Vascular Endothelial Growth Factor Immunostaining Research Articles

Prognostic Significance of Vascular Endothelial Growth Factor, Tumor Necrosis, and Mitotic Activity Index in Malignant Pleural Mesothelioma

Tumor growth and metastasis are angiogenesis-dependent events, and several prognostic factors have been determined in malignant mesothelioma. In this study, we investigated the prognostic significance of vascular endothelial growth factor (VEGF), tumor necrosis (TN), and mitotic activity index (MAI) in malignant mesothelioma. For the first time (to our knowledge), we also demonstrated the association between VEGF staining, TN, and MAI in malignant mesothelioma. Decortication and VATS materials of 40 patients were investigated. The routinely processed formalin-fixed, paraffin-embedded, hematoxylin-eosin-stained tumor sections that had been used for the original diagnostic purposes were retrieved. Hematoxylin-eosin sections were selected for VEGF immunostaining, TN, and mitotic count. Clinicopathologic data, stage of disease, and survival of patients were all determined. The correlations between variables were evaluated by Spearman rank correlation test. Both univariate analysis using the log-rank test and multivariate analysis using Cox regression model were performed for survival analysis. There was a significant correlation between VEGF staining and TN (r = 0.42, p = 0.006). In univariate analysis, both VEGF staining (p = 0.0002) and TN (p = 0.0055) showed significant correlation with short survival. Also, there was a positive correlation between VEGF and tumor stage (p = 0.046). In multivariate analysis, only VEGF was determined as an independent prognostic factor in malignant mesothelioma (p = 0.001). There was no association between MAI and survival (p = 0.504). VEGF, known as an important angiogenic peptide, is an independent prognostic factor in malignant pleural mesothelioma. TN stimulates angiogenesis, and we observed a significant correlation between VEGF and TN. However, further studies are needed to evaluate the prognostic significance of angiogenic properties in malignant mesothelioma.

Influence of different hormonal regimens on endometrial microvascular density and VEGF expression in women suffering from breakthrough bleeding

The aim of this study was to quantify blood vessel density (BVD) and immunoreactive vascular endothelial growth factor (VEGF) levels in endometrial biopsies taken from women suffering breakthrough bleeding (BTB) under different exogenous hormonal regimes. Endometrial biopsies from women in Melbourne with BTB were divided into four groups: combined-continuous hormone therapy (HT) (estrogen and progestin taken daily), cyclical HT (daily estrogen with progestin for 14 days each cycle), progestin-only, or no HT. Subjects from Barcelona were using the Mirena intrauterine levonorgestrel-releasing system for contraceptive purposes, with menstrual diaries for classification into four groups (amenorrhea, infrequent, regular and prolonged). Control biopsies from Melbourne were included in the study. Endometrial samples were immunostained for VEGF and blood vessel localization using an antibody to CD34. Results showed that BVD was significantly reduced in the progestin-only treated group compared with the other three treatment groups (P = 0.028). In addition, all four Mirena BTB groups had significantly reduced BVD compared with controls. Considerable heterogeneity was observed in VEGF immunostaining within and between individual samples with no major differences between HT or Mirena. These results provide strong evidence that unopposed progestins reduce endometrial BVD and that there is no link between VEGF immunostaining and BVD or BTB.

Prognostic significance of VEGF immunohistochemical expression and tumor angiogenesis in head and neck squamous cell carcinoma

Tumor angiogenesis is crucial for both the growth of the primary tumor and the development of metastases. Among the factors causing tumor angiogenesis, vascular endothelial growth factor (VEGF) is considered as a leading candidate. We aimed to assess the prognostic significance of VEGF and tumor angiogenesis in head and neck squamous cell carcinoma (HNSCC). We performed a retrospective study of 69 patients with HNSCC, in order to investigate whether VEGF immunohistochemical expression and tumor angiogenesis correlate with clinicopathological parameters and outcome. Tumor angiogenesis was estimated by determining microvessel density (MVD), and VEGF expression was assessed quantitatively. Vascular endothelial growth factor and MVD correlated statistically significant with the clinical stage, but not with the presence of lymph node metastasis at the time of diagnosis. Tumors located in the oral cavity and larynx more often expressed high VEGF immunostaining compared with tumors located in the lower lip. High VEGF expression was associated with higher clinical stage and worse overall survival in this cohort of patients. Vascular endothelial growth factor expression may have prognostic significance for patients with HNSCC.

Pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in aged human choroid and eyes with age-related macular degeneration

The purpose of this study was to examine the localization and relative levels of vascular endothelial growth factor (VEGF; an angiogenic factor) and pigment epithelium-derived factor (PEDF; an antiangiogenic factor) in aged human choroid and to determine if the localization or their relative levels changed in age-related macular degeneration (AMD). Ocular tissues were obtained from eight aged control donors (age range, 75–86 years; mean age, 79.8 years) with no evidence or history of chorioretinal disease and from 12 donors diagnosed with AMD (age range, 61–105 years; mean age, 83.9 years). Tissues were cryopreserved and streptavidin alkaline phosphatase immunohistochemistry was performed with rabbit polyclonal anti-human VEGF and rabbit polyclonal anti-human PEDF antibodies. Binding of the antibodies was blocked by preincubation of the antibody with an excess of recombinant human PEDF or VEGF peptide. Choroidal blood vessels were identified with mouse anti-human CD-34 antibody in adjacent tissue sections. Three independent observers graded the immunohistochemical reaction product. The most prominent sites of VEGF and PEDF localization in aged control choroid were RPE–Bruch's membrane–choriocapillaris complex including RPE basal lamina, intercapillary septa, and choroidal stroma. There was no significant difference in immunostaining intensity and localization of VEGF and PEDF in aged control choroids. The most intense VEGF immunoreactivity was observed in leukocytes within blood vessels. AMD choroid had a similar pattern and intensity of VEGF immunostaining to that observed in aged controls. However, PEDF immunoreactivity was significantly lower in RPE cells ( p=0.0073), RPE basal lamina ( p=0.0141), Bruch's membrane ( p<0.0001), and choroidal stroma ( p=0.0161) of AMD choroids. The most intense PEDF immunoreactivity was observed in disciform scars. Drusen and basal laminar deposits (BLDs) were positive for VEGF and PEDF. In aged control subjects, VEGF and PEDF immunostaining was the most intense in RPE-Bruch's membrane-choriocapillaris complex. In AMD, PEDF was significantly lower in RPE cells, RPE basal lamina, Bruch's membrane and choroidal stroma. These data suggest that a critical balance exists between PEDF and VEGF, and PEDF may counteract the angiogenic potential of VEGF. The decrease in PEDF may disrupt the balance and be permissive for the formation of choroidal neovascularization (CNV) in AMD.

Possible Involvement of Adipocyte-Derived Leucine Aminopeptidase via Angiotensin II in Endometrial Carcinoma

Objective: It has recently been appreciated that a local autocrine or paracrine renin-angiotensin system (RAS) may exist in a number of tissues. Angiotensin II (AngII) is a potent RAS-derived vasoconstrictor peptide, and it is involved in tumor angiogenesis. We have cloned human adipocyte-derived leucine aminopeptidase (A-LAP), which degrades Ang II. This study investigated whether the expression of A-LAP, Ang II, angiotensin type I receptor (AT1R) and vascular endothelial growth factor (VEGF) correlates with clinicopathologic factors and prognosis in patients with endometrial endometrioid adenocarcinoma. Methods: Histologic sections of formalin-fixed, paraffin-embedded specimens from 94 primary endometrial carcinomas were stained for A-LAP, AngII, AT1R and VEGF using each antibody. Disease-free survival (DFS) and other clinicopathologic characteristics were analyzed according to the intensity of each staining. Results: Of 94 cases, 91 (96.8%) showed specific A-LAP immunostaining. A-LAP expression demonstrated negative correlations with myometrial invasion (p = 0.01) and vascular infiltration (p = 0.01). Of 94 cases, 77 (81.9%) showed specific AngII immunostaining. We found a positive correlation between AngII expression and surgical stage (p = 0.01). Of 94 cases, 56 (59.6%) showed specific AT1R immunostaining and 73 (77.7%) specific VEGF immunostaining. We found a positive correlation between VEGF expression and lymph node metastasis (p = 0.05). AngII and AT1R expression predicted a significantly poorer prognosis. Contrarily, A-LAP expression indicated a significantly more favorable prognosis in endometrial endometrioid adenocarcinoma patients. Multivariate analysis demonstrated that A-LAP expression (odds ratio, 0.12; 95% confidence interval, 0.025–0.618; p = 0.01) was an independent prognostic factor. Conclusions: In this study, we demonstrated the existence of local RAS and A-LAP in endometrial endometrioid adenocarcinoma as prognostic predictors of clinical outcome. These findings suggest that the assessment of RAS and A-LAP status provides clinically useful prognostic information in patients with endometrial carcinoma.

VEGF and VEGFR-2 immunohistochemistry in human melanocytic naevi and cutaneous melanomas

Vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2) play a key role in vasculogenesis and angiogenic sprouting, which are crucial for tumour development and metastasis. In order to determine their possible role in the acquisition of metastatic potential throughout melanocytic tumour progression, VEGF and VEGFR-2 immunohistochemical expression were evaluated in 36 human melanocytic tumours of the skin (24 malignant melanomas and 12 common naevi). Different VEGFR-2 immunostaining patterns were detected in the vast majority of melanomas (21/24; 88%). A nuclear membrane-like pattern was mainly associated with in situ and microinvasive melanomas, whereas a combined cytoplasmic membrane and nuclear membrane-like pattern was seen in invasive melanomas. A nuclear membrane-like pattern was also observed in 83% (10/12) of common naevi. Cytoplasmic immunostaining for VEGF was observed in 72% (8/11) of in situ/microinvasive melanomas, 84% (11/13) of invasive melanomas and 91% (11/12) of naevi. CD31 was also investigated as an immunohistochemical marker for microvessel density (MVD) evaluation. No associations were found between MVD and VEGF/VEGFR-2 expression. Taken together, these data indicate that VEGF production is a common event in benign melanocytic tumours, whereas VEGFR-2 expression, co-localized in the cytoplasmic and nuclear membrane, is associated with progression towards invasive melanoma. The role exerted by VEGF/VEGFR-2, however, seems to be independent of the development of a tumour-related capillary network.

Vascular Endothelial Growth Factor Level as a Prognostic Determinant of Small Cell Lung Cancer in Japanese Patients

To clarify the clinical significance of vascular endothelial growth factor (VEGF) in Japanese patients with small cell lung cancer (SCLC). We measured serum VEGF levels using an enzyme-linked immunosorbent assay in 45 patients with SCLC before treatment and in 38 patients with benign pulmonary disease and in 32 healthy subjects (71 non-malignant subjects). VEGF immunostaining was performed in tissue biopsies obtained from 23 SCLC patients during bronchoscopic examination. Median serum VEGF level was 332 pg/ml in patients with SCLC and 160 pg/ml in non-malignant subjects, respectively. The 95% cut-off level to exclude non-malignant subjects was 500 pg/ml. An elevated VEGF level (>500 pg/ml) was found more frequently in patients with extensive disease of SCLC than in those with the limited disease (p<0.01). A significant positive correlation was found between the serum VEGF level and platelet count in SCLC patients (r=0.389; p=0.0083). Serum VEGF level also correlated with serum lactate dehydrogenase in SCLC patients (r=0.381; p=0.0098). However, it did not correlate with serum neuron-specific enolase and pro-gastrin-releasing peptide level. Patients with the elevated VEGF levels had significantly shorter progression-free time than those with the normal VEGF levels (p<0.05). Patients with the elevated VEGF levels had a significantly shorter overall survival time than those with the normal VEGF levels in univariate survival analysis (p<0.05). Further, the elevated VEGF level remained as a significant determinant of poor survival in multivariate analysis (p<0.01). Serum VEGF level was significantly higher in patients with positive VEGF protein immunoreactivity in tumor tissue in SCLC. Elevated serum VEGF levels were associated with poor outcome in SCLC.

The Expressions of Hypoxia-Inducible Factor-1 and Vascular Endothelial Growth Factor in Osteosarcoma and Chondrosarcoma

Purpose: The purpose of this study is to investigate the impact of the vascular endothelial growth factor (VEGF) and the hypoxia-inducible factor-1 (HIF-1) expression on clinicopathologic factors in osteosarcomas and chondrosarcomas. Materials and Methods: Thirty-five osteosarcomas and 9 chondrosarcomas were assessed for HIF-1 and VEGF expressions with monoclonal antibodies using immunohistochemistry. The pathologic tumor grades are followed the WHO classification. Results: Nineteen out of 35 osteosarcomas (54.3%) and 8 out of 9 chondrosarcomas (88.9%) showed HIF-1 expressions. The HIF-1 expressions increased with the pulmonary metastasis (p=0.020) and the tumor grades (p=0.016) in only osteosarcomas. Twenty-four out of 35 osteosarcomas (68.6%) and 6 out of 9 chondrosarcomas (66.7%) showed VEGF expressions. The extents of VEGF expression in only osteosarcomas showed positive correlation with the pulmonary metastasis (p=0.024). Conclusion: The HIF-1 and VEGF appear to be an important role for the development and progression of these two tumors. The HIF-1 expression in osteosarcomas seems to be poor prognostic value including aggressive tumor grades and pulmonary metastasis. The extents of VEGF immunostaining might indicate the possibility of pulmonary metastasis in osteosarcomas. However, the two gene expressions could not indicate the significant prognostic value in chondrosarcomas.

Immunolocalization of von Willebrand Factor and Vascular Endothelial Growth Factor during Follicular Atresia in the Swamp Buffalo Ovary

The aim of this study was to investigate the distribution pattern of von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF) in the healthy antral and atretic follicles of Philippine swamp buffaloes (SB) in comparison with Holstein-Friesian cows (HF). Paraffin sections of healthy follicles and atretic follicles at various stages were immunostained with vWF antibody and VEGF antibody. The density of vWF-positive capillary vessels in the theca interna significantly increased as atresia progressed in SB, whereas the density significantly decreased in late atretic follicles compared with advanced ones in HF. On the other hand, the area of vWF-positive capillary vessels in the theca interna significantly increased as atresia progressed in both SB and HF. Immunoreactions of VEGF in the granulosa cells (in all follicle types) were observed in both SB and HF. In the granulosa layer, a reduction in the VEGF immunoreaction was noted as follicles progressed from healthy to advanced atretic follicles in both animals. Granulosa cells (in both SB and HF) showed a higher immunopositive staining than theca cells. In the theca interna, VEGF immunostaining diminished as follicles progressed to the late atretic follicles in both animals. These results indicate that during atresia, changes of vWF expression are the opposite of VEGF expression in SB. Both vWF and VEGF are suggested to be associated with follicular atresia in SB.

Vascular endothelial growth factor expression in serous ovarian carcinoma: relationship with topoisomerase IIα and prognosis

Objective.This study investigated the expression of vascular endothelial growth factor (VEGF) and topoisomerase IIα (TPIIα) in a cohort of serous ovarian carcinomas by immunohistochemistry with regard to outcome and clinicopathologic variables. Methods.Formalin-fixed, paraffin-embedded archival tissue sections of 10 benign serous cystadenomas and 41 serous ovarian carcinomas were immunostained with antibodies to VEGF and TPIIα. Immunostaining for VEGF was scored with regard to quantity and intensity of positively stained cells as weak, focal strong, and diffuse strong. TPIIα labeling indices (LI) were quantitated as the percentage of positively stained nuclei in 1000 tumor cells. Results.VEGF expression was weak or negative in serous cystadenomas. In contrast, focal or diffuse strong immunostaining was observed in 21 cases (51%) of serous carcinomas. VEGF immunoreactivity was positively related with TPIIα LI (P = 0.0055), adverse outcome (P = 0.0052), high International Federation of Gynecology and Obstetrics (FIGO) stage (P = 0.0158), and high tumor grade (P = 0.0303). TPIIα LI increased with FIGO stage (P = 0.0076) as well as tumor grade (P = 0.0034) and indicated poor prognosis (P = 0.0182). Conclusions.VEGF immunoreactivity is related with TPIIα LI and prognostically relevant. Expression of VEGF protein and TPIIα may serve as markers of aggressive tumor behavior in serous adenocarcinomas.

Sodium hyaluronate regulating angiogenesis during Achilles tendon healing

The aim of this study was to evaluate the effect of SH (sodium hyaluronate-NaHA) on vascular endothelial growth factor (VEGF) and type IV collagen expression during the Achilles-tendon healing process. Adult New Zealand white rabbits (n=32) aged 4 months and weighing 2.7-3.9 kg were used. The rabbits were randomly divided into two groups, and each group was divided into two subgroups and monitored for 6 and 12 weeks. Tendo calcanei were incised transversely and repaired. An injection of 0.5 ml NaHA (15 mg/ml) was administered between the tendon and paratenon of the right leg and repeated twice at one-week intervals. Equal numbers of animals were sacrificed at the 6th and 12th weeks, and the repaired tissue was examined macroscopically and histologically for the presence of VEGF and type IV collagen expression every week. The decrease in the amount of adhesion tissue and the acceleration of tendon healing in the NaHA group were significantly high when compared with control groups at 6 and 12 weeks (p<0.001, p<0.05). In the NaHA group, due to vascular proliferation VEGF immunostaining was strongly positive in the 6th week (p<0.05), and remained positive in the 12th week (p<0.05). Similar immunostaining findings were detected for type IV collagen in the 6th week. However, there was a significant decline in immunostaining rate in the 12th week (p<0.05). The increases in VEGF and type IV collagen expression following SH administration might be an indication that SH may partly be involved in regulation of angiogenesis.

Vascular endothelial growth factor expression and survival in node positive breast cancer patients treated with adjuvant therapy

653 Background: Vascular endothelial growth factor (VEGF) expression may affect tumor angiogenesis, with most studies finding worse survival associated with VEGF positivity. Methods: An immunohistochemical analysis for VEGF expression was performed on a cohort of 125 primary breast cancer patients diagnosed between 1989 and 1998 with 4 or more positive axillary lymph nodes, treated with adjuvant therapy. Patients were followed via registry for treatment, recurrence, and vital status current as of 2003. VEGF immunostaining, using the C1 clone on deparaffinized, formalin fixed tissue, was scored via a semi-quantitative system based on the intensity of immunostaining signal. Correlations were measured with factors associated with better and worse outcomes such as tumor size, number of positive lymph nodes, estrogen/progesterone receptor status, and HER-2/neu over expression. Log rank test for survival differences by VEGF status were calculated using Kaplan Meier plots. Results: 37% of the sample tested positive (2+) for VEGF (n=46). Fifty seven percent of the cases were over 50 years of age, 65% had 4–9 positive lymph nodes and 35%>=10, and median follow up was six years. VEGF positive patients had worse breast cancer survival (log rank test = 8.93, p = .003), overall survival (log rank test = 7.54, p = .006), and relapse free survival (log rank test = 5.61, p = .018). Patients who had 4–9 versus 10 or more positive lymph nodes, were estrogen receptor positive, or were HER-2/neu negative had significantly worse disease specific survival if they were VEGF positive. ER+/VEGF+ patients had significantly worse survival that ER+/VEGF- patients. Conclusions: In this study high levels of expression of VEGF were associated with significantly worse survival in breast cancer patients, even in subgroups expected to have a better prognosis. No significant financial relationships to disclose.

Vascular endothelial growth factor expression and survival in node positive breast cancer patients treated with adjuvant therapy

653 Background: Vascular endothelial growth factor (VEGF) expression may affect tumor angiogenesis, with most studies finding worse survival associated with VEGF positivity. Methods: An immunohistochemical analysis for VEGF expression was performed on a cohort of 125 primary breast cancer patients diagnosed between 1989 and 1998 with 4 or more positive axillary lymph nodes, treated with adjuvant therapy. Patients were followed via registry for treatment, recurrence, and vital status current as of 2003. VEGF immunostaining, using the C1 clone on deparaffinized, formalin fixed tissue, was scored via a semi-quantitative system based on the intensity of immunostaining signal. Correlations were measured with factors associated with better and worse outcomes such as tumor size, number of positive lymph nodes, estrogen/progesterone receptor status, and HER-2/neu over expression. Log rank test for survival differences by VEGF status were calculated using Kaplan Meier plots. Results: 37% of the sample tested positive (2+) for VEGF (n=46). Fifty seven percent of the cases were over 50 years of age, 65% had 4–9 positive lymph nodes and 35%>=10, and median follow up was six years. VEGF positive patients had worse breast cancer survival (log rank test = 8.93, p = .003), overall survival (log rank test = 7.54, p = .006), and relapse free survival (log rank test = 5.61, p = .018). Patients who had 4–9 versus 10 or more positive lymph nodes, were estrogen receptor positive, or were HER-2/neu negative had significantly worse disease specific survival if they were VEGF positive. ER+/VEGF+ patients had significantly worse survival that ER+/VEGF- patients. Conclusions: In this study high levels of expression of VEGF were associated with significantly worse survival in breast cancer patients, even in subgroups expected to have a better prognosis. No significant financial relationships to disclose.

Vascular endothelial growth factor, p53, Rb, Bcl-2 expression and response to chemotherapy in advanced non-small cell lung cancer

Vascular endothelial growth factor (VEGF) increases microvascular permeability and stimulates endothelial cell growth. p53 Overexpression has been associated with resistance to cisplatin-based chemotherapy in patients (pts) with NSCLC. The aim of this study was to evaluate the predictive role of VEGF for chemotherapy response, its relationship with p53, Rb, Bcl-2 and hemoglobin levels and its impact on overall survival in pts with advanced NSCLC. Bronchial or fine-needle biopsy specimens from 85 pts with NSCLC obtained before chemotherapy were analyzed using an immunohistochemical method for VEGF, p53, Rb and Bcl-2. There were 73 males and 12 females with a median age of 62.6 years. The majority of pts (48%) had squamous cell histology. Ten pts had stage IIIA, 25 stage IIIB and 50 stage IV. Thirty six (43%) pts had positive immunostaining for VEGF, 37 (44%) had positive p53, 53 (62%) had negative Rb and 4 (5%) had positive Bcl-2. VEGF was negatively correlated with Rb ( r s=0.26; P=0.015), positively with Bcl-2 ( r s=0.22; P=0.042), whereas no statistically significant correlation with p53, age, stage and histological type was found. In a logistic regression model, adjusting for treatment, VEGF expression was not associated with chemotherapy response (odds ratio (OR)=1.01; P=0.085), unlike p53 positivity and Rb negativity (OR=4.0, P=0.005; OR=2.6, P=0.016, respectively). A statistically significant higher VEGF expression was detected in the subgroups defined, using as cut-off value Hb median level (13.3 g/dl) ( χ 2=5.00; one d.f.; P=0.025). At a median follow-up time of 8.4 years, 2-year survival was 21%. After adjustment for stage and chemotherapy treatment, VEGF expression was not associated with a better overall survival (OR=1.06; P=0.80), unlike Bcl-2 positivity showed a statistically significant effect (OR=0.28; P=0.02). Our results suggest that VEGF is weakly correlated with regulators of apoptosis and has not been shown to be an independent predictive factor for resistance to cisplatin-based chemotherapy and prognostic for survival.

Immunohistochemical expression of vascular endothelial growth factor (VEGF) and C-KIT in cutaneous melanocytic lesions.

Vascular endothelial growth factor (VEGF) and C-KIT are involved in tumor progression in several human neoplasms. The aim of the present study has been to investigate their immunohistochemical expression in melanocytic lesions. We examined 11 compound nevi, 12 dysplastic nevi, and 18 melanomas. Immunostaining for VEGF was observed only in melanomas; c-kit expression was detected in melanomas (higher in radial than in vertical growth phase) and in nevi (predominantly in the junctional component). Our data indicate that assessment of VEGF expression might aid in the differential diagnosis between dysplastic nevi and melanomas. Moreover, VEGF might be a candidate for targeted therapy. The loss of c-kit expression might contribute to melanoma progression.

248EXPRESSION AND LOCALIZATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 2 IN EQUINE FOLLICLES

Ovarian follicles undergo pronounced morphological changes, alternating between periods of growth and regression. The equine follicle will grow to an average of 45mm in diameter at ovulation, and during the phase of growth, there is an increase in blood supply to the follicle. Vascular endothelial growth factor (VEGF) is a cytokine that interacts with tyrosine kinase receptors to stimulate angiogenesis, endothelial cell proliferation and vascular permeability. The aim of the study was to evaluate the expression and localization of VEGF and the VEGF-receptor 2 (VEGF-R2) in equine follicles. Ovaries were collected from a slaughterhouse. Granulosa cells from follicles were pooled regardless of the size of the follicles. Western blots were performed using protein extracted from granulosa cells and follicular fluid. Blots were probed with rabbit anti-human VEGF and rabbit anti-mouse VEGF-R2 antibodies and visualized with chemiluminescence. Total RNA was extracted from granulosa cells and integrity of the RNA samples was tested by the amplification of β-actin. Complementary DNA was synthesized by reverse transcription, followed by polymerase chain reaction amplification of cDNA encoding with bovine primer sequences for VEGF and VEGF-R2. The PCR product was resolved on 1% agarose gel and the resulting VEGF and VEGF-R2 bands were sequenced. Immunostaining for VEGF and VEGF-R2 was performed on fixed, paraffin-embedded sections of follicle wall from follicles larger than 30mm. Western blot analysis of granulosa cell lysates revealed 22kDa bands for VEGF, and 210kDa bands for VEGF-R2. VEGF protein was present in follicular fluid, whereas VEGF-R2 was not detectable. RT-PCR experiments revealed the presence of VEGF and VEGF-R2 mRNA in isolated granulosa cells. Sequencing demonstrated 93% and 99% homology to known sequences of equine VEGF and VEGF-R2, respectively. Immunofluorescence experiments performed on dissected equine follicles localized VEGF to the granulosa cell layer and sporadically to the theca cell layer. VEGF-R2 co-localized with VEGF in the granulosa cells, and was relatively absent in the theca layer. The present study detected novel expression patterns for VEGF and VEGF-R2 in equine ovarian follicles. The results of these experiments suggest an extra-vascular role for the VEGF family in follicle development. Future studies will be directed at studying the genomic and proteonomic profiles of follicles during the selection of the dominant follicle in mares.

Inhibition of Lung Carcinogenesis and Effects on Angiogenesisand Apoptosis in A/J Mice by Oral Administration of Green Tea

Oral administration of tea (Camellia sinensis) has been shown to inhibit the formation and growth of several tumor types in animal models. The present study investigated the effects of treatment with different concentrations of green tea on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in female A/J mice. Two days after a single dose of NNK (100 mg/kg body weight, i.p.), the mice were given 0.1, 0.2, 0.4, and 0.6% green tea solution (1, 2, 4, and 6 g of tea solids, respectively, dissolved in 1 l of water), 0.02% caffeine, or water as the sole source of drinking fluid until the termination of the experiment. Only the treatment with 0.6% tea preparation significantly reduced lung tumor multiplicity (mean ± SE, 6.07 ± 0.77 vs. 8.60 ± 0.50 tumors per mouse, P = 0.018). Treatment with 0.6% tea also inhibited angiogenesis, as indicated by the lower microvessel density (number of blood vessels/mm2) based on immunostaining for the von Willebrand factor antigen (81.9 ± 9.5 vs. 129.4 ± 8.2, P = 0.0018) and anti-CD31 antibody staining (465.3 ± 61.4 vs. 657.1 ± 43.6, P = 0.0012). Significantly lower vascular endothelial growth factor immunostaining scores were also observed in the 0.6% tea-treated group (0.98 ± 0.17 vs. 1.43 ± 0.07, P = 0.006). The apoptosis index was significantly higher in lung adenomas from 0.6% tea-treated mice based on morphological analysis of cell apoptosis (2.51 ± 0.18% vs. 1.57 ± 0.11%, P = 0.00005), and the result was further confirmed using the TUNEL method. Inhibition of angiogenesis and the induction of apoptosis by green tea may be closely related to the inhibition of pulmonary carcinogenesis.

Profiling of genes expressed by PTEN haploinsufficient neural precursor cells

PTEN is a lipid phosphatase, and PTEN mutations are associated with gliomas, macrocephaly, and mental deficiencies. We have used PTEN +/− and PTEN +/+ mice to prepare subventricular zone (SVZ) precursor cells. Using DNA microarrays, we compared the expression profiles of PTEN +/+ and PTEN +/− cells and identified 91 differentially expressed genes in PTEN +/− precursor cells. Many of the PTEN-regulated genes are involved with signaling, cytoskeleton, extracellular matrix, metabolism, and transcription factors. Some of these changes are likely mediated by the transcription factor, HIF-1. We confirmed a subset of these changes by real-time PCR. In addition, we examined protein levels for two of the PTEN-up-regulated genes, vascular endothelial growth factor (VEGF) and doublecortin (DCX). PTEN haploinsufficiency increases immunostaining for VEGF for both cultured precursor cells and sections of the SVZ. PTEN haploinsufficiency shifted most of the DCX-positive cells from the SVZ to the olfactory bulb. These observations indicate that even a small decrease in PTEN levels results in substantial changes in gene expression and precursor cell function.

Profiling of genes expressed by PTEN haploinsufficient neural precursor cells

PTEN is a lipid phosphatase , and PTEN mutations are associated with gliomas , macrocephaly , and mental deficiencies. We have used PTEN +/− and PTEN +/+ mice to prepare subventricular zone (SVZ) precursor cells. Using DNA microarrays , we compared the expression profiles of PTEN +/+ and PTEN +/− cells and identified 91 differentially expressed genes in PTEN +/− precursor cells. Many of the PTEN-regulated genes are involved with signaling, cytoskeleton , extracellular matrix , metabolism, and transcription factors. Some of these changes are likely mediated by the transcription factor, HIF-1. We confirmed a subset of these changes by real-time PCR. In addition, we examined protein levels for two of the PTEN-up-regulated genes, vascular endothelial growth factor (VEGF) and doublecortin (DCX). PTEN haploinsufficiency increases immunostaining for VEGF for both cultured precursor cells and sections of the SVZ. PTEN haploinsufficiency shifted most of the DCX-positive cells from the SVZ to the olfactory bulb . These observations indicate that even a small decrease in PTEN levels results in substantial changes in gene expression and precursor cell function.

Expression of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in sickle cell retina and choroid

Pigment epithelium-derived factor (PEDF) has been shown to be an inhibitor of angiogenesis as well as a multipotent neurotrophic factor in the mammalian eye. Changes in PEDF levels have been correlated with development of retinal neovascularization in oxygen-induced retinopathy. The purpose of this study was to determine the localization and relative level of PEDF in human retinas and choroids using immunohistochemistry and evaluate the changes in PEDF and vascular endothelial growth factor (VEGF) localization and their relation to the progression of proliferative sickle cell retinopathy. Cryopreserved tissues from eyes of normal subjects and subjects with non-proliferative or proliferative sickle cell retinopathy were used with streptavidin peroxidase immunohistochemistry. A rabbit polyclonal antibody was made against recombinant human PEDF. Binding of the antibody was blocked by preincubation of the antibody with excess human recombinant PEDF. Relative levels of immunoreactivity were scored with a seven-point grading system and by microdensitometric analysis. The most prominent sites of PEDF localization in the normal eye were the vitreous condensed at the internal limiting membrane and RPE–Bruch's membrane–choriocapillaris complex. PEDF was also prominent in choroidal stroma. There was limited immunoreactivity in some cells of the neural retinas, in blood vessels and in the interphotoreceptor matrix (IPM). There was no difference in ratio (1·47 vs. 1·44) of PEDF/VEGF or the relative levels of either growth factor in the retinal vasculatures of the control subjects and perfused area of non-proliferative sickle cell retinas. The ratio was increased in the non-perfused area of the non-proliferative sickle cell retinas (2·24). In eyes with proliferative sickle cell retinopathy, elevated PEDF and VEGF immunostaining was present in viable vessels of sea fan neovascular formations as well as feeder vessels of sea fans. The PEDF/VEGF ratio in sea fans was 1·0. Immunoreactivity for PEDF was prominent in retinal vessels in non-perfused regions and in atrophic sea fans, while VEGF immunoreactivity was weak or absent in these structures. In conclusion, PEDF and VEGF were both significantly elevated in viable sea fan formations in sickle cell disease ( p<0·05) but only PEDF was present in non-viable sea fans. The highest levels of PEDF in all eyes were associated with extracellular matrices (vitreous, choroidal stroma, IPM, and walls of blood vessels). PEDF might play an important role in inhibiting angiogenesis and inducing the regression of sea fans. Progression of angiogenesis may be dependent on the ratio of PEDF/VEGF.