Hypoxia-inducible factor-1 mediates activation of cultured vascular endothelial cells by inducing multiple angiogenic factors.

Hypoxia-inducible factor-1 (HIF-1) mediates transcriptional activation of vascular endothelial growth factor (VEGF) and other hypoxia-responsive genes. Transgenic expression of a constitutively stable HIF-1alpha mutant increases the number of vascular vessels without vascular leakage, tissue edema, or inflammation. This study aimed to investigate the molecular basis by which HIF-1 mediates the angiogenic response to hypoxia. In primary human endothelial cells, hypoxia, desferrioxamine, or infection with Ad2/HIF-1alpha/VP16, an adenoviral vector encoding a constitutively stable hybrid form of HIF-1alpha, increased the mRNA and protein levels of VEGF, angiopoietin-2 (Ang-2), and angiopoietin-4 (Ang-4). Infection with Ad2/CMVEV (a control vector expressing no transgene) had no effect. Angiopoietin-1 (Ang-1) expression was not detected in human endothelial cells. Ang-4 was also induced by hypoxia or Ad2/HIF-1alpha/VP16 in human cardiac cells, whereas Ang-1 expression remained unchanged. Recombinant Ang-4 protein protected endothelial cells against serum starvation-induced apoptosis and increased cultured endothelial cell migration and tube formation. Ad2/HIF-1alpha/VP16 stimulated endothelial cell proliferation and tube formation. Hypoxia- or Ad2/HIF-1alpha/VP16-induced tube formation was significantly reduced by a Tie-2 inhibitor. These results suggest that HIF-1 mediates the angiogenic response to hypoxia by upregulating the expression of multiple angiogenic factors. Ang-4 can function similarly as Ang-1 and substitute for Ang-1 to participate in hypoxia-induced angiogenesis. Activation of the angiopoietin/Tie-2 system may play a role in the ability of HIF-1 to induce hypervascularity without excessive permeability.

Vegfb gene knockout mice display reduced pathology and synovial angiogenesis in both antigen-induced and collagen-induced models of arthritis.

OBJECTIVE: To determine the role of vascular endothelial growth factor B (VEGF-B) in 2 mouse models of arthritis, antigen-induced arthritis (AIA) and collagen-induced arthritis (CIA). METHODS: For AIA studies, monarticular AIA was induced by methylated bovine serum albumin (mBSA) priming of Vegfb gene knockout (Vegfb(-/-)) and wild-type (Vegfb(+/+)) mice, followed by intraarticular injection of mBSA or saline control 8 days later. CIA was induced in Vegfb(-/-) and Vegfb(+/+) mice by intradermal injection of chick type II collagen in adjuvant. Arthritis was monitored in both models using defined criteria (clinical and histologic). Angiogenesis was measured by synovial vessel density in diseased and control joints. RESULTS: In AIA studies, Vegfb(+/+) mice displayed significant knee joint swelling and synovial inflammation 7 days after intraarticular injection of antigen. Synovial inflammation was associated with angiogenesis, since vessel density in AIA synovium was significantly higher in arthritic than in control joints from the same animal. Knee joint swelling, synovial inflammation, and inflammation-associated vessel density in arthritic joints were reduced in Vegfb(-/-) mice compared with arthritic joints from Vegfb(+/+) mice. Similarly, in CIA, both disease incidence and mean clinical severity scores were significantly reduced in Vegfb(-/-) mice compared with Vegfb(+/+) mice. Mean histologic severity scores and mean synovial vessel density were reduced in diseased joints from Vegfb(-/-) mice when compared with diseased joints from Vegfb(+/+) mice. CONCLUSION: The reduction in inflammation-associated synovial angiogenesis in Vegfb(-/-) mice implicates VEGF-B in pathologic vascular remodeling in inflammatory arthritis. VEGF-B may be an attractive target in the design of anti-angiogenic therapies for rheumatoid arthritis.

Expression of the VEGF and angiopoietin genes in endometrial atypical hyperplasia and endometrial cancer.

Angiogenesis is critical for the growth and metastasis of endometrial cancer and is therefore an important therapeutic target. Vascular endothelial growth factor-A (VEGF-A) is a key molecule in angiogenesis, but the identification of related molecules and the angiopoietins suggests a more complex picture. We investigated the presence of transcripts for VEGF-A, VEGF-B, VEGF-C, VEGF-D, Angiopoietin-1 and Angiopoietin-2 in benign endometrium, atypical complex hyperplasia (ACH) and endometrioid endometrial carcinoma using in situ hybridisation. We confirmed the presence of VEGF-A mRNA in the epithelial cells of cancers examined (13 out of 13), but not in benign endometrium or ACH. We also demonstrate, using quantitative polymerase chain reaction, that levels of VEGF-B mRNA are significantly lower in endometrial cancer than benign endometrium. We conclude that loss of VEGF-B may contribute to the development of endometrial carcinoma by modulating availability of receptors for VEGF-A.

Altered expression of angiopoietins during blood-brain barrier breakdown and angiogenesis.

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) belong to a novel family of endothelial growth factors that function as ligands for the endothelial-specific receptor tyrosine kinase, Tie-2. Ang-1 reduces endothelial permeability of noncerebral vessels and has a major role in vascular stabilization and maturation, whereas Ang-2 is thought to be an endogenous antagonist of the action of Ang-1 at Tie-2. Expression of these ligands at the mRNA and protein level were studied during both blood-brain barrier (BBB) breakdown and cerebral angiogenesis occurring in the rat cortical cold-injury model by RT-PCR analysis and immunohistochemistry respectively, during a time course of 6 hours to 6 days. In addition, immunohistochemical detection of fibronectin was used to detect BBB breakdown at the lesion site and dual labeling was used to determine whether the vessels demonstrating BBB breakdown expressed endothelial Ang-1 or Ang-2. Endothelial Ang-1 and Tie-2 proteins were present in all cerebral vessels of normal brain including those of the choroid plexuses, whereas both these proteins as well as Ang-2 were present in choroid plexus epithelium and in ependymal cells, suggesting that angiopoietins have an autocrine effect on these cell types as well. In contrast, in the early phase after injury during the known period of BBB breakdown, increased Ang-2 mRNA and protein and decreased endothelial Ang-1 and Tie-2 proteins were observed. Two to 6 days after injury, the progressive increase in Ang-1 mRNA and protein and the decrease in Ang-2 coincided with cerebrovascular angiogenesis. Confocal microscopy showed colocalization of both Ang-1 and Ang-2 in endothelium of lesion vessels, and our observation of colocalization of Ang-1 and Ang-2 in polymorphonuclear leukocytes and macrophages has not been reported previously. This study demonstrates that Ang-1 is an important factor in maintaining normal homeostasis in the brain. Thus Ang-1 therapy may have therapeutic potential in reducing BBB breakdown and the ensuing edema after massive brain injury.

Contrasting effects of VEGF gene disruption in embryonic stem cell-derived versus oncogene-induced tumors.

Previous gene targeting studies have implicated an indispensable role of vascular endothelial growth factor (VEGF) in tumor angiogenesis, particularly in tumors of embryonal or endocrine origin. In contrast, we report here that transformation of VEGF-deficient adult fibroblasts (MDF528) with ras or neu oncogenes gives rise to highly tumorigenic and angiogenic fibrosarcomas. These aggressive VEGF-null tumors (528ras, 528neu) originated from VEGF(-/-) embryonic stem cells, which themselves were tumorigenically deficient. We also report that VEGF production by tumor stroma has a modest role in oncogene-driven tumor angiogenesis. Both ras and neu oncogenes down-regulated at least two endogenous inhibitors of angiogenesis [pigment epithelium derived factor (PEDF) and thrombospondin 1 (TSP-1)]. This is functionally important as administration of an antiangiogenic TSP-1 peptide (ABT-526) markedly inhibited growth of VEGF(-/-) tumors, with some ingress of pericytes. These results provide the first definitive genetic demonstration of the dispensability of tumor cell-derived VEGF in certain cases of 'adult' tumor angiogenesis, and thus highlight the importance of considering VEGF-independent as well as VEGF-dependent pathways when attempting to block this process pharmacologically.

Localization of mRNA for vascular endothelial growth factor (VEGF), angiopoietins and their receptors during the peri-implantation period and early pregnancy in marmosets (Callithrix jacchus).

Implantation of a blastocyst into a receptive endometrium is a prerequisite for successful pregnancy. Angiogenesis is a key event in this process but the mechanisms by which localized changes in vascular permeability and angiogenesis occur have yet to be elucidated. Vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 have been implicated as key players in vascular remodelling and placentation. Angiopoietins also appear to have a significant role in regulation of blood vessel growth, maturation and regression. The aim of this study was to describe the molecular regulation of angiogenesis in the first month of pregnancy in marmosets and to address the putative physiological roles for these factors. Uteri were studied at weeks 2, 3 and 4 of pregnancy and compared with late secretory non-pregnant endometrium. Implantation in marmosets occurs at day 11 of pregnancy; hence, these time points were chosen so that the peri-implantation period and very early pregnancy could be studied. mRNAs for VEGF, VEGFR-1 and VEGFR-2, angiopoietin 1, angiopoietin 2 and their receptor Tie-2 were localized and quantified by in situ hybridization. Endothelial cells were identified by CD31 immunocytochemistry. VEGF mRNA was present in all compartments except endothelial cells, and its expression generally increased throughout pregnancy except in upper zone glandular epithelium and luminal epithelium, where a decrease in expression was observed. VEGF receptor mRNAs were found in endothelial cells of the upper zones immediately surrounding glandular epithelium. Angiopoietin 1 mRNA was localized to glandular epithelium of the upper and lower zones throughout pregnancy, and increased in stroma at week 4. Expression of angiopoietin 2 mRNA was localized exclusively to endothelial cells of large luminal vessels and was higher in endometrium from marmosets at week 4 of pregnancy than in endometrium from all other stages. These data provide comprehensive evidence that VEGFR-1 and -2, and angiopoietin 1, angiopoietin 2 and Tie-2 interactions may be involved in the preparation of endometrium for implantation, remodelling of the maternal vasculature and trophoblast invasion during the peri-implantation period in this primate species.

[Induction of angiogenesis in ischemic myocardium by adenovirus mediated angiopoietin-1 gene transfer, an experimental study].

OBJECTIVE: To investigate the effect on angiogenesis in ischemic myocardium of adenovirus mediated angiopoietin-1 gene transfer. METHODS: Ang-1 cDNA was obtained from human spleen by RT-PCR and then cotransfected into 293 host cells together with Adv5-CAG, E1 and E3 substituted adenovirus genome, thus constructing recombinant adenovirus Adv5-CAG/Ang-1. Recombinant adenovirus Adv5-CAG/LacZ containing LacZ gene was constructed too. Thirty six male New Zealand white rabbits were randomly divided into 3 group of 12 rabbits: DMEM group, Ang-1 group, and LacZ group and underwent high-positioned double-ligation of the left anterior descending coronary artery and then myocardial injection of DMEM, Adv5-CAG/Ang-1, or Adv5-CAG/LacZ respectively. Fourteen days after the operation 2 rabbits in each group were killed, the myocardial tissues where injected was given were taken to detect the expression of Ang-1 by RT-PCR. Coronary angiography was conducted 28 days postoperatively upon 5 rabbits in each group to observe the angiogenesis in the ischemic myocardium. Five rabbits in each group were killed at the 14 th and 28 th postoperative days to observe the capillary density by immunohistochemical staining. RESULTS: The Ang-1 cDNA cloned in the laboratory was 1,515 bp in length containing the signal peptide structure in accordance with the report in literature. Fourteen days after operation, Ang-1 gene was positive in the myocardium of Ang-1 group and negative in the other 2 groups. New vessel formation was obvious at the 28 th postoperative day in the Ang-1 group and not in the other 2 groups. Capillary density increased after operation in all 3 groups, however, more significant in the Ang-1 group, especially 28 days after. CONCLUSION: Adenovirus-mediated angiopoietin-1 gene effectively promotes the neovascularization in ischemic myocardium of rabbits.

Tumor necrosis factor alpha induces angiogenic factor up-regulation in malignant glioma cells: a role for RNA stabilization and HuR.

Malignant glioma (MG) cells up-regulate angiogenic factor expression in response to different extracellular signals such as hypoxia and cytokines. This up-regulation in turn promotes angiogenesis and tumor progression. Posttranscriptional gene regulation has been implicated as one mechanism for this tumor response, and we have previously shown that HuR, a protein associated with RNA stabilization, is overexpressed in MGs (L. B. Nabors et al., Cancer Res., 61: 2154-2161, 2001). Here, we demonstrate a marked up-regulation (RNA and protein) of tumor necrosis factor alpha (TNF-alpha), interleukin 8, and, to a lesser extent, vascular endothelial growth factor in U251 glioma cells after stimulation with TNF-alpha. RNA kinetic studies indicated that TNF-alpha induced the stabilization of all three transcripts. Using a luciferase reporter assay, we demonstrate that the AU-rich elements (AREs) in the 3'-untranslated region of these genes significantly contribute to this posttranscriptional regulation. UV cross-linking and immunoprecipitation with glioma extracts indicate that HuR binds to all three AREs. When HuR is overexpressed in glioma cells, there is enhanced RNA stabilization of all three angiogenic factor transcripts with a concomitant increase in mRNA and protein expression (up to 7-fold). These findings indicate that TNF-alpha up-regulates angiogenic factor expression in MG cells and that RNA stabilization, via the AREs in the 3'-untranslated region, contributes to this up-regulation.

Angiopoietin-1 inhibits vascular permeability, angiogenesis, and growth of hepatic colon cancer tumors.

Angiopoietin (Ang)-1 and -2 are critical regulators of embryonic and postnatal neovascularization. Ang-1 activates the endothelial cell-specific tyrosine kinase receptor Tie-2, which in turn leads to enhanced endothelial cell survival and stabilization. The effects of Ang-1 on tumor angiogenesis remain controversial; although we have previously demonstrated that Ang-1 overexpression in colon cancer cells leads to a decrease in s.c. tumor growth, others have shown that Ang-1 may be proangiogenic. Few studies have addressed the role of the Angs in tumors growing in the organ of metastatic growth. We hypothesized that overexpression of Ang-1 may inhibit the growth of colon cancers growing in the liver by inhibition of angiogenesis. We also wanted to investigate the mechanisms by which Ang-1 affects angiogenesis in vivo. Human colon cancer cells (HT29) were stably transfected with an Ang-1 construct or an empty vector (pcDNA) and injected directly into the livers of nude mice. After 37 days, livers were harvested and weighed, and tumor sizes were measured. In an additional experiment, to validate the paracrine effect of Ang-1, various mixtures of control cells and Ang-1-transfected cells were injected into livers, and tumor growth was assessed. Direct effects of recombinant Ang-1 on angiogenesis were studied with an in vivo Gelfoam angiogenesis assay. The impact of Ang-1 on vascular permeability was investigated using an intradermal Miles assay with conditioned media from transfected cells. Liver weights (P < 0.05), tumor volumes (P < 0.05), vessel counts (P < 0.01), and tumor cell proliferation (P < 0.01) in the Ang-1 group were significantly lower than those in the control (pcDNA) group. Tumor vessels in the Ang-1 group developed a significantly higher degree of pericyte coverage (P < 0.02) than vessels in pcDNA tumors. In the cell mixture experiment, even as few as a 1:10 mixture of Ang-1-transfected cells/control cells resulted in a significant reduction of hepatic tumor volumes (P < 0.04). In the angiogenesis assay, vessel counts in Gelfoam implants were significantly decreased by the addition of Ang-1 (P < 0.01). Finally, conditioned medium from Ang-1-transfected cells decreased vascular permeability more than that from control cells (P < 0.05). Our results suggest that Ang-1 is an important regulator of angiogenesis and vascular permeability and that this effect may be secondary to increasing periendothelial support and vessel stabilization. Thus, Ang-1 could potentially serve as an antineoplastic or anti-permeability agent for patients with metastatic colorectal cancer.

Tumor-derived vascular endothelial growth factor up-regulates angiopoietin-2 in host endothelium and destabilizes host vasculature, supporting angiogenesis in ovarian cancer.

Vascular remodeling in host tissues surrounding growing tumors is implicated in the successful development of tumor neovasculature. Cooperation between vascular endothelial growth factor (VEGF) and angiopoietins (Angs) is considered to be critical in this context. However, the mechanisms regulating the coordinated expression of these molecules remain, to date, elusive. In this study, we used a murine ovarian cancer angiogenesis model induced by overexpression of VEGF, as well as 52 human ovarian cancer specimens and 36 established cancer cell lines to characterize the expression and regulation of Ang-2 in the context of tumor angiogenesis. Using a combination of immunohistochemistry, laser capture microdissection and real-time quantitative reverse transcription-PCR, we showed that tumor-derived VEGF significantly up-regulated the expression of Ang-2 in host stroma endothelial cells, resulting in markedly increased Ang-2/Tie-2 mRNA copy number ratio in vivo. In vitro experiments showed that VEGF directly up-regulated Ang-2, which is mediated via VEGF receptor-2/flk-1/KDR pathway, in cultured endothelial cells through transcriptional activation rather than the enhanced mRNA stability. In human ovarian cancer, Ang-2 was primarily expressed in stroma endothelial cells and detectable in tumor cells of only 12% tumor specimens; however, it was not detected in the majority of established ovarian cancer cell lines. In addition, a significant correlation was observed between VEGF and Ang-2 mRNA expression (P < 0.01) but not between VEGF and Ang-1 or Tie-2 in human ovarian cancer specimens. In the mouse ovarian cancer model, up-regulation of Ang-2 in host stroma endothelial cells was significantly associated with pericyte loss and instability of the host vasculature surrounding the tumor. Our study suggests a novel mechanism by which tumor-derived VEGF interacts with Angs/Tie-2 system in host stroma endothelial cells and induces in a paracrine manner the remodeling of host vasculature to support angiogenesis during tumor growth.

Placental growth factor, a member of the VEGF family, contributes to the development of choroidal neovascularization.

PURPOSE: VEGF has been shown to be necessary, but not sufficient alone, for the development of subretinal pathologic angiogenesis. In the current study, the influence of placental growth factor (PlGF), a member of the VEGF family, in human and experimental choroidal neovascularization (CNV) was investigated. METHODS: The presence of VEGF family member mRNA was evaluated by RT-PCR in neovascular membranes extracted during surgery. The spatial and temporal pattern of VEGF isoforms and PlGF mRNA expression were explored by using the laser capture catapulting technique and RT-PCR in a murine laser-induced model and in vitro. PlGF expression was also studied in human donor eyes. The influence of endogenous PlGF was evaluated in deficient mice (PlGF(-/-)) and by antibody-mediated neutralization of the PlGF receptor. RESULTS: Human neovascular membranes consistently expressed VEGF-A, -B, and -C; PlGF; and VEGFR-1 and -2. The VEGF(120) isoform mRNA was primarily induced in early stages of angiogenesis in vivo and in vitro. PlGF mRNA expression was present in the intact choroid and significantly upregulated during the course of experimental CNV. Both deficient PlGF expression in PlGF(-/-) mice and PlGF receptor neutralization in wild-type mice prevented the development of choroidal neovascularization induced by laser. CONCLUSIONS: These observations demonstrate the participation of PlGF in experimental CNV. They identify therefore PlGF as an additional promising target for ocular antiangiogenic strategies.

Novel expression of vascular endothelial growth factor receptor (VEGFR)-3 and VEGF-C on corneal dendritic cells.

Vascular endothelial growth factor-3 (VEGFR-3) plays a critical role in embryonic cardiovascular development and is thought to be expressed exclusively on the lymphatic endothelium, high endothelial venules, and rarely on adult vascular endothelium. Recent evidence also suggests expression of VEGFR-3 on some tumor-associated macrophages. We have studied the expression of VEGFR-3, its ligand VEGF-C and the co-receptor neuropilin-2, in normal and inflamed corneas and characterized the phenotype and distribution of VEGFR-3(+) cells. Our data demonstrate, for the first time, the expression of VEGFR-3 on corneal dendritic cells (DC) and its up-regulation in inflammation. VEGFR-3(+) DC are CD11c(+)CD45(+)CD11b(+), and are mostly major histocompatibility (MHC) class II(-)CD80(-)CD86(-), indicating immature DC of a monocytic lineage. During inflammation, there is rapid increase in the number of VEGFR-3(+) DC in the cornea associated with heightened membranous expression as compared to a mostly intracellular expression in uninflamed tissue. VEGFR-3(+) DC in normal corneas are VEGF-C(-)neuropilin-2(-), but express VEGF-C in inflammation. Interestingly, similar cells are absent both in the normal and inflamed skin. These data demonstrate, for the first time, the expression of VEGFR-3 and VEGF-C on tissue DC, which implicate a novel potential relationship between lymphangiogenesis and leukocyte trafficking in the eye.

Angiogenic activity of an Onchocerca volvulus Ancylostoma secreted protein homologue.

Angiogenesis is an important step in the development of ocular onchocercaisis. In previous studies, it has been demonstrated that Onchocerca volvulus homologues of the Ancylostoma secreted protein family have pronounced angiogenic activity. The overall goal of the current study was to determine if this angiogenic effect is exerted through a direct or indirect mechanism. These studies focused on one member of this family, OvASP-2, as this protein is expressed in microfilaria, the stage of the parasite that causes ocular onchocercaisis. Clones encoding truncated and full length open reading frames were expressed as fusion proteins with Escherichia coli maltose binding protein (MBP), and angiogenic activity was compared in vitro and in vivo with MBP alone. Truncated constructs expressing only the first 105 amino acids of OvASP-2 were as active as the full length protein in inducing new blood vessel formation. The full length fusion protein did not stimulate proliferation or production of vascular endothelial growth factor in vascular endothelial cells in vitro, indicating that OvASP-2 does not directly stimulate angiogenesis. Sequence analysis demonstrated that the gene encoding OvASP-2 contained five introns. Sequence comparisons of the genomic loci from West African blinding and non-blinding strains of O. volvulus revealed that some polymorphism existed among the various isolates tested. However, none of these polymorphisms could be used to differentiate the parasite strains, suggesting that qualitative variation in OvASP-2 could not explain the difference in ocular pathogenic potential of the two parasite strains.

Modulation of the angiogenesis response through Ha-ras control, placenta growth factor, and angiopoietin expression in mouse skin carcinogenesis.

Tumor angiogenesis is governed by a complex balance of positive and negative angiogenic factors. Development of chemically-induced mouse skin tumors appears to be highly dependent on an early burst of neovascularization. We have previously shown that Ha-ras-driven vascular endothelial growth factor (VEGF) expression plays a pivotal role in this process. However, the status of other critical positive and negative angiogenic factors throughout skin tumorigenesis has not been studied to the same extent. In the present study, we show that another VEGF family member, placenta growth factor (PlGF), was highly upregulated at all tumor stages in a ras-dependent manner. The study of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), ligands of receptor tyrosine kinase 2 (Tie-2), showed that while stroma-derived Ang-2 was increased, epidermal Ang-1 expression was completely abolished at early papilloma formation. Studies using epidermal tumor cell lines suggest that the disappearance of Ang-1 also depends on ras activation, extending the plethora of events controlled by this oncogene in mouse skin carcinogenesis. Our results indicated that tumor development occurred in a strong angiogenesis-prone scenario in which PlGF and Ang-2 acted cooperatively with VEGF, whereas the negative or stabilizing effect of Ang-1 was abrogated. A time-course sequence of expression of angiogenic factors expressed throughout tumor growth, as well as the identification of key signaling molecules triggering the angiogenic response, may contribute to the development and testing of antiangiogenic therapeutic strategies with this in vivo tumor model.

Differential expression of the angiogenic factor genes vascular endothelial growth factor (VEGF) and endocrine gland-derived VEGF in normal and polycystic human ovaries.

Angiogenesis is a key aspect of the dynamic changes occurring during the normal ovarian cycle. Hyperplasia and hypervascularity of the ovarian theca interna and stroma are also prominent features of the polycystic ovary syndrome (PCOS), a leading cause of infertility. Compelling evidence indicated that vascular endothelial growth factor (VEGF) is a key mediator of the cyclical corpus luteum angiogenesis. However, the nature of the factor(s) that mediate angiogenesis in PCOS is less clearly understood. Endocrine gland-derived (EG)-VEGF has been recently identified as an endothelial cell mitogen with selectivity for the endothelium of steroidogenic glands and is expressed in normal human ovaries. In the present study, we compared the expression of EG-VEGF and VEGF mRNA in a series of 13 human PCOS and 13 normal ovary specimens by in situ hybridization. EG-VEGF expression in normal ovaries is dynamic and generally complementary to VEGF expression in both follicles and corpora lutea. A particularly high expression of EG-VEGF was detected in the Leydig-like hilus cells found in the highly vascularized ovarian hilus. In PCOS ovaries, we found strong expression of EG-VEGF mRNA in theca interna and stroma in most of the specimens examined, thus spatially related to the new blood vessels. In contrast, VEGF mRNA expression was most consistently associated with the granulosa cell layer and sometimes the theca, but rarely with the stroma. These findings indicate that both EG-VEGF and VEGF are expressed in PCOS ovaries, but in different cell types at different stages of differentiation, thus suggesting complementary functions for the two factors in angiogenesis and possibly cyst formation.

Angiogenic actions of angiopoietin-1 require endothelium-derived nitric oxide.

Angiopoietin1 (Ang1) is a novel angiogenic factor with important actions on endothelial cell (EC) differentiation and vascular maturation. Ang1 has been shown to prevent EC apoptosis through activation of PI3-kinase/Akt, a pathway that is also known to activate endothelium nitric oxide synthase (eNOS). Therefore, we hypothesized that the angiogenic effects of Ang1 would also be dependent on the PI3-kinase/Akt pathway, possibly mediated by increased eNOS activity and NO release. Treatment of human umbilical vein endothelial cells with recombinant Ang1* (300 ng/ml) for 15 minutes resulted in PI3-kinase-dependent Akt phosphorylation, comparable to that observed with vascular endothelial growth factor (VEGF) (50 ng/ml), and increased NO production in a PI3-kinase/Akt-dependent manner. Capillary-like tube formation induced by Ang1* in fibrin matrix at 24 hours (differentiation index, DI: 13.74 +/- 0.76 versus control 1.71 +/- 0.31) was abolished in the presence of the selective PI3-kinase inhibitor, LY294002 (50 micro mol/L) (DI: 0.31 +/- 0.31, P < 0.01) or the NOS inhibitor, L-NAME (3 mmol/L) (DI: 4.10 +/- 0.59, P < 0.01). In subcutaneous Matrigel implants in vivo, addition of recombinant Ang1* or wild-type Ang1 from conditioned media of COS-1 cells transfected with a pFLAG Ang1 expression vector, induced significant neovascularization to a degree similar to VEGF. Finally, angiogenesis in vivo in response to both Ang1 and VEGF was significantly reduced in eNOS-deficient compared with wild-type mice. In summary, our results demonstrate for the first time that endothelial-derived NO is required for Ang1-induced angiogenesis, and that the PI3-kinase signaling mediates the activation of eNOS and NO release in response to Ang1.

Increased expression of angiopoietin-2 and Tie2 receptor in a rat model of myocardial ischaemia/reperfusion.

The angiopoietins and Tie receptors are involved in blood vessel formation. The role of the angiopoietin/Tie receptor system in myocardial ischaemia/reperfusion is not well known. To investigate the participation of angiopoietins and Tie receptors in myocardial ischaemia/reperfusion, adult Wistar rats were studied in which the left coronary artery was ligated for 30 min, followed by reperfusion. Angiopoietin-1 (Ang1), angiopoietin-2 (Ang2), Tie1 and Tie2 were measured immediately after relief of occlusion, and 1, 6, 24 and 72 h after reperfusion, by Northern blot, Western blot and immunohistochemical staining. Ang2 mRNA was increased significantly at 24 h and 48 h after reperfusion, and returned to baseline levels at 72 h, in the jeopardized myocardium. Tie2 mRNA increased 3.4-fold immediately after the relief of occlusion, reached a maximum 8-fold increase at 24 h after reperfusion and remained elevated up to 72 h. Ang2 protein levels also increased after reperfusion, reaching a maximum 2.2-fold increase at 48 h after reperfusion. Tie2 protein increased immediately after relief of ischaemia, and showed a significant increase from 6 h to 72 h after reperfusion as compared with the sham control. Ang1 and Tie1 mRNA and protein did not show significant changes after ischaemia/reperfusion. Immunohistochemical studies also showed increased immunoreactivity of Ang2 and Tie2 in the jeopardized myocardium after ischaemia/reperfusion. In conclusion, expression of both Ang2 and Tie2 increased after ischaemia/reperfusion in the rat ventricular myocardium, while the expression of Ang1 and Tie1 did not.

Mouse endocrine gland-derived vascular endothelial growth factor: a distinct expression pattern from its human ortholog suggests different roles as a regulator of organ-specific angiogenesis.

We recently described human endocrine gland-derived vascular endothelial growth factor (EG-VEGF) as an endothelial cell mitogen with a novel selective activity and an expression pattern essentially limited to steroidogenic glands. Herein we present the identification and characterization of the mouse ortholog. The mouse cDNA and predicted amino acid sequences are, respectively, 86% and 88% identical with the human. Surprisingly, the mouse EG-VEGF transcript is predominantly expressed in liver and kidney. A comparison of human and mouse EG-VEGF promoter sequences revealed a potential binding site for NR5A1, which is known to be a pivotal element for steroidogenic-specific transcription, in the human but not mouse promoter. In situ hybridization studies localized expression of mouse EG-VEGF mRNA to hepatocytes and renal tubule cells. Interestingly, capillary endothelial cells in these sites share several common structural features with those found in steroidogenic glands. Within liver and kidney, EG-VEGF receptor expression was largely restricted to endothelial cells. Mouse EG-VEGF promoted proliferation and survival of endothelial cells. We propose that mouse EG-VEGF, like human EG-VEGF, plays a role in regulating the phenotype and growth properties of endothelial cells within distinct capillary beds.

Synergistic effect of angiopoietin-1 and vascular endothelial growth factor on neoangiogenesis in hypercholesterolemic rabbit model with acute hindlimb ischemia.

Vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) are essential for vascular integrity and development. The purpose of the study was to test the hypothesis that Ang1 will promote angiogenic response to VEGF in the spontaneous Watanabe heritable hypercholesterolemic (WHHL) rabbit model of acute hindlimb ischemia. Immediately after the ligation of the external iliac artery and the excision of the common and superficial femoral artery in one female WHHL rabbit, 250 microg of phVEGF(165) (n = 8), 500 microg of pAng1* (n = 8), or 250 microg of phVEGF(165) plus 500 microg of pAng1* (n = 8) was injected intramuscularly into the ischemic hindlimb muscles. Gross appearance of ischemic limb, collateral vessel formation and limb perfusion were assessed 30 days after treatment. The incidence of ischemic limb necrosis was higher in the animals treated by phVEGF(165) or by pAng1* than in those treated by phVEGF(165) plus pAng1* (100%, 75% and 14.3%, respectively; P = 0.002). Animals in the combination therapy group had a significantly higher calf blood pressure ratio at day 30 (VEGF plus Ang1* = 0.84 +/- 0.06; VEGF = 0.54 +/- 0.01; Ang1* = 0.59 +/- 0.05; P < 0.01). A combination therapy of VEGF plus Ang*1 had a significantly higher (P < 0.01) angiographic score than either therapy alone. Capillary density (P < 0.05) and capillary/muscle fiber ratio (P < 0.01) of the combination therapy group were also significantly higher than that of either therapy alone. In conclusion, Ang1 can potentiate the angiogenic response to VEGF in the hyperlipidemic rabbit model of acute hindlimb ischemia. Intramuscular administration of cytokines on revascularization of the ischemic hindlimb model of hyperlipidemic rabbit is feasible.

Evidence for hypoxia-induced neuronal-to-chromaffin metaplasia in neuroblastoma.

We present evidence that in neuroblastoma, a pediatric malignancy of embryonal sympathetic origin, hypoxia, underlies a phenotypic switch from a primitive neuronal to a chromaffin cell type. This conclusion is based on morphological and molecular data on 116 clinical tumors and is supported by data on the phenotypic effects of hypoxia on neuroblastoma cell lines when studied in monolayer culture and as tumor xenografts. In the clinical material, extensive chromaffin features were seen in regions of chronic tumor hypoxia. This was the exclusive form of intra-tumoral maturation of stroma-poor tumors and was also seen in stroma-rich tumors, either exclusively or in combination with ganglion-like cells. In neuroblastoma cell lines, hypoxia induced changes in gene expression associated with the chromaffin features observed in vivo. We therefore propose tumor hypoxia as a major cue determining phenotype in sympathetic tumors of neuroblastic origin. Because it appears to be reversible upon reoxygenation in monolayer culture, we suggest the term metaplasia for the phenomenon.