Ang-2 mRNA Research Articles

Differential expression of the angiogenic Tie receptor family in arthritic and normal synovial tissue.

Angiopoietins (Ang) are vascular endothelial cell-specific growth factors that play important roles principally during the later stages of angiogenesis. We have compared the distribution of the receptor tyrosine kinase (Tie) and the Ang ligands in synovial tissues from normal subjects and those with rheumatoid arthritis (RA) and osteoarthritis (OA). Immunohistochemical analysis was used to determine the expression of Ang-1, Ang-2, Tie1 and Tie2 in synovial tissue of normal subjects and those with RA and OA. Ang-1, Ang-2, Tie1 and Tie2 mRNA and protein expression were quantified in synovial tissues and RA synovial tissue fibroblasts with real-time reverse transcription polymerase chain reaction and western blot analysis. In RA, Ang-1 positive immunostaining on lining cells, macrophages and endothelial cells was significantly higher than in OA and normal synovial tissue. The expression pattern of Ang-2 in synovial tissue was similar in RA and OA, whereas the Ang-2 expression was low in normal tissue. Synovial tissue from subjects with RA and OA showed a significant upregulation of Tie1 on lining cells, macrophages and endothelial cells compared to that from normal subjects. Tie2 was significantly upregulated in the RA and OA synovial tissue lining cells, macrophages and smooth muscle cells compared to normal synovial tissue. Generally Ang-1, Ang-2, Tie1 and Tie2 mRNA levels were higher in RA synovial tissue compared to normal and OA synovial tissues, and RA synovial tissue fibroblasts. Western blot analysis also demonstrated greater Tie1 and Tie2 protein expression in RA and OA synovial tissue compared to RA synovial tissue fibroblasts. In conclusion, the dominance of Ang-1 mRNA and protein expression over Ang-2 is in agreement with an active neovascularization in RA synovial tissue.

Thrombin induces increased expression and secretion of angiopoietin-2 from human umbilical vein endothelial cells

Angiogenesis is required for tumor growth and metastasis. It has recently been suggested that thrombin is a potent promoter of angiogenesis. We therefore examined the possibility that thrombin could be inducing the expression of angiopoietin-2 (Ang-2), necessary for remodeling. Human umbilical vein endothelial cells were incubated with or without thrombin (1 U/mL) for 1 to 24 hours and then examined for messenger RNA (mRNA) by Northern analysis. Enhanced mRNA expression (about 4-fold over baseline) was noted at 4 hours. Enhanced expression of Ang-2 mRNA was secondary to enhanced transcription (about 4-fold), with no effect on stabilization. Enhanced Ang-2 mRNA transcription was inhibited by H7 and PD98059, indicating the requirement of serine/threonine kinases as well as the mitogen-activated protein kinase pathway. Up-regulation of mRNA was associated with enhanced Ang-2 protein synthesis and secretion as assayed by immunoblot. Thrombin-induced secreted Ang-2 inhibited the binding of recombinant (35)S-Ang-1 to its Tie-2-Fc receptor, demonstrating functionality. Hirudin reversed this effect, demonstrating thrombin specificity. Thus, thrombin-induced tumorigenesis and metastasis is associated with enhanced Ang-2 protein synthesis and secretion via enhanced transcription of Ang-2. This could help explain how thrombin promotes angiogenesis.

Hemochorial placentation in the primate: expression of vascular endothelial growth factor, angiopoietins, and their receptors throughout pregnancy.

Vascular development and its transformation are necessary for successful hemochorial placentation, and vascular endothelial growth factor (VEGF), angiopoietins, and their receptors may be involved in the molecular regulation of this process. To determine the potential role of these putative regulators in a widely studied primate, the common marmoset, we investigated their mRNA expression and protein location in the placenta throughout pregnancy using in situ hybridization, Northern blot analysis, and immunocytochemistry. VEGF was localized in decidual and cytotrophoblast cells, and its highest expression was found in the maternal decidua. The Flt receptor was exclusively detected in the syncytial trophoblast with increasing expression in placentae from 10 wk to term. Soluble Flt (sFlt) was also detectable by Northern blot analysis. KDR receptor expression was restricted to mesenchymal cells during early placentation and to the fetoplacental vasculature during later placentation. KDR expression increased throughout pregnancy. Angiopoietin-1 (Ang-1) was localized in the syncytial trophoblast, being highly expressed in the second half of gestation. Ang-2 mRNA localized exclusively to maternal endothelial cells, and was highly expressed in 10-wk placentae. The Tie-2 receptor was found in cytotrophoblast cells and in fetal and maternal vessels. High Tie-2 levels were detected in the wall of chorion vessels at 14-wk, 17-wk, and term placentae. These results suggest that the processes of trophoblast invasion, maternal vascular transformation, and fetoplacental vascular differentiation and development are regulated by the specific actions of angiogenic ligand-receptor pairs. Specifically, 1) VEGF/Flt and Ang-1/Tie-2 may promote trophoblast growth, 2) VEGF/KDR and Ang-1/Tie-2 may support fetoplacental vascular development and stabilization, 3) sFlt may balance VEGF actions, and 4) Ang-2/Tie-2 may remodel the maternal vasculature.

Down-Regulation of Angiopoietin-1 Expression in Menorrhagia

Angiogenesis is an essential component of endometrial repair and regeneration following menses. Perturbation of this process is associated with menorrhagia, a common gynecological disorder that results in excessive menstrual bleeding. Angiopoietin-1 (Ang-1) promotes vascular maturation via the Tie-2 receptor, while angiopoietin-2 (Ang-2) is its natural antagonist that destabilizes vessels and initiates neovascularization in the presence of vascular endothelial growth factor. To test the hypothesis that menorrhagia arises as a result of poor signal for vascular maturation, we have examined the expression of Ang-1, Ang-2, and Tie-2 in endometrium throughout the menstrual cycle from 30 normal women and 28 patients with menorrhagia. Ribonuclease protection assay and Western blot analysis showed Ang-2 expression was consistently higher than Ang-1 in normal endometrium throughout the cycle. However, with menorrhagia Ang-1 mRNA and protein were not detected or down-regulated, while Ang-2 was observed at similar levels in both normal and menorrhagic endometrium resulting in a greater than a 50% decrease in the ratio of Ang-1 to Ang-2 protein. In situ hybridization and immunohistochemical studies supported these findings and revealed cyclical changes in the expression of Ang-1 and Ang-2. These results suggest that the angiopoietin/Tie-2 system promotes vascular remodeling in endometrium and loss of normal Ang-1 expression may contribute to the excessive blood loss observed in menorrhagia.

Regulation of angiopoietin and Tie-2 receptor expression in non-reproductive tissues by estrogen.

Estrogen promotes endothelial cell proliferation and survival in the vasculture of non-reproductive organs. The main mechanisms through which estrogen exerts its effects on endothelial cells remain unknown. Angiopoietins are newly described modulators of endothelial cell survival and they exert their effects through the activation of endothelial cell-specific Tie-2 receptors. In this study, we evaluated whether estrogen modulates the activity and expression of Tie-2 receptors, Ang-1 and its endogenous antagonist; angiopoietins-2 (Ang-2) in non-reproductive organs. Using RT-PCR, we found that daily administration of 17-beta-estradiol for 8 days in ovariectomized rats results in a significant reduction in tissue Ang-1 mRNA expression. By comparison, estrogen therapy produced a significant increase in Ang-2 mRNA in estrogen-treated rats with heart, kidney and lung Ang-2 mRNA levels reaching 169%, 152% and 224% of those of oil-treated animals, respectively. We also observed that tyrosine phosphorylation of Tie-2 receptors is significantly attenuated in ovariectomized rats treated with 17-beta-estradiol. Our results suggest that the effects of estrogen on the vasculature of non-reproductive organs require the inhibition of angiopoietin-1-Tie-2 receptor pathway and that this inhibition is achieved through simultaneous down-regulation of Ang-1 and Tie-2 expression and elevation in Ang-2 expression.

Tie-2 and angiopoietin-1 expression in human thyroid tumors.

Tie-2 is an endothelial cell-specific receptor tyrosine kinase involved in vascular maturation and remodeling. Although its expression is considered to be restricted to vascular endothelial cells and hematopoietic progenitors, our immunohistochemical and in situ hybridization studies showed that Tie-2 and its ligand, angiopoietin (Ang)-l were expressed not only in benign and malignant human thyroid tumor cells but also in hyperplastic regions of adenomatous goiter. To confirm the expression in these tissues further, we used a laser capture microdissection system to isolate epithelial tumor cells from tissue specimens selectively, and demonstrated the expression of Tie-2 and Ang-1 mRNAs in tumor cells by RT-PCR analysis. Furthermore, Tie-2 and Ang-1 mRNAs and proteins were also detected in rat thyroid cell lines, FRTL-5 and PCCL-3. Our results suggest that Ang-1/Tie-2 signaling may be involved in the proliferation of thyroid epithelial cells.

Expression of Angiopoietin-1 & -2 mRNA in Invasive Cervical Cancer

Objective : The angiopoietins (Ang) are recently described as one of growth factors for vascular endothelium. This study was aimed to investigate the Ang-1 and Ang-2 mRNA expression between in cervical cancer and in normal cervix and to assess the relationships among their expression and other prognostic factors of cervical cancer. Patients and Methods : Mg-1 and Ang-2 mRNA expression were investigated by qualitative competitive PCR between in cervical cancer of 34 cases and in normal cervix of 14 cases at Ewha Womans University Mokdong Hospital from March 1998 to June 2000. Results : Both in cervical cancer and normal cervical tissue, 612 bp sized Mg-I mRNA and 518 bp sized Ang-2 mRNA were expressed. In comparison to normal cervix, their expression was significantly elevated in cervical cancer (p

Increased Tie2 Expression, Enhanced Response to Angiopoietin-1, and Dysregulated Angiopoietin-2 Expression in Hemangioma-Derived Endothelial Cells

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.

The Regulation and Localization of Angiopoietin-1, -2, and Their Receptor Tie2 in Normal and Pathologic Human Placentae

Angiopoietin-1 (Ang-1) and its antagonist angiopoietin-2 (Ang-2) act on the endothelial cell Tie-2 receptor to regulate vascular integrity and remodeling. The local balance of these factors and the level of other angiogenic factors determine whether blood vessels grow, are maintained or regress. Profound angiogenesis and vascular remodeling occur in the placenta and this is altered in preeclampsia, a major cause of maternal and fetal morbidity and mortality. The mRNAs encoding Ang-1, Ang-2, and Tie-2 were detected and localized in human placentae throughout gestation. The mechanism of regulation angiopoietin mRNAs level was determined by explant culture in ambient and reduced oxygen, and in the presence of actinomycin D. In situ hybridization showed that Ang-2 mRNA was abundant in the syncytiotrophoblast in the first trimester of human pregnancy. Ang-1 mRNA could not be detected by in situ hybridization, but was by reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blotting. Placental vascular structure is altered in preeclampsia and intrauterine growth restriction, conditions where feto-placental oxygenation is perturbed. In villous explant cultures, a reduction in oxygen tension significantly raised the levels of Ang-2 mRNA, and this was dependent on transcription. However, similar experiments showed that the stability of the Ang-1 message was greatly reduced under these conditions. Thus, hypoxia has opposite effects on Ang-1 and Ang-2 mRNA levels. Placentae obtained from women with preeclampsia had reduced levels of Ang-2 mRNA compared to gestationally matched controls. There was no difference in the levels of Ang-1 mRNAs. These data show that the relative levels of Ang-1 and Ang-2 mRNA are regulated by local oxygen tension by different mechanisms and that this may be important during normal human placentation.

Vascular Endothelial Growth Factor and Angiopoietin in Liver Regeneration

Liver architecture remodeling following partial hepatectomy (PHx) involves the formation of a complex network of liver sinusoids through which the blood flows. The present study examines the involvement of vascular endothelial growth factor (VEGF) and angiopoietin-1 (ang-1) during liver regeneration. Following PHx, VEGF and ang-1 mRNA levels increase, followed by gradual return to baseline levels. RT-PCR analysis of VEGF mRNA reveals three isoforms, VEGF120, VEGF164 and VEGF188. Of the three, VEGF188 is the predominant isoform, VEGF120 being the less abundant. Although VEGF mRNA fluctuates following PHx, the relative expression of each isoform remains the same throughout the recovery process. The level of neuropilin-1, an accessory receptor of VEGF to main receptor corresponds with that of VEGF and ang-1. We have previously demonstrated the capacity of exogenous VEGF165 to stimulate liver cell proliferation following PHx. We now report similar effect using VEGF121, further demonstrating the benefit of manipulating growth factors where such an intervention is required.

Cell type specific expression of vascular endothelial growth factor and angiopoietin-1 and -2 suggests an important role of astrocytes in cerebellar vascularization.

The vascularization of the central nervous system occurs by angiogenic sprouting, a process in which different factors like vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-1/2 must act in a coordinated fashion. We investigated how these factors participate in capillarization of the cerebellum, an area experiencing marked reorganization processes during its postnatal development. VEGF and Ang-1 mRNA were predominantly expressed by astrocytes, while Ang-2 mRNA was specifically induced at the tip of invading endothelial cell cords. Similar to the cerebral cortex, vascularization of the cerebellum occurred in an inside-out pattern, following closely the generation and differentiation of each cerebellar layer. VEGF and Ang-1/2 expression patterns shifted in a similar inside-out fashion, supporting their proposed function in vessel growth and maturation.

Abnormal balance in the angiopoietin-tie2 system in human brain arteriovenous malformations.

Brain arteriovenous malformations (BAVMs) are congenital vascular lesions that often present as cerebral hemorrhage in young adults. The variable nature of the clinical course, especially with respect to spontaneous hemorrhage, recurrence, growth, and regression, suggests that BAVMs are lesions with active angiogenesis and vascular remodeling. We examined mRNA and protein expression of angiopoietin 1 (Ang1) and Ang2 by semiquantitative reverse transcriptase-polymerase chain reaction, in situ hybridization, and Western blot in BAVMs and control brains obtained from temporal lobectomy for medically intractable seizures. Although Ang1 mRNA levels were similar in BAVMs and controls, Ang1 protein levels were 30% lower in BAVMs than in controls. Ang2 mRNA levels were 40% higher and Ang2 protein levels were 8-fold higher in BAVMs than in controls. In situ hybridization showed that the Ang2 mRNA was localized to the perivascular area in BAVMs. This abnormal balance in the Ang-Tie2 system may, in part, explain the aberrant vascular phenotype in BAVMs.

Angiogenesis during follicular development in the primate and its inhibition by treatment with truncated Flt-1-Fc (vascular endothelial growth factor Trap(A40)).

The aims of this study were to 1) quantify changes in angiogenesis during follicular growth in a primate model; 2) investigate the molecular regulation using in situ hybridization of vascular endothelial growth factor (VEGF), its receptor, Flt-1, the angiopoietins (Ang-1 and Ang-2), and their receptor, Tie-2; 3) elucidate the role of VEGF in follicular angiogenesis by blocking its action by treatment with a soluble truncated form of the Flt-1 receptor, (VEGF Trap(A40)). Changes in angiogenesis were quantified using bromodeoxyuridine to obtain a proliferation index, and CD31 immunocytochemistry to visualize endothelial cell area. Percentage of proliferating endothelial cells was calculated by double labeling for bromodeoxyuridine and CD31. Vascularization was first observed in follicles containing four granulosa cell layers. A significant increase in proliferation in the thecal layer was observed from the early to late secondary stage, and dual staining showed that 25% of proliferating cells were of endothelial cell origin. VEGF messenger RNA (mRNA) was expressed in granulosa cells with an increase of grain density from late secondary to tertiary follicles. Ang-1 was weakly expressed in the theca of tertiary follicles. Ang-2 mRNA was not detected in any follicles. The mRNA for the Flt-1 and Tie-2 receptors was localized in endothelial cells of the theca. Unexpectedly, Tie-2 mRNA was also found in granulosa cells of early follicular stages and its translation was confirmed by immunocytochemistry. VEGF trap treatment for 3 days resulted in an 87% decrease of proliferation in the theca of secondary and tertiary follicles, a reduction in endothelial cell area and a marked decline in Flt-1 mRNA expression. Granulosa cell proliferation also decreased. These results show that onset and establishment of the follicle vasculature takes place early during follicular development. The ability of VEGF trap treatment to severely restrict follicular angiogenesis establishes that VEGF is the major regulator of this process in the primate ovary.

Tie-2 is expressed on thyroid follicular cells, is increased in goiter, and is regulated by thyrotropin through cyclic adenosine 3',5'-monophosphate.

Angiogenesis is coordinated with follicular cell growth in goitrogenesis. The angiopoietins, Ang-1 and Ang-2, are angiogenic growth factors acting through Tie-2, a tyrosine kinase receptor. We have examined the expression and regulation of the angiopoietins and Tie-2 in human and rat thyroids. In human goiters there was increased Tie-2 immunostaining, compared with that in normal thyroids, on both follicular and endothelial cells. In an induced goiter in rats, in situ hybridization showed increased expression of messenger ribonucleic acids (mRNAs) for Tie-2 and Ang-1 in follicular cells. As Tie-2 has previously been believed to be restricted to cells of endothelial lineage in adults, we examined its expression further in isolated follicular cells. Tie-2 and Ang-1 mRNA expression in human thyrocytes was confirmed by ribonuclease protection assay. Ang-2 mRNA was not detected in human cultures or rat thyroids. In both human follicular cell cultures and FRTL-5 cells, immunoblotting showed that Tie-2 expression was increased by TSH and agents that increased intracellular cAMP. In conclusion, we have demonstrated the expression of Tie-2 and Ang-1 in thyroid epithelial and endothelial cells, and have shown the regulation of Tie-2 by TSH and cAMP in follicular cells. Tie-2 expression is increased in goiter in both humans and rats, consistent with a role in goitrogenesis.

Angiotensin II induces expression of the Tie2 receptor ligand, angiopoietin-2, in bovine retinal endothelial cells.

Recent studies have shown that angiopoietins (Angs) and their receptor, Tie2, play a role in vascular integrity and neovascularization. The renin-angiotensin system has been hypothesized to contribute to the development of diabetic retinopathy. In this study, we investigated the effect of angiotensin II (AII) on Ang1 and Ang2 expression in cultured bovine retinal endothelial cells (BRECs). AII stimulated Ang2 but not Ang1 mRNA expression in a dose- and time-dependent manner. This response was inhibited completely by angiotensin type 1 receptor (AT1) antagonist. AII increased the transcription of Ang2 mRNA, but did not change the half-life. Protein kinase C (PKC) inhibitor completely inhibited AII-induced Ang2 expression, and the mitogen-activated protein kinase (MAPK) inhibitor also inhibited it by 69.4+/-15.6%. In addition, we confirmed the upregulation of Ang2 in an AII-induced in vivo rat corneal neovascularization model. These data suggest that AII stimulates Ang2 expression through AT1 receptor-mediated PKC and MAPK pathways in BREC, and AII may play a novel role in retinal neovascularization.

Angiogenic growth factor messenger ribonucleic acids in uterine natural killer cells.

Angiogenesis is essential for endometrial growth and repair, and disruption of this process may lead to common disorders of women, including menorrhagia and endometriosis. In pregnancy, failure of the endometrial spiral arterioles to undergo remodeling leads to preeclampsia. Here we report that in addition to vascular endothelial growth factor A (VEGF-A), human endometrium expresses messenger ribonucleic acids (mRNAs) encoding VEGF-C, placenta growth factor (PlGF), the angiopoietins, angiopoietin 1 (Ang1) and Ang2, and the receptors VEGFR-3 (Flt-4), Tie 1, and Tie 2. Levels of VEGF-C, PlGF, and Tie 2 changed during the menstrual cycle. Intense hybridization for VEGF-C and PlGF mRNAs was found in uterine nature killer cells in secretory phase endometrium and for Ang2 mRNA in the same cells in the late secretory phase. Interleukin-2 (IL-2) and IL-15 up-regulated VEGF-C, but not PlGF or Ang2, mRNA levels in isolated NK cells. Conditioned medium from decidual NK cells did not induce human umbilical vein endothelial cell apoptosis. These results indicate that human endometrium expresses a wide range of angiogenic growth factors and that uterine nature killer cells may play an important role in the abnormal endometrial angiogenesis that underlies a range of disorders affecting women.

Expression of angiopoietin-1 in human glioblastomas regulates tumor-induced angiogenesis: in vivo and in vitro studies.

To define a role for the angiopoietin/Tie2 system in astrocytoma angiogenesis, we examined the expression of angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) in these tumors by immunohistochemistry and in situ hybridization. Furthermore, we studied in vitro the effects elicited by glioblastoma cell-secreted Ang1 or by recombinant Ang1 on functions of endothelial cells (ECs). Our observations of astrocytomas show that a stage-specific induction of angiopoietins occurs and is correlated with angiogenic phases of different intensity. Ang1 expression was found in a few astrocytes scattered in the tumor at all stages of astrocytoma progression. In blood vessels, Ang1 mRNA increased progressively in high-grade glioblastomas, in which the number of vessels was higher than in low-grade tumors. Ang2 was detected in tumor cells and in ECs in high-grade astrocytomas, whereas its expression was negligible in low-grade tumors. Coculture of glioblastoma cell lines producing Ang1 with endothelium demonstrated a key role of this ligand in the control of EC network organization. We found that recombinant Ang1 in vitro induces EC spreading and reorganization of the cell monolayer into cordlike structures. These results suggest that Ang1 directly acts on ECs by modulating cell-cell and cell-matrix associations and promoting the differentiation phase of angiogenesis.

The angiopoietin-tie2 system in coronary artery endothelium prevents oxidized low-density lipoprotein-induced apoptosis.

A healthy, intact coronary artery endothelium is important because most common coronary artery diseases result from loss of endothelial integrity. In this study, we explored the biological significance of the angiopoietin-Tie2 system in porcine coronary artery. Cultured porcine coronary artery endothelial cells and explanted coronary arteries were used. Immunohistochemical analyses indicated that Ang1 is selectively expressed in vascular muscular cells, whereas angiopoietin-2 (Ang2) and Tie2 are selectively expressed in endothelial cells. Accordingly, Ang1 mRNA is mainly expressed in cultured porcine coronary artery vascular smooth muscle cells, whereas Ang2 and Tie2 mRNAs are mainly expressed in cultured porcine coronary artery endothelial cells (PCAECs). Ang1 (200 ng/ml) induced Tie2 phosphorylation, while Ang2 (200 ng/ml) did not produce Tie2 phosphorylation. Ang1 increased the survival of cultured PCAECs during apoptosis induced by oxidized low-density lipoprotein (OxLDL). This survival effect was does-dependent and PI. Furthermore, Ang1 also protected endothelial cells of explanted coronary artery against OxLDL-induced apoptosis artery. These results suggest that adult coronary artery contains Ang1-Tie2 components that enhance endothelial cell survival to help maintain the normal integrity of the coronary artery endothelium.

Angiotensin AT(1) and AT(2) receptors differentially regulate angiopoietin-2 and vascular endothelial growth factor expression and angiogenesis by modulating heparin binding-epidermal growth factor (EGF)-mediated EGF receptor transactivation.

Angiotensin II (Ang II)-mediated signals are transmitted via heparin binding epidermal growth factor (EGF)-like growth factor (HB-EGF) release followed by transactivation of EGF receptor (EGFR). Although Ang II and HB-EGF induce angiogenesis, their link to the angiopoietin (Ang)-Tie2 system remains undefined. We tested the effects of Ang II on Ang1, Ang2, or Tie2 expression in cardiac microvascular endothelial cells expressing the Ang II receptors AT(1) and AT(2). Ang II significantly induced Ang2 mRNA accumulations without affecting Ang1 or Tie2 expression, which was inhibited by protein kinase C inhibitors and by intracellular Ca(2+) chelating agents. Ang II transactivated EGFR via AT(1), and inhibition of EGFR abolished the induction of Ang2. Ang II caused processing of pro-HB-EGF in a metalloproteinase-dependent manner to stimulate maturation and release of HB-EGF. Neutralizing anti-HB-EGF antibody blocked EGFR phosphorylation by Ang II. Ang II also upregulated vascular endothelial growth factor (VEGF) expression in an HB-EGF/EGFR-dependent manner. AT(2) inhibited AT(1)-mediated Ang2 expression and phosphorylation of EGFR. In an in vivo corneal assay, AT(1) induced angiogenesis in an HB-EGF-dependent manner and enhanced the angiogenic activity of VEGF. Although neither Ang2 nor Ang1 alone induced angiogenesis, soluble Tie2-Fc that binds to angiopoietins attenuated AT(1)-mediated angiogenesis. These findings suggested that (1) Ang II induces Ang2 and VEGF expression without affecting Ang1 or Tie2 and (2) AT(1) stimulates processing of pro-HB-EGF by metalloproteinases, and the released HB-EGF transactivates EGFR to induce angiogenesis via the combined effect of Ang2 and VEGF, whereas AT(2) attenuates them by blocking EGFR phosphorylation. Thus, Ang II is involved in the VEGF-Ang-Tie2 system via HB-EGF-mediated EGFR transactivation, and this link should be considerable in pathological conditions in which collateral blood flow is required.

Hypoxia up-regulates angiopoietin-2, a Tie-2 ligand, in mouse mesangial cells

Angiopoietins are secreted factors modulating endothelial survival and morphogenesis. Our previous studies demonstrated angiopoietin-2 (Ang-2) promoter activity in vivo in maturing kidney vascular smooth muscle and mesangial cells, with Tie-2 expressed by adjacent endothelia, including glomerular capillaries. In this study we investigated Ang-2 expression in immortalized mouse mesangial cell lines and studied the response to hypoxia. Using reverse transcription-polymerase chain reaction, Ang-2 and Ang-3 mRNA were detected but Ang-1 and Tie-2 transcripts were absent. As assessed by Northern and slot blotting, 8 to 24 hours hypoxia (3% O(2)) significantly increased Ang-2 mRNA levels versus normoxic (21% O(2)) cells and the rate of Ang-2 mRNA degradation was similar in both conditions, consistent with increased transcription. Hypoxia also increased immunoreactive Ang-2 in cell lysates. Hypoxic stimulation of Ang-2 mRNA was significantly reduced by inhibitors of tyrosine kinase (genistein) and protein kinase C (GF109203X), but not by a mitogen-activated protein kinase 1 inhibitor (PD98059). Furthermore, hypoxia coincidentally up-regulated levels of vascular endothelial growth factor (VEGF) mRNA in these cells. Finally, in vivo, immunoreactive Ang-2 was observed in the cores of immature glomeruli of neonatal mice, but immunostaining in this location was absent in four-week postnatal mice. This is the first demonstration that isolated mesangial cells express Ang-2 mRNA and protein and up-regulate Ang-2 in response to hypoxia. We speculate that hypoxia-induced, mesangial-derived Ang-2 and VEGF may have synergistic paracrine roles in the growth of glomerular endothelia during normal development and diseases.