Evaluation of angiogenic apelin/apelin receptor axis in normal prostate, high grade prostatic intraepithelial neoplasia and prostatic adenocarcinoma.

INTRODUCTION AND OBJECTIVES: Prostate cancer is one of the most commonly diagnosed cancers in American men. Apelin is an endogenous peptide identified as the ligand of the G protein-associated apelin receptor. Apelin and apelin receptor have many tissues distribution and they participate in pathological processes, such as cancer. Apelin stimulates cancer angiogenesis. However, there are insufficient data in the literature regarding the role of apelin/apelin receptor in normal tissue, highgrade prostatic intraepithelial neoplasia, and prostatic adenocarcinoma tissues. Therefore, this study aimed to investigate the apelin and apelin receptor expression levels in tissues of normal prostate tissue, high-grade prostatic intraepithelial neoplasia, and prostatic adenocarcinoma. MATERIALS AND METHODS: In this study, 38 samples of patients undergoing radical prostatectomy were used. Among 38 samples; 20 patients were with prostatic adenocarcinoma, 18 patients were with high-grade prostatic intraepithelial neoplasia and adjacent normal prostatic tissue areas. The immunolocalisation of apelin and apelin receptor in these tissues were determined immunohistochemically. RESULTS: Apelin and apelin receptor expressions were higher in prostatic adenocarcinoma than normal prostate tissue and high-grade prostatic intraepithelial neoplasia. Apelin receptor expression was also increased in high-grade prostatic intraepithelial neoplasia compared to normal tissue. CONCLUSION: Apelin and apelin receptor are increase in the process of prostate carcinogenesis. This increase may adversely affect the clinical course of prostate cancer patients by stimulating angiogenesis, which is important for invasion and metastasis in prostate cancer.

Correlations between intratumoral vascular network and tumoral architecture in prostatic adenocarcinoma.

The authors made a preliminary assessment of possible correlations between the intratumoral vascular density (IVD) and the architectural tumoral patterns described by Gleason. The studied material consisted of samples obtained by transurethral resection from 34 patients diagnosed with prostatic adenocarcinoma. Ten fields, five for dominant and five for secondary identified patterns of each case, with no necrosis were selected randomly from CD34 immunomarked sections using ×20 objective. IVD increased with Gleason pattern both for the entire group, but also for "solid" phenotype group of subtypes up to pattern 4, respectively subtype 4B. In "necrotizing" phenotype group of subtypes, IVD had a decreasing trend from the better-differentiated subtypes to the poorest one. These preliminary data showed that the intratumoral vascular network reacts differently to the loss of tumoral differentiation in the two groups of Gleason subtypes suggesting the existence of two different populations of malignant cells.

Sorafenib's inhibition of prostate cancer growth in transgenic adenocarcinoma mouse prostate mice and its differential effects on endothelial and pericyte growth during tumor angiogenesis.

OBJECTIVE: To evaluate Sorafenib's efficacy (60 mg/kg/d per os) in preventing the transformation of high grade prostate intraepithelial neoplasia (HGPIN) into adenocarcinoma (ADC) and in inhibiting the onset and progression of poorly differentiated carcinoma (PDC) in transgenic adenocarcinoma mouse prostate (TRAMP) mice. STUDY DESIGN: Forty-four TRAMP mice were randomly divided into 2 groups of 22 and assigned to daily treatment by gavage with vehicle only or Sorafenib from the 10th to the 26th week of age. At 26 weeks of age the mice were killed, and their genitourinary apparatus was removed and examined by histology, immunohistochemistry and confocal microscopy. RESULTS: Sorafenib reduced HGPIN growth and progression to ADC and was probably also effective in PDC inhibition. The major effect of Sorafenib was on tumor angiogenesis. Interestingly a dissociation between endothelial cells and pericytes was noted in treated PDC since inhibition of pericyte recruitment was less complete than that of endothelial cells. CONCLUSION: Sorafenib's potent antiangiogenic action may be supposed to be exerted primarily by inhibiting endothelial proliferation and sprouting, whereas its inhibition of pericyte recruitment and maturation is less complete. These observations suggest that Sorafenib's effects could be improved by the joint employment of substances capable of interfering with the recruitment and organization of pericytes.

Tissue factor and VEGF expression in prostate carcinoma: a tissue microarray study.

Tissue factor (TF) is the principal physiologic initiator of coagulation. It also plays an important role in tumor growth and metastasis possibly by contributing to angiogenesis. We evaluated the expression of TF in benign and malignant prostate tissue and correlated it with the expression of the pro-angiogenic protein, vascular endothelial growth factor (VEGF). We used a tissue microarray (TMA) constructed from 80 archival prostatectomy specimens. Core samples were collected from benign prostate tissue (BP) (n= 77), high-grade prostatic intraepithelial neoplasia (PIN) (n= 26), and carcinoma (PCa) (n= 93). TMA sections were stained with an immunopurified polyclonal TF antibody and a rabbit polyclonal VEGF. Two pathologists manually scored staining in epithelial cells using the German Immunoreactive Score. Positive staining for TF was seen predominantly in PCa with rare positive glands in BP and PIN. TF expression was significantly lower in BP versus PCa specimens (p< .001) and in PIN versus PCa specimens (p< .001). Positive staining for VEGF was seen in PCa, BP, and PIN. Rates of VEGF expression were also significantly lower in BP versus PCa specimens (p= .003) but not in PCa versus PIN (p= .430). The majority of PCa samples positive for TF were also positive for VEGF (p< .001). Our findings reinforce the link between angiogenesis and TF expression in PCa. We suggest further exploration of TF-mediated pathways leading to increased tumor aggressiveness in PCa, and the possible use of anti-TF agents in PCa.

Stage-specific inhibitory effects and associated mechanisms of silibinin on tumor progression and metastasis in transgenic adenocarcinoma of the mouse prostate model.

Herein, using transgenic adenocarcinoma of the mouse prostate (TRAMP) model, we assessed the "stage-specific" efficacy of silibinin feeding against prostate cancer (PCa) initiation, progression, angiogenesis and metastasis, and associated molecular events involved in silibinin effects during these stages. Male TRAMP mice starting at ages 4, 12, 20, and 30 weeks of age were fed with control or 1% silibinin-supplemented diet for 8 to 15 weeks in stage-specific manners. At the end of studies, silibinin-fed mice showed less severe prostatic lesions compared with positive controls. During early stages of prostate tumor development, silibinin mediated its efficacy mostly via antiproliferative mechanisms. Feeding of silibinin to animals burdened with higher stages of prostate tumor significantly decreased tumor grade via antiproliferative effect, and inhibition of angiogenesis as evidenced by decreased expressions of platelet endothelial cell adhesion molecule-1/CD-31, vascular endothelial growth factor, and associated receptor, vascular endothelial growth factor R2, hypoxia-inducible factor-1alpha, and inducible nitric oxide synthase. Metastasis to distant organs was decreased in silibinin-fed mice, which was associated with a decreased expression of matrix metalloproteinases, mesenchymal markers snail-1, and fibronectin in the prostatic tissue and retention of epithelial characteristics. Together, these findings are both novel and highly significant in establishing the dual efficacy of silibinin where it inhibits progression of primary prostatic tumor and also shows protective efficacy against angiogenesis and late stage metastasis. These effects of silibinin could have potential implications to improve the morbidity and survival in PCa patients.

[Expression of hypoxia-inducible factor 1 alpha and vascular endothelial growth factor in prostate cancer and its significance].

OBJECTIVE: To study the expression of hypoxia-inducible factor 1 alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF) in prostate cancer (PCa) and its biological significance. METHODS: Specimens from 75 patients with PCa (32 cases), benign prostatic hyperplasia (BPH, 16 cases), high-grade prostate intraepithelial neoplasia (PIN, 15 cases) and normal prostate (NP, 12 cases) were comprised in the study. The PCa was divided into high Gleason score group (Gleason score > or = 7) and low Gleason score group (Gleason score < 7). Immunohistochemistry was performed with antibodies against HIF-1 alpha, VEGF and CD34. Microvessel density (MVD) was analyzed by staining with antibodies to CD34. RESULTS: The positive rates of HIF-1alpha were significantly higher in PCa (62.5%) and PIN (60.0%) than those in BPH (6.3%) and NP(0) (P < 0.05), respectively. The positive rates of VEGF were significantly higher in PCa (78.1%) and PIN (73.3%) than those in BPH (18.7%) and NP (8.3%) (P < 0.05), respectively. A higher concentration of MVD was observed in PCa (66.9 +/- 18.0) compared with those in BPH (28.3 +/- 6.9) and NP (15.3 +/- 2.9) (P < 0.05). MVD and the positive rates of HIF-1alpha and VEGF were significantly higher in high Gleason score group than those in low Gleason score group. CONCLUSION: The results suggest that the overexpression of HIF-1alpha and VEGF are closely related to PCa, and the son score group. up-regulation of HIF-1alpha and VEGF are early events in PCa.

Angiogenesis with pericyte abnormalities in a transgenic model of prostate carcinoma.

BACKGROUND: Previous studies of the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model vasculature suggest that, as tumors develop, vessels invade the glandular epithelium. However, changes in the vasculature are difficult to study in conventional thin tissue sections. The authors used a new approach to characterize morphologic and architectural changes of blood vessels and pericytes during tumor development in TRAMP mice. METHODS: Eighty-micron cryostat sections of normal prostate and three histopathologic stages of TRAMP tumor sections, classified by epithelial cell E-cadherin immunoreactivity, were immunostained with vascular endothelial cell and pericyte receptor antibodies and evaluated by confocal microscopy. RESULTS: In the normal mouse prostate, capillaries were most abundant in the fibromuscular tunica between the epithelium and smooth muscle of the ductules. In the prostatic intraepithelial neoplasia (PIN) stage, vessels accompanied epithelial cell protrusions into the ductule lumen but remained in the connective tissue at the basal side of the epithelium. Well differentiated tissues had extensive angiogenesis with five times the normal mean vascularity outside ductules. Vessels were of variable diameter, were associated with an increased number of pericytes, and some had endothelial sprouts. Angiogenic blood vessels from poorly differentiated adenocarcinomas were tortuous, variable in caliber, and lacked the normal hierarchy. Pericytes on these vessels had an abnormal phenotype manifested by alpha-smooth muscle actin expression and loose association with endothelial cells. Angiogenesis and loss of vascular hierarchy were also found in human prostate carcinoma. CONCLUSIONS: Vascular abnormalities, which begin at the PIN stage and intensify in well differentiated and poorly differentiated tumors, may be useful readouts for early detection and treatment assessment in prostate carcinoma.

Microvessel density as a molecular marker for identifying high-grade prostatic intraepithelial neoplasia precursors to prostate cancer.

BACKGROUND: Existing clinical data have shown that high-grade prostatic intraepithelial neoplasia (HGPIN) is the most likely precursor to prostate cancer (CaP). Criteria to distinguish HGPIN that progress to CaP from those that do not remain poorly defined. Our objective was to evaluate microvessel density as a molecular marker for distinguishing HGPINs that have the potential of progressing to cancer. MATERIALS AND METHODS: Human prostatic tissue samples were collected randomly from 50 prostatectomy and cystoprostatectomy patients. Formalin-fixed and paraffin-embedded sections were used for immunohistochemical localization of rabbit anti-human von Willebrand factor VIII (vWF) IgG, mouse anti-high molecular weight cytokeratin 34BE-12 in basal cells, and mouse anti-heparan sulphate proteoglycan (HSPG) IgGs in basement membranes associated with benign prostatic hyperplasia (BPH), PIN associated with some BPH (isolated PIN), and PIN associated with CaP. RESULTS: Analysis of immunostaining data showed that PINs could be categorized according to their distributions within and outside 2 standard deviations (SD) of the mean for microvessel density. The average number of microvessels was significantly higher (P < 0.0001) in PINs associated with Gleason score 7 tumors than those associated with Gleason scores 4-6 (P < 0.1328) or 8 and 9 tumors (P < 0.1708). Morphologically, PINs within 2 SD were composed of low- and high-grade type, whereas those outside 2 SD of microvessel density were predominantly of high-grade type. Cytokeratin and HSPG localization patterns also showed differences in PINs found within and outside 2 SD of microvessel density. We found localization of cytokeratin 34BE-12 in basal cells of specimens with BPH alone, isolated PIN, and PIN associated with CaP within 2 SD, whereas many PINs outside 2 SD showed disruptions in cytokeratin localization. The basement membranes of PINs within 2 SD of microvessel density were relatively intact, whereas those outside 2 SD were fragmented. CONCLUSIONS: Our immunostaining data indicates that once HGPIN is found in the initial prostatic biopsy, it should be evaluated for microvessel density by localization of vWF. Our data indicate that characteristics of HGPIN can be augmented by evaluations of cytokeratin and HSPG molecular markers to assess the potential of HGPIN progression to malignancy. When biopsy samples show HGPIN with increased microvessel density and disrupted cytokeratin and HSPG markers, the patient may be a candidate for repeat biopsy. Since our study is limited to 50 prostate tissue samples, we emphasize that our conclusion is tentative and ought to be confirmed in a study with a larger sample size. This is the first report to show that microvessel density may distinguish HGPIN that is a precursor to prostate cancer.

Inducible prostate intraepithelial neoplasia with reversible hyperplasia in conditional FGFR1-expressing mice.

Accurate determination of the contributions of oncogenes toward tumor progression requires their regulation. Herein, we created transgenic mice with prostate-specific expression of ligand-inducible FGFR1 or FGFR2, based on lipid-permeable dimerizing molecules, called chemical inducers of dimerization. Despite extensive homology and equivalent expression by both chimeric receptors in the ventral prostate gland, only FGFR1 triggers detectable nuclear translocation of Erk and progression to prostatic intraepithelial neoplasia (PIN). Induction of PIN grade I-II, indicated by multiple layers of atypical cells, is seen consistently by 12 weeks of chemical inducers of dimerization treatment. By 6 months, more extensive nuclear atypia, thickened "reactive" stroma, and basement membrane herniation occurs, corresponding to PIN IV. By timed removal of FGFR1 signaling, we show that induced hyperplasia is reversible until extensive intraductal vascularization occurs, but continued progression requires prolonged FGFR1 signaling. Additionally, by highlighting differences between the two receptors and creating the foundation for controlling FGFR1 signaling during prostate cancer progression, a model of early stage prostate cancer is established for developing targeted intervention directed toward the FGFR signaling axis.

A potential autocrine role for vascular endothelial growth factor in prostate cancer.

Vascular endothelial growth factor (VEGF) is a peptide growth factor specific for the tyrosine kinase receptors VEGF receptor-1 and -2 (VEGFR-1 and R-2). Whereas VEGF has well-defined actions on the vasculature, including the stimulation of endothelial cell growth and motility and blood vessel permeability, the function of the VEGF/receptor pathway in other cell types is largely unknown. Recently, VEGFR-1 and R-2 expression has been reported in prostate tumor cells. In this study, we demonstrate that these receptors colocalize with VEGF in prostate tumor cells, prostatic intraepithelial neoplasia, and the basal cells of normal glands. Furthermore, in comparison with normal glands, the expression of VEGFR-1 and R-2 is increased in prostatic intraepithelial neoplasia and malignant cells in well and moderately differentiated prostate cancer but is decreased in poorly differentiated cancer. Culture of the prostate cancer cell line LNCaP in the presence of recombinant human VEGF165 resulted in a 50% increase in [(3)H]thymidine uptake by these cells and recruitment of quiescent cells into the cell cycle. This effect of recombinant human VEGF165 was abolished by neutralizing antisera to VEGFR-2. These data suggest that VEGF may not only mediate neovascularization associated with prostate cancer progression but may also directly stimulate prostate tumor cells via VEGFR-2-dependent autocrine and/or paracrine mechanisms.

Angiogenesis as a potential biomarker in prostate cancer chemoprevention trials.

Prostate cancer is a multistep process in which progression rather than initiation may be the rate-limiting step. A strong possibility is that prostatic intraepithelial neoplasia lesions that switch to angiogenic phenotype eventually progress to cancer. However, it is a challenging task to quantitate angiogenesis in preneoplastic lesions. A promising approach to measuring angiogenesis involves real-time TaqMan polymerase chain reaction to quantitate mRNAs encoding a panel of angiogenesis markers. This highly sensitive molecular technique has potential for quantitating angiogenesis in clinical settings and can be used as a high-throughput screening procedure in prostate cancer clinical trials.

Angiogenesis and metastasis marker of human tumors.

Tumor growth and metastasis are dependent on angiogenesis. Therefore, certain angiogenesis markers may be useful as metastasis markers and/or the targets for antiangiogenic therapy. We and others have been studying endoglin(EDG; CD105) for such purposes. EDG is a proliferation-associated antigen of endothelial cells and essential for angiogenesis. In addition, EDG is a component of the transforming growth factor(TGF)-beta receptor complex. Expression of EDG is up-regulated in tumor-associated angiogenic vasculature compared with normal tissue vasculature. Microvessel density detected for EDG expression in breast cancer tissues showed a statistically significant correlation with overall and disease-free survival. In addition, elevated serum EDG was associated with metastasis in patients with colorectal, breast, and other solid tumors. On the other hand, We have been targeting EDG on tumor vasculature to suppress tumor growth and metastasis by systemic(i.v.) administration of anti-EDG monoclonal antibodies(mAbs) and immunoconjugates(IMCs). To thid end, we have been using three animal models, i.e., severe combined immunodeficient(SCID) mouse model of MCF-7 human breast cancer, human skin/SCID mouse chimera model bearing MCF-7 tumor, and syngeneic metastasis model of colon-26 adenocarcinoma cells in BALB/c mice. In addition, antiangiogenic activities of anti-EDG mAbs and IMCs were evaluated in mice using the dorsal air sac assay. The IMCs were prepared by coupling deglycosylated ricin A-chain or 125I to individual anti-EDG mAbs. These anti-EDG IMCs and mAbs showed substantial antitumor efficacy and antimetastatic activities without showing severe toxicity. Recently, we generated a recombinant human/mouse chimeric anti-EDG mAb to facilitate clinical application of the mAb.

Vascular endothelial growth factor expression and capillary architecture in high-grade PIN and prostate cancer in untreated and androgen-ablated patients.

BACKGROUND: Recent studies have demonstrated that angiogenesis is a potent prognostic indicator for patients with prostate cancer (PCa) and have pointed out that the evaluation of vascular endothelial growth factor (VEGF) is useful in assessing the angiogenic phenotype in PCa. The aim of the study was to investigate immunohistochemically the expression of VEGF and its correlation with the pattern of capillary architecture in prostate cancer and high-grade prostatic intraepithelial neoplasia (PIN), in untreated and androgen-ablated patients. METHODS: Forty-five patients who underwent radical prostatectomy (RP) for localized prostate carcinoma were recruited for this study. The study population included two groups: 35 patients who did not receive chemo-, hormone, or radiation therapy before surgery, and 10 patients who were under complete androgen blockade (CAB) for 3 months at time of surgery. VEGF was examined by immunohistochemistry, and its tissue expression was compared with the pattern of capillary architecture evaluated by immunostaining the endothelial antigen CD34. The relationship of VEGF expression to chromogranin A-positive (e.g., neuroendocrine) cells was investigated. RESULTS: In normal tissue, the intensity of the VEGF immunoreactivity in the cytoplasm of secretory cells ranged from negative to low. Very few basal cells stained for VEGF. All prostate cancer specimens stained positively, the intensity of the immunoreaction ranging from low to strong and being correlated with the Gleason score. Strongly positive VEGF immunoreactivity was detected in vascular endothelial cells and in stromal cells surrounding blood vessels. Two discrete immunostaining patterns were observed in high-grade PIN. VEGF expression of low-to-moderate intensity was defined as pattern A. The other, characterized by a strong cytoplasmic immunoreaction similar to that of poorly differentiated tumors, was defined as pattern B. The capillary architecture in high-grade PIN with pattern A was similar to the orderly vascular network seen in normal prostates, whereas in the pattern B it had the characteristics of microvessels usually seen in PCa. The degree of vascularization in the stroma adjacent to intensely VEGF-stained cells (neuroendocrine phenotype) was higher than that noted in association with secretory cells. CAB before surgery downregulated the expression of VEGF and decreased the degree of vascularization, except in the cell areas with neuroendocrine (NE) features. CONCLUSIONS: Our immunohistochemical results indicate that significant levels of VEGF are present in prostate cancer and in a population of PIN lesions, expression being highest in association with NE cells. VEGF expression is downregulated by hormonal manipulation, except in the population of NE cells.

CD34 immunohistochemical assessment of angiogenesis as a prognostic marker for prostate cancer recurrence after radical prostatectomy.

PURPOSE: We assess the neovascularity of clinically localized prostate cancer by immunohistochemistry using the monoclonal antibody CD34 in an attempt to identify associations between angiogenesis and disease progression following radical prostatectomy. MATERIALS AND METHODS: Microvascularity was evaluated using the CD34 monoclonal antibody in archival paraffin embedded radical prostatectomy specimens from 149 patients followed from 3 to 10 years (mean 6.6). Vessels were quantified by counting a minimum of 2 selected microscopic fields (200x, 0.754 mm.2) from each tumor, area of prostatic intraepithelial neoplasia and prostatic hyperplasia, and given a numerical value representing the microvessel density count. RESULTS: Mean microvessel density count did not vary significantly with age or race. There was a significant association between the count and nuclear grade, Gleason sum and pathological stage. Cox survival analysis shows that microvessel density is significantly related to time to recurrence when considered as a continuous variable (p=0.03) as well as dichotomous variable (p=0.007) (microvessel density count less than 90 and 90 or greater). The 5-year recurrence-free survival was significantly higher for patients with a count less than 90 (71%) than for those with a count 90 or greater (51%) (p=0.006). The 5-year recurrence-free survival was also significantly different when microvessel density was used as a continuous variable (p=0.02). Controlling for stage, Gleason sum, race and nuclear grade, microvessel density remained significant in predicting recurrence (p=0.03) but when pretreatment prostate specific antigen was included in the model the count was no longer significant. The microvessel density count in the tumor area significantly increased with increasing Gleason sum and nuclear grade but it did not increase significantly in the adjacent benign prostate or areas of prostatic intraepithelial neoplasia in the same specimen. CONCLUSIONS: Microvascularity or neovascularity as measured by the CD34 antigen may be a prognostic marker of recurrence for prostate cancer patients after radical prostatectomy but more study in prostate specific antigen era patients with sufficient followup is needed.

Analysis of the capillary architecture in the precursors of prostate cancer: recent findings and new concepts.

OBJECTIVE: To report on recent findings and new concepts in the remodeling of the capillary architecture in the precursors of prostate cancer. METHODS: Immunohistochemical methods have been adopted in prostate cancer and in its precursors (prostatic intra-epithelial neoplasia) to investigate capillary pattern changes-which were mainly analyzed as capillary frequency- and the degree of endothelial cell proliferation. Several features related to the capillary architecture have been considered. Manual, semiautomatic, and automatic (machine vision) types of evaluation have been used to quantify the features. RESULTS: The data available indicate that: (1) Going from normal prostate through prostatic intra-epithelial neoplasia up to invasive adenocarcinoma, an increasing proportion of capillaries becomes shorter, with open lumen and undulated external contour and with greater proliferation of the endothelial cells and greater expression of type IV collagenase. The highest proportion of touching capillaries is seen in normal prostate, while the lowest is found in invasive adenocarcinoma, being intermediate in prostatic intra-epithelial neoplasia. (2) When total androgen ablation is induced, there is no proliferation of the endothelium, whereas the capillaries are reduced in frequency and represented by small vessels lined by flat endothelial cells and with an open lumen. (3) Automation in the evaluation of the capillary architecture is feasible with a machine vision system. CONCLUSIONS: The progression in prostate carcinogenesis is associated with changes in the capillary architecture. There are some preliminary data indicating that total androgen ablation can inhibit the angiogenesis in precursors of prostate cancer.