Presence and regulation of endocrine gland vascular endothelial growth factor/prokineticin-1 and its receptors in ovarian cells.

Endocrine gland vascular endothelial growth factor (EG-VEGF) is a novel angiogenic mitogen selective for endothelial cells (EC) in endocrine glands. EG-VEGF is identical to a protein previously cloned and termed prokineticin (PK)-1. The present study examined the expression of EG-VEGF/PK-1 and its receptors in ovarian steroidogenic cells and EC and compared the regulation of EG-VEGF/PK-1 and VEGF expression in SV40 transformed luteinized human granulosa cell line (SVOG). Normal granulosa or SVOG cells expressed EG-VEGF/PK-1 mRNA. Incubation of SVOG cells with forskolin augmented EG-VEGF/PK-1 expression in a dose-dependent manner. Chemical hypoxia induced by CoCl(2) and desferrioxamine mesylate (100 micro M each) markedly reduced EG-VEGF/PK-1. In contrast, hypoxia significantly elevated VEGF mRNA (VEGF165, 189) and protein secretion. Thrombin, like hypoxia, also induced an opposite effect on VEGF and EG-VEGF/PK-1. Whereas EG-VEGF/PK-1 and VEGF were inversely regulated, steroidogenesis and EG-VEGF/PK-1 were positively correlated in SVOG cells. A distinct pattern of ovarian PK receptor (PK-R) expression was observed in which steroidogenic cells predominantly express PK-R1 receptors, whereas corpus luteum-derived EC express high levels of both PK-R1 and PK-R2. Therefore, acting via either PK-R2 or PK-R1, EG-VEGF/PK-1 may have angiogenic as well as nonangiogenic functions in the ovary.

Lactate dehydrogenase-5 (LDH-5) overexpression in non-small-cell lung cancer tissues is linked to tumour hypoxia, angiogenic factor production and poor prognosis.

Lactate dehydrogenase-5 (LDH-5) catalyses the reversible transformation of pyruvate to lactate, having a principal position in the anaerobic cellular metabolism. Induction of LDH-5 occurs during hypoxia and LDH-5 transcription is directly regulated by the hypoxia-inducible factor 1 (HIF1). Serum LDH levels have been correlated with poor prognosis and resistance to chemotherapy and radiotherapy in various neoplastic diseases. The expression, however, of LDH in tumours has never been investigated in the past. In the present study, we established an immunohistochemical method to evaluate the LDH-5 overexpression in tumours, using two novel antibodies raised against the rat muscle LDH-5 and the human LDH-5 (Abcam, UK). The subcellular patterns of expression in cancer cells were mixed nuclear and cytoplasmic. In direct contrast to cancer cells, stromal fibroblasts were reactive for LDH-5 only in a minority of cases. Serum LDH, although positively correlated with, does not reliably reflect the intratumoral LDH-5 status. Lactate dehydrogenase-5 overexpression was directly related to HIF1alpha and 2alpha, but not with the carbonic anhydrase 9 expression. Patients with tumours bearing high LDH-5 expression had a poor prognosis. Tumours with simultaneous LDH-5 and HIF1alpha (or HIF2alpha) overexpression, indicative of a functional HIF pathway, had a particularly aggressive behaviour. It is concluded that overexpression of LDH-5 is a common event in non-small-cell lung cancer, can be easily assessed in paraffin-embedded material and provides important prognostic information, particularly when combined with other endogenous markers of hypoxia and acidity.

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.

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.

Proangiogenic function of CD40 ligand-CD40 interactions.

Angiogenesis is a characteristic component of cell-mediated immune inflammation. However, little is known of the immunologic mediators of angiogenesis factor production. Interactions between CD40 ligand (CD40L) and CD40 have been shown to have pluripotent functions in inflammation, including the production of cytokines, chemokines, as well as the angiogenesis factor, vascular endothelial growth factor (VEGF), by endothelial cells. In this study we found that treatment of cultured human endothelial cells with an anti-CD40 Ab (to ligate CD40) resulted in the expression of several other angiogenesis factors, including fibroblast growth factor-2 and the receptors Flt-1 and Flt-4. To determine the proangiogenic effect of CD40L in vivo, human skin was allowed to engraft on SCID mice for 6 wk. These healed human skins express CD40 on resident endothelial cells and monocyte/macrophages, but not on CD20-expressing B cells. Skins were injected with saline, untransfected murine fibroblasts, or murine fibroblasts stably transfected with human CD40L. We found that the injection of CD40L-expressing cells, but not control cells, resulted in the in vivo expression of several angiogenesis factors (including VEGF and fibroblast growth factor) and a marked angiogenesis reaction. Mice treated with anti-VEGF failed to elicit an angiogenesis reaction in response to injection of CD40L-expressing cells, suggesting that the proangiogenic effect of CD40L in vivo is VEGF dependent. These observations imply that ligation of CD40 at a peripheral inflammatory site is of pathophysiological importance as a mediator of both angiogenesis and inflammation.

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.

Protective role of angiopoietin-1 in experimental pulmonary hypertension.

Angiopoietin-1 (Ang-1), a newly discovered ligand of the endothelial-specific tyrosine kinase receptor Tie-2, has been found to promote cell survival, vascular maturation, and stabilization. We hypothesized that Ang-1 gene transfer to the pulmonary microcirculation would improve pulmonary hemodynamics and vascular remodeling in experimental pulmonary hypertension. Rat pulmonary artery smooth muscle cells were transfected with Ang-1 cDNA or null (pFLAG-CMV-1) vector. Syngeneic Fisher 344 rats were treated with monocrotaline (MCT) (75 mg/kg IP) with or without delivery of 5x10(5) Ang-1-transfected cells into the right jugular vein. After 28 days, plasmid-derived Ang-1 mRNA was consistently and robustly detected by reverse transcriptase-polymerase chain reaction in lungs from all animals receiving Ang-1 gene therapy. Tie-2 receptor expression was markedly downregulated in rats treated with MCT, and this was partially restored by gene therapy with Ang-1. Animals receiving MCT exhibited 77% mortality by 28 days. In contrast, in pAng-1-treated animals, the 28-day mortality was only 14% (P<0.0001). In addition, right ventricular systolic pressure was reduced from 52+/-1.3 mm Hg in the MCT-treated group to 38+/-1.3 mm Hg by Ang-1 gene transfer (P<0.01), whereas the measurement of right to left ventricular plus septal weight ratio was also reduced from 0.41+/-0.03 to 0.31+/-0.01 (P<0.05). Moreover, MCT resulted in increased apoptosis, mainly in the microvasculature, and reduced endothelial NO synthase mRNA expression, both of which were prevented by Ang-1 gene transfer. Thus, cell-based gene transfer with Ang-1 improved survival and pulmonary hemodynamics in experimental pulmonary hypertension by a mechanism involving the inhibition of apoptosis and protection of the pulmonary microvasculature.

CDT6-expression can alter tumor sensitivity to chemotherapy.

BACKGROUND: Cornea-derived transcript 6 (CDT6 = AngX) has been shown to have an anti-tumor effect. MATERIALS AND METHODS: We transfected the murine melanoma cell line B16-F10 with the CDT6 gene and compared the sensitivity to cytostatic drugs of the resulting cell line, B16-CDT6, to that of the empty vector-transfected control cell line B16-CMV. RESULTS: The B16-CDT6 line showed a significantly increased sensitivity to doxorubicin as well as a tendency towards a decreased sensitivity to cisplatin. To further resolve the mechanism of the increase in sensitivity to doxorubicin, we also tested the cytotoxic agents vincristine, etoposide and taxol. However no difference in sensitivity for these drugs was found between the B16-CDT6 and the control cell line. CONCLUSION: CDT6 increased the sensitivity to doxorubicin in a mechanism probably not related to interference with the efflux pumps MRP, Pgp or the DNA-associated target enzyme topoisomerase II. Altered gene expression induced by gene therapy might influence tumor sensitivity to chemotherapy.

Altered angiogenesis and survival in human tumor-derived endothelial cells.

Knowledge on the functional properties of tumor-derived endothelial cells (TEC) can be relevant for the development of antiangiogenic therapeutic strategies. In the present study, we obtained and characterized endothelial cell lines from human renal carcinomas. TEC did not undergo senescence and showed constant expression of markers of endothelial activation and angiogenesis. In vitro, TEC, in contrast to normal endothelial cells, were resistant to apoptosis, proadhesive for renal carcinoma cells, and able to grow and organize in the absence of serum in persistent capillary-like structures. In vivo, TEC were able to grow in immunodeficient mice and to form vascular structures connected with the circulation. At a molecular level, gene array analysis showed an increased expression of genes involved in survival and cell adhesion compared with expression in normal microvascular endothelial cells. Moreover, expression of angiopoietin-1 and vascular endothelial growth factor (VEGF)-D and the Akt survival pathway were up-regulated. Inhibition of interaction of VEGFR-2 or VEGFR-3 with VEGF-D but not of Tie-2-angiopoietin-1 interaction with soluble receptors abrogated Akt activation and survival of TEC. These results indicate that at least some of the TEC within a tumor display abnormal characteristics in terms of survival and angiogenic properties and also indicate the presence of a functional autocrine pathway related to VEGF-D.

Expression of VEGF and angiopoietins-1 and -2 during ischemia-induced coronary angiogenesis.

The mechanisms underlying coronary capillary growth in response to ischemia are undefined. We hypothesized that the expression of vascular endothelial growth factor (VEGF) and angiopoietin (Ang)/Tie-2 were involved in capillary growth as an adaptation to ischemia. To test this hypothesis we measured capillary density, and the expressions of VEGF, Ang-1, Ang-2, and the Tie-2 receptor and its phosphorylation state during repetitive episodes of myocardial ischemia in chronically instrumented canines. Repetitive episodes of ischemia were induced by multiple (once/hour; 8/day), brief (2 min) occlusions of the left anterior descending coronary artery for 1, 7, 14, or 21 days. A sham group received the same instrumentation as the experimental groups but not the occlusion protocol. Collateral blood flow (microspheres) progressively increased from 9 +/- 3 to 83 +/- 10 ml. min-1. 100 g-1 on day 21. Capillary density increased at day 7 from 2378 +/- 53 (sham) to 2962 +/- 60/mm2, but it decreased to 2594 +/- 39/mm2 at day 21. Both VEGF and Ang-2 expression in myocardial interstitial fluid (Western analyses) peaked at day 3 of the repetitive occlusions but waned thereafter. In contrast the expression of Ang-1 remained relatively constant at all times in the occlusion groups. In shams, the expression of VEGF and Ang-2 was low and constant at all times. Tie-2 phosphorylation myocardial decreased decreased at day 7 but increased at 21 days of occlusions (P < 0.05). Our results indicate that capillary density was augmented by myocardial ischemia, but after development of collaterals and restoration of flow to the ischemic zone, capillary density returned to control levels. The change in capillary density paralleled with VEGF and Ang-2 expression but was inversely related to Tie-2 phosphorylation. We speculate the coronary angiogenesis is a coordinated event involving the expression of both VEGF and Ang-2 and that therapeutic angiogenic strategies may ultimately require treatment with more than a single factor.

Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation.

Fracture healing is a specialized post-natal repair process that recapitulates aspects of embryological skeletal development. While many of the molecular mechanisms that control cellular differentiation and growth during embryogenesis recur during fracture healing, these processes take place in a post-natal environment that is unique and distinct from those which exist during embryogenesis. This Prospect Article will highlight a number of central biological processes that are believed to be crucial in the embryonic differentiation and growth of skeletal tissues and review the functional role of these processes during fracture healing. Specific aspects of fracture healing that will be considered in relation to embryological development are: (1) the anatomic structure of the fracture callus as it evolves during healing; (2) the origins of stem cells and morphogenetic signals that facilitate the repair process; (3) the role of the biomechanical environment in controlling cellular differentiation during repair; (4) the role of three key groups of soluble factors, pro-inflammatory cytokines, the TGF-beta superfamily, and angiogenic factors, during repair; and (5) the relationship of the genetic components that control bone mass and remodeling to the mechanisms that control skeletal tissue repair in response to fracture.

Proangiogenic properties of human myeloma cells: production of angiopoietin-1 and its potential relationship to myeloma-induced angiogenesis.

Patients with multiple myeloma (MM) have increased bone marrow (BM) angiogenesis; however, the proangiogenic properties of myeloma cells and the mechanisms of MM-induced angiogenesis are not completely clarified. The angiopoietin system has been identified as critical in the regulation of vessel formation. In this study we have demonstrated that myeloma cells express several proangiogenic factors, and, in particular, we found that angiopoietin-1 (Ang-1), but not its antagonist Ang-2, was expressed by several human myeloma cell lines (HMCLs) at the mRNA and the protein levels. In a transwell coculture system, we observed that myeloma cells up-regulated the Ang-1 receptor Tie2 in human BM endothelial cells. Moreover, in an experimental model of angiogenesis, the conditioned medium of HMCLs significantly stimulated vessel formation compared with control or vascular endothelial growth factor (VEGF) treatment. The presence of anti-Tie2 blocking antibody completely blunted the proangiogenic effect of XG-6. Finally, our in vitro results were supported by the in vivo finding of Ang-1, but not Ang-2, mRNA and protein expression in purified MM cells obtained from approximately 47% of patients and by high BM angiogenesis in patients with MM positive for Ang-1, suggesting that the angiopoietin system could be involved, at least in part, in MM-induced angiogenesis.

Mast cell-mediated stimulation of angiogenesis: cooperative interaction between A2B and A3 adenosine receptors.

Adenosine is released during tissue injury, ischemia and tumor growth, and promotes angiogenesis. Because mast cells accumulate in the proximity of new blood vessel development, we examined if they may contribute to adenosine-induced angiogenesis. We found that HMC-1 human mast cells express A2A, A2B, and A3 adenosine receptors. The adenosine agonist NECA (100 micromol/L) increased interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and angiopoietin-2 mRNA expression. NECA-induced secretion of IL-8 and VEGF was verified by ELISA. A2B receptors mediate VEGF and IL-8 secretion because neither CGS21680 (selective A2A agonist) nor IB-MECA (selective A3 agonist) produced this effect, and it was inhibited by the selective A2B antagonist IPDX but not by the selective A2A antagonist SCH58261 or the selective A3 antagonist MRS1191. In contrast, the selective A3 agonist IB-MECA (EC50 1 nmol/L) stimulated angiopoietin-2 expression. Conditioned media from NECA-activated HMC-1 stimulated human umbilical vein endothelial cell proliferation and migration, and induced capillary tube formation. Capillary formation induced by mast cell-conditioned media was maximal if both HMC-1 A2B and A3 receptors were activated, whereas activation of A2B receptor alone was less effective. Thus, adenosine A2B and A3 receptors act in a functional cooperative fashion to promote angiogenesis by a paracrine mechanism involving the differential expression and secretion of angiogenic factors from human mast cells.

Signaling molecules in nonfamilial pulmonary hypertension.

BACKGROUND: Biochemical, genetic, and clinical evidence indicates that smooth-muscle proliferation around small pulmonary vessels is an essential part of the pathogenesis of pulmonary hypertension. Mutations in the bone morphogenetic protein receptor type 2 (BMPR2) have been linked to familial cases of pulmonary hypertension, but the molecular basis of the common nonfamilial forms is unknown. METHODS: We evaluated the pattern of expression of angiopoietin-1, a protein involved in the recruitment of smooth-muscle cells around blood vessels; TIE2, the endothelial-specific receptor for angiopoietin-1; and bone morphogenetic protein receptor type 1A (BMPR1A) and BMPR2 in lung-biopsy specimens from patients with pulmonary hypertension and from normotensive control patients. The effect of angiopoietin-1 on the modulation of BMPR expression was also evaluated in subcultures of human pulmonary arteriolar endothelial cells. RESULTS: The expression of angiopoietin-1 messenger RNA and the protein itself and the phosphorylation of TIE2 were strongly up-regulated in the lungs of patients with various forms of pulmonary hypertension, correlating directly with the severity of disease. A mechanistic link between familial and acquired pulmonary hypertension was demonstrated by the finding that angiopoietin-1 shuts off the expression of BMPR1A, a transmembrane protein required for BMPR2 signaling, in pulmonary arteriolar endothelial cells. Similarly, we found that the expression of BMPR1A was severely reduced in the lungs of patients with various forms of acquired as well as primary nonfamilial pulmonary hypertension. CONCLUSIONS: These findings suggest that all forms of pulmonary hypertension are linked by defects in the signaling pathway involving angiopoietin-1, TIE2, BMPR1A, and BMPR2 and consequently identify specific molecular targets for therapeutic intervention.

Aldose reductase inhibitor fidarestat prevents retinal oxidative stress and vascular endothelial growth factor overexpression in streptozotocin-diabetic rats.

The study addressed the role for aldose reductase (AR) in 1) retinal oxidative stress and vascular endothelial growth factor (VEGF) overexpression in early diabetes, and 2) high glucose-induced oxidative stress in retinal endothelial cells. In vivo experiments were performed on control rats and diabetic rats treated with or without low or high dose of the AR inhibitor (ARI) fidarestat (2 or 16 mg. kg(-1). day(-1)). In vitro studies were performed on bovine retinal endothelial cells (BREC) cultured in either 5 or 30 mmol/l glucose with or without 1 micro mol/l fidarestat. Intracellular reactive oxygen species were assessed using the 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA) probe and flow cytometry. Both low and high doses of fidarestat (i.e., the doses that partially and completely inhibited sorbitol pathway hyperactivity) arrested diabetes-induced retinal lipid peroxidation. This was achieved due to upregulation of the key antioxidative defense enzyme activities rather than changes in reduced glutathione, oxidized glutathione, ascorbate and dehydroascorbate concentrations, and the glutathione and ascorbate redox states. Diabetes-associated 2.1-fold VEGF protein overexpression (enzyme-linked immunosorbent assay; ELISA) was dose-dependently prevented by fidarestat, whereas total VEGF mRNA and VEGF-164 mRNA (RT-PCR) abundance were not affected by either diabetes or the ARI. In BREC, fidarestat corrected hyperglycemia-induced increase in H(2)DCFDA fluorescence but not oxidative stress caused by three different pro-oxidants in normoglycemic conditions. In conclusion, increased AR activity contributes to retinal oxidative stress and VEGF protein overexpression in early diabetes. The findings justify the rationale for evaluation of fidarestat on diabetic retinopathy.

Platelet endothelial cell adhesion molecule-1 and angiogenic factor expression in idiopathic membranous nephropathy.

BACKGROUND: Idiopathic membranous nephropathy (IMN), a principal disease of glomerular capillaries, was investigated for some aspects of glomerular capillary injury and repair (angiogenesis). METHODS: Fifteen cases of IMN were studied immunohistochemically for expression of the endothelial cell antigen platelet endothelial cell adhesion molecule-1 (PECAM-1[CD31]) and the angiogenesis-stimulating factors vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP). An equal number of normal control kidneys of fetal and mature origin were tested for the same antigens. RESULTS: Normal tissues expressed PECAM-1 in both glomerular and interstitial endothelial cells, whereas VEGF and TP were expressed in the tubular epithelium. IMN was characterized by complete or partial loss of PECAM-1 expression from glomerular capillaries and a parallel gain/expression of this antigen by the tubular epithelium. In addition, VEGF and TP expression was lost or considerably reduced from tubular cells of IMN. CONCLUSION: We hypothesize that PECAM-1 expression by tubular epithelial cells represents uptake of CD31(+) cell-surface fragments released by glomerular endothelial cells after glomerular damage. The damage is confounded by the failure of angiogenic mechanisms to promote glomerular angiogenesis (repair) because both VEGF and TP stimulation by the tubular epithelium is eliminated. It is suggested that immunohistochemical detection of VEGF or TP in the tubular epithelium may be useful in understanding the pathogenesis of IMN.

Expression of endocrine gland-derived vascular endothelial growth factor in ovarian carcinoma.

The first tissue-specific angiogenic molecule, endocrine gland-derived vascular endothelial growth factor (EG-VEGF), was identified recently in human ovary, raising hopes of developing tumor type-specific angiogenesis inhibitors. In the present study, we analyzed the expression of EG-VEGF mRNA in normal human tissues and ovarian neoplasms by quantitative real-time reverse transcription-PCR. EG-VEGF mRNA was expressed in all ovarian neoplasms examined. No significant difference was identified among benign, low malignant potential neoplasms or stage I ovarian cancer, all of which exhibited 2-fold lower mRNA levels compared with normal premenopausal ovaries. EG-VEGF mRNA levels further decreased in late stage compared with early stage carcinomas (P < 0.05) and were consistently lower in laser capture microdissected tumor islets compared with surrounding stroma. EG-VEGF was undetectable by reverse transcription-PCR in 17 established epithelial ovarian cancer cell lines or in cultured human ovarian surface epithelial cells, whereas it was detected in peripheral blood as well as tumor-infiltrating T lymphocytes. Finally, in contrast to VEGF, EG-VEGF mRNA levels did not correlate with clinical outcome in advanced ovarian carcinoma. These results suggest that EG-VEGF is most likely derived from nonepithelial components of ovarian carcinomas and may play a marginal role in promoting angiogenesis in advanced ovarian carcinoma. We postulate that EG-VEGF-targeted antiangiogenic therapy may prove useful in early stage but not in advanced stage ovarian carcinoma.

Expression of angiogenic factors including VEGFs and the effects of hypoxia and thalidomide on human myeloma cells.

Angiogenic factors are major causes of tumor progression in hematological malignancies, particularly multiple myeloma, as well as solid tumors. The introduction of thalidomide as an anti-angiogenic agent in myeloma treatment has demonstrated the importance of angiogenic factors in the progression of myeloma. However, the direct effects of angiogenic factors, particularly VEGFs, hypoxia, and thalidomide, on myeloma cells are not been documented. In this study, we demonstrate increased expression and production levels of VEGF in myeloma compared to non-myelomatous hematological lines, resistance to hypoxia and enhancement of VEGF-A production by hypoxia in myeloma, and direct growth inhibition of myeloma cells due to apoptosis and G1 arrest caused by TNFalpha upregulation induced by thalidomide. These findings may encourage the clinical use of anti-angiogenic agents for their cytostatic effects and the prevention of progression.

Angiopoietin-2 expression in breast cancer correlates with lymph node invasion and short survival.

Angiogenic factors produced by tumor cells are essential for tumor growth and metastasis. In our study, the expression of Angiopoietin-1 (ANG1) and Angiopoietin-2 (ANG2) mRNA in archival human breast cancer tumor samples and in 6 breast cancer cell lines was investigated. Total RNA from biopsies of 38 breast cancer patients was extracted and ANG1 and ANG2 mRNA expression was measured by means of quantitative real-time RT-PCR (Taqman). Matching data with available clinicopathologic and biochemical data revealed a significant association between ANG2 expression and axillary lymph node invasion. Univariate and multivariate survival analysis, by means of Kaplan-Meier method and Cox's proportional hazards model, showed significant and independent association between ANG2 mRNA level and both disease-free (p < 0.0001) and overall survival (p < 0.0003). An important fact is that, notwithstanding the small number of cases examined, this association was confirmed also in the group of lymph node-negative patients (DFS, p < 0.003; OS, p < 0.020). Immunohistochemical analysis demonstrated that Ang2 is expressed by both tumor cells and endothelial elements. Expression in tumor cells was confirmed by studying a panel of human breast carcinoma cell lines in culture by RT-PCR. In ZR75.1 and T47D cells, expression of ANG2 mRNA was increased up to 10-fold by treatment with estrogen within 24 hr. Although preliminary, these data suggest a possible role of ANG2 as a prognostic factor for primary breast cancer.