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 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.

Pigment epithelium-derived factor targets endothelial and epithelial cells in Wilms' tumor.

PURPOSE: Loss of pigment epithelium-derived factor (PEDF), a potent inhibitor of angiogenesis, has been linked to progression of angiogenesis-dependent diseases. We postulated that decreased levels of endogenous PEDF in the kidney creates a tumor permissive environment for Wilms' tumor. METHODS: Fresh and frozen Wilms' tumor (n = 28), adjacent (n = 3), and normal kidney (n = 8) were immunostained and graded. The Wilms' tumor cells (SK-NEP-1), renal epithelial cells (NRK-52), and fresh tumor samples were grown in culture. Condition media were collected and analyzed by an in vitro angiogenesis assay and Western blot. The SK-NEP-1 cells were treated with PEDF and cell viability assessed. RESULTS: Wilms' tumors expressed less PEDF than normal and adjacent kidney. Pigment epithelium-derived factor protein secretion was abundant in NRK-52 cells but significantly decreased in Wilms' tumor. Pigment epithelium-derived factor acted as blockade to angiogenesis and it had a dose-dependent cytotoxic effect on Wilms' tumor epithelial cells. CONCLUSION: Renal tubular epithelial cells are a rich source of PEDF in the normal kidney. Reduced levels of PEDF in Wilms' tumor remove a critical endogenous renal barrier to angiogenesis and tumor cell survival. Therapeutic replacement of PEDF may prove to be an effective strategy to combat Wilms' tumor progression.

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.

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.

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.

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.

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.

Captopril suppresses post-transplantation angiogenic activity in rat allograft coronary vessels.

BACKGROUND: The development of transplant coronary artery disease is associated with neovascularization in the thickened neointima. We previously reported that captopril inhibits neointimal proliferation in a rat allograft model. We postulated that angiogenic inducers are upregulated post-transplantation and captopril ameliorates transplant coronary artery disease by suppressing the angiogenic activity of coronaries. METHODS: Animals received no treatment or captopril (50 mg/kg/day). Allograft hearts were analyzed at post-transplantation Days 0, 14, and 21 and angiogenic inducer, plasma platelet-activating factor, determined. The conditioned media from coronaries and myocardium were tested for vascular endothelial growth factor, thrombospondin-1 and angiogenic activity using an endothelial migration assay and rat corneal neovascularization assay. RESULTS: The captopril-treated group had reduced plasma platelet-activating factor and coronary media revealed earlier upregulation of thrombospondin-1 secretion, diminished vascular endothelial growth factor and no angiogenic activity. At Day 0, the coronary and myocardial conditioned medium had inhibitory activity due to thrombospondin-1, and circulating levels of platelet-activating factor were negligible. By 21 days post-transplantation, plasma platelet-activating factor was elevated and the conditioned medium from untreated coronaries had significantly higher angiogenic activity due to increased vascular endothelial growth factor whereas the myocardium remained non-angiogenic. CONCLUSIONS: After transplantation, coronary vessels switch to an angiogenic phenotype and vascular endothelial growth factor contributes to the high angiogenic activity, possibly exacerbated by high circulating levels of platelet-activating factor. The ability of captopril to modulate angiogenic mediators and maintain the allograft coronary to its normal anti-angiogenic phenotype may be one mechanism by which it suppresses transplant coronary artery disease.

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.

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.

Wilms' tumor growth is suppressed by antiangiogenic pigment epithelium-derived factor in a xenograft model.

BACKGROUND/PURPOSE: Pigment epithelium-derived factor (PEDF), a potent endogenous inhibitor of angiogenesis, is highly expressed in the kidney. The authors postulated that systemic administration of PEDF would decrease Wilms' tumor growth in a xenograft model, and increased renal vascularity would result in a mouse null for PEDF. METHODS: Tumors were induced in athymic mice using human anaplastic Wilms' tumor cells. Purified PEDF protein or vehicle was administered for 7 days beginning 2 to 3 weeks after inoculation. Tumors were stained with anti-PEDF and anti-Factor VIII antibodies. Mitoses and microvascular density (MVD) were counted per high-power field (hpf). PEDF-null mice were generated on a SV129/C57Bl6 background. Wild-type and null kidneys were assessed for MVD. RESULTS: Mean tumor weight in the 2-week group was 60% less than controls (P <.05). The MVD and mitotic count in treated tumors were significantly less than controls (P <.05). PEDF stained strongly in normal kidneys but was minimal to absent in Wilms' tumor. PEDF-null kidneys had increased MVD compared with wild-type (P <.05). CONCLUSIONS: PEDF is expressed strongly in normal murine kidney, and loss of its angioinhibitory activity may contribute to pathologic angiogenesis in Wilms' tumor. Systemic PEDF suppresses WT growth by targeting both the tumor cells and its associated vasculature.

Coactivator PRIP, the peroxisome proliferator-activated receptor-interacting protein, is a modulator of placental, cardiac, hepatic, and embryonic development.

Nuclear receptor coactivator PRIP (peroxisome proliferator-activated receptor (PPAR gamma)-interacting protein) and PRIP-interacting protein with methyltransferase activity, designated PIMT, appear to serve as linkers between cAMP response element-binding protein-binding protein (CBP)/p300-anchored and PBP (PPAR gamma-binding protein)-anchored coactivator complexes involved in the transcriptional activity of nuclear receptors. To assess the biological significance of PRIP, we disrupted the PRIP gene in mice by homologous recombination. Mice nullizygous for PRIP died between embryonic day 11.5 and 12.5 (postcoitum) due in most part to defects in the development of placenta, heart, liver, nervous system, and retardation of embryonic growth. Transient transfection assays using fibroblasts isolated from PRIP(-/-) embryos revealed a significant decrease in the capacity for ligand-dependent transcriptional activation of retinoid X receptor alpha and to a lesser effect on PPAR gamma transcriptional activity. These observations indicate that PRIP like PBP, CBP, and p300 is an essential and nonredundant coactivator.

Serum vascular endothelial growth factor as a surveillance marker for cellular rejection in pediatric cardiac transplantation.

BACKGROUND: Early detection and treatment of acute rejection in cardiac transplant recipients significantly improves long-term survival. Endomyocardial biopsy is used routinely for diagnosing allograft rejection; however, in young children, this procedure carries some risk. We evaluated serum vascular endothelial growth factor (VEGF) as a potential surveillance marker of acute cellular rejection. METHODS: Blood samples (n=62) were analyzed from 23 patients and compared with controls (n=18) using an ELISA for VEGF. Results were correlated with endomyocardial biopsy rejection grades. RESULTS: Mean baseline VEGF levels of the transplant population were consistently higher than controls. Serum VEGF levels were significantly higher during acute cellular rejection when compared with the non-rejecting transplant group (700.7+/-154 pg/ml vs. 190.5+/-29 pg/ml). VEGF decreased two- to eightfold after immunosuppressive therapy in 9 of 11 rejection episodes. CONCLUSIONS: These data suggest that VEGF may play a role in the pathogenesis of acute allograft rejection and it may serve as a reliable serologic surveillance marker.

Defects of the heart, eye, and megakaryocytes in peroxisome proliferator activator receptor-binding protein (PBP) null embryos implicate GATA family of transcription factors.

Peroxisome proliferator activator receptor (PPAR)-binding protein (PBP) is an important coactivator for PPARgamma and other nuclear receptors. It has been identified as an integral component of a multiprotein thyroid hormone receptor-associated protein/vitamin D(3) receptor-interacting protein/activator-recruited cofactor complexes required for transcriptional activity. Here, we show that PBP is critical for the development of placenta and for the normal embryonic development of the heart, eye, vascular, and hematopoietic systems. The primary functional cause of embryonic lethality at embryonic day11.5 observed with PBP null mutation was cardiac failure because of noncompaction of the ventricular myocardium and resultant ventricular dilatation. There was a paucity of retinal pigment, defective lens formation, excessive systemic angiogenesis, a deficiency in the number of megakaryocytes, and an arrest in erythrocytic differentiation. Some of these defects involve PPARgamma and retinoid-sensitive sites, whereas others have not been recognized in the PPAR-signaling pathway. Phenotypic changes in four organ systems observed in PBP null mice overlapped with those in mice deficient in members of GATA, a family of transcription factors known to regulate differentiation of megakaryocytes, erythrocytes, and adipocytes. We demonstrate that PBP interacts with all five GATA factors analyzed, GATA-1, GATA-2, GATA-3, GATA-4, and GATA-6, and show that the binding of GATA-1, GATA-4, and GATA-6 to PBP is not dependent on the nuclear receptor recognition sequence motif LXXLL (where L is leucine and X is any amino acid) in PBP. Coexpression of PBP with GATA-3 markedly enhanced transcriptional activity of GATA-3 in nonhematopoietic cells. These observations identify the GATA family of transcription factors as a new interacting partner of PBP and demonstrate that PBP is essential for normal development of vital organ systems.