Low Vascular Endothelial Growth Factor Expression Research Articles

A phase 2 clinical study of SU5416 in patients with refractory acute myeloid leukemia

AbstractNeoangiogenesis has been shown to play an important role in the pathogenesis of acute myeloid leukemia (AML). Autocrine and paracrine secretion of angiogenic and hematopoietic growth factors such as vascular endothelial growth factor (VEGF) and stem cell factor (SCF) in the bone marrow microenvironment may promote proliferation and survival of leukemic blasts. This concept represented the rationale for the initiation of a multicenter phase 2 trial of SU5416, a small molecule inhibitor of phosphorylation of VEGF receptors 1 and 2, c-kit, the SCF receptor, and fms-like tyrosine kinase-3 (FLT3) in patients with advanced AML. Entered into the study were 43 patients with refractory AML or elderly patients not judged medically fit for intensive induction chemotherapy; 42 patients received at least one dose of study drug. Treatment was generally well tolerated, with nausea, headache, and bone pain the most frequent treatment-related side effects. One patient had a morphologic remission (French-American-British [FAB] criteria of complete response without normalization of blood neutrophil and platelet counts) lasting for 2 months. There were 7 patients who achieved a partial response (reduction of blasts by at least 50% in bone marrow and peripheral blood) lasting 1 to 5 months. Patients with AML blasts expressing high levels of VEGF mRNA by quantitative polymerase chain reaction (PCR) had a significantly higher response rate and reduction of bone marrow microvessel density than patients with low VEGF expression consistent with the antiangiogenic effects of SU5416.

VEGF in 105 pheochromocytomas: enhanced expression correlates with malignant outcome.

Pheochromocytomas are rare sympathoadrenal tumors that are highly vascular. Their malignancy is extremely difficult to estimate on the basis of histopathological features. Vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors involved in both tumor growth and metastasis. In our search for new prognostic markers, we investigated the expression of VEGF in normal adrenal gland, in 105 primary pheochromocytomas, and in 6 metastases by using immunohistochemistry and Northern blot analysis. We also calculated the microvessel density of these tumors by staining the endothelial cells with monoclonal CD34 antibody. VEGF messenger ribonucleic acid was found in all pheochromocytomas studied. Immunohistochemically, VEGF was not found in normal adrenal medullary cells. Interestingly, all malignant pheochromocytomas (n=8), regardless of their primary location, had strong or moderate VEGF immunoreactivity, while most benign adrenal pheochromocytomas (26 of 37, 70.3%) were either negative or only weakly positive. The staining was heterogenous in extraadrenal pheochromocytomas as well as in a group of tumors that had histologically suspicious features but had not metastasized, here called borderline tumors (n=29). The microvessel density varied greatly in all of the tumor groups, and no statistical difference was found between these groups. Here we report moderate to strong VEGF expression in malignant pheochromocytomas, and negative or weak expression in benign adrenal pheochromocytomas. Normal medullary cells are immunohistochemically negative. Thus, low VEGF expression in pheochromocytomas favors a benign diagnosis.

Hypoxia and expression of VEGF-A protein in relation to capillary growth in electrically stimulated rat and rabbit skeletal muscles.

To investigate the role of hypoxia as a stimulus to the early upregulation of vascular endothelial growth factor (VEGF) in fast skeletal muscles during chronic low frequency stimulation, blood flow, oxygen consumption, VEGF expression and capillary : fibre ratio were measured in chronically stimulated tibialis anterior and extensor digitorum longus (EDL) muscles in rabbits and rats. No differences were found in blood flow, oxygen consumption and extraction between rabbit muscles stimulated for 2 or 4 days (8 h on-16 h off) and controls. Muscle P(O(2)) polarographically measured immediately at the end of stimulation on day 2 was also no different from control under resting conditions (10.7 +/- 1.6 vs. 9.5 +/- 1.2 Torr, n.s.). Unlike control muscles, however, P(O(2)) in 2 day stimulated muscles did not increase immediately after a further acute bout of contractions. This difference was not apparent after similar acute contractions in 4 day stimulated muscles. The involvement of VEGF in early angiogenesis in stimulated muscles was studied in serial cryosections of rat EDL. The proportion of capillaries positively immunostained for VEGF increased from 25 +/- 1 % to 40 +/- 1 % (P < 0.05) in muscles removed on day 2 immediately at the end of chronic stimulation; it decreased slightly after 16 h rest, and increased again after 4 days of stimulation. Capillary : fibre ratio was unchanged throughout the experiment. Capillary cell proliferation increased only after the rest period on day 2 (20-fold increase) and day 4 (12-fold increase), indicating angiogenesis in progress. Thus the timing of transient hypoxia and increase in capillary-linked VEGF in stimulated muscles, albeit in different species, was similar, and increased VEGF staining and capillary cell proliferation occurred even after the hypoxia had resolved. This suggests (1) a connection between hypoxia and VEGF during the early stages of stimulation, although ensuing capillary proliferation may thereafter rapidly correct for local hypoxia, and (2) that the subsequent angiogenesis and VEGF expression are dependent on factors other than hypoxia.

Vascular endothelial growth factor and prognosis in patients with node-negative breast cancer.

Vascular endothelial growth factor (VEGF) has emerged as one of the most important angiogenic growth factors from experimental in vitro and in vivo studies. In the present study, we investigated the relationship between VEGF expression and microvessel density (MVD) and defined their prognostic relevance on a series of 242 patients with node-negative breast cancer, using immunohistochemical methods. In parallel, estrogen and progesterone receptors were quantitatively assessed using the dextran-charcoal technique and cell proliferation was evaluated as S-phase cell fraction according to (3)H-thymidine-labeling index (TLI). The percentage of VEGF-expressing cells varied from 0-95% in the different tumors and was unrelated to menopausal status, tumor size or steroid receptor status. Conversely, a significant inverse relation was observed with patient age or tumor cell proliferation, albeit with very poor correlation coefficients. A significant relation was observed between VEGF expression and MVD (r(s) = 0.55, p < 0.001). Clinical outcome analyzed as a function of high and low VEGF expression showed slight differences in terms of both disease-free survival (DFS) and overall survival (OS) that never reached statistical significance. Moreover, the trend was paradoxically in favor of patients with highly VEGF-expressing tumors. Finally, DFS and OS curves, when analyzed as a function of VEGF expression or MVD, were superimposable. In conclusion, our study did not highlight a prognostic relevance of VEGF expression in patients with node-negative breast cancer, as already observed for MVD.

Interleukin 8 and vascular endothelial growth factor — prognostic factors in human gastric carcinomas?

Gastric carcinoma cells express potent angiogenic factors including vascular endothelial growth factor (VEGF). We previously reported that interleukin-8 (IL-8) acts as an angiogenic factor for human gastric carcinomas. More recently, we found that IL-8 upregulates matrix metalloproteinase-9 (MMP-9) expression and increases invasive activity of gastric carcinoma cells. The purpose of this study was to determine whether the expression of IL-8 and VEGF correlates with clinicopathological parameters in human gastric carcinomas. IL-8 and VEGF expression levels were measured by an enzyme-linked immunosorbent assay (ELISA) in 56 gastric carcinomas and the surrounding normal mucosa. Macroscopic and histopathological tumour findings, presence of metastasis and prognosis were obtained from the patient records and endoscopic, surgical and pathological reports. IL-8 protein levels were higher in most neoplasms than in the corresponding normal mucosal tissue. In contrast, VEGF expression in the tumours was similar to that in normal mucosa. The IL-8 level in the neoplasms correlated significantly with the depth of invasion, venous invasion and lymphatic invasion. VEGF expression in the tumours correlated well with the depth of invasion and lymph node metastasis. No correlation between IL-8 and VEGF expression in the tumours was observed. The survival rates of patients with tumours displaying high IL-8 and VEGF expression levels were significantly lower ( P<0.05) than those of patients with tumours displaying low IL-8 and VEGF expression. The results suggest that IL-8 and VEGF may be independent and important prognostic factors in human gastric carcinomas.

Vascular endothelial growth factor and other biological predictors related to the postoperative survival rate on non-small cell lung cancer

Objective: An analysis of postoperative 1–5 year-survival rates related to vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGF-r), microvascular density (MVD), P53, H-ras, CerbB2, proliferative index (PI), divisional index (DI) were carried out on 127 cases of non-small cell lung cancer (NSCLC) treated with curative resection and aim to find out more sensitive molecular prognostic predictors for the reference of medicine and molecular pathology. Methods: All the cases were staged strictly using the UICC criteria of 1997 and regional lymphonodes seen by the naked eye should be dissected and sent to pathology with lung specimens. Immunohistochemical analyses were used for those biological predictors. Kaplan–Meier curve, Cox univariance and multivariance analysis were used for the survival and prognostic predictors. Results: 52 cases (40.9%) with high expression of VEGF showed a worse postoperative year-survival rate than cases with low expression, but no statistical difference. The difference of survival on stage I case with high and low expression were closed to be significant value ( P=0.0643). P53, PI and DI were related to postoperative survival, P=0.0341, 0.0005 and 0.0162, respectively. Co-expression of VEGF combined with P53, PI and DI was calculated for the difference of year-survival rate by Cox multivariance analysis. The survival rates of cases with both negative VEGF +P53 or VEGF+PI co-expression rate were better than those with both positive or either positive, P values were 0.0159 and 0.0154, respectively. Conclusion: The post-operative year-survival of NSCLC was of no statistical difference between with high expression and low expression of VEGF, but in stage I case it was closed to be significant difference, it speculated that the neoangiogenesis is more obviously in early stage NSCLC, but in the later stage of NSCLC, it may be covered by more complicated molecular–biological factors such as P53 and other oncogens. Co-expression of VEGF combined with p53 or PI was more meaningful than a single biological predicator on survival rate of NSCLC, it is worth to do further studies.

Microvascular Permeability of Human Melanoma Xenografts to Macromolecules: Relationships to Tumor Volumetric Growth Rate, Tumor Angiogenesis, and VEGF Expression

The effective microvascular permeability of human melanoma xenografts to albumin–Evans blue was measured and related to tumor volumetric growth rate, rate of tumor angiogenesis, and expression of vascular endothelial growth factor (VEGF) in an attempt to identify mechanisms regulating the microvascular permeability of tumors to macromolecules. Three melanoma lines (A-07, R-18, and U-25) were included in the study. Effective microvascular permeability was assessed by using the indicator diffusion method. Intradermal and intratumor angiogenesis assays were used to measure the rate of tumor angiogenesis. VEGF expression was studied by ELISA, immunohistochemistry, Western blotting, and measurement of tumor-induced formation of ascitic fluid. The effective microvascular permeabilities of albumin–Evans blue were determined to be (1.5 ± 0.2) × 10−6 cm/s (A-07), (1.1 ± 0.4) × 10−6 cm/s (R-18), and (0.9 ± 0.3) × 10−6 cm/s (U-25). These values are high compared with those measured for other tumor lines and are not significantly different. Correlations between the effective microvascular permeability of albumin–Evans blue and tumor volumetric growth rate, rate of tumor angiogenesis, or VEGF expression were not found. The three last-mentioned parameters differed significantly among the melanoma lines and covered a broad range of values relative to those of other experimental tumors. Our study suggests that the effective microvascular permeability of macromolecules can be high even in slowly growing tumors, poorly angiogenic tumors, and tumors showing low VEGF expression.

The association of K-ras gene mutation and vascular endothelial growth factor gene expression in pancreatic carcinoma.

Previously, the authors reported the role of the vascular endothelial growth factor (VEGF) as an angiogenic factor in 40 patients with pancreatic carcinoma. In this study, they investigated the mechanism underlying the regulation of VEGF gene expression and evaluated VEGF expression and K-ras gene status in 48 patients with pancreatic carcinoma. The authors used quantitative reverse transcriptase-polymerase chain reaction analysis and direct sequencing techniques for a retrospective study of VEGF gene expression and K-ras gene status in tumor tissue samples from 48 patients with pancreatic carcinoma. Immunohistochemistry also was used to investigate VEGF protein expression. Thirty-one tumors (64.6%) were evaluated with high VEGF expression, and 17 tumors (35.4%) were evaluated with low VEGF expression. Of the 48 primary pancreatic tumors studied, 33 tumors (68.8%) contained mutations of the K-ras gene. There was a significant correlation between VEGF expression and K-ras status. Twenty-five of 33 tumors (75.8%) with mutant K-ras genes showed high VEGF expression, whereas only 6 of 15 tumors with the wild type K-ras (40.0%) showed high VEGF expression (P = 0.038). The mean (+/- standard error) VEGF conservation rate for the 33 tumors with mutant K-ras was 1.839 +/- 1.241, and that for the 15 tumors with wild type K-ras was 1.057 +/- 0.983 (P = 0.037). Furthermore, the median survival for patients with mutant K-ras was shorter than for those with wild type K-ras (10.6 months vs. 27.6 months, respectively; P = 0.026), whereas the median survival for patients with high VEGF expression was shorter compared with that for patients with low VEGF expression (9.5 months vs. 26.4 months, respectively; P = 0.002). Cox regression model analysis indicated that only the VEGF status was a significant factor for prognosis (P = 0.024). Other variables, i.e., K-ras status, histopathologic tumor grade, tumor status, lymph node status, metastatic status, gender, and age at surgery, were not significant. The results of this study suggest that K-ras oncogene mutation may be associated with VEGF expression and that patients with pancreatic carcinoma who have high VEGF expression are associated with a poor prognosis.

Vascular endothelial growth factor expression in stage I non-small cell lung cancer correlates with neoangiogenesis and a poor prognosis.

Vascular endothelial growth factor (VEGF) plays an important role in tumor growth and metastasis. We investigated the prognostic significance of VEGF overexpression, intratumoral microvessel density (MVD), and angiolymphatic invasion in stage Ia-b non-small cell lung cancer (NSCLC). Eighty-five patients undergoing complete surgical resection of pathologic stage Ia-b NSCLC were evaluated. The mean and median clinical follow-up were 37.1 and 39.0 months (range, 30-44 months), respectively. Paraffin-embedded tumor specimens were stained with VEGF and CD31 (a specific endothelial marker) using immunohistochemical methods. VEGF staining was evaluated, by combining both percentage of positive tumor cells and staining intensity, as low (negative and < 20% of tumor cells showing weak positivity), or high (> 20% of tumor cells showing strong positivity). CD31 staining was expressed as MVD per high power field at 400x magnification. Angiolymphatic invasion was expressed as either presence or absence. Low VEGF expression was seen in 25 (29%) patients, and high VEGF expression was seen in 60 (71%) patients. The survival rate in patients with low VEGF expression was significantly higher (80%) than that in those with high VEGF expression (48%, P = .018). The mean MVD in the low VEGF group was 23.7 +/- 5.7 vs. 34.4 +/- 9.3 in the high VEGF group (P = .001). Patients with high MVD also had a significantly lower survival rate than did those with low MVD count (46% vs. 73%, P = .0053). Age, sex, tumor type, and tumor differentiation were not found to be associated with overall survival. The presence of angiolymphatic invasion and T2 stage (i.e., tumor size > 3 cm) were associated with decreased survival. High VEGF expression, tumor size, and angiolymphatic invasion emerged as three independent factors predicting worsening prognosis using multivariate analysis. High VEGF expression within stage I NSCLC is closely associated with high intratumoral angiogenesis and poor prognosis. Immunohistochemical evaluation of T stage and VEGF expression along with examination of angiolymphatic invasion perioperatively may aid in predicting prognosis. Adjuvant therapies aimed at retarding tumor angiogenesis may be considered for stage I NSCLC patients with high VEGF levels.

Prognostic significance of vascular endothelial growth factor expression in human ovarian carcinoma.

The influence of vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) on prognosis and the relationship between VEGF expression and MVD in ovarian carcinoma are not well defined. We studied VEGF expression in parallel with MVD by immunohistochemistry in 94 ovarian tumours (64 malignant, 13 borderline, and 17 benign) and correlated the results with the clinicopathologic prognostic factors of the disease to clarify their significance in this disease. Assessment of VEGF mRNA isoforms by RT-PCR was also performed. Of the malignant, borderline, and benign ovarian tumours respectively, two (3%), four (31%) and 16 (94%) were negative, 31 (48%), seven (54%) and one (6%) had low expressions, and 31 (48%), two (15%) and none (0%) had high expressions of VEGF. There were significant associations between the VEGF expression and disease stage (P = 0.002), histologic grade (P = 0.0004), and patient outcome (P = 0.0002). MVD did not correlate significantly with the clinicopathologic parameters. Likewise, no correlation was found between MVD and VEGF expression. The survival of patients with high VEGF expression was significantly worse than that of patients with low and negative VEGF expression (P = 0.0004). Multivariate analysis revealed that disease stage and VEGF expression were significant and independent prognostic indicators of overall survival time (P = 0.008 and P = 0.006 respectively). These findings suggest that in conjunction with the established clinicopathologic prognostic parameters of ovarian carcinoma, VEGF expression may enhance the predictability of patients at high risk for tumour progression who are potential candidates for further aggressive therapy. © 2000 Cancer Research Campaign

High expression of vascular endothelial growth factor is associated with liver metastasis and a poor prognosis for patients with ductal pancreatic adenocarcinoma.

Vascular endothelial growth factor (VEGF), a recently identified growth factor with significant angiogenic properties, is a multifunctional angiogenic cytokine that is expressed in many tumors. High VEGF expression has been shown to correlate with the incidence of metastasis and poor prognosis in various cancers. In this study, the authors investigated VEGF expression and microvessel density (MVD) in ductal pancreatic adenocarcinoma and examined the correlations among VEGF expression, clinicopathologic factors, and clinical outcome. The authors especially focused on the correlation between VEGF expression and liver metastasis. Paraffin embedded tumor specimens of 142 surgically resected pancreas carcinoma were immunohistochemically stained for VEGF and MVD. The correlations among VEGF expression and MVD, clinicopathologic factors, and clinical outcome were then statistically analyzed. One hundred thirty-two (93%) of 142 ductal pancreatic adenocarcinomas were positive for VEGF protein by immunohistochemistry. A significant correlation was observed between VEGF positivity and MVD (P < 0.0001). Multivariate logistic regression analysis indicated a significant association between high VEGF expression and liver metastasis (P = 0.010) but no other factors, such as age, tumor size, histologic type, lymph node metastasis, venous invasion, neural invasion, peritoneal metastasis, or local recurrence. Patients with tumors that showed moderate or high VEGF expression had significantly shorter survival than patients with low VEGF expression or none at all in their tumors (P < 0.05). These results indicate that VEGF expression is closely correlated with MVD and seems to be an important predictor for both liver metastasis and poor prognosis in ductal pancreatic adenocarcinoma.

Correlation of vascular endothelial growth factor content with recurrences, survival, and first relapse site in primary node-positive breast carcinoma after adjuvant treatment.

To determine the predictive value of vascular endothelial growth factor (VEGF) for relapse-free survival (RFS) and overall survival (OS) in primary node-positive breast cancer (NPBC) after adjuvant endocrine treatment or adjuvant chemotherapy. VEGF was quantitatively measured in tumor cytosols from 362 consecutive patients with primary NPBC using an enzyme immunoassay for human VEGF(165). Adjuvant treatment was given to all patients, either as endocrine therapy (n = 250) or chemotherapy (n = 112). The median follow-up time was 56 months. Univariate analysis showed VEGF to be a significant predictor of RFS (P =.0289) and OS (P =.0004) in the total patient population and in patients who received adjuvant endocrine treatment (RFS, P =.0238; OS, P =.0121). In the group of patients who received adjuvant chemotherapy, no significant difference was seen in RFS, but a difference was seen in OS (P =.0235). Patients with bone recurrences tended to have lower VEGF expression (median, 2.17 pg/microg DNA) than patients with visceral metastasis (4.41 pg/microg), brain metastasis (8.29 pg/microg), or soft tissue recurrences (3.16 pg/microg). Multivariate analysis showed nodal status (P =.0004), estrogen receptor (ER) status (P <.0001), and tumor size (P =.0085) to be independent predictors of RFS. VEGF was found to be an independent predictor of OS (P =.0170; relative risk [RR] = 1.82), as were ER (P <.0001; RR = 5.19) and nodal status (P =.0002; RR = 2.58). For patients receiving adjuvant endocrine treatment, multivariate analysis showed VEGF content to be an independent predictor of OS (P =.0420; RR = 1.90) but not of RFS. The results suggest that VEGF(165) content in tumor cytosols is a predictor of RFS and OS in primary NPBC. VEGF content might also predict outcome after adjuvant endocrine treatment, but further studies in a prospective setting with homologous treatments are required.

The K-ras gene regulates vascular endothelial growth factor gene expression in non-small cell lung cancers.

Tumor angiogenesis is an essential step for tumor cell growth, progression and metastasis. Vascular endothelial growth factor (VEGF) is mitogen specific for endothelial cells, and therefore is believed to play a key role in tumor angiogenesis. However, the mechanisms underlying the regulation of VEGF expression remain virtually unknown and the only major regulator of VEGF expression has been reported to be hypoxia. Recently, it was reported that a mutant p53 in#duced the expression of VEGF mRNA, and that wild-type p53 down-regulated endogenous VEGF mRNA levels. In contrast, it has also been reported that mutant ras oncogenes were associated with the marked up-regulation of VEGF in transformed epithelial cells. Based on these results, we performed a retrospective study of the p53 and K-ras genes status and VEGF gene expression in the tumor tissues from 181 patients with non-small cell lung cancer using SSCP, sequencing, RT-PCR and immunohistochemical techniques. Forty-six carcinomas (25.4%) were evaluated as having high VEGF expression, and 135 tumors (74.6%) had low VEGF expression. Of the 181 primary NSCLC studied, 63 carcinomas (34.8%) contained mutations of p53, whereas only 14 carcinomas (7.7%) had mutations of K-ras. There were no significant relationships between VEGF expression and p53 status or each mutant exon of p53. In contrast, a significant difference was found between VEGF expression and K-ras status. Of the 14 tumors with mutant K-ras genes, 7 cases (50.0%) had high VEGF expression whereas only 39 of the 167 tumors with wild-type K-ras (23.4%) had high VEGF expression (p=0.0278). The mean VEGF conservation rate for the 14 tumors with mutant K-ras genes was 0.77+/-0.58 and the rate of the 167 tumors with wild-type K-ras genes was 0.49+/-0.46 (p=0. 0350). Moreover, the overall survival rate of patients with high VEGF expression was lower than patients with low VEGF expression (45.7% vs 60.7%, p=0.0419). On the other hand, there was no significant difference in the overall survival rate between patients with a mutant p53 and those with a wild-type p53; there was also no difference in the overall survival between patients with a mutant K-ras and those with a wild-type K-ras. The Cox regression model analysis indicated that three variables, VEGF status, K-ras status and nodal status, were found to be significant indicators for prognosis (p=0.0236, p=0.0172 and p<0.0001, respectively). Our data suggest that a high expression of VEGF in lung cancer may be associated with a poor prognosis. This may be a clue to improving lung cancer diagnoses and therapies aimed at inhibiting tumor angiogenesis due to VEGF.

Macrophage infiltration is associated with VEGF and EGFR expression in breast cancer

Angiogenesis is esential for tumour growth and metastasis. Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and is an important component of the angiogenic stimulus in a range of human neoplasias. In addition to its mitogenic activities, VEGF has also been found to stimulate migration in macrophages via the flt-1 VEGF receptor. It has previously been shown that increased focal tumour macrophage infiltration is associated with increased angiogenesis and worsened relapse-free and overall survival in breast cancer. Macrophages are able to stimulate angiogenesis by their production of a range of factors including VEGF, tumour necrosis factor-α (TNF-α), and thymidine phosphorylase (TP). Thus, in breast cancer, VEGF could have a dual role in the regulation of angiogenesis, by direct mitogenic stimulation of endothelial cells, and also indirectly by attracting macrophages into avascular tumours. The purpose of this study was to localize VEGF protein in a series of 96 consecutive primary breast carcinomas and to determine its relationship to focal macrophage infiltration (macrophage index). These two variables were also compared with the pathological features of the tumours, as well as oestrogen receptor (ER), epidermal growth factor receptor (EGFR), microvessel density, macrophage index, and survival. An inverse relationship (p=0.0006) was noted between VEGF and EGFR, with high VEGF expression correlating with low EGFR levels. In the EGFR-negative group of cases (n=56), positive associations were observed between VEGF expression and macrophage index (p=0.005), ER (p=0.05), p53 (p=0.006), tumour grade (p=0.02), and tumour necrosis (p=0.03). Macrophage counts were higher in EGFR-positive tumours (p=0.0006) and no associations were found between VEGF expression and increased microvessel density. These results show that in breast cancers there are two types of macrophage infiltrates, one associated with the presence of EGFR and low VEGF expression in tumours and the other with high VEGF expression in EGFR-negative tumours. VEGF expression may be an important factor in the recruitment of tumour-associated macrophages into breast carcinomas and may thus have an additional, indirect, pathway of angiogenic stimulation in this type of tumour. Copyright © 2000 John Wiley & Sons, Ltd.

Macrophage infiltration is associated with VEGF and EGFR expression in breast cancer.

Angiogenesis is esential for tumour growth and metastasis. Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and is an important component of the angiogenic stimulus in a range of human neoplasias. In addition to its mitogenic activities, VEGF has also been found to stimulate migration in macrophages via the flt-1 VEGF receptor. It has previously been shown that increased focal tumour macrophage infiltration is associated with increased angiogenesis and worsened relapse-free and overall survival in breast cancer. Macrophages are able to stimulate angiogenesis by their production of a range of factors including VEGF, tumour necrosis factor-alpha (TNF-alpha), and thymidine phosphorylase (TP). Thus, in breast cancer, VEGF could have a dual role in the regulation of angiogenesis, by direct mitogenic stimulation of endothelial cells, and also indirectly by attracting macrophages into avascular tumours. The purpose of this study was to localize VEGF protein in a series of 96 consecutive primary breast carcinomas and to determine its relationship to focal macrophage infiltration (macrophage index). These two variables were also compared with the pathological features of the tumours, as well as oestrogen receptor (ER), epidermal growth factor receptor (EGFR), microvessel density, macrophage index, and survival. An inverse relationship (p=0.0006) was noted between VEGF and EGFR, with high VEGF expression correlating with low EGFR levels. In the EGFR-negative group of cases (n=56), positive associations were observed between VEGF expression and macrophage index (p=0.005), ER (p=0.05), p53 (p=0. 006), tumour grade (p=0.02), and tumour necrosis (p=0.03). Macrophage counts were higher in EGFR-positive tumours (p=0.0006) and no associations were found between VEGF expression and increased microvessel density. These results show that in breast cancers there are two types of macrophage infiltrates, one associated with the presence of EGFR and low VEGF expression in tumours and the other with high VEGF expression in EGFR-negative tumours. VEGF expression may be an important factor in the recruitment of tumour-associated macrophages into breast carcinomas and may thus have an additional, indirect, pathway of angiogenic stimulation in this type of tumour.

Vascular endothelial growth factor and its correlation with angiogenesis and p53 expression in prostate cancer.

Previously it was demonstrated that in prostate tumors, angiogenesis measured as microvessel density (MVD) is associated with tumor stage as well as WHO grade and is an independent predictor of clinical outcome. Vascular endothelial growth factor (VEGF) is a major inducer of angiogenesis. There is some evidence that P53 mutations cause overexpression of VEGF. We studied VEGF expression, p53 overexpression, and P53 mutations in prostate cancer (PCA) to investigate the role of VEGF as an angiogenic marker and the possible deregulation of VEGF as a result of P53 mutations in PCA. Immunohistochemical staining with a polyclonal VEGF antibody was performed in 55 paraffin-embedded PCA, in which MVD had previously been determined, as well as in 5 prostatic adenomas (PA) and 20 adjacent normal prostate tissues. In addition, 37 PCA and 5 PAs were examined for p53 expression by immunohistochemistry. Temperature gradient gel electrophoresis (TGGE) was performed in 13 of these PCA to screen for P53 mutations. VEGF expression, p53 expression, and mutations were then correlated with tumor stage, grade, MVD, and clinical outcome. While PA and normal prostate tissue generally showed no or only low VEGF expression, there was a significant increase in VEGF expression with tumor stage, grade, and MVD in PCA. During clinical follow-up (mean, 31.9 months), 9 of 55 patients had tumor progression. Significant differences in VEGF expression were found between patients with tumor progression and those without (P = 0.0004). Of the 37 PCA evaluated for p53 expression, 12 exhibited p53 overexpression. TGGE revealed P53 mutations in 3 of 13 PCA. However, there was no correlation between VEGF expression, p53 overexpression, and P53 mutation, respectively. VEGF seems to be an important, clinically relevant inducer of angiogenesis in PCA. VEGF expression was shown to correlate positively with tumor stage, grade, MVD, and clinical outcome. However, regulation of VEGF in PCA appears to be independent of p53 expression.

Expression of vascular endothelial growth factor in primary non-small cell lung carcinoma

Objective: Tumor growth depends on angiogenesis. The aim of this paper is to clarify the relationship between the expression of vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) and the angiogenesis, or growth, or invasion and prognostic value in Non-Small Cell Lung Cancer (NSCLC). Methods: Microvessel quantification and expression of VEGF was performed immunohistochemically, using multiclonal antibodies against endothelial protein factor VIII-related antigen (F–VIII antigen, or factor VIII) for evaluating the angiogenesis; against VEGF antigen for the expression of VEGF. Results: A total of 53 patients with NSCLC after the radical resection were evaluated. The patients with high and low expression of VEGF were 33 and 20, respectively. A significant higher microvessel density (MVD) was observed in the tumors with high expression of VEGF compared with the tumors with low expression of it (12.17±2.57/mm2 vs 6.01±1.161/mm2, rank sum test, P<0.01). There were 29 patients with lymphonodes metastasis in the high expression VEGF/VPF (29/33, 87.88%) group, and 9 patients in the low (9/20, 45%) group. There was good correlation between MVD and expression of VEGF (chisquare tests, P<0.001). The overall 5 years survival for 53 patients was 20.75±5.78%; that of the high expression of the VEGF group was 3.03±2.98%; that of the low group was 36.36±13.94%, by Log rank test, P=0.0001. The difference between them had a high significance. There was good correlation between the survival and the expression of VEGF. By the COX’s proportional hazard model analysis, the expression of VEGF and MVD was considered to be an independent marker of the prognosis in non-small cell lung cancer. Conclusion: the expression of VEGF has a significant correlation with MVD, growth, invasion, and lymph node metastasis. The increasing of the node metastasis and the size of tumor accompanied the increasing of VEGF/VPF. The cancer patient with higher VEGF and MVD expression might have worse prognosis. By multivariate analysis, VEGF/VPF or MVD may be taken as the independent predictors of prognosis for NSCLC.

Characterization of a Novel Form of Angiopoietin-2 (Ang-2B) and Expression of VEGF and Angiopoietin-2 during Chicken Testicular Development and Regression

The complex process of angiogenesis is controlled by the vascular endothelial growth factor (VEGF) and its receptors and by the recently isolated angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) that signal through the transmembrane endothelial receptor tyrosine kinase Tie2. We report here the characterization of a novel form of Ang-2 (Ang-2B) with a truncated amino-terminal domain resulting from an alternative splicing of the gene. While previous reports have found the expression of Ang-2 limited to the embryo, female reproductive organs, and tumor tissues, we have observed striking changes in Ang-2 expression during chicken testicular development and regression. The expression of Ang-2 and VEGF is abundant in prepuberal testis and low in quiescent adult testis. Testicular regression is accompanied by high expression of Ang-2 and very low expression of VEGF. These observations are in accordance with the proposal that Ang-2 induces angiogenesis in the presence of VEGF and vascular regression in its absence.

Hypoxia-induced angiogenesis and vascular endothelial growth factor secretion in human melanoma.

Tumour cells exposed to hypoxia in vitro can show increased expression of several selected genes, including the gene encoding the vascular endothelial growth factor (VEGF), suggesting that hypoxia followed by reoxygenation might promote the malignant progression of tumours. An in vitro/in vivo study was conducted to investigate whether hypoxia can increase the angiogenic potential of tumour cells through increased VEGF secretion. Four human melanoma cell lines (A-07, D-12, R-18, U-25) were included in the study. Cell cultures were exposed to hypoxia (oxygen concentration <10 p.p.m.) in vitro using the steel chamber method. Rate of VEGF secretion was measured in vitro in aerobic and hypoxic cell cultures by ELISA. Angiogenesis was assessed in vivo using the intradermal angiogenesis assay. Aliquots of cells harvested from aerobic cultures or cultures exposed to hypoxia for 24 h were inoculated intradermally in the flanks of adult female BALB/c-nu/nu mice. Tumours developed and angiogenesis was quantified by scoring the number of capillaries in the dermis oriented towards the tumours. The number of tumour-oriented capillaries did not differ significantly between tumours from hypoxic and aerobic cultures for A-07 and U-25, whereas tumours from hypoxic cultures showed a larger number of tumour-oriented capillaries than tumours from aerobic cultures for D-12 and R-18. The VEGF secretion under aerobic conditions and the absolute increase in VEGF secretion induced by hypoxia were lower for D-12 and R-18 than for A-07 and U-25, whereas the relative increase in VEGF secretion induced by hypoxia was higher for D-12 and R-18 than for A-07 and U-25. VEGF is not a limiting factor in the angiogenesis of some tumours under normoxic conditions. Hypoxia can increase the angiogenic potential of tumour cells by increasing the secretion of VEGF, but only of tumour cells showing low VEGF secretion under normoxia. Transient hypoxia might promote the malignant progression of tumours by temporarily increasing the angiogenic potential of tumour cells showing low VEGF expression under normoxic conditions.

Vessel counts and vascular endothelial growth factor expression in pancreatic adenocarcinoma.

Angiogenesis is essential for growth and metastasis of solid malignancies. In several tumours, tumour vessel count and expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, have been associated with prognosis. To determine if vessel count and VEGF expression are prognostic factors in pancreatic cancer, we examined these parameters in resected tumour specimens from 22 patients who did not receive pre-operative therapy. Paraffin-embedded tumour specimens were immunohistochemically stained for factor VIII (surrogate for vessels) and VEGF. Vessel counts and VEGF expression were evaluated without knowledge of patient outcome. The median follow-up for the entire group had not been reached as of 23.1 months (range 10-69 months). The mean vessel count and VEGF expression were no different between those patients who had recurrences and those who did not. By linear regression analysis, the correlation of VEGF expression with vessel count did not reach statistical significance (P = 0.0685). Survival and time to recurrence were similar in patients with high and low vessel counts and VEGF expression of 1, 2 or 3. Tumour differentiation or lymph node positivity had no effect on either VEGF expression or vessel count. Our data suggest that, in contrast to findings in other solid malignancies, vessel count and VEGF expression are not predictors of survival or recurrence in patients with resectable adenocarcinoma of the pancreas.