FOXO3 growth inhibition of colonic cells is dependent on intraepithelial lipid droplet density.

Forkhead transcription factor FOXO3 plays a critical role in suppressing tumor growth, in part, by increasing the cell cycle inhibitor p27kip1, and Foxo3 deficiency in mice results in marked colonic epithelial proliferation. Here, we show in Foxo3-deficient colonic epithelial cells a striking increase in intracytoplasmic lipid droplets (LDs), a dynamic organelle recently observed in human tumor tissue. Although the regulation and function of LDs in non-adipocytes is unclear, we hypothesize that the anti-proliferative effect of FOXO3 was dependent on lowering LD density, thus decreasing fuel energy in both normal and colon cancer cells. In mouse colonic tumors, we found an increased expression of LD coat protein PLIN2 compared with normal colonic epithelial cells. Stimulation of LD density in human colon cancer cells led to a PI3K-dependent loss of FOXO3 and a decrease in the negative regulator of lipid metabolism in Sirtuin6 (SIRT6). Foxo3 deficiency also led to a decrease in SIRT6, revealing the existence of LD and FOXO3 feedback regulation in colonic cells. In parallel, LD-dependent loss of FOXO3 led to its dissociation from the promoter and decreased expression of the cell cycle inhibitor p27kip1. Stimulation of LD density promoted proliferation in colon cancer cells, whereas silencing PLIN2 or overexpression of FOXO3 inhibited proliferation. Taken together, FOXO3 and LDs might serve as new targets for therapeutic intervention of colon cancer.

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.

Peritumoral expression of adipokines and fatty acids in breast cancer.

BACKGROUND: Adipokines in the tumor microenvironment may contribute to cancer growth. We hypothesized that peritumoral fat can be a source of lipid-derived energy for tumors by increasing adipose triglyceride lipase (ATGL)-mediated lipolysis and down-regulating a negative regulator of adipogenesis, pigment epithelium-derived factor (PEDF). METHODS: In a pilot study, tissue from mastectomies (n = 19) was collected from sites both adjacent (peritumoral) and distant to the tumor for comparison of ATGL, PEDF, and leptin expression levels using immunohistochemistry. Statistical analysis was performed by Student's t test to determine significance. RESULTS: Mean tumor size was 2.4 cm, and 10 (59 %) patients had tumor-positive nodes. Mean body mass index (BMI) was 28.1 kg/m(2). ATGL expression was significantly increased in obese patients (BMI ≥ 30 kg/m(2)) compared with the nonobese group (P < 0.04). Leptin expression was increased in the peritumoral stroma of obese patients compared with distant sites (P = 0.03). Peritumoral PEDF and the leptin/PEDF ratio were significantly affected by tumor size and node status. Tumors ≥ 2 cm had lower peritumoral stromal expression of PEDF than tumors <2 cm (P = 0.01). In node-positive cases, expression of PEDF was significantly decreased in the peritumoral stroma compared with node-negative cases (1.22 vs. 1.80, P < 0.04). The leptin/PEDF ratio was markedly elevated in the peritumoral region of node-positive cases versus node-negative cases (2.17 vs. 1.18, P < 0.001). CONCLUSIONS: Peritumoral expression of adipokines was altered in both obesity and more advanced breast tumors, suggesting a role for adipokines in enhancing tumor growth. Future studies should focus on the use of adipokines as biomarkers.

Concurrent PEDF deficiency and Kras mutation induce invasive pancreatic cancer and adipose-rich stroma in mice.

BACKGROUND AND AIMS: Pigment epithelium-derived factor (PEDF), a non-inhibitory SERPIN with potent antiangiogenic activity, has been recently implicated in metabolism and adipogenesis, both of which are known to influence pancreatic cancer progression. Increased pancreatic fat in human pancreatic tumour correlates with greater tumour dissemination while PEDF deficiency in mice promotes pancreatic hyperplasia and visceral obesity. Oncogenic Ras, the most common mutation in pancreatic ductal adenocarcinoma (PDAC), has similarly been shown to promote adipogenesis and premalignant lesions. METHODS: In order to determine whether concurrent loss of PEDF is sufficient to promote adipogenesis and tumorigenesis in the pancreas, the authors ablated PEDF in an EL-Kras(G12D) mouse model of non-invasive cystic papillary neoplasms. RESULTS: EL-Kras(G12D)/PEDF deficient mice developed invasive PDAC associated with enhanced matrix metalloproteinase (MMP)-2 and MMP-9 expression and increased peripancreatic fat with adipocyte hypertrophy and intrapancreatic adipocyte infiltration (pancreatic steatosis). In support of increased adipogenesis, the stroma of the pancreas of EL-Kras(G12D)/PEDF deficient mice demonstrated higher tissue levels of two lipid droplet associated proteins, tail-interacting protein 47 (TIP47, perilipin 3) and adipose differentiation-related protein (ADRP, Pperilipin 2), while adipose triglyceride lipase, a key factor in lipolysis, was decreased. In patients with PDAC, both tissue and serum levels of PEDF were decreased, stromal TIP47 expression was higher and the tissue VEGF to PEDF ratio was increased (p<0.05). CONCLUSIONS: These data highlight the importance of lipid metabolism in the tumour microenvironment and identify PEDF as a critical negative regulator of both adiposity and tumour invasion in the pancreas.

Pigment epithelium-derived factor regulates early pancreatic fibrotic responses and suppresses the profibrotic cytokine thrombospondin-1.

Pigment epithelium-derived factor (PEDF) is important for maintaining the normal extracellular matrix. We hypothesized that the initiation of pancreatic fibrosis is dependent on the loss of PEDF. Pancreatic PEDF expression was assessed in wild-type mice fed either a control or ethanol diet using an intragastric feeding model. Pancreatitis responses were elicited with either a single episode or a repetitive cerulein-induced (50 µg/kg, 6 hourly i.p. injections) protocol in wild-type and PEDF-null mice. Quantitative real-time PCR and immunoblotting were performed to assess fibrogenic responses. In wild-type animals, PEDF expression increased with pancreatitis and was more pronounced in mice fed ethanol. Compared with wild-type mice, α-smooth muscle actin staining and expression levels of fibrogenic markers (eg, transforming growth factor-ß1, platelet-derived growth factor, collagen I, and thrombospondin-1) were higher in PEDF-null mice at baseline. Sirius red staining revealed more fibrosis in PEDF-null versus wild-type pancreas 1 week after pancreatitis. Differences in tissue fibrosis resolved with longer recovery periods. PEDF overexpression suppressed thrombospondin-1 levels in vitro. Ethanol feeding and experimental pancreatitis increased PEDF expression in wild-type mice. PEDF-null mice, however, demonstrated enhanced early fibrotic responses compared with wild-type mice with pancreatitis. These findings indicate that PEDF acts as a compensatory antifibrotic cytokine in pancreatitis.

The vacuolar-ATPase modulates matrix metalloproteinase isoforms in human pancreatic cancer.

The vacuolar-ATPase (v-ATPase) is a proton transporter found on many intracellular organelles and the plasma membrane (PM). The v-ATPase on PMs of cancer cells may contribute to their invasive properties in vitro. Its relevance to human cancer tissues remains unclear. We investigated whether the expression and cellular localization of v-ATPase corresponded to the stage of human pancreatic cancer, and its effect on matrix metalloproteinase (MMP) activation in vitro. The intensity of v-ATPase staining increased significantly across the range of pancreatic histology from normal ducts to pancreatic intraepithelial neoplasms (PanIN), and finally pancreatic ductal adenocarcinoma (PDAC). Low-grade PanIN lesions displayed polarized staining confined to the basal aspect of the cell in the majority (86%) of fields examined. High-grade PanIN lesions and PDAC showed intense and diffuse v-ATPase localization. In pancreatic cancer cells, PM-associated v-ATPase colocalized with cortactin, a component of the leading edge that helps direct MMP release. Blockade of the v-ATPase with concanamycin or short-hairpin RNA targeting the V1E subunit reduced MMP-9 activity; this effect was greatest in cells with prominent PM-associated v-ATPase. In cells with detectable MMP-2 activities, however, treatment with concanamycin markedly increased MMP-2's most activated forms. V-ATPase blockade inhibited functional migration and invasion in those cells with predominantly MMP-9 activity. These results indicate that human PDAC specimens show loss of v-ATPase polarity and increased expression that correlates with increasing invasive potential. Thus, v-ATPase selectively modulates specific MMPs that may be linked to an invasive cancer phenotype.

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.

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.

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.

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.

Adipose Triglyceride Lipase (ATGL) Expression Is Associated with Adiposity and Tumor Stromal Proliferation in Patients with Pancreatic Ductal Adenocarcinoma.

BACKGROUND: Obesity is an established risk factor for the development of pancreatic ductal adenocarcinoma (PDAC). However, the pathophysiology of how increased adiposity increases the risk for PDAC has not been fully elucidated. Adipose triglyceride lipase (ATGL) is a lipase that catabolizes triglyceride hydrolysis and has been implicated in the development of breast cancer. We hypothesized that overweight patients with PDAC would demonstrate higher tumor ATGL expression compared to non-overweight patients with PDAC. MATERIALS AND METHODS: Immunohistochemical analysis for ATGL expression was performed on PDAC tissues from 44 patients after Whipple procedure or distal pancreatectomy. Correlation of ATGL expression with clinicopathological features was evaluated. RESULTS: A total of 23/44 (52.2%) PDACs showed low level ATGL immunoreactivity, while 21/44 (47.8%) showed a high level, with moderate to strong positive ATGL immunoreactivity in more than 50% of the tumor cells. Chi-squared testing revealed a statistically significant association between high ATGL expression and both BMI >25 kg/m2 (χ2=5.74, p=0.017) and increased tumor stroma (χ2=19.14, p<0.001). Chi-squared testing failed to reveal a statistically significant association when comparing ATGL expression by lymph node metastasis, histological grade, tumor size, patient age, patient sex and presence of fat invasion. CONCLUSION: Our results suggest that increased ATGL expression is associated with increased adiposity and stromal proliferation in patients with PDAC, making it a possible key protein in how obesity increases the risk of PDAC.

Topical VEGF enhances healing of thoracic aortic anastomosis for coarctation in a rabbit model.

BACKGROUND: Recurrent stenosis after extended end-to-end anastomosis for aortic coarctation is the primary indication for further interventions in children. Tension because of the extended resection and local arterial wall hypoxia are possible pathogenetic mechanisms. We hypothesized that (1) tension interferes with healing and (2) that vascular endothelial growth factor (VEGF), a hypoxia sensitive angiogenic inducer, may enhance healing of the vascular anastomosis. METHODS AND RESULTS: In a model of coarctation repair, rabbits underwent thoracic aortic end-to-end anastomosis after transection (no-tension; n=15), resection of an aortic ring (tension; n=14) or resection and topical VEGF treatment (0.75 microg VEGF165; tension+VEGF; n=14). Gross and histologic characteristics of the aortic wall were assessed at 1 week, 1 and 2 months. In the tension only group at 1 month, the severity of vascular remodeling was increased with fibrosis and calcification compared with controls. At 2 months, this group also revealed more luminal stenosis (29% versus 19%; P<0.001). Exogenous VEGF resulted in significantly less fibrosis, calcification and chondroid metaplasia at 1 month (P<0.05) and luminal area was only reduced 3% at 2 months (P<0.001 versus tension group). CONCLUSIONS: In a rabbit model of coarctation repair, the addition of tension on the vascular anastomosis resulted in poor healing and luminal stenosis. Topical VEGF maintained luminal integrity by decreasing fibrosis and calcification. These findings suggest that topical VEGF may be a promising new strategy to enhance healing and improve the outcome of vascular anastomoses for coarctation of the aorta.

Osteopontin expression and adventitial angiogenesis induced by local vascular endothelial growth factor 165 reduces experimental aortic calcification.

BACKGROUND: Vascular calcification is a common pathologic and precisely regulated process involving bone-associated proteins such as osteopontin. In this study, we investigated mechanisms by which recombinant human vascular endothelial growth factor 165 protects the arterial wall from severe vascular remodeling, including calcification, a newly discovered biologic action of vascular endothelial growth factor. METHODS: In a rabbit model of thoracic aortic end-to-end anastomosis that simulates cardiovascular intervention, recombinant human vascular endothelial growth factor 165 at a dose of 0.75 mug (n = 19) or albumin (control; n = 19) was delivered intraluminally and on the serosal surface. Animals were killed, and aortic tissue was evaluated by Western blotting, immunohistochemistry, and immunofluorescence at 4, 8, and 24 hours; 1 week; and 1 month after surgery. RESULTS: All controls revealed extensive aortic medial calcification at 1 month, whereas calcification was significantly reduced or absent with vascular endothelial growth factor treatment. Compared with controls, vascular endothelial growth factor treatment resulted in an earlier infiltration of macrophages in the vessel media (at 8 hours: 5.7 +/- 2.3 macrophages per high-power field in control vs 32.1 +/- 7.5 in vascular endothelial growth factor-treated aortas; P < .001), whereas controls showed an increase in macrophages starting at 1 week (24.1 +/- 6.9 vs 4.3 +/- 1.8; P < .001). Osteopontin expression was transiently increased and detected in macrophages and endothelial cells in vascular endothelial growth factor-treated vessels, and adventitial microvascular density was significantly increased by 1 week (9.5 +/- 0.43 vs 25.0 +/- 1.3; P < .001). CONCLUSIONS: Our data suggest that exogenous vascular endothelial growth factor is capable of increasing adventitial angiogenesis and shifting macrophage infiltration and osteopontin expression in the media to an earlier time point, thereby promoting prompt repair and diminishing vascular remodeling and calcification after acute vascular injury.

Local delivery of osteopontin attenuates vascular remodeling by altering matrix metalloproteinase-2 in a rabbit model of aortic injury.

OBJECTIVE: Vascular remodeling, often accelerated after cardiovascular procedures, may result in stenosis or aneurysm formation. The bone-associated protein osteopontin has been suggested to be involved in vascular remodeling, yet the effect of locally applied osteopontin in an acute vascular injury model of aortic calcification has not been examined. METHODS: Vascular healing of rabbit thoracic aortas treated locally with recombinant osteopontin (dose: 1 microg; n = 16) or albumin (control, n = 16) after acute injury created by end-to-end anastomosis was evaluated. Matrix metalloproteinase-2 level was quantified by gelatin zymography. Proliferation of smooth muscle cells was detected by immunostaining for proliferative cell nuclear antigen. RESULTS: Osteopontin-treated aortas showed significantly diminished vascular remodeling with less calcification (P = .001) and reduced anastomotic luminal stenosis (4% vs 28%, P = .002) compared with controls 2 months postsurgery. Moreover, osteopontin-treated aortas revealed a thickened adventitia with increased fibrosis (P = .006). Matrix metalloproteinase-2 level was up-regulated 2-fold with osteopontin treatment compared with control at 1 week, returning to baseline by 1 month. Staining for proliferation cell nuclear antigen disclosed an increase in proliferation cell nuclear antigen-positive smooth muscle cells in the media of osteopontin-treated aortas at 1 week, normalizing by 1 month. CONCLUSIONS: These data suggest a beneficial effect of locally applied osteopontin after acute injury possibly by altering matrix metalloproteinase-2 activity and smooth muscle cell proliferation. Brief application of osteopontin may effectively enhance vascular healing by reducing calcification and thus maintaining luminal integrity. The role of the osteopontin-related increase in adventitial fibrosis on vascular healing has to be explored.

Neointimal inflammation and adventitial angiogenesis correlate with severity of cardiac allograft vasculopathy in pediatric recipients.

BACKGROUND: Chronic inflammation and angiogenesis have been implicated in the pathogenesis of both cardiac allograft vasculopathy (CAV) and age-related vasculopathy. Because concurrent atherosclerosis does not complicate assessment of CAV in children, we sought to characterize the spectrum of coronary lesions in this population and determine whether inflammatory infiltrates and angiogenesis correlate with severity of CAV. METHODS: In 18 pediatric heart specimens CAV was graded 1 to 4 (none to severe). Each case was assigned to either: Group I, no inflammation; Group II, perivascular inflammation; or Group III, perivascular and neointimal inflammation. Inflammatory infiltrates were immunophenotyped using anti-CD3, anti-CD20 and HAM 56. Angiogenesis was assessed by determining microvascular density (MVD) in 5 high-power fields (HPFs) per section. RESULTS: CAV was evident in 94% of cases, and inflammation in 61%. Cases with neointimal inflammation had significantly more severe CAV compared with cases without inflammation (2.7 +/- 0.16 vs 1.9 +/- 0.2, p = 0.002). MVD was significantly higher in both inflammation groups (Groups II and III) compared with Group I (4.1 +/- 0.5 per HPF and 5.9 +/- 0.5 vs 3.1 +/- 0.7, p = 0.018 and p = 0.002) and correlated with the degree of CAV (p = 0.007). The perivascular infiltrates (Group II, n = 5) contained lymphocytes, macrophages and plasma cells, and 67% of neointimal infiltrates (Group III, n = 6) also contained eosinophils. CONCLUSIONS: CAV in children is more common than previously reported. Our data indicate that CAV is often associated with inflammation and that adventitial angiogenesis correlated with the severity of CAV.

Altered pigment epithelium-derived factor and vascular endothelial growth factor levels in lymphangioma pathogenesis and clinical recurrence.

OBJECTIVE: To determine the role of angiogenesis in the clinical behavior and pathogenesis of lymphangioma tumors. DESIGN: A retrospective study. Median follow-up period was 44.5 months. SETTING: Children's Memorial Hospital, Chicago, Ill. PATIENTS: Tumor specimens from 12 pediatric patients who underwent surgical excision of cervicofacial lymphangioma were examined for expression of angiogenic inducer vascular endothelial growth factor (VEGF) and angiogenic inhibitor pigment epithelium-derived factor (PEDF) using immunohistochemical analysis. Specimens were divided into recurrent and nonrecurrent tumors based on clinical information. MAIN OUTCOME MEASURES: Staining patterns of VEGF and PEDF were evaluated in lymphangioma specimens. Staining patterns were then compared in both recurrent and nonrecurrent groups and graded in a blinded fashion. Histological evidence of increased angiogenesis including microvascular density, stromal fibrosis, and inflammation were graded in each group and correlated with recurrence. RESULTS: Lymphangioma specimens demonstrated histological evidence of increased angiogenic activity including multiple areas of increased VEGF staining combined with little PEDF staining. Sex, age at onset, or tumor location did not correlate with recurrence. Furthermore, recurrent specimens had increased histological evidence of angiogenesis as well as increased VEGF and decreased PEDF activity compared with nonrecurrent lesions. CONCLUSIONS: Lymphangiomas exhibit tumorlike pathogenesis owing to the high expression of angiogenic inducers compared with the low expression of inhibitors. Recurrence may be influenced by this imbalance of angiogenic mediators. Further research with antiangiogenic therapy using agents such as PEDF analogues or anti-VEGF receptor antibodies is indicated because they may stabilize or suppress the growth of these neoplasms.

Pigment Epithelium-Derived Factor (PEDF) Inhibits Wnt/ß-catenin Signaling in the Liver.

BACKGROUND & AIMS: Pigment epithelium-derived factor (PEDF) is a secretory protein that inhibits multiple tumor types. PEDF inhibits the Wnt coreceptor, low-density lipoprotein receptor-related protein 6 (LRP6), in the eye, but whether the tumor-suppressive properties of PEDF occur in organs such as the liver is unknown. METHODS: Wnt-dependent regulation of PEDF was assessed in the absence and presence of the Wnt coreceptor LRP6. Whole genome expression analysis was performed on PEDF knockout (KO) and control livers (7 months). Interrogation of Wnt/ß-catenin signaling was performed in whole livers and human hepatocellular carcinoma (HCC) cell lines after RNA interference of PEDF and restoration of a PEDF-derived peptide. Western diet feeding for 6 to 8 months was used to evaluate whether the absence of PEDF was permissive for HCC formation (n = 12/group). RESULTS: PEDF levels increased in response to canonical Wnt3a in an LRP6-dependent manner but were suppressed by noncanonical Wnt5a protein in an LRP6-independent manner. Gene set enrichment analysis (GSEA) of PEDF KO livers revealed induction of pathways associated with experimental and human HCC and a transcriptional profile characterized by Wnt/ß-catenin activation. Enhanced Wnt/ß-catenin signaling occurred in KO livers, and PEDF delivery in vivo reduced LRP6 activation. In human HCC cells, RNA interference of PEDF led to increased levels of activated LRP6 and ß-catenin, and a PEDF 34-mer peptide decreased LRP6 activation and ß-catenin signaling, and reduced Wnt target genes. PEDF KO mice fed a Western diet developed sporadic well-differentiated HCC. Human HCC specimens demonstrated decreased PEDF staining compared with hepatocytes. CONCLUSIONS: PEDF is an endogenous inhibitor of Wnt/ß-catenin signaling in the liver.

Chemopreventive apigenin controls UVB-induced cutaneous proliferation and angiogenesis through HuR and thrombospondin-1.

Plant flavonoid apigenin prevents and inhibits UVB-induced carcinogenesis in the skin and has strong anti-proliferative and anti-angiogenic properties. Here we identify mechanisms, by which apigenin controls these oncogenic events. We show that apigenin acts, at least in part, via endogenous angiogenesis inhibitor, thrombospondin-1 (TSP1). TSP1 expression by the epidermal keratinocytes is potently inhibited by UVB. It inhibits cutaneous angiogenesis and UVB-induced carcinogenesis. We show that apigenin restores TSP1 in epidermal keratinocytes subjected to UVB and normalizes proliferation and angiogenesis in UVB-exposed skin. Importantly, reconstituting TSP1 anti-angiogenic function in UVB-irradiated skin with a short bioactive peptide mimetic representing exclusively its anti-angiogenic domain reproduced the anti-proliferative and anti-angiogenic effects of apigenin. Cox-2 and HIF-1α are important mediators of angiogenesis. Both apigenin and TSP1 peptide mimetic attenuated their induction by UVB. Finally we identified the molecular mechanism, whereby apigenin did not affect TSP1 mRNA, but increased de novo protein synthesis. Knockdown studies implicated the RNA-binding protein HuR, which controls mRNA stability and translation. Apigenin increased HuR cytoplasmic localization and physical association with TSP1 mRNA causing de novo TSP1 synthesis. HuR cytoplasmic localization was, in turn, dependent on CHK2 kinase. Together, our data provide a new mechanism, by which apigenin controls UVB-induced carcinogenesis.