MRS measured fatty acid composition of periprostatic adipose tissue correlates with pathological measures of prostate cancer aggressiveness.

PURPOSE: To investigate the association between magnetic resonance (MR) spectroscopically measured fatty acid composition of periprostatic adipose tissue and pathological markers of prostate cancer aggressiveness. MATERIALS AND METHODS: Periprostatic adipose (PPA) and subcutaneous adipose (SQA) tissue from prostate cancer patients undergoing radical prostatectomy were examined ex vivo by proton MR spectroscopy at 14.1T (n = 31). Fractions of monounsaturated, polyunsaturated, total unsaturated, and saturated fatty acids, as well as T2 relaxation times were measured from the spectra. Univariate and multivariate analyses based on receiver operating characteristic (ROC) and support vector machines (SVM) were used to evaluate the association between differential measures of fatty acid levels in the PPA and SQA tissues and Gleason score and extracapsular extension (ECE), which are pathological measures of prostate cancer aggressiveness. RESULTS: Both pathological markers for aggressive prostate cancer have separable patterns in the MRS features space. The association between ECE and PPA tissue fatty acid composition is linear (area under receiver operating characteristic curve (AROC) and 95% confidence intervals [CIs]: 1.00, [1.00, 1.00]), along the Δ(fM /fS ) measure, and is marked by elevated monounsaturated and reduced saturated fatty acids in the PPA tissue relative to SQA. In contrast, the association between Gleason score and PPA tissue fatty acid composition is nonlinear (classifier AROC and 95% CIs: 0.86, [0.71, 1.00]). CONCLUSION: Fatty acid composition is altered in the PPA tissue of patients with aggressive prostate cancer. Ex vivo MR spectroscopy may be a useful tool in studying the altered fatty acid metabolism in prostate cancer.

Elevated neonatal insulin-like growth factor I is associated with fetal hypertrophic cardiomyopathy in diabetic women.

OBJECTIVE: We sought to determine if fetal hypertrophic cardiomyopathy (HCM) or cardiac dysfunction is associated with elevated maternal or neonatal insulin-like growth factor (IGF)-I levels in women with diabetes. STUDY DESIGN: In a prospective cohort study, fetal echocardiogram findings at 36 weeks' gestation in women with pregestational diabetes mellitus were compared to those in women without diabetes mellitus. HCM was defined as septal or free wall thickness ≥5 mm and cardiac dysfunction as a modified myocardial performance index ≥0.43. Cord serum IGF-I levels at delivery were measured with enzyme-linked immunosorbent assay. Neonates with abnormal fetal echocardiogram were followed up until resolution or 6 months of life. RESULTS: In all, 75 participants completed fetal echocardiography (55 diabetics and 20 controls). In the diabetic group, 33 of 55 (60%) had abnormal fetal echocardiograms with cardiac dysfunction in 21 of 55 (38.2%) and HCM in 8 of 55 (14.5%) and both in 4 of 55 (7.3%). At 6 months of age, 1 of 12 (8%) had persistent HCM. None in the comparison group had abnormal findings. There were no significant clinical differences in those diabetic women with normal vs abnormal fetal echocardiograms. However, among diabetic women, mean neonatal IGF-I was significantly higher in fetuses with HCM (80 ± 16 ng/mL) as compared to those without HCM (61 ± 18 ng/mL), (P < .001). CONCLUSION: Elevated neonatal IGF-I appears to be associated with fetal HCM in fetuses of diabetic women.

TGF-ß: duality of function between tumor prevention and carcinogenesis.

Several mechanisms underlying tumor progression have remained elusive, particularly in relation to transforming growth factor beta (TGF-ß). Although TGF-ß initially inhibits epithelial growth, it appears to promote the progression of advanced tumors. Defects in normal TGF-ß pathways partially explain this paradox, which can lead to a cascade of downstream events that drive multiple oncogenic pathways, manifesting as several key features of tumorigenesis (uncontrolled proliferation, loss of apoptosis, epithelial-to-mesenchymal transition, sustained angiogenesis, evasion of immune surveillance, and metastasis). Understanding the mechanisms of TGF-ß dysregulation will likely reveal novel points of convergence between TGF-ß and other pathways that can be specifically targeted for therapy.

Periprostatic adipose tissue from obese prostate cancer patients promotes tumor and endothelial cell proliferation: a functional and MR imaging pilot study.

BACKGROUND: Obesity, particularly visceral adiposity, confers a worse prognosis for prostate cancer (PCa) patients, and increasing periprostatic adipose (PPA) tissue thickness or density is positively associated with more aggressive disease. However, the cellular mechanism of this activity remains unclear. Therefore, in this pilot study, we assessed the functional activity of PPA tissue secretions and established a biochemical profile of PPA as compared to subcutaneous adipose (SQA) tissues from lean, overweight and obese PCa patients. METHODS: Adipose tissues were collected from PCa patients undergoing surgical prostate removal. Tissues were analyzed by histologic and magnetic resonance (MR) techniques. Explant tissue culture secretions were used in proliferation assays on PCa and endothelial cells. RESULTS: PPA secretions obtained from obese patients were significantly more pro-proliferative in both PCa and endothelial cells as compared to PPA obtained from lean or overweight men and SQA tissues. Consistent with this, PPA microvessel density was increased, and the T2 relaxation time was decreased, compared to SQA tissues, and we observed a modest, inverse correlation between the T2 and tumor stage. Moreover, the ratio of unsaturated to saturated fatty acids, obtained using MR spectroscopy, showed a modest, inverse correlation with Gleason score. CONCLUSIONS: These pilot data show that PPA stimulates PCa cell proliferation and angiogenesis and that obesity intensifies this activity, thus generating a mechanistic hypothesis to explain the worse prognosis observed in obese PCa patients. Our pilot study also shows that MR technology may be useful in further elucidating the relationship between obesity and PCa progression.

Mutations in SERPINF1 cause osteogenesis imperfecta type VI.

Osteogenesis imperfecta (OI) is a spectrum of genetic disorders characterized by bone fragility. It is caused by dominant mutations affecting the synthesis and/or structure of type I procollagen or by recessively inherited mutations in genes responsible for the posttranslational processing/trafficking of type I procollagen. Recessive OI type VI is unique among OI types in that it is characterized by an increased amount of unmineralized osteoid, thereby suggesting a distinct disease mechanism. In a large consanguineous family with OI type VI, we performed homozygosity mapping and next-generation sequencing of the candidate gene region to isolate and identify the causative gene. We describe loss of function mutations in serpin peptidase inhibitor, clade F, member 1 (SERPINF1) in two affected members of this family and in an additional unrelated patient with OI type VI. SERPINF1 encodes pigment epithelium-derived factor. Hence, loss of pigment epithelium-derived factor function constitutes a novel mechanism for OI and shows its involvement in bone mineralization.

Overexpression of transforming growth factor ß1 in malignant prostate cells is partly caused by a runaway of TGF-ß1 auto-induction mediated through a defective recruitment of protein phosphatase 2A by TGF-ß type I receptor.

OBJECTIVES: To elucidate the mechanism of transforming growth factor (TGF)-ß1 overexpression in prostate cancer cells. METHODS: Malignant (PC3, DU145) and benign (RWPE1, BPH1) prostate epithelial cells were used. Phosphatase activity was measured using a commercial kit. Recruitment of the regulatory subunit, Bα, of protein phosphatase 2A (PP2A-Bα) by TGF-ß type I receptor (TßRI) was monitored by coimmunoprecipitation. Blockade of TGF-ß1 signaling in cells was accomplished either by using TGF-ß-neutralizing monoclonal antibody or by transduction of a dominant negative TGF-ß type II receptor retroviral vector. RESULTS: Basal levels of TGF-ß1 in malignant cells were significantly higher than those in benign cells. Blockade of TGF-ß signaling resulted in a significant decrease in TGF-ß1 expression in malignant cells, but not in benign cells. Upon TGF-ß1 treatment (10 ng/mL), TGF-ß1 expression was increased in malignant cells, but not in benign cells. This differential TGF-ß1 auto-induction between benign and malignant cells correlated with differential activation of extracellular signal-regulated kinase (ERK). Following TGF-ß1 treatment, the activity of serine/threonine phosphatase and recruitment of PP2A-Bα by TßRI increased in benign cells, but not in malignant cells. Inhibition of PP2A in benign cells resulted in an increase in ERK activation and in TGF-ß1 auto-induction after TGF-ß1 (10 ng/mL) treatment. CONCLUSIONS: These results suggest that TGF-ß1 overexpression in malignant cells is caused, at least in part, by a runaway of TGF-ß1 auto-induction through ERK activation because of a defective recruitment of PP2A-Bα by TßRI.

Thrombospondin-1 regulates the normal prostate in vivo through angiogenesis and TGF-beta activation.

Castration experiments in rodents show that the stromal vasculature is critical to the androgen-mediated prostate growth regulation. However, the role of angiogenesis inhibitors, such as thrombospondin-1 (TSP-1), in this process is unclear. TSP-1 is a multifunctional glycoprotein that can function as a potent angiogenesis inhibitor and an in vivo activator of latent transforming growth factor-beta (TGF-beta) in some tissues. On the basis of these observations, we hypothesized that TSP-1 regulated androgen withdrawal-induced prostate regression and that this process was mediated not only through antiangiogenic activity but also through TGF-beta activation. To test this, we evaluated angiogenic activity in human prostate epithelial and stromal cells treated with androgens and hypoxia in vitro. TSP-1 knockout mice were characterized to investigate the in vivo functions of TSP-1. In vitro, we found that androgens and hypoxia differentially regulated TSP-1 and angiogenic activity. Androgens stimulated normal epithelial cell, but inhibited normal stromal cell, angiogenic activity. Conversely, hypoxia stimulated stromal while inhibiting epithelial activity. Thus, in vivo, net angiogenic activity must reflect cellular interactions. And, we found that media conditioned by epithelial cells grown under normoxic conditions stimulated stromal cell angiogenic activity, and if epithelial cells were grown under hypoxic conditions, stromal activity was further increased. TSP-1 levels, however, were unchanged. In vivo, TSP-1 loss in a mouse model led to prostate epithelial hyperplasia by 3 months of age with only a modest stromal effect. Androgens suppressed TSP-1 as expression increased after castration both in normal mouse prostate and in human prostate cancer tissues. In addition, TSP-1 expression corresponded to increased TGF-beta activation in mouse tissues, specifically in the stromal compartment. These data show a critical role for TSP-1 in prostate epithelial and stromal growth regulation through angiogenic inhibition and activation of latent TGF-beta. Therefore, loss of TSP-1 during tumorigenesis would eliminate two barriers to cancer progression.

Pigment epithelium-derived factor stimulates tumor macrophage recruitment and is downregulated by the prostate tumor microenvironment.

Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis but whether it has additional effects on the tumor microenvironment is largely unexplored. We show that overexpression of PEDF in orthotopic MatLyLu rat prostate tumors increased tumor macrophage recruitment. The fraction of macrophages expressing inducible nitric oxide synthase, a marker of cytotoxic M1 macrophages, was increased, suggesting that PEDF could enhance antitumor immunity. In addition, PEDF overexpression reduced vascular growth both in the tumor and in the surrounding normal tissue, slowed tumor growth, and decreased lymph node metastasis. Contrary, extratumoral lymphangiogenesis was increased. PEDF expression is, for reasons unknown, often decreased or lost during prostate tumor progression. When AT-1 rat prostate tumor cells, expressing high levels of PEDF messenger RNA (mRNA) and protein, were injected into the prostate, PEDF is markedly downregulated, suggesting that factors in the microenvironment suppressed its expression. One such factor could be macrophage-derived tumor necrosis factor alpha (TNFalpha). A fraction of the accumulating macrophages expressed TNFalpha, and TNFalpha treatment downregulated the expression of PEDF protein and mRNA in prostate AT-1 tumor cells in vitro and in the rat ventral prostate in vivo. PEDF apparently has multiple effects in prostate tumors: it suppresses angiogenesis and metastasis, but it also causes macrophage accumulation. Accumulating macrophages may inhibit tumor growth, but they may also suppress PEDF and enhance lymph angiogenesis and, in this way, eventually enhance tumor growth.

Ethanol exposure depletes hepatic pigment epithelium-derived factor, a novel lipid regulator.

BACKGROUND & AIMS: Ethanol abuse can lead to hepatic steatosis and evolve into cirrhosis and hepatocellular carcinoma. Pigment epithelium-derived factor (PEDF) is a multifunctional secreted glycoprotein that is expressed by hepatocytes. Proteomic, experimental, and clinical studies implicate PEDF's role in lipid regulation. Because matrix metalloproteinase (MMP)-2/9 activity regulates PEDF levels, we investigated whether PEDF degradation by MMPs has a permissive role in ethanol-induced hepatic steatosis. METHODS: PEDF levels were examined in liver biopsy specimens from patients with ethanol-induced steatosis. Hepatic PEDF levels and MMP activity were assessed in 2 animal models of ethanol feeding (rats on an alcohol-containing liquid diet and mice given intragastric infusion of ethanol). The consequences of PEDF depletion in the liver were examined in PEDF-null mice. RESULTS: Liver biopsy samples from patients with ethanol-induced steatosis had reduced PEDF levels, compared with normal liver samples. Ethanol-fed animals had histologic steatosis and increased liver triglyceride content (P< .05), as well as reduced levels of hepatic PEDF and increased MMP-2/9 activity. Ethanol-exposed hepatic lysates degraded PEDF in a MMP-2/9-dependent manner, and liver sections demonstrated abundant MMP-2/9 activity in situ. Addition of recombinant PEDF to PEDF-null hepatocytes, reduced their triglyceride content. CONCLUSIONS: Ethanol exposure leads to marked loss of hepatic PEDF in human livers and in 2 animal models of ethanol feeding. Loss of PEDF contributes to the accumulation of lipids in ethanol-induced hepatic steatosis.

Decreased expression of pigment epithelium derived factor (PEDF), an inhibitor of angiogenesis, in placentas of unexplained stillbirths.

Normal placental vascular development depends upon the complex interactions between angiogenic inducers and inhibitors within the placenta. Alterations within the placental microenvironment can promote an imbalance in angiogenic mediators which may be associated with adverse perinatal outcomes. The purpose of this study was to investigate the placentas of infants with unexplained stillbirth as compared to live-born infants and to determine whether alterations in angiogenic inducer vascular endothelial growth factor (VEGF) or inhibitor pigment epithelium-derived factor (PEDF) are associated with altered angiogenesis, vascular remodeling and stillbirth. Placentas of 22 unexplained stillbirths and 44 age-matched live-born controls were scored for microvascular density (MVD), vasculopathy and microvascular permeability. A subset was scored for expression of angiogenic inducer VEGF and inhibitor pigment epithelium-derived factor. Stillborn placentas demonstrated higher MVD than controls (mean+SD: 116.6+/-46.3 v. 60.8+/-13.5, respectively, p<0.001). Vasculopathy was present in 10/22 (45%) stillbirths compared to 0/44 (0%) controls (p<0.001); increased vascular permeability was present in 15/22 (68%) cases and 5/44 (11%) controls (p<0.001). PEDF expression was significantly lower in stillborn placentas (1.7+/-0.3) than live-born controls (3.6+/-0.8, p<0.01) while VEGF expression was similar (3.3+/-0.7 v. 3.7+/-0.4, respectively, p>0.05). In conclusion, we found that unexplained stillbirth is associated with loss of angiogenic inhibitor PEDF, vasculopathy and heightened angiogenesis in the placenta.

Pigment epithelium-derived factor is an angiogenesis and lipid regulator that activates peroxisome proliferator-activated receptor alpha.

Pigment epithelium-derived factor (PEDF) is an endogenous antiangiogenic protein that also possesses antitumor activity. The mechanisms by which PEDF exerts its actions remains poorly understood. We sought to understand the role of PEDF in hepatocellular carcinoma (HCC), a hypervascular malignancy that has been shown to upregulate enzymes involved in fatty acid synthesis. PEDF expression occurs in two HCC cell lines and is oxygen dependent. Migration studies confirm PEDF's role as an endogenous inhibitor of angiogenesis in HCC cells. Loss of PEDF in an animal model leads to hepatocyte lipid accumulation, proliferation, and cellular atypia. To investigate potential interactions with transcription factors that are involved in fatty acid metabolism and cellular proliferation, we examined PEDF's interaction with PPARalpha in vitro and its functional activity through transactivation assays. We show that PEDF binds to PPARalpha but minimally to PPARgamma. In the presence of the ligand, ciprofibrate, PEDF binding to PPARalpha decreases whereas the presence of troglitazone does not alter PEDF interactions with PPARgamma. Transfection of the PEDF gene in the presence of the PPARalpha/RXR heterodimer demonstrates transcriptional activation of PPARalpha by PEDF. These data show that PEDF regulates lipid metabolism through activation of the transcription factor PPARalpha.

Anti-angiogenic pigment epithelium-derived factor regulates hepatocyte triglyceride content through adipose triglyceride lipase (ATGL).

BACKGROUND/AIMS: Anti-angiogenic pigment epithelium-derived factor (PEDF) is a 50 kDa secreted glycoprotein that is highly expressed in hepatocytes. Adipose triglyceride lipase (ATGL), a novel lipase critical for triglyceride metabolism, is a receptor for PEDF. We postulated that hepatocyte triglyceride metabolism was dependent on interactions between PEDF and ATGL, and loss of PEDF would impair mobilization of triglycerides in the liver. METHODS: Immunoprecipitation studies were performed in PEDF null and control hepatocytes with recombinant PEDF (rPEDF) as bait. Immunofluorescent microscopy was used to localize ATGL. Triglyceride content was analyzed in hepatocytes and in whole liver with and without rPEDF. ATGL was blocked using an inhibitor, (R)-bromoenol lactone. RESULTS: PEDF co-immunoprecipitated with ATGL in hepatic and HCC lysates. All PEDF deficient livers demonstrated steatosis. Triglyceride content was significantly increased in PEDF null livers compared to wildtype (p<0.05) and in isolated hepatocytes (p<0.01). Treatment of PEDF null hepatocytes with rPEDF decreased TG content (p<0.05) and this activity was dependent on ATGL. CONCLUSIONS: Our results identify a novel role for PEDF in hepatic triglyceride homeostasis through binding to ATGL and demonstrate that rPEDF and ATGL localize to adiposomes in hepatocytes. Dysregulation of this pathway may be one mechanism underlying fatty liver disease.

Alternatively spliced human tissue factor promotes tumor growth and angiogenesis in a pancreatic cancer tumor model.

INTRODUCTION: Tissue Factor (TF) expression is observed in many types of cancer, associated with more aggressive disease, and thrombosis. Alternatively-spliced human tissue factor (asHTF) has recently been identified in which exon 5 is deleted. asHTF is soluble due to the substitution of the transmembrane and cytoplasmic domains of exon 6 with a unique COOH-terminal domain. MATERIALS AND METHODS: We examine the expression and function of asHTF and full-length Tissue Factor ((FL)TF) in six human pancreatic cancer cells. Further, we transfected asHTF, (FL)TF, and control expression vectors into a non-expressing, human pancreatic cancer line (MiaPaCa-2). We studied the procoagulant activity of asHTF and (FL)TF and the effect on tumor growth in mice. RESULTS: asHTF is expressed in 5 of 6 human pancreatic cancer cell lines, but not in normal human fibroblasts, nor the MiaPaCa-2 line. (FL)TF conferred procoagulant activity, but asHTF did not. Transfected cells were injected subcutaneously in athymic mice. Interestingly, compared with control transfection, (FL)TF expression was associated with reduced tumor growth (mean 7 mg vs 85 mg), while asHTF-expression was associated with enhanced tumor growth (mean 389 mg vs. 85 mg). asHTF expression resulted in increased mitotic index and microvascular density. CONCLUSIONS: These data suggests that asHTF expression promotes tumor growth, and is associated with increased tumor cell proliferation and angiogenesis in vivo. Our results raise a new perspective on the understanding of the relationship between TF expression and cancer growth, by showing a dissociation of the procoagulant activity of (FL)TF and the cancer-promoting activity of asHTF.

Differential binding of plasminogen, plasmin, and angiostatin4.5 to cell surface beta-actin: implications for cancer-mediated angiogenesis.

Angiostatin4.5 (AS4.5) is the product of plasmin autoproteolysis and consists of kringles 1 to 4 and approximately 85% of kringle 5. In culture, cancer cell surface globular beta-actin mediates plasmin autoproteolysis to AS4.5. We now show that plasminogen binds to prostate cancer cells and that the binding colocalizes with surface beta-actin, but AS4.5 does not bind to the cell surface. Plasminogen and plasmin bind to immobilized beta-actin similarly, with a Kd of approximately 140 nmol/L. The binding is inhibited by epsilon-aminocaproic acid (epsilonACA), indicating the requirement for a lysine-kringle domain interaction. Using a series of peptides derived from beta-actin in competitive binding studies, we show that the domain necessary for plasminogen binding is within amino acids 55 to 69 (GDEAQSKRGILTLKY). Substitution of Lys61 or Lys68 with arginine results in the loss of the ability of the peptide to block plasminogen binding, indicating that Lys61 and Lys68 are essential for plasminogen binding. Other actin peptides, including peptides with lysine, did not inhibit the plasminogen-actin interaction. AS4.5 did not bind actin at concentrations up to 40 micromol/L. Plasminogen, plasmin, and AS4.5 all contain kringles 1 to 4; however, kringle 5 is truncated in AS4.5. Isolated kringle 5 binds to actin, suggesting intact kringle 5 is necessary for plasminogen and plasmin to bind to cell surface beta-actin, and the truncated kringle 5 in AS4.5 results in its release from beta-actin. These data may explain the mechanism by which AS4.5 is formed locally on cancer cell surfaces and yet acts on distant sites.

Gene transfer of pigment epithelium-derived factor suppresses tumor growth and angiogenesis in a hepatoblastoma xenograft model.

Normal hepatocytes express pigment epithelium-derived factor (PEDF), an endogenous antiangiogenic factor. We hypothesized that decreased PEDF expression may be one mechanism driving hepatoblastoma growth, and in vivo gene transfer of PEDF could suppress neovascularization and limit tumor growth. PEDF functional activity was determined in vitro using endothelial cell migration assays and in vivo using a subcutaneous tumor model. HUH-6 human hepatoblastoma tumors were treated with hybrid adenoviral/adeno-associated viral expression vectors for PEDF (Hyb-PEDF, n = 4) or beta-galactosidase (Hyb-betagal, n = 4) daily for 4 d. Mitotic figures, microvascular density (MVD), PEDF, and VEGF expression were assessed. Hyb-PEDF treatment inhibited in vivo tumor growth (p < 0.008) and decreased MVD (p < 0.001), the number of mitotic figures (p < 0.001), and VEGF expression when compared with Hyb-betagal-treated tumors. HUH-6 expression of PEDF was dramatically reduced when cultured under hypoxic conditions and also when grown in vivo, and the addition of neutralizing anti-PEDF antibody increased the already high baseline angiogenic activity of the HUH-6 cell secretions in vitro (p < 0.04). PEDF is an important endogenous regulator of the liver vasculature. Augmenting intra-tumoral PEDF levels inhibits tumor growth by reducing angiogenesis and VEGF expression. Potent inhibitors of angiogenesis, such as PEDF, may be an effective alternative treatment for children with hepatoblastoma.

In vivo generation of angiostatin isoforms by administration of a plasminogen activator and a free sulfhydryl donor: a phase I study of an angiostatic cocktail of tissue plasminogen activator and mesna.

PURPOSE: Angiostatin4.5 (AS4.5), the endogenous human angiostatin, is derived from plasminogen in a two-step process. A plasminogen activator converts plasminogen to plasmin, then plasmin undergoes autoproteolysis to AS4.5. A free sulfhydryl donor can mediate plasmin autoproteolysis. To translate this process to human cancer therapy, we conducted a phase I trial of administration of a tissue plasminogen activator (tPA) with a free sulfhydryl donor (mesna). PATIENTS AND METHODS: Fifteen patients with advanced solid tumors were treated. The dose of tPA was escalated (cohorts; 1, 2, 3, 5, and 7.5 mg/h for 6 hours). Mesna was administered as a 240 mg/m2 bolus followed by an infusion of 50 mg/h, concurrent with tPA. Both tPA and mesna were administered 3 consecutive days every 14 days. RESULTS: No dose-limiting toxicity was observed. Two AS4.5 isoforms were generated, Lys-AS4.5 and Glu-AS4.5. Mean baseline Lys-AS4.5 level was 20.4 nmol/L (SE, 2.9). In the 5 mg/h tPA cohort, Lys-AS4.5 levels increased by an average of 143% or 24 nmol/L (SE, 4.9) above baseline. Glu-AS4.5 (M(r) approximately 62,000) was also generated (additional 77 amino acids at amino terminus compared with Lys-AS4.5). Glu-AS4.5 level at baseline was undetectable in four of five patients in the 5 mg/h tPA cohort, but at end of infusion, was approximately 67 nmol/L (SE, 20). Two patients in the 5 mg/h tPA cohort experienced decreases in tumor markers with treatment, although no clinical objective responses were observed. CONCLUSION: This study shows that in vivo generation of AS4.5 is safe in humans and may provide a practical approach to achieve antiangiogenic therapy.

Decreased pigment epithelium-derived factor is associated with metastatic phenotype in human and rat prostate tumors.

Pigment epithelium-derived factor, a potent angiogenesis inhibitor in the eye, is also expressed in the prostate. Prostate size and angiogenesis is increased in pigment epithelium-derived factor knockout mice, and pigment epithelium-derived factor is down-regulated in some prostate cancers. To investigate whether pigment epithelium-derived factor expression correlates with tumor progression, we examined 5 Dunning rat prostate sublines with different growth rates, differentiation, androgen dependence, vascular density, and metastatic ability and 26 human prostate cancers of Gleason score 8-10 obtained from patients at transurethral resection selected to represent two groups, with and without metastases at diagnosis. By Western blot, real-time quantitative reverse transcription-PCR, and immunostaining, pigment epithelium-derived factor was detected in highly differentiated, nonmetastatic, androgen-sensitive Dunning tumors and in the anaplastic, androgen insensitive but nonmetastatic Dunning tumors. In contrast, the metastatic Dunning tumor sublines showed very low pigment epithelium-derived factor expression levels. In human cancer tissues, by immunohistochemistry and real-time quantitative reverse transcription-PCR, patients without metastases at diagnosis had higher tumor pigment epithelium-derived factor levels than tumors from patients with metastases at diagnosis. In both the rat model and in the human tumors, the proliferation index and vascular count, as determined by Ki-67 staining and endoglin and/or factor VIII-related antigen staining, inversely correlated with pigment epithelium-derived factor mRNA levels. These observations indicate that loss of pigment epithelium-derived factor expression could be associated with the progression toward a metastatic phenotype in prostate cancer.

Pigment epithelium-derived factor, a human testis epididymis secretory product, promotes human prostate stromal cell growth in culture.

PURPOSE: We identified and characterized unrecognized testicular secretory proteins that impact human prostate growth. MATERIALS AND METHODS: Human spermatocele fluid served as a source of testicular epididymal secretions and prostatectomy specimen benign prostatic hyperplasia stromal cells as the in vitro prostate growth promoting effects indicator. RMPI plus medium supplemented with 10% fetal bovine serum MALDI-TOF, MS FBS and ITS+ (Collaborative Research-Becton Dickinson, Bedford, Massachusetts) served as positive and negative controls, respectively. Whole and fractionated spermatocele fluid or specific proteins without and with select polyclonal or monoclonal antibodies were added to routine 6-day cultures. The observation of significantly increased 6-day cell counts compared with appropriate controls (p <0.05) was judged to reflect cell growth. Amino acid microsequencing and MALDI-TOF MS sequence analysis were done on persistent protein bands from active spermatocele fluid fractions. RESULTS: Whole and fractionated human spermatocele fluid increased stromal cell culture numbers significantly. Sequence analysis of 47 and 17 kDa 1-dimensional gel bands in the final active fraction identified a major peptide with sequence homology to human pigment epithelium-derived factor (PEDF). The presence of PEDF was confirmed by Western blot analysis. Addition of recombinant PEDF to incomplete medium significantly increased stromal cell culture number. PEDF antibodies neutralized or markedly decreased the stromal stimulating effect of spermatocele fluid and PEDF. CONCLUSIONS: The observations presented provide evidence for human testis/epididymis secretion of PEDF and for a PEDF in vitro growth promoting effect on benign prostatic hyperplasia stroma. The concept that testicular epididymal secretory proteins may influence normal and abnormal prostate growth warrants continued consideration.

Pigment epithelium-derived factor regulates the vasculature and mass of the prostate and pancreas.

Angiogenesis sustains tumor growth and metastasis, and recent studies indicate that the vascular endothelium regulates tissue mass. In the prostate, androgens drive angiogenic inducers to stimulate growth, whereas androgen withdrawal leads to decreased vascular endothelial growth factor, vascular regression and epithelial cell apoptosis. Here, we identify the angiogenesis inhibitor pigment epithelium-derived factor (PEDF) as a key inhibitor of stromal vasculature and epithelial tissue growth in mouse prostate and pancreas. In PEDF-deficient mice, stromal vessels were increased and associated with epithelial cell hyperplasia. Androgens inhibited prostatic PEDF expression in cultured cells. In vivo, androgen ablation increased PEDF in normal rat prostates and in human cancer biopsies. Exogenous PEDF induced tumor epithelial apoptosis in vitro and limited in vivo tumor xenograft growth, triggering endothelial apoptosis. Thus, PEDF regulates normal pancreas and prostate mass. Its androgen sensitivity makes PEDF a likely contributor to the anticancer effects of androgen ablation.