Veronica Stellmach

Thrombospondin-1 Is a Major Activator of TGF-β1 In Vivo

The activity of TGF-beta1 is regulated primarily extracellularly where the secreted latent form must be modified to expose the active molecule. Here we show that thrombospondin-1 is responsible for a significant proportion of the activation of TGF-beta1 in vivo. Histological abnormalities in young TGF-beta1 null and thrombospondin-1 null mice were strikingly similar in nine organ systems. Lung and pancreas pathologies similar to those observed in TGF-beta1 null animals could be induced in wild-type pups by systemic treatment with a peptide that blocked the activation of TGF-beta1 by thrombospondin-1. Although these organs produced little active TGF-beta1 in thrombospondin null mice, when pups were treated with a peptide derived from thrombospondin-1 that could activate TGF-beta1, active cytokine was detected in situ, and the lung and pancreatic abnormalities reverted toward wild type.

How tumors become angiogenic.

Publisher Summary As normal cells progress to tumorigenicity, they must develop two distinct new characteristics not possessed by the normal cells from which they arise: they must become able to multiply without restraint and they must be able to create in vivo an environment where this newly acquired growth potential can be realized. Normal cells are antiangiogenic, because they secrete only low levels of inducers of angiogenesis and high levels of molecules that inhibit neovascularization. These cells develop into successful tumors that are potently angiogenic and secrete high levels of a cocktail of molecules that induce neovascularization. It is possible to halt the production and/or release of angiogenic activity by tumor cells themselves using retinoic acid and similar compounds. Endothelial cells that form the vessels that support tumor growth can be disabled in two distinct ways. Antiangiogenic agents have distinct advantages over conventional cytotoxic cancer therapies. Although the importance of angiogenesis to the growth of tumors is now well established, the mechanism by which tumor cells develop this crucial ability to attract new blood vessels is just beginning to be explored. Available data suggest that the angiogenic phenotype develops gradually as a result of many of the same genetic changes in oncogenes and tumor suppressor genes that are also responsible for the deregulation of cell growth. The inhibitors of angiogenesis offer a new and promising avenue for tumor therapy.

Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor

Aberrant blood vessel growth in the retina that underlies the pathology of proliferative diabetic retinopathy and retinopathy of prematurity is the result of the ischemia-driven disruption of the normally antiangiogenic environment of the retina. In this study, we show that a potent inhibitor of angiogenesis found naturally in the normal eye, pigment epithelium-derived growth factor (PEDF), inhibits such aberrant blood vessel growth in a murine model of ischemia-induced retinopathy. Inhibition was proportional to dose and systemic delivery of recombinant protein at daily doses as low as 2.2 mg/kg could prevent aberrant endothelial cells from crossing the inner limiting membrane. PEDF appeared to inhibit angiogenesis by causing apoptosis of activated endothelial cells, because it induced apoptosis in cultured endothelial cells and an 8-fold increase in apoptotic endothelial cells could be detected in situ when the ischemic retinas of PEDF-treated animals were compared with vehicle-treated controls. The ability of low doses of PEDF to curtail aberrant growth of ocular endothelial cells without overt harm to retinal morphology suggests that this natural protein may be beneficial in the treatment of a variety of retinal vasculopathies.

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.

The modulation of thrombospondin and other naturally occurring inhibitors of angiogenesis during tumor progression

SummaryFifteen different natural inhibitors of angiogenesis have now been identified that are produced by mammalian cells and are able to blockin vivo neovascularization. The majority of these are able to inhibit endothelial cell activitiesin vitro and all those tested have demonstrated significant antitumor activity. Most normal cells produce inhibitors of neovascularization that must be downregulated before the cells can develop into angiogenic, malignant tumors. In several cases the production of inhibitors ceases when tumor suppressor genes are inactivated. In the BT549 human breast carcinoma cell line, the reintroduction of a wild type p53 tumor suppressor gene resulted in the stimulation of the secretion of an inhibitor of angiogenesis, thrombospondin-1, and as a result the cells lost their angiogenic phenotype and became able to suppress angiogenesis induced by the parental tumor line. These results provide a new example of tumor suppressor gene control of a natural inhibitor of angiogenesis and add support to the concept that thrombospondin loss may play an important role in the development of some human breast cancers.

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 (PPARγ)-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 (PPARγ-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 α and to a lesser effect on PPARγ transcriptional activity. These observations indicate that PRIP like PBP, CBP, and p300 is an essential and nonredundant coactivator.

Increased expression of transforming growth factor-β1 and thrombospondin-1 in congenital hepatic fibrosis : Possible role of the hepatic stellate cell

BACKGROUND Congenital hepatic fibrosis is a rare disease characterized by portal tract fibrosis and biliary duct ectasia. It is associated with autosomal recessive polycystic kidney disease and rarely progresses to cirrhosis. The activated stellate cell has been implicated in the pathogenesis of alcohol- or inflammation-mediated cirrhosis through fibrogenic proteins such as transforming growth factor-beta1; however, the role of the stellate cell in pure, noninflammatory fibrosis is unknown. It has been hypothesized that fibrosis in congenital hepatic fibrosis may be caused by upregulation of transforming growth factor-beta1 and thrombospondin-1, and that the hepatic stellate cell may be the mediator of these proteins. METHODS Human liver tissue samples from patients with congenital hepatic fibrosis (n = 9) and from normal patients (n = 3) were analyzed. Tissue homogenates from both groups were analyzed for transforming growth factor-beta1 protein and mRNA by Western blot analysis and in situ hybridization, respectively. Immunolocalization studies were performed in fixed tissue sections from both groups. Stellate cells were cultured from livers exhibiting congenital hepatic fibrosis and confirmed by desmin staining. The cells were cultured in serum-free medium for 48 hours, and media were collected and analyzed by Western blot analysis for thrombospondin-1 and transforming growth factor-beta1. RESULTS Congenital hepatic fibrosis liver tissue homogenates had higher levels of thrombospondin-1 and transforming growth factor-beta1 protein than in normal livers. In congenital hepatic fibrosis tissue, transforming growth factor-beta1 was more highly expressed in the ectatic biliary epithelium and the perisinusoidal space, whereas thrombospondin-1 localized most intensely to the hepatocytes and spared the bile ducts. Congenital hepatic fibrosis-derived stellate cells secreted both thrombospondin-1 and transforming growth factor-beta1, in vitro. CONCLUSIONS Transforming growth factor-beta1 and thrombospondin-1 may play a role in the pathogenesis of liver fibrosis in patients with congenital hepatic fibrosis. One potential source of these fibrogenic proteins is the hepatic stellate cell.

Tumour suppressor genes and angiogenesis: the role of TP53 in fibroblasts.

‘Department of Microbiology-Immunology; ‘Department of Pathology; ‘R.H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611; 4Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02 115; and ‘Howard Hughes Medical Institute, Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, U.S.A.

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.

Thrombospondin-1 regulates the normal prostate in vivo through angiogenesis and TGF- β 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-β (TGF-β) 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-β 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-β 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-β. Therefore, loss of TSP-1 during tumorigenesis would eliminate two barriers to cancer progression.