Ang-2 mRNA Research Articles

Correction: Circulating Ang-2 Mrna Expression Levels: Looking ahead to a New Prognostic Factor for NSCLC

Correction: Circulating Ang-2 Mrna Expression Levels: Looking ahead to a New Prognostic Factor for NSCLC

Circulating Ang-2 mRNA Expression Levels: Looking Ahead to a New Prognostic Factor for NSCLC

Non-small cell lung cancer (NSCLC) is the most common cancer and the leading cause of death from cancer worldwide. Antiangiogenic strategies directed towards tumor stroma are becoming gold standard in NSCLC treatment and researchers have been searching for biomarkers to identify patients for whom therapy with antiangiogenic inhibitors may be most beneficial and the importance of these as prognostic factors in NSCLC. The purpose of this study was to evaluate the prognostic value of circulating Ang-2 mRNA levels prior to treatment in NSCLC patients. The mRNA levels were determined by quantitative real-time PCR in the peripheral blood of 92 NSCLC patients. Our results demonstrate that patients with high circulating Ang-2 mRNA levels have diminished overall survival when compared to those with low mRNA levels (20.3 months vs 34.3 months, respectively; Log Rank Test, p = 0.016), when considering all NSCLC stages and this difference is even bigger when considering only patients with stage IV (15.9 months vs 31.3 months, respectively; Log Rank Test, p = 0.036). Moreover, circulating Ang-2 mRNA levels independently determine overall survival, and the concordance (c) index analysis showed that the definition of a nomogram that contains information regarding tumor stage, patients' smoking status and circulating Ang-2 mRNA levels present an increased capacity to predict overall survival in NSCLC patients (c-index 0.798). These results suggest that this nomogram could serve as a unique and practical tool to determine prognosis in NSCLC, not relying on the availability of adequate surgical or biopsy specimens of NSCLC. Attending to our results, the circulating Ang-2 mRNA levels should also be included in the design of preclinical studies and clinical trials involving antiangiogenic drugs targeting Ang-2, to guide adequate patient stratification and dose selection and increasing the likelihood of benefit to a level that is acceptable to patients and clinicians.

Therapeutic Effect of Alprostadil in Diabetic Nephropathy: Possible Roles of Angiopoietin-2 and IL-18

Background/Aims: To investigate the role of angiopoietin-2 (Ang-2) and IL-18 in the pathogenesis of diabetic nephropathy (DN) and the molecular mechanisms through which alprostadil protects renal function. Methods: DN was induced by streptozotocin and intraperitoneal injection of alprostadil was given to diabetic mice. After 2, 4 and 8 weeks of alprostadil treatment, the mRNA and protein expression of kidney Ang-2 and IL-18 were detected by reverse transcription PCR, Western blot and immunohistochemistry analyses. Mouse glomerular endothelial cells (GEnCs) were cultured in high glucose and treated with alprostadil. After transfection with an Ang-2-pcDNA and Ang-2-siRNA, both Ang-2 and IL-18 expression were measured by Western blot analyses. Results: Alprostadil treatment caused a significant decrease in the renal damage parameters. Both Ang-2 and IL-18 were significantly increased in DN mice and in GEnCs cultured in high glucose; however, their expression was greatly reduced by alprostadil treatment. Ang-2 could also increase IL-18 expression in cultured endothelial cells under high glucose, and this response was partially blocked by Ang-2 siRNA. Conclusions: Ang-2 and IL-18 may be associated with the development and progression of DN in mice. Alprostadil treatment can protect renal function by reducing proteinuria. These effects are mediated, at least in part, through down-regulation of Ang-2 and IL-18 expression.

Simultaneous silencing of VEGF and KSP by siRNA cocktail inhibits proliferation and induces apoptosis of hepatocellular carcinoma Hep3B cells

BackgroundVascular endothelial growth factor (VEGF) is involved in the growth of new blood vessels that feed tumors and kinesin spindle protein (KSP) plays a critical role in mitosis involving in cell proliferation. Simultaneous silencing of VEGF and KSP, an attractive and viable approach in cancer, leads on restricting cancer progression. The purpose of this study is to examine the therapeutic potential of dual gene targeted siRNA cocktail on human hepatocellular carcinoma Hep3B cells.ResultsThe predesigned siRNAs could inhibit VEGF and KSP at mRNA level. siRNA cocktail showed a further downregulation on KSP mRNA and protein levels compared to KSP-siRNA or VEGF-siRNA, but not on VEGF expression. It also exhibited greater suppression on cell proliferation as well as cell migration or invasion capabilities and induction of apoptosis in Hep3B cells than single siRNA simultaneously. This could be explained by the significant downregulation of Cyclin D1, Bcl-2 and Survivin. However, no sigificant difference in the mRNA and protein levels of ANG2, involving inhibition of angiogenesis was found in HUVECs cultured with supernatant of Hep3B cells treated with siRNA cocktail, compared to that of VEGF-siRNA.ConclusionSilencing of VEGF and KSP plays a key role in inhibiting cell proliferation, migration, invasion and inducing apoptosis of Hep3B cells. Simultaneous silencing of VEGF and KSP using siRNA cocktail yields promising results for eradicating hepatocellular carcinoma cells, a new direction for liver cancer treatment.

Antiangiogenic effects of ganetespib in colorectal cancer mediated through inhibition of HIF-1α and STAT-3

Hypoxia-inducible factors (HIFs) and STAT-3 play essential roles in angiogenesis. HIF-1α and STAT-3 are clients of the heat shock protein 90 (HSP90). We hypothesized that ganetespib, a potent HSP90 inhibitor, would disrupt angiogenesis in colorectal cancer (CRC) through inhibition of HIF-1α and STAT-3. CRC cell lines (HCT116 and HT29) were used in all the experiments. Egg CAM and HUVEC assays revealed decreased angiogenesis in ganetespib treated cell lines. Ganetespib inhibited matrigel plug vascularization and tumor growth of xenografts. Significant inhibition of PDGFA, FGF2, Ang-1, Ang-2, TGFβ1, VEGF, HIF-1α and STAT-3 expression was observed in both cell lines treated ganetespib. HIF-1α overexpression resulted in the increase VEGF and STAT-3 expression and this was inhibited by ganetespib. HIF-1α knockdown inhibited VEGF and STAT-3 expression. STAT-3 knockdown inhibited VEGF but not HIF-1α expression. HSP90, STAT-3 and VEGF expression was significantly higher in CRC compared to adjacent normal tissue. Significant downregulation of PDGFA, FGF2, Ang-1, Ang-2, TGFβ1, VEGF, STAT-3 and HIF-1α mRNA was observed in the post ganetespib treatment tumor samples from patients with rectal cancer. These results collectively suggest that inhibition of HSP90 is a promising antiangiogenic strategy in CRC. HSP90 angiogenic effects are mediated through HIF-1α and STAT-3.

Soluble Tie2 fusion protein decreases peritoneal angiogenesis in uremic rats

Angiogenesis is considered to be one of the most common mechanisms leading to ultrafiltration failure (UFF) in long-term peritoneal dialysis (PD) patients. The angiopoietin (Ang)/Tie system was found to play a role in the initiation of pathological neoangiogenesis and is also involved in peritoneal angiogenesis caused by peritoneal fluid. This aim of this study was to investigate the effects of the soluble Tie2 fusion protein (sTie2/Fc) on peritoneal angiogenesis in PD-treated uremic rats. The rats were divided into 6 groups: normal, sham surgery, uremic rats without PD, uremic PD-treated rats, uremic rats treated with PD and sTie2/Fc (0.25µg/100g) and uremic rats treated with PD and sTie2/Fc (0.5µg/100g). PD rats were treated once a day for 28days prior to testing. Real-time polymerase chain reaction (RT-PCR) or tissue immunohistochemical staining was used to detect Ang-2 mRNA or protein expression in the peritoneal tissues of each group. The microvessel density (MVD) of the peritoneum was detected and quantified by immunohistochemical staining using the anti-CD34 antibody. Compared with the control group, Ang-2 mRNA and protein expression was significantly upregulated in the uremic and PD groups (P<0.05). MVD in the experimental group increased compared with the control group. sTie2/Fc treatment decreased the levels of Ang-2 mRNA and protein expression (P<0.05) in a dose-dependent manner and decreased PD-induced MVD in the peritoneum. In conclusion, angiogenesis of the peritoneum induced by PD was inhibited using sTie2/Fc in a uremic rat model.

Inhibition of hepatocellular carcinoma growth and angiogenesis by dual silencing of NET-1 and VEGF

Simultaneous silencing of multiple up-regulated genes is an attractive and viable strategy to treat many incurable diseases including cancer. Herein we used dual gene targeted siRNA (DGT siRNA) conjugate composed of NET-1 and VEGF siRNA sequences in the same backbone could inhibit growth and angiogenesis HCC. DGT siRNA showed a further down regulation on VEGF mRNA and protein levels compared with NET-1 siRNA or VEGF siRNA, but not on NET-1 expression. It also exhibited greater suppression on proliferation and trigger of apoptosis in HepG2 cells than NET-1 siRNA or VEGF siRNA; this could be explained by the significant down regulation of cyclin D1 and Bcl-2. A lower level of ANG2 mRNA and protein was detected in HUVEC cultured with supernatant of HepG2 cells treated with DGT siRNA than that of VEGF siRNA or NET-1 siRNA, resulting in much more inhibited angiogenesis of HUVEC. Tumor growth was inhibited and microvessel density dropped in the xenograft tumor models compared to the untreated controls. NET-1 and VEGF silencing play a key role in inhibiting hepatocellular cell proliferation, promoting apoptosis, and reducing angiogenesis. Simultaneous silencing of NET-1 and VEGF using DGT siRNA construct may provide an advantageous alternative in development of therapeutics for Hepatocellular carcinoma.

Influence of granulocyte macrophagecolony stimulating factor on the expression of angiopoietin-1 in deep partial thickness burn wound in rats

Objective To investigate the influence of granulocyte macrophage colony stimulatingfactor （ GM-CSF） on the expression of angiopoietin-1 （Ang-1） in deep partial thickness bum wound in 1rats. Methods The models of deep partial thickness bum wounds on the back in Spregue-Dawley （ SD）rats were established. The paired wounds on the same rats were randomly divided into GM-CSF treatment（test） and control groups by self-control. Tissue samples were harvested from the wounds on the 1st,3rd,7th, 14th and 21st day respectively after bums. The mRNA expression of Ang-1 was detected by using re-al-time reverse transcriptase-polymerase chain reaction （ FQ-PCR） . And the protein expression of Ang-1was detected by immunohistochemical and western blot, the protein expression of CD31 by using immuno-histochemical staining,and the microvascular density was calculated. Results The mRNA expression levelof Ang-1 in the bum wounds was decreased slightly on the 1st and 3rd postbum days and that in test group（1. 44 ±0. 20,0. 30 ± 0. 17 ） was lower than that in control group （2. 36 ± 0. 18 , 1. 34 ± 0. 24） （P 〈0.05）. Then the Ang-1 mRNA expression was gradually increased on the 7 th and 14 th postbum days anddecreased slightly on the 21st postbum day, and the expression level in the test group at the above timepoints （4. 52 ±0. 21,8. 47 ±0. 14, 7. 52 ±0. 20） was higher than that in control group （2. 71 ±0. 12,3.69 ±0. 24,3. 07 ±0. 17）（p 〈0. 05）. The protein expression of Ang-1 was declined remarkably on the 1stand 3rd postburn days, which in test group （0. 491 ±0. 055, 0. 181 ±0. 025） was lower than that in control group （0. 735 ± O. 080, O. 436 ± 0. 071 ） （ P 〈 O. 05 ）. Then the Ang-1 protein expression was gradually in- creased and reached the peak on the 14th postburn day and the expression in the test group （0. 821 ±0. 060, 1. 257 ± 0. 225, 1. 126 ±0. 131 ） was higher than that in the control group （0. 701 ± 0. 078, 0. 825 ± 0. 072, 0. 733 ±0. 073 ） （P 〈 0. 05 ）. The microvascular number with positive CD31 expression was increased evi- dently from the 3rd postburn day in test group, and the counting of MVD was remarkably higher in the test group （9.5±1.8, 23.4±2.9, 38.4±3. 1,24.6±2.5） than in the control group （4.0±1.9, 10.5±2.0, 22. 9 ±2. 7, 15.1 ±2. 6） on the 3rd, 7th, 14th and 21st days respectively after burns （P 〈0. 05）. Conclu- sion GM-CSF inhibits the mRNA and protein expression of Ang-1 during the early stage of wound healing, which can be beneficial to the angiogenesis. And GM-CSF boosts Ang-1 expression during the middle and late stages of wound healing. This would be helpful to wound vascular maturation and wound healinz. Key words: Granulocyte macrophage colony stimulating factor; Angiopoietin; Burn; Woundhealing

Effect of Bushen Yiqi Huoxue recipe on placental vasculature in pregnant rats with fetal growth restriction induced by passive smoking

Interactions of vascular endothelial growth factor (VEGF) with receptors VEGFR1/Flt1 and VEGFR2/Flk1, and those of angiopoietins (Ang-1, Ang-2) with receptor Tie2 play important roles in placental angiogenesis. This study investigated vascular morphology and expression of these angiogenic factors in rat placenta on the day 15, 18, 21 of gestation (D15, D18 and D21). The rats were randomly assigned into 3 groups: normal group, model group [fetal growth restriction (FGR) model], and Bushen Yiqi Huoxue (BYHR) recipe treatment group (BYHR group, the pregnant rats with FGR were treated with BYHR recipe). Morphological analysis indicated that during initial villous formation, fetal nucleated erythrocytes (FNEs) appeared in maternal blood sinus (MBS). Subsequently, FNEs were surrounded by endothelial cells to form fetal capillary (FC) and then by trophoblast cells to form villi. As pregnancy proceeded, FC density increased progressively with increasing endothelial identification staining (EIS) in normal and BYHR groups. Whereas, villous formation was suppressed, normal increase in FC density was impaired and EIS was weakened in model group. Quantitative PCR analysis showed that VEGF and Flk1 mRNA increased over gestation in all groups, indicating that VEGF might play a pivotal role in FC growth during late gestation. VEGF mRNA was increased on D15, while decreased on D21 in model group as compared with normal group and BYHR group. Immunohistochemically, Ang-2 protein was highly expressed in FNEs, gradually disappeared as villi matured, and decreased over gestation in all groups, indicating that Ang-2 might play a pivotal role in villous formation, which was further supported by decreased Ang-2 mRNA and protein expression in model group on D15. Ang-1 mRNA, Tie2 mRNA and Ang-1/Ang-2 ratio increased from D15 to D18 in all groups as placenta matured. Ang-1 mRNA, Tie2 mRNA and Ang-1/Ang-2 ratio were decreased on D18 in model group as compared with normal and BYHR groups, indicating delayed maturity of FGR placenta. Alterations in angiogenic factors may result in altered placental vasculature and cause placental insufficiency. BYHR recipe could balance the angiogenic factors to promote the formation and maturation of FGR placental vasculature.

Human Mesenchymal Stem Cells Increases Expression of &amp;alpha;-Tubulin and Angiopoietin 1 and 2 in Focal Cerebral Ischemia and Reperfusion

Angiogenesis is associated with improved neurologic recovery after cerebral ischemia. Human bone marrow mesenchymal stem cells (hMSCs) have been successfully used to treat ischemic stroke and were shown to induce the expression of a number of neurotrophic factors including VEGF, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) in a rat middle cerebral artery occlusion (MCAO) ischemia model. In this study, we aimed to understand the mechanism underlying the improvement of neurological function following hMSCs transplantation into MCAO rats. We established a rat MCAO model and used immunofluorescence to evaluate α-tubulin expression in the hippocampus. We used RT-PCR to determine the expression of Ang-1 and Ang-2 mRNAs after transplantation of hMSCs into MCAO rats. We showed a significant decrease in α-tubulin expression in rats with cerebral ischemia, suggesting that α-tubulin is a protective protein in cerebral ischemia Transplantation of hMSCs significantly upregulated α-tubulin levels in the hippocampus. Transplantation of hMSCs also resulted in a significant upregulation of Ang-1 and Ang-2 mRNAs in MCAO rats. Ang-2 expression was upregulated earlier than Ang-1, suggesting that (1) transplantation of hMSCs promotes angiogenesis and that (2) Ang-2 may be an initiator of angiogenesis. Our results provide a theoretical basis for the therapeutic use of hMSCs in cerebral ischemia.

Canonical Wnt/ β‐catenin Signaling Pathway mediates Shear Stress‐Activated Angiopoeitin‐2 expression and vasculogenesis

It is well known that fluid shear stress is intimately involved in vascular stability and differentiation. In particular, low or oscillatory shear stress (OSS) upregulates Angiopoietin‐2 (Ang‐2) in human aortic endothelia cells (HAEC) via VEGF‐dependent mechanism. We hypothesized that Wnt signaling is implicated in shear stress activated vasculogenesis via modulating Ang‐2. Activation of Wnt signaling was assessed with TOPflash lentivirus reporter. After 8 hours of oscillatory flow, HAECs infected with lentivirus of Wnt reporter demonstrated markedly increased luciferase activity compared to cells under static condition, suggesting an activation of canonical Wnt signaling pathway with OSS. We further demonstrated that Wnt3a, a Wnt activator, upregulated Ang‐2 mRNA expression, whereas DKK‐1, a Wnt inhibitor, inhibited Ang‐2 expression in both HAEC cell lines and Tg(hsp70:dkk1‐GFP) zebrafish embryos 72 hours post‐fertilization (hpf). Knock‐down of Ang‐2 by siRNA in HUVEC resulted in impaired tube formation. Similarly, Ang‐2 knock‐down with morpholino oligomers in 72 hpf Tg(flk1:GFP) zebrafish embryos caused impaired systematic vasculature development. Taken together, our findings suggest that Wnt/β‐catenin signaling pathway mediates shear stress‐activated endothelial Ang‐2 and regulates vasculogenesis. These studies were supported by NIH HL083015 and HD069305.Grant Funding Source: NIH

Abstract 149: Endothelial NADPH Oxidase 4 Worsens Ischemic Stroke by Suppressing eNOS Activity

NADPH oxidase (Nox) family proteins are a major source of reactive oxygen species (ROS) and play various important roles in immune systems, arteriosclerosis, angiogenesis and aging. We have previously demonstrated that among the Nox proteins, Nox4 is abundantly expressed and is a major source of ROS in endothelial cells; however, its roles in endothelial cells are not fully understood. In the present study, we investigated the roles of endothelial Nox4 in brain ischemia, using mice with endothelial cell-specific Nox4 overexpression (Tg-EC-Nox4) and cultured human brain microvascular endothelial cells (HMEC) overexpressing Nox4. Nox4 was abundantly expressed and was significantly upregulated in response to hypoxia at O2 1% (2.0-fold increase, p&lt;0.001) in HMEC. Immunofluorescent double staining demonstrated that in a middle cerebral occlusion model (MCAO) of wild type mice, Nox4 was markedly upregulated in endothelial cells in peri-infarct area. We subjected Tg-EC-Nox4 mice and their littermates to either transient (ischemia 90 min and reperfusion) or permanent MCAO. Triphenyltetrazolium chloride staining demonstrated that infarct volume at 1 day after MCAO was significantly larger in Tg-EC-Nox4 than wild-type mice in both the models (transient MCAO: 151 %, permanent MCAO: 132 %, p&lt;0.05). We overexpressed Nox4 by adenovirus in HMEC. In the endothelial cells overexpressing Nox4, angiopoietin-2 (Ang-2) was drastically upregulated (30-fold increase, p&lt;0.0001) at mRNA levels and the phosphorylation of Akt at Ser-473 (47 %, p&lt;0.01) and endothelial nitric oxide synthase (eNOS) at Ser-1177 (43 %, p&lt;0.01) were significantly attenuated. Treatment with Ang-2 suppressed the phosphorylation of Akt and eNOS induced by angiopoietin-1 in a dose-dependent manner in HMEC. Consistently, Ang-2 mRNA was significantly increased (2.0-fold, p&lt;0.05), and the phosphorylation of Akt (37 %, p&lt;0.05) and eNOS (41 %, p=0.12) were suppressed in the ischemic hemisphere of Tg-EC-Nox4, compared to those of wild-type mice. In conclusion, the overexpression of endothelial Nox4 may increase infarct volume by suppressing eNOS activity through the upregulation of Ang-2 in brain ischemia.

Effect of valproic acid against angiogenesis of Kasumi-1 xenograft tumor in nude mice

This study was aimed to investigate the effect of valproic acid (VPA), a histone deacetylase inhibitor, on angiogenesis of acute myeloid leukemia in vivo and vitro, and to explore its molecular mechanism. Human t (8;21) AML cell line Kasumi-1 cells were treated with VPA at different concentration for 3 d, the mRNA and protein expression levels of Ang1 and Ang2 were determined by semi-quantitative RT-PCR and Western blot respectively. Nude mice model with xenograft Kasumi-1 tumor was established by subcutaneous inoculation of Kasumi-1 cells. The CD34, Ang1 and Ang2 protein levels were analyzed by immunohistochemistry method. The mRNA and protein expression levels of Ang1, Ang2 and VEGF were determined by semi-quantitative RT-PCR and Western blot. The results showed that in vitro, VPA at 3 mmol/L downregulated the Ang mRNA relative expression level for Ang1 from 0.360 ± 0.116 to 0.040 ± 0.008, Ang2 from 0.540 ± 0.049 to 0.146 ± 0.038. The animal experiment further verified that VPA 500 mg/kg, ip, for 14 d, reduced the relative expression of Ang1, Ang2 and VEGF mRNA and proteins in Kasumi-1 tumor of nude mice, and reduced microvascular density in xenograft tumor of nude mice (8.470 ± 0.300 vs 2.600 ± 0.200). It is concluded that VPA significantly inhibits tumor angiogenesis through the regulation of angiopoietins, thereby inhibits the proliferation and metastasis of leukemia cells.

Angiogenetic axis angiopoietins/Tie2 and VEGF in familial breast cancer

Angiogenesis leads to the formation of blood vessels from pre-existing ones, allowing tumor growth. Vascular endothelial growth factor (VEGF) and Angiopoietins (Ang-1, Ang-2) have a pivotal role in tumor angiogenesis but few data regarding their role in hereditary breast cancer are available. The aim of the present study was to analyze Ang-1, Ang-2, tyrosine-protein kinase receptor Tie2 and VEGF expression and their correlation in a cohort of familial and sporadic breast cancers in order to verify whether the presence of germline mutations in BRCA may have a role in tumor microenvironment regulation. Tumor samples from a cohort of 41 patients with a first diagnosis and a family history of breast cancer and 19 patients with sporadic breast cancers were enrolled. The expression of Tie2, Ang-1, Ang-2 and VEGF were analyzed by quantitative real-time PCR. Patients harboring BRCA mutations had higher levels of Ang-1 (P=0.05), Ang-2 (P=0.02) and VEGF (P=0.04) mRNA compared with those without BRCA mutations (BRCAX). The same was observed in triple-negative breast cancer (TNBC). Moreover, a positive correlation between Ang-2 and VEGF was found in both the familial breast cancer group (BRCA carriers: r=0.83; P<0.0001 and BRCAX: r=0.58; P=0.008) and in TNBC (r=0.62; P=0.007). The higher levels of Ang-1, Ang-2 and VEGF mRNA found in BRCA carriers and TNBCs suggest that they could be attractive angiogenic therapeutic targets in these breast cancers.

Interfering growth of malignant melanoma with Ang2-siRNA

To investigate the intervention therapy effect on the growth of malignant melanoma, we have made an observation of expression levels of Ang2 in malignant melanoma cells, which was transduced by small interfering RNA (Ang2-siRNA) of Ang2 in vitro and in vivo. We successfully constructed Ang2-siRNA lent virus, and constructed nude mice model by transplanting malignant melanoma. Ang2-siRNA lent virus inhibited Ang2 mRNA of malignant melanoma in vitro and in vivo, and inhibited malignant melanoma tumor growth at the same time. Ang2-siRNA lent virus can interfere expression levels of Ang2 in malignant melanoma cells, inhibit tumor growth, and silent Ang2 gene expression, which may pave a new way for clinical gene therapy of malignant melanoma.

Expression and regulation of ang-2 in murine ovaries during sexual maturation and development of corpus luteum

The aim of this study was to examine the expression and regulation of angiopoietin-2 (Ang-2) in murine ovaries during sexual maturation, gonadotropin treatment and luteal development by in situ hybridization and RT-PCR. By in situ hybridization Ang-2 mRNA was mainly localized in granulosa cells, thecal cells and corpus luteum, otherwise in oocytes. Moreover, Ang-2 mRNA was highly expressed in corpus luteum and granulosa cells of atretic follicles. According to RT-PCR data, Ang-2 mRNA was lowly expressed on day 10 after birth, then expression levels gradually increased and reached their highest values on day 25 after birth. In the superovulated model of immature mice, Ang-2 expression was strongly induced by equine chorionic gonadotropin (eCG) 48 h post the eCG injection, and was high from 0.5 to 13 h after hCG treatment. In situ hybridization showed that Ang-2 mRNA was highly expressed in corpus luteum from day 2 to 9 post the hCG injection, then the expression levels gradually declined on days 11 and 13 after hCG treatment. According to RT-PCR data, the levels of Ang-2 mRNA expression showed a decline after the hCG injection, with a nadir on day 3, followed by an increase, reaching the highest level on day 9 post-hCG injection. Then again Ang-2 expression gradually declined from day 11 to 15 after hCG injection. These results suggest that Ang-2 may be involved in follicular development, atresia, ovulation, and corpus luteum formation and regression.

Endothelins reciprocally regulate VEGF‐A and angiopoietin‐1 production in cultured rat astrocytes: Implications on astrocytic proliferation

Vascular endothelial growth factors (VEGFs) and angiopoietins (ANGs) are involved in pathophysiological responses in damaged nerve tissues. Astrocytes produce VEGFs and ANGs upon brain ischemia and traumatic injury. To clarify the extracellular signals regulating VEGF and ANG production, effects of endothelins (ETs), a family of endothelium-derived peptides, were examined in cultured rat astrocytes. ET-1 (100 nM) and Ala(1,3,11,15)-ET-1 (100 nM), an ET(B) receptor agonist, increased VEGF-A mRNA levels in cultured astrocytes, while ANG-1 mRNA was decreased by ETs. ET-1 did not affect astrocytic VEGF-B, placental growth factor (PLGF), and ANG-2 mRNA levels. The effects of ET-1 on VEGF-A and ANG-1 mRNAs were inhibited by BQ788, an ET(B) antagonist. Release of VEGF-A proteins from cultured astrocytes was increased by ET-1. In contrast, ET-1 reduced release of astrocytic ANG-1. Exogenous ET-1 (100 nM) and VEGF(165) (100 ng/mL), an isopeptide of VEGF-A, stimulated bromodeoxyuridine (BrdU) incorporation into cultured astrocytes. Treatment with ET-1 and VEGF(165) increased the numbers of cyclin D1-positive astrocytes. Exogenous ANG-1 (250 ng/mL) did not stimulate the BrdU incorporation. Increases in BrdU incorporation by ET-1 and VEGF(165) were not affected by ANG-1. In 60-70% confluent cultures, SU4312 (10 μM), a VEGF receptor tyrosine kinase inhibitor, partially reduced the effects of ET-1 on BrdU incorporation and cyclin D1 expression. ET-induced BrdU incorporation and cyclin D1 expression were reduced by a neutralizing antibody against VEGF-A. Our findings suggest that ET-1 is a factor regulating astrocytic VEGF-A and ANG-1, and that increased VEGF-A production potentiates ET-induced astrocytic proliferation by an autocrine mechanism.

Methylseleninic acid restricts tumor growth in nude mice model of metastatic breast cancer probably via inhibiting angiopoietin-2

BackgroundAngiopoietin-2 (Ang-2) plays critical roles in vascular morphogenesis and its upregulation is frequently associated with various tumors. Previous studies showed that certain selenium compounds possess anti-tumor effects. However, the underlining mechanism has not been elucidated in detail. Plus, results of research on the anti-tumor effects of selenium compounds remain controversial.MethodsWe investigated levels of Ang-2 and vascular endothelial growth factor (VEGF) on the estrogen-independent bone metastatic mammary cancer (MDA-MB-231) cells in response to treatment by methylseleninic acid (MSeA), and further examined the effects of MSeA oral administration on xenograft mammary tumors of athymic nude mice by RT-PCR, Western, radioimmuno assay, and Immunohistochemistry.ResultsTreatment of MDA-MB-231 cells with MSeA caused significant reduction of Ang-2 mRNA transcripts and secretion of Ang-2 proteins by the cells. Level of VEGF protein was accordingly decreased following the treatment. Compared with the controls, oral administration of MSeA (3 mg/kg/day for 18 days) to the nude mice carrying MDA-MB-231 induced tumors resulted in significant reduction in xenograft tumor volume and weights, significant decrease in microvascular density, and promotion of vascular normalization by increasing pericytes coverage. As expected, level of VEGF was also decreased in MSeA treated tumors.ConclusionsOur results point out that MSeA exerts its anti-tumor effects, at least in part, by inhibiting the Ang-2/Tie2 pathway, probably via inhibiting VEGF.

Immunoregulatory Protein Profiles of Necrotizing Enterocolitis versus Spontaneous Intestinal Perforation in Preterm Infants

Necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP) are the most common acute surgical emergencies associated with high morbidity and mortality in preterm infants. We aimed to compare the profiles of immunoregulatory proteins and identify novel mediators in plasma of NEC and SIP infants. We also investigated the expression of target genes in resected intestinal tissues and an enterocyte cell line. Using Cytokine Antibody Array assay, we reported the first comparative profiles of immunoregulatory proteins in plasma of NEC and SIP infants, and showed that dysregulated proteins belonged to functionally diversified categories, including pro- and anti-inflammation, angiogenesis, cell growth, wound healing, anti-apoptosis, cell adhesion and extracellular matrix reorganization. Validation by ELISA confirmed significantly higher concentrations of interleukin (IL)-6, angiopoietin (Ang)-2, soluble type II interleukin-1 receptor (sIL-1RII), and soluble urokinase-type plasminogen activator receptor (suPAR) in NEC infants compared with gestational age-matched control, and a lower level of an epidermal growth factor receptor, secreted form of receptor tyrosine-protein kinase ErbB3 (sErbB3), compared with SIP infants. mRNA expressions of IL1-RII and uPAR were up-regulated in resected bowel tissues from NEC infants, indicating that immunoregulation also occurred at the cellular level. In FHs-74 Int cells, Ang-2, IL1-RII and uPAR mRNA expressions were significantly induced by the combined treatment with lipopolysaccharide (LPS) and platelet activating factor (PAF). Our study provided plasmatic signatures of immunoregulatory proteins in NEC and SIP infants, and demonstrated involvement of multiple functional pathways. The magnitude of changes in these proteins was significantly more extensive in NEC infants, reflecting the different nature of injury and/or severity of inflammation. We speculate that dysregulation of IL-6, Ang-2, IL-1RII and uPAR occurred at both systemic and cellular levels, and probably mediated via LPS and endogeneous PAF signals. Such exaggerated immunologic responses may account for the high morbidity and mortality in NEC compared with SIP patients.

Shear Stress‐Activated Angiopoeitin‐2 Modulates Endothelial Cell Repairs and Vasculogenesis via Wnt/β‐catenin Signaling Pathway

Fluid shear stress is intimately involved in vascular endothelial cell repairs. It is recognized that pulsatile shear stress up‐regulated Angiopoetin‐2 expression (Ang‐2) in human umbilical vein endothelial cells (HUVEC). We hypothesized that Ang‐2 was implicated in HUVEC repairs and vasculogenesis via Wnt/β‐catenin signaling pathway. Knock‐down of Ang‐2 expression with siRNA resulted in an impaired HUVEC migration and tube formation. Wnt3a, a Wnt activator, up‐regulated Ang‐2 mRNA expression (p &lt; 0.05 vs. no addition, n = 3), whereas DKK‐1, a Wnt inhibitor, inhibited Ang‐2 expression (p &lt; 0.05 vs. PBS treated, n = 3), leading to impaired migration and tubule formation. We further demonstrated that subintestinal vessels (SIVs) were absent in heat shock inducible DKK‐1:gfp zebrafish at 72 hpf. Knock‐down of Ang‐2 with morpholino oligomers in the transgenic zebrafish embryo (TG(flk1:GFP)) at 1‐cell stage resulted in impaired vasculogenesis to the entire vasculature at 48 hpf. Treatment with ionomycine, a Calcium (Ca2+) ionophore, also resulted in both impaired HUVEC repairs and suppression of SIV formation in TG(flk1:GFP) lines at 72 hpf as evidenced by down‐regulation of Ang‐2 mRNA expression (p &lt; 0.05 vs. DMSO treatment, n = 3). Taken together, our findings suggest that shear stress‐activated Ang‐2 is implicated in endothelial cell repairs and vasculogenesis via Wnt/β‐catenin signaling pathway.Grant Funding Source : NIH