High Vascular Endothelial Growth Factor Expression Research Articles

Inhibition effect of complement factor-small interference RNA on the angiogenesis of choroidal neovascularization induced by photocoagulation in rats

Objective To observe the inhibitory effects of construction of complement factor (CFB)small interference RNA (siRNA) on choroidal neovascularization(CNV)induced by photocoagulation in rats.Methods We constructed an expression vector of CFB-siRNA by cutting CFB-siRNA and plasmid pRNATU6.1/Neo.Experimental CNV was induced by photocoagulation in 42 Brown Norway rats.After the model was set up,the rats were randomly divided into tail intravenous injection,vitreous injection,subretinal injection,and control group;each group except the control one had a corresponding blank plasmid control group.CFB-siRNA was injected 1,3,and 5 days respectively after photocoagulation in the injection groups;the dosage was 50,20,and 10 μg in tail intravenous injection,vitreous injection,and subretinal injection group respectively,while no injection was give to the control group after photocoagulation.Before and 14 days after photocoagulation,fundus fluorescein angiograoph (FFA) was performed and CNV development was judged by the leakage;the expression of vascular endothelial growth factor (VEGF) and factor Ⅷ were detected by immunohistochemistry.Results The leakage of fluorescein was obvious lower in tail intravenous injection group than that in the control group (X~2=15.1620,P 0.05);while was apparently lower than that in the other groups at different time points (F=77.96,55.68;P<0.05).All of the groups,except tail intravenous injection group,had higher expression of VEGF and factor Ⅷ 14 days after photocoagulation compared with that 7 days after photocoagulation (F=60.89,61.12;P<0.05).Conclusions Constructed CFB-siRNA can inhibite CNV by down-regulating the expression of VEGF and factor Ⅷ. Key words: Choroidal neovascularization/ therapy; Gene therapy; RNA,small interfering; Disease models,animal

The preliminary study of molecular imaging of colorectal cancer cells with superparamagnetic iron oxide-based MR targeting probe containing vascular endothelial growth factor in vitro

Objective To develop a superparamagnetic iron oxide (SPIO)-based MR probe containing vascular endothelial growth factor(VEGF) to investigate their biological and chemical properties and targeting effect of colorectal cancer cells in vitro. Methods The anti-VEGF-SPIO probe was fabricated with VEGF antibody and SPIO through chemical method. Its biological and chemical properties and reflexivity were tested with SDS-PAGE and MRL The SW620 cells incubated with anti-VEGF-SPIO probe for 30, 60 and 90 minutes respectively and compared with marrow mesenchymal stem cell at 37℃. The comparison among groups was conducted by using analysis of variance and LSD-t test. The MRI results were confirmed by the Prussian blue staining. The comparison among groups was performed by analysis of variance and factorial experiment. Results SPIO-based MR probe containing VEGF was successfully contributed and isolated. The reflexivity of anti-VEGF-SPIO probe was 0.0426×10~6 mol/s. The immunofluorescence and prussia blue stain proved high expression of VEGF in SW620 cells. Anti-VEGF-SPIO probe and SW620 cellscombined at 37℃ in vitro MRI proved the SW620 cells incubated with anti-VEGF-SPIO probe appeared hypointense on T_2WI and T_2~* WI. MR signal were 392±7,91±8,264±10 for 30, 60 and 90 minutes respectively, which were statistically different from that before incubation 679±12 (F=4735.489, P 0.05). Conclusion Nanoscale iron particles containing the anti-vascular endothelial growth factor molecular probe can evaluate tumor angiogenesis at the receptor level, which provides a new way of the tumor angiogenesis diagnosis and anti-angiogenesis therapy. Key words: Molecular probes; Receptors; vascular endothelial growth factor; Magnetic resonance imaging; Colonic neoplasms

Angiogenesis in Atherosclerotic Plaque Obtained From Carotid Endarterectomy: Association Between Symptomatology and Plaque Morphology

Carotid plaque with hemorrhage leads to cerebral embolism and ischemic stroke. Plaque angiogenesis and angiogenetic factors such as vascular endothelial growth factor (VEGF) are critical in the progression of atherosclerotic carotid plaque and intraplaque hemorrhage. The correlation between plaque angiogenesis and presence of clinical symptoms was studied in 41 specimens obtained during carotid endarterectomy from 20 symptomatic and 21 asymptomatic patients treated for carotid artery stenosis. Histological findings using hematoxylin-eosin and immunohistochemical staining against von Willebrand factor and VEGF were examined. Intraplaque hemorrhage, calcification, necrosis, and invasion of foam cells were frequently observed in the carotid plaques from symptomatic patients compared with asymptomatic patients. Higher microvessel density was found in the carotid plaques with necrosis and invasion of foam cells compared with plaques without necrosis and/or foam cell invasion, and higher expression of VEGF was found from symptomatic patients compared with asymptomatic patents. These results suggest that plaque angiogenesis and higher level of VEGF expression may enhance the progression of ischemic symptoms in patients with carotid artery stenosis. Invasive macrophages in the plaque of symptomatic patients increase levels of VEGF and might enhance plaque angiogenesis and atherosclerosis progression.

Fighting on the border

Colorectal cancer (CRC) is a public healthproblem inmost develped countries. It is potentially curable in its early stages, but a elevant proportion of patients still presents with or ultimately evelops metastatic disease. Over the past 10 years, significant advances have been made in he molecular profiling and clinico-pathological characterization f CRC. In particular, the expanding body of information on cancer iology may provide the biological rationale for new therapeutic trategies. The interesting manuscript published in this issue of igestive and Liver Disease by Cianchi et al. further demonstrates ow molecular studies may help therapy to be tailored to cancer’s iology [1]. COX2 gene expression is virtually undetectable in the normal ntestine, but frequently upregulated in CRC [2]. In vivo and in itro studies have demonstrated that COX2 gene product promotes ngiogenesis, supporting both tumour growth and metastatic mplants [3]. A direct correlation between COX2 expression and icrovessel density has also been documented. As in several ther epithelial malignancies, COX2 protein over-expression conistently correlates with an unfavourable prognosis. In a study on large cohort of CRC patients, Soumaoro et al. associated COX2ositive cancers with a significantly shorter overall survival, and his unfavourable outcomewas also confirmedwhen different caner stages were considered separately [4]. In the Soumaoro study, OX2 over-expression was consistently found in primary tumours, s well in all liver metastases and almost in all metastatic regional ymph nodes. Vascular endothelial growth factor (VEGF) is a glycoproteinwith itogenic effects on endothelium and its activation results in sigalling cascades that promote both new endothelial cell growth nd the survival of existing vessels. The role of VEGF in cancer has een extensively studied and a higher expression of VEGF has been ssociated with COX2 upregulation [2]. Cianchi et al. provide original, interesting data on this hot issue 1]. They tested the expression of a panel of well-known angio-

PTEN Regulates VEGF, VEGFR1 Expression and Its Clinical Significance in Myeloid Leukemia.

Abstract 1001Poster Board I-23Phosphatase and tensin homology deleted on chromosome ten (PTEN) as a novel tumor suppressor gene, plays an important role in regulating proliferation, apoptosis, invasion and migration of many cancer cells. PTEN also modulates angiogenesis mediated by vascular endothelial growth factor (VEGF) via down-regulating the activity of PI3K/Akt pathway in many solid tumors. However, in myeloid leukemia, the effects of PTEN on VEGF and VEGFR1 (FLT1) mediated angiogenesis, migration, invasion of leukemia cells and its clinical significance are still unknown. Therefore, in the present study, we investigated the effect of PTEN on the activity of PI3K/Akt and VEGF/FLT1 pathways. Wild type PTEN gene was transfected into K562 cells, a cell line establish from a chronic myelogenous leukemia in blast crisis, to induce high expression of wild-type PTEN gene and protein by the cells. The correlation between the expression levels of PTEN and VEGF/FLT1 and its clinical significance in myeloid leukemia patients were also observed. We found that the expression reconstitution of wild-type PTEN had significance effect on inhibiting proliferation, migration and invasion ability of K562 cells via down-regulation of Akt phosphorylation and inhibition of VEGF/FLT1 expression. In myeloid leukemia patients, a negative correlation was found between the expression level of PTEN mRNA and that of VEGF and FLT1 mRNA. Low expression of PTEN mRNA and high expression of VEGF and FLT1 mRNA indicated a higher tendency of extramedullary disease in acute myeloid leukemia patients. Taken together, our findings indicated that PTEN could modulate the function of VEGF/VEGFR signaling pathway via down regulating Akt phosphorylation and that PTEN would be a candidate target for the treatment of myeloid leukemia. Disclosures:Pan:Nature science foundation of Hebei Province: Research Funding; Research Fund for the Doctoral Program of Higher Education of China: Research Funding; Emphases follow up pregram of Health Bureau of Hebei Province: Research Funding.

Vascular endothelial growth factor in children with neuroblastoma: a retrospective analysis

BackgroundDespite aggressive therapy, advanced stage neuroblastoma patients have poor survival rates. Although angiogenesis correlates with advanced tumour stage and plays an important role in determining the tumour response to treatment in general, clinical data are still insufficient, and more clinical evaluations are needed to draw conclusions. The aim of this study was to evaluate vascular endothelial growth factor (VEGF) expression in patients with neuroblastoma, determine whether it correlates with other prognostic factors and/or therapeutic response, and to assess should VEGF be considered in a routine diagnostic workup.Materials and methodsVEGF expression was determined by immunohistochemistry using anti-VEGF antibody in paraffin embedded primary tumour tissue from 56 neuroblastoma patients. Semiquantitative expression of VEGF was estimated and compared with gender, age, histology, disease stage, therapy, and survival. Statistical analyses, including multivariate analysis, were performed.ResultsVEGF expression correlated with disease stage and survival in neuroblastoma patients. Combination of VEGF expression and disease stage as a single prognostic value for survival (P-value = 0.0034; odds ratio (OR) (95%CI) = 26.17 (2.97-230.27) exhibited greater correlation with survival than individually. Hematopoietic stem cell transplantation significantly improved survival of the advanced stage patients with high VEGF expression.ConclusionVEGF expression should be considered in a routine diagnostic workup of children with neuroblastoma, especially in those more than 18 months old and with advanced disease stage. High VEGF expression at the time of disease diagnosis is a bad risk prognostic factor, and can be used to characterize subsets of patients with an unfavourable outcome.

Effects of Increased Renal Tubular Vascular Endothelial Growth Factor (VEGF) on Fibrosis, Cyst Formation, and Glomerular Disease

The role of vascular endothelial growth factor (VEGF) in renal fibrosis, tubular cyst formation, and glomerular diseases is incompletely understood. We studied a new conditional transgenic mouse system [Pax8-rtTA/(tetO)(7)VEGF], which allows increased tubular VEGF production in adult mice. The following pathology was observed. The interstitial changes consisted of a ubiquitous proliferation of peritubular capillaries and fibroblasts, followed by deposition of matrix leading to a unique kind of fibrosis, ie, healthy tubules amid a capillary-rich dense fibrotic tissue. In tubular segments with high expression of VEGF, cysts developed that were surrounded by a dense network of peritubular capillaries. The glomerular effects consisted of a proliferative enlargement of glomerular capillaries, followed by mesangial proliferation. This resulted in enlarged glomeruli with loss of the characteristic lobular structure. Capillaries became randomly embedded into mesangial nodules, losing their filtration surface. Serum VEGF levels were increased, whereas endogenous VEGF production by podocytes was down-regulated. Taken together, this study shows that systemic VEGF interferes with the intraglomerular cross-talk between podocytes and the endocapillary compartment. It suppresses VEGF secretion by podocytes but cannot compensate for the deficit. VEGF from podocytes induces a directional effect, attracting the capillaries to the lobular surface, a relevant mechanism to optimize filtration surface. Systemic VEGF lacks this effect, leading to severe deterioration in glomerular architecture, similar to that seen in diabetic nephropathy.

Vascular Endothelial Growth Factor Expression in Invasive Papillary Thyroid Carcinoma

The vascular endothelial growth factor (VEGF) is a major promoter of endothelial growth and migration. Some studies have shown a correlation between expression of this growth factor and prognosis in several cancers, including well-differentiated thyroid cancer. We studied VEGF expression, local invasiveness, and other prognostic factors in papillary thyroid carcinoma (PTC) to test the hypothesis that the expression of VEGF is correlated with the degree of invasion of PTC. Clinical and pathological data of 76 patients with PTC were retrospectively reviewed. Group 1 consisted of patients with gross locally invasive tumors, group 2 consisted of patients with only invasion of the thyroid capsule, and group 3 consisted of patients with noninvasive PTC. VEGF expression was noted within the tumor in all groups of PTC patients but was absent in the surrounding normal tissue. Older patients had higher expression of VEGF than younger patients. The age of patients with strong reaction to VEGF was 46 +/- 14 (mean +/- standard deviation), and that in patients with a weaker reaction was 39 +/- 16 (p < 0.05). Only 20% of patients with a follicular variant of PTC had a strong reaction to VEGF compared with 68% of patients with classical PTC (p < 0.01). VEGF expression appears to be an early event in the development of PTC. Whether VEGF expression promotes the progression of PTC is not known, but the answer to this question may be important in view of its greater expression in older patients, a group whose prognosis in PTC is worse.

Absence of VHL gene alteration and high VEGF expression are associated with tumour aggressiveness and poor survival of renal-cell carcinoma

Background:The von Hippel–Lindau gene (VHL) alteration, a common event in sporadic clear-cell renal-cell carcinoma (CCRCC), leads to highly vascularised tumours. Vascular endothelial growth factor (VEGF) is the major factor involved in angiogenesis, but the prognostic significance of both VHL inactivation and VEGF expression remain controversial. The aims of this study were to analyse the relationship between VHL genetic and epigenetic alterations, VHL expression and VEGF tumour or plasma expression, and to analyse their respective prognostic value in patients with CCRCC.Methods:A total of 102 patients with CCRCC were prospectively analysed. Alterations in VHL were determined by sequencing, Multiplex Ligation-dependent Probe Amplification (MLPA) and methylation-specific MLPA. Expression of pVHL and VEGF was determined by immunohistochemistry. Plasma VEGF was measured by enzyme-linked immunosorbent assay (ELISA).Results:VHL mutation, deletion and promoter methylation were identified in 70, 76 and 14 cases, respectively. Overall, at least one VHL-gene alteration occurred in 91 cases (89.2%). Both VEGF tumour and plasma expression appeared to be decreased in case of VHL alteration. Median progression-free survival and CCRCC-specific survival were significantly reduced in patients with wild-type VHL or altered VHL and high VEGF expression, which, therefore, represent two markers of tumour aggressiveness in CCRCC.Conclusion:Stratifying CCRCCs according to VHL and VEGF status may help tailor therapeutic strategy.

The Relationship of Vascular Endothelial Growth Factor and Coagulation Factor (Fibrin and Fibrinogen) Expression in Clear Cell Renal Cell Carcinoma

To investigate the relationship between angiogenesis and coagulation markers in tumor tissues of primary renal cell carcinoma (RCC). Tumors stimulate angiogenesis and activate the coagulation cascade. The importance of the interplay between these pathways for RCC is unknown. In all, 69 clear cell RCC specimens were analyzed by immunohistochemical staining applied to tissue microarrays. The expression of vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1alpha, fibrinogen and fibrin, and microvessel density were visually analyzed. Finally, staining patterns were related to clinical variables and survival. The VEGF expression was detected in 100% of tumors, with 68% showing a high expression, whereas hypoxia-inducible factor-1alpha staining was low (only 26% had a moderate to high staining). Fibrinogen was expressed adjacent to the tumor cells in 26% of cases, whereas in 84% it was expressed around the blood vessels. In 30% of tumors, expression of fibrin was detected. High tumor VEGF expression correlated with high fibrin staining (P = .05). From a multivariate analysis, microvessel density (P = .033) and fibrinogen adjacent to tumor cells (P = .046) were independent factors related to VEGF expression. In this study, we found clinical evidence for the permeability activity of VEGF as reflected by extravascular fibrinogen expression adjacent to tumor cells in the extracellular matrix. In addition, VEGF and fibrin expression were associated, indicative for concomitant activation of the coagulation cascade and angiogenesis in RCC. Taken together, these data indicate that activation of angiogenesis and coagulation are related in RCC.

Expression of hypoxia-inducible factor-1 alpha and vascular endothelial growth factor in malignant melanoma

To evaluate the expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF) and their relationship in malignant melanoma. 32 cases of malignant melanoma and 11 samples of normal skin were examined for HIF-1 alpha and VEGF expression by immunohistochemistry. The expression rate of HIF-1 alpha and VEGF was 62.5% (20/32) and 84.8% (27/32), respectively, in malignant melanoma, and 9.1% (1/11) and 18.2% (2/11), respectively, in normal skin, showing a significant difference between the two groups. Positive relationship between the expression of HIF-1 alpha and VEGF was found in malignant melanoma (P < 0.05). 1) The high expression of HIF-1 alpha and VEGF in malignant melanoma indicates that it may play an important role in the generation and development of malignant melanoma. 2) The positive correlation between the expression of HIF-1 alpha and VEGF in malignant melanoma suggests that HIF-1 alpha may be involved in the regulation of VEGF in malignant melanoma.

Shorter survival-times following adjuvant endocrine therapy in oestrogen- and progesterone-receptor positive breast cancer overexpressing HER2 and/or with an increased expression of vascular endothelial growth factor

To investigate the possible correlation between expression of HER2 and vascular endothelial growth factor (VEGF), and to determine the predictive value of these factors in patients receiving adjuvant endocrine therapy including the group with a breast cancer (BC) positive for both oestrogen receptor (ER) and progesterone receptor (PgR). By enzyme immuno-sorbent assays (ELISA) tumour levels of HER2 and VEGF proteins were determined in 679 consecutive primary BC patients, median age 63 years, median follow-up time 92 months. A total of 404 patients received adjuvant endocrine therapy, mainly tamoxifen, out of them 295 had an ER and PgR positive BC. In 160 patients, HER2 status was also determined by immunohistochemistry (IHC) using the monoclonal antibody CB11. Overexpression of HER2 by IHC was found in 15% of the patients. Overexpression of HER2 by ELISA correlated with HER2 by IHC (P < 0.001) and a higher VEGF expression (P = 0.004). Patients receiving adjuvant endocrine therapy with high VEGF (RFS P = 0.0087, BCCS P = 0.0012) or over-expressing HER2 (RFS P = 0.0116, BCCS P = 0.0036) had significantly shorter survival. Factors retaining statistical significance in multivariate analyses for recurrence-free survival (RFS) were nodal status (P < 0.001), tumour size (P = 0.005) and VEGF (P = 0.032) and for breast cancer corrected survival (BCCS) nodal status (P < 0.001), tumour size (P = 0.001), ER status (P = 0.022), and VEGF (P = 0.016). Both factors were significantly correlated with survival in the group with a BC positive for both ER and PgR; VEGF (RFS P = 0.0177, BCCS P = 0.0321) and HER2 (RFS P = 0.0143, BCCS P = 0.0311). In multivariate analyses, nodal status (P < 0.001) and VEGF (P = 0.021) were independent factors for RFS. Nodal status (P < 0.001) and tumour size (P = 0.016) retained independent factors for BCCS. Combined analysis identified a high-risk group (HER2 positive and high VEGF) with significantly reduced survival. The results from this retrospective analysis suggest that overexpression of HER2 and higher VEGF expression may add information on patient's outcome after adjuvant endocrine therapy in ER and PgR positive BC.

Involvement of vascular endothelial growth factor, CD44 and CD133 in periodontal disease and diabetes: an immunohistochemical study

The aim of this study was to investigate the relationship between expression of angiogenic and regeneration markers and periodontal disease in subjects with/without diabetes mellitus. Immunohistochemical detection of vascular endothelial growth factor (VEGF), CD44 and CD133 was performed in 16 samples each of (1) healthy gingiva from non-diabetic subjects (controls), (2) gingiva from non-diabetic subjects with periodontitis, (3) gingiva from subjects with type 1 diabetes and periodontitis, (4) gingiva from subjects with type 2 diabetes and periodontitis. Diseased gingivae from patients with diabetes and periodontitis had greater clinical measures of periodontal disease than those with periodontitis only. VEGF expression was significantly enhanced in epithelial and endothelial cells from patients with periodontitis compared with controls (p<0.05). Epithelial CD44 expression was strong in all groups, while CD44 was significantly enhanced (p<0.05) in connective tissue cells from both diabetic groups. Epithelial and endothelial CD133 expression was comparable in all patients except those with type 2 diabetes and periodontitis, where it was not detected. Stromal CD133 expression was significantly lower in patients with type 2 diabetes and periodontitis and was increased in periodontitis patients (p<0.05). The involvement and high expression of VEGF, CD44 and CD133 in periodontal disease may predict a greater regeneration capacity of gingival tissue.

Analysis of VEGF, Flt-1, Flk-1, nestin and MMP-9 in relation to astrocytoma pathogenesis and progression

Astrocytomas, particularly high grade astrocytoma, are brain tumors with potent angiogenic activity. Our immnunohistochemical study assessed vascular endothelial growth factor (VEGF), VEGF receptors (Flk-1, and Flt-1), the intermediate filamental protein nestin which plays a role in central nervous system development, and MMP-9, which belongs the family of matrix metalloproteinases implicated in tumor invasion and angiogenesis regulation. We investigated the expression of VEGF, its receptors, nestin and MMP-9 in astrocytomas and their correlation with tumor grade. We used paraffin-embedded samples from 66 patients, 29 with low grade (WHO-grade II) and 37 with high grade (WHO-grade III and IV) astrocytomas. Antibodies against VEGF, Flk-1, Flt1, nestin, CD34 and MMP-9 were used, followed by standard indirect immunohistochemical methods. Expression of Flt-1 and Flk-1 showed no significant differences between low and high grade tumor groups. Expression of VEGF and MMP-9 was increased in the high grade group (p equal to or less than 0.026 and 0.024). Nestin expression in tumor astrocytes and endothelial cells increased in high grade group (p same 0.007 and 0.003). Higher expression of VEGF in high grade astrocytomas may subsequently lead to activation of survival, angiogenesis and migration. Expression of nestin and MMP-9 also suggest their likely role in astrocytoma vascular development and proliferation.

Prognostic significance of cellular vascular endothelial growth factor (VEGF) expression in the course of chronic myeloid leukaemia

Increased angiogenesis in bone marrow is one of the characteristics of chronic myeloid leukaemia (CML), a clonal myeloproliferative disorder that expresses a chimeric bcr/abl protein. Vascular endothelial growth factor (VEGF) is one of the most potent and a specific regulator of angiogenesis which principally targets endothelial cells and regulates several of their functions, including mitogenesis, permeability and migration. The impact of elevated VEGF expression on the course of chronic myeloid leukaemia is unknown. The aim of this study was the follow-up of VEGF expression during the course of CML. We studied VEGF expression of 85 CML patients (median age 50 years, range 16-75 years). At the commencement of the study, 29 patients were in chronic phase (CP), 25 in an accelerated phase (AP), and 31 in the blast crisis (BC). The temporal expression (percentage positivity per 1000 analysed cells) VEGF proteins over the course of CML were studied using the immunohistochemical technique utilizing relevant monoclonal antibodies. It was correlated with the laboratory (Hb, WBC and platelet counts, and the percentage of blasts) and clinical parameters (organomegaly, duration of CP, AP, and BC) of disease progression. The expression ofVEGF protein was most pronounced in AP (ANOVA, p=0.033). The level of VEGF expression correlated inversely with the degree of splenomegaly (Pearson, r=-0.400, p=0.011). High expression of VEGF correlated with a shorter overall survival (log rank, p=0.042). Immunohistochemically confirmed significance of the expression of VEGF in dependence of the CML stage could be of clinical importance in deciding on the timing therapy. These data suggest that VEGF plays a role in the biology of CML and that VEGF inhibitors should be investigated in CML.

Nanosphere-mediated delivery of vascular endothelial growth factor gene for therapeutic angiogenesis in mouse ischemic limbs

Abstract summary In this study poly(lactic-co-glycolic acid) (PLGA) nanospheres were investigated to serve as a vascular endothelial growth factor (VEGF) gene carrier to ischemic limb. Loading of VEGF gene into PLGA nanospheres sustained the release of the gene maintaining its structural and functional integrity and showing low toxicity. In mouse ischemic limbs, the PLGA nanosphere system showed significantly higher VEGF expression and more extensive neovascularization compared to naked pDNA and polyethylenimine (PEI). These results suggest that PLGA nanospheres might be useful as gene carrier for skeletal muscle gene delivery applications.

Quantitative Analysis of Vascular Endothelial Growth Factor in Liver Metastases from Pancreatic Carcinoma as a Predictor of Chemotherapeutic Effect and Prognosis

In pancreatic carcinoma, vascular endothelial growth factor (VEGF) expression at the primary site has been suggested to be a prognostic parameter. We quantitatively analyzed VEGF expression in liver metastases from pancreatic carcinoma and examined the correlation among VEGF expression in liver metastases, clinicopathologic factors, and clinical outcome. The subjects consisted of 23 patients with pancreatic adenocarcinoma who had liver metastases and were treated with S-1 and gemcitabine as the first-line treatment. VEGF expression was quantitated by enzyme immunoassay in biopsy specimens of liver metastases and nontumorous liver tissue, and in plasma. In 10 of the 23 patients, VEGF expression was also quantitated in biopsy specimens of the primary pancreatic tumor. All samples were collected before treatment. The VEGF level in nontumorous liver tissue was 36.6 +/- 10.0 pg/mg protein versus 376.8 +/- 106.1 pg/mg protein in liver metastases (P = 0.0016). Pretreatment VEGF levels in plasma and in primary pancreatic carcinoma did not correlate with VEGF levels in the corresponding liver metastases. The median VEGF level in liver metastases (138.9 pg/mg protein) was used as the cutoff value between high and low VEGF expression in liver metastases. Patients showing high VEGF expression had a significantly longer progression-free survival and overall survival than patients showing low VEGF expression in liver metastases (P = 0.0219 and P = 0.0074, respectively). Evaluation of VEGF levels in liver metastases might be useful in assessing the prognosis of patients with metastatic pancreatic carcinoma who are under systemic chemotherapy.

Hypoxia-Inducible Factor-2α Correlates to Distant Recurrence and Poor Outcome in Invasive Breast Cancer

Differential regulation as well as target gene specificity of the two hypoxia-inducible factor (HIF)-alpha subunits HIF-1alpha and HIF-2alpha in various tumors and cell lines have been suggested. In breast cancer, the prognostic significance of HIF-1alpha is not clear-cut and that of HIF-2alpha is largely unknown. Using IHC analyses of HIF-1alpha, HIF-2alpha, and vascular endothelial growth factor (VEGF) expression in a tissue microarray of invasive breast cancer specimens from 512 patients, we investigated the expression patterns of the 2 HIF-alpha subunits in relation to established clinicopathologic variables, VEGF expression, and survival. HIF-1alpha and HIF-2alpha protein levels and their effect on survival were additionally analyzed in a second cohort of 179 patients. To evaluate the individual role of each subunit in the hypoxic response and induction of VEGF, HIF-alpha protein and HIF-alpha and VEGF mRNA levels were further studied in cultured breast cancer cells after hypoxic induction and/or knockdown of HIF-alpha subunits by siRNA by Western blot and Quantitative Real-Time PCR techniques. We showed that although HIF-1alpha and HIF-2alpha protein levels in breast cancer specimens were not interrelated, high levels of both HIF-1alpha and HIF-2alpha associated to high VEGF expression. HIF-2alpha expression was an independent prognostic factor associated to reduced recurrence-free and breast cancer-specific survival, whereas HIF-1alpha did not exhibit these correlations. In cultured cells, acute hypoxia induced both HIF-proteins. At prolonged hypoxia, HIF-2alpha remained accumulated, whereas HIF-1alpha protein levels decreased, in agreement with the oxygen level and time-dependent induction of HIFs recently reported in neuroblastoma.

Transgenic Expression of VEGF in Intestinal Epithelium Drives Mesenchymal Cell Interactions and Epithelial Neoplasia

Vascular endothelial growth factor (VEGF) is expressed robustly in human colon neoplasia and is a major new "rational" target of therapy for cancers of the colon and other organs. Nonetheless, the mechanism(s) of action of VEGF-targeted therapies and the biologic roles of VEGF in tumorigenesis have not been well defined. We used a transgenic approach to directly test the hypothesis that augmented VEGF expression can drive progression of intestinal neoplasia. Transgenic mouse lines were generated with moderate (vilVEGF1) and high (vilVEGF2) VEGF expression from the intestinal epithelium. vilVEGF1 mice were bred to Min mice (adenomatous polyposis coli [APC] +/-). Colon epithelial cells from an APC patient were cocultured with endothelial cells and fibroblasts. vilVEGF mice were generally healthy but displayed red small intestines. Vessels were larger and more numerous in the submucosa but not the mucosa. The mucosa showed striking stromal and epithelial hypercellularity, with increased epithelial proliferation. Many crypts formed cysts composed of relatively undifferentiated epithelial cells surrounded by cells with endothelial and myofibroblast markers. Compared with Min controls, vilVEGF1-Min mice developed 6-fold more intestinal adenomas of all sizes, with more advanced histologic features. Polycystic masses were also observed. Coculture of human colonocytes with endothelial cells and fibroblasts directly stimulated colonocyte proliferation. Augmented VEGF expression from intestinal epithelium potently stimulated cross talk with mesenchymal cells and proliferation of normal and neoplastic epithelium. These effects of VEGF, largely occurring prior to the canonical angiogenic switch in tumors, may be in part independent of angiogenesis.

Differential expression of angiopoietin‐2 and vascular endothelial growth factor in androgen‐independent prostate cancer models

To investigate the relationship between microvessel density (MVD), blood vessel morphology and the expression of angiopoietin (Ang)-1, Ang-2, tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (Tie)-2, and vascular endothelial growth factor (VEGF) in androgen-dependent (AD) and androgen-independent (AI) prostate cancer models, to gain insight into the regulation of angiogenesis at different stages of prostate cancer. MVD and blood vessel morphology were evaluated by CD34 immunohistochemical staining. The mRNA and protein secretion of the Angs, Tie-2 and VEGF were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assays, respectively, in LNCaP (AD) and LNCaP-19, C4-2, C4-2B4 and PC-3 (AI) prostate cancer xenografts in mice. LNCaP, C4-2 and C4-2B4 xenografts had high expression of Ang-2 and VEGF, similar MVD and blood vessel morphology. However, the most angiogenic cell line LNCaP-19 expressed low levels of both factors and had different vessel morphology. PC-3 xenografts had a similar MVD to LNCaP, C4-2 and C4-2B4, but the Ang-2 and VEGF expression as well as the vessel morphology were similar to LNCaP-19. The differences in MVD, blood vessel morphology and the expression of Ang-2 and VEGF show that prostate cancer cells display angiogenic heterogeneity, which indicates different roles of these factors in the regulation of angiogenesis in different stages of prostate cancer.