low Ang-1/Ang-2 gene expression ratio and high VEGF and high Tie2 gene expression and those in others ( P = 0.0025). FIGO stage ( P = 0.014), residual disease ( P = 0.042), histological grade ( P = 0.028), Ang-1/Ang-2 gene expression ratio ( P = 0.010), and combination of Ang-1/Ang-2 gene expression ratio and VEGF gene expression ( P = 0.019), were found to be significantly associated with a poor prognosis in univariate Cox regression analysis. Multivariate Cox regression analysis revealed that FIGO stage is an independent prognostic factor ( P = 0.035). Low Ang-1/Ang-2 gene expression ratio had a tendency to be an independent prognostic factor ( P = 0.061). Conclusion. Angiogenesis occurred by angiopoietin/Tie2 system in concert with VEGF in epithelial ovarian cancer did not affect patients' survival. However, gene expression of Ang-1 and Ang-2 might present a pertinent diagnostic tool to select a high-risk group of patients independent of clinical–pathological parameters and a new insight to understand the biology of epithelial ovarian cancer.

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.

Increased renal vascular endothelial growth factor and angiopoietins by angiotensin II infusion is mediated by both AT1 and AT2 receptors.

A link between angiotensin II and cell proliferation has previously been reported. However, there remains controversy as to the role of the individual angiotensin II receptor subtypes in mediating these effects and their link to angiogenic cytokines and their receptors. Male Sprague-Dawley rats were infused with either angiotensin II or vehicle for 14 d at a dose of 58.3 ng/min. Angiotensin II-infused rats received no treatment, an AT(1) receptor antagonist valsartan (30 mg/kg per d), or an AT(2) receptor antagonist PD123319 (830 ng/min). Gene expression of vascular endothelial growth factor (VEGF) and receptor VEGF-R2, as well as Tie-2 and its ligands angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) were assessed by reverse transcription-PCR. Protein expression was assessed by Western blotting and immunohistochemistry. Gene and protein expression of VEGF, Ang-1, and Ang-2 were increased by angiotensin II infusion. Valsartan and PD123319 attenuated angiotensin II-associated increases in VEGF gene and protein expression. Ang-1 and Ang-2 gene but not protein expression were reduced by both treatments. These changes occurred in the context of attenuation of angiotensin II-induced glomerular cell proliferation by both valsartan and PD123319. In situ hybridization and immunohistochemical studies localized VEGF, Ang-1, and Ang-2 expression to the epithelial cells of the glomerulus, and VEGF-R2 and Tie-2 receptors to the endothelial cells of the kidney. These findings extend the increasing evidence that the AT(2) receptor, in addition to the AT(1) receptor subtype, plays an important role in mediating the proliferative actions of angiotensin II in the kidney.

Evaluation of the antiangiogenic effect of Taxol in a human epithelial ovarian carcinoma cell line.

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are major ligands for the endothelium-specific tyrosine kinase receptor Tie-2 and are important regulators of endothelial cell survival. In the presence of vascular endothelial growth factor (VEGF), vessel destabilization by Ang-2 has been hypothesized to induce an angiogenic response, but in the absence of VEGF, Ang-2 leads to vessel regression. In the present study, a human ovarian cancer cell line was used to investigate the possibility that Taxol might affect the expression of Ang-1, Ang-2, and VEGF. KF 28, a single-cell clone of a human ovarian epithelial carcinoma cell line, was used. The expression of Ang-1, Ang-2, and VEGF was assessed by quantitative real-time RT-PCR and Western blot analysis or enzyme-linked immunosorbent assay. Conditioned medium was used in the in vitro angiogenesis assay. The concentration of Taxol that inhibited the growth of cells to the level of 50% of control cell growth was 4.65+/-0.35 nM. Quantitative real-time RT-PCR indicated that Ang-1 gene expression was significantly decreased by exposure to 2 nM Taxol for 168 h ( P<0.05 vs control cells). Western blot analysis confirmed that the Ang-1 protein level was decreased by exposure to 2 nM Taxol for 168 h. Ang-2 gene expression did not significantly differ between control cells and those exposed to Taxol for any of the indicated times. The Ang-1/ Ang-2 gene expression ratio was significantly decreased by exposure to Taxol for 168 h ( P<0.05 vs control cells). VEGF gene expression was significantly decreased by exposure to Taxol for 168 h ( P<0.05). The VEGF concentration in the conditioned medium was also significantly reduced by exposure to Taxol for 168 h ( P<0.05). Conditioned medium collected following Taxol treatment for 168 h significantly inhibited endothelial tubule formation ( P<0.05). Cell growth did not significantly differ between control cells and those exposed to Taxol for any of the indicated times. Our results show that exposure of ovarian cancer cells to a low concentration of Taxol may inhibit the initiating event in angiogenesis, namely, vascular regression. This information might be valuable in the development of new therapeutic interventions for epithelial ovarian cancer.

Angiopoietin-1 and vascular endothelial growth factor expression in human esophageal cancer

Angiopoietin-1 (Ang-1) has been shown to act as an angiogenic promoter in embryonic angiogenesis by promoting vascular branching, pericyte recruitment and endothelial survival. Ang-1 expression has not been examined in human esophageal cancer. We examined Ang-1 and vascular endothelial growth factor (VEGF) gene expression in tumors from 45 esophageal cancer patients who underwent surgical resection. Forty (88.9%) of the 45 esophageal cancers revealed Ang-1 gene expression. VEGF121, VEGF165 and VEGF189 isoforms were detected in 93.3 (42/45), 55.6 (25/45) and 26.7% (12/45) of the cases, respectively. Ang-1 gene expression was significantly correlated with VEGF121 and VEGF165 gene expression (P=0.0289 and P=0.0127, respectively, Fisher's test). The results suggest that Ang-1 is associated with neovascularization in the cancer stroma through VEGF net-works in esophageal cancer.

Expression of the angiopoietins and their receptor Tie2 in human renal clear cell carcinomas; regulation by the von Hippel-Lindau gene and hypoxia.

Angiogenesis is essential for tumour growth and metastasis, and is co-ordinated by several classes of angiogenic factors. To determine the significance and regulation of the angiopoietin (Ang) pathway in highly vascular human renal cell carcinomas (RCCs), this study has investigated the expression of the Ang-1, Ang-2, Ang-4, and Tie2 genes in a series of normal (n = 26) and neoplastic (n = 45; clear cell n = 35, papillary n = 10) human kidney tissues, examined the pattern of Ang-2 and Tie2 protein expression, and correlated expression with clinicopathological variables. The effect of the von Hippel-Lindau (VHL) gene and hypoxia in the renal cell lines RCC786-0 and RCC4 has also been investigated. Ang-1, Ang-2 and Tie2, but not Ang-4 mRNA, were detected in normal and tumour samples. A significant increase in Ang-2 (p < 0.001) and a decrease in Tie2 receptor mRNA (p = 0.001) were observed, but no significant difference was observed in Ang-1 mRNA abundance between normal kidney and RCC (p = 0.37). Immunohistochemistry for Ang-2 showed strong expression in vascular endothelium and weak expression in tumour cells, whereas Tie2 was expressed exclusively on endothelium. Tie2 gene expression was positively correlated with Ang-2 expression in cancers (p = 0.001) and showed a borderline significant association with Ang-1 (p = 0.06), but there was no significant relationship between Ang-1 and Ang-2 (p = 0.69). No significant relationships were observed in clear cell carcinomas between Ang-1, Ang-2 and Tie2 mRNA abundance and patient sex, patient age, or tumour size (p > 0.05). However, there was significantly greater Ang-1 (p = 0.02), Ang-2 (p = 0.03), and Tie2 (p = 0.04) mRNA abundance in clear cell than in chromophil RCCs. Ang-2 gene expression was down-regulated by hypoxia in VHL wild-type RCC786-0 and RCC4 transfectants (p = 0.0002 and p = 0.04, respectively), mirroring the low expression in human tumour cells. These data suggest that it is endothelial induction of Ang-2 in tumours that regulates vessel stability and supports targeting Tie2 as an effective novel anti-angiogenic therapy in clear cell RCCs.

Expression of Angiopoietin-1, Angiopoietin-2, and Tie2 Genes in Normal Ovary with Corpus luteum and in Ovarian Cancer

Objective: The recent discovery of angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) has provided novel and important insights into the molecular mechanisms of blood vessel formation. Ang1 and Ang2 bind with similar affinity to the endothelial cell tyrosine kinase receptor Tie2. Our purpose was to assess the potential role of the Ang/Tie2 system in physiological and pathological angiogenesis in the ovary. Methods:Ang1, Ang2, and Tie2 gene expression in 14 normal ovaries with corpus luteum (CL) and in 19 cases of ovarian cancer were analyzed by polymerase chain reaction of RNA after reverse transcription. The level of each gene expression was presented by the relative yield of each gene to the β₂-microglobulin gene, respectively. Furthermore, cellular distribution of Ang1 and Ang2 mRNA was examined by in situ hybridization, and localization of Tie2 was studied by immunochemistry. Results: The Ang1, Ang2, and Tie2 gene expression in normal ovary with CL ranged from 0.18 to 1.06 (median 0.54), 0.31–2.64 (median 1.01), and 0.10–0.47 (median 0.20), respectively. The expression of these same genes in ovarian cancer ranged from 0.06 to 0.75 (median 0.14), 0.69–1.59 (median 1.12), and 0.04–0.35 (median 0.15), respectively. Ang1 gene expression in normal ovary with CL was significantly higher than that in ovarian cancer (p = 0.0004). The gene expression levels of Ang2 and Tie2 were statistically the same in both groups. There was a significant correlation between Ang1 gene expression and Tie2 gene expression in normal ovary with CL (r = 0.619, p = 0.018). No such significant correlation was found in ovarian cancer. Moreover, Ang2 gene expression showed no significant correlation with the Tie2 gene expression either in normal ovary with CL or in ovarian cancer. Transcripts for Ang1 were observed in CL cells and endothelial cells around CL, and in tumor cells and endothelial cells at the periphery of tumor invasion. Ang2 transcripts were expressed in the same patterns. Tie2 expression was positive primarily in the endothelial cells around CL and in those at the periphery of tumor invasion. Conclusion: Our results indicate that there is a difference in the Ang/Tie2 gene expression between physiological and pathological angiogenesis in the ovary. This finding may aid in the development of new therapeutic interventions for ovarian cancer.

Gene expression of angiogenesis related factors in glioma.

Angiogenesis plays an important role in growth and proliferation of cancer. Various angiogenic and angiostatic factors regulate angiogenesis. In this study, we examined gene expression of the angiopoietin family including angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2) in 39 gliomas and 5 glioma-xenografts by RT-PCR. Ang1 and Ang2 genes were expressed in 54%, and 77% of gliomas, respectively. The expression of Ang1 was significantly correlated with the expression of Ang2. Both Ang1 and Ang2 were shown to be expressed in the glioma cells. Ang2 gene expression was correlated with VEGF gene expression. Angiopoietin molecules may synergistically cooperate in growth and vascularization in glioma.