Aberrant neuronal and paracellular deposition of endostatin in brains of patients with Alzheimer's disease.
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Cerebrovascular pathology is common in Alzheimer's disease (AD) and is considered to contribute to cerebral malfunction. However, distinct antiangiogenic proteins that accumulate in AD brains have not yet been identified. Endostatin is a 20 kDa C-terminal fragment of collagen XVIII that, when added exogenously, inhibits endothelial proliferation and migration in vitro and angiogenesis and tumor growth in vivo by inducing apoptosis in endothelial cells. We produced a monoclonal antibody directed against endostatin and observed significantly more (p < 0.0001) immunoreactive cortical neurons in AD brains compared with age-matched neuropathologically unaltered controls. High numbers of extracellular and frequently perivascular endostatin deposits were detected in the cerebral hemispheres. Double-labeling experiments revealed colocalization of endostatin in amyloid-beta(1-40) (Abeta(1-40)), tau protein, and periodic acid-Schiff stain-positive plaques that were surrounded by focal gliosis. Western blotting revealed more 20 kDa endostatin in an AD patient compared with a control. In unstimulated SKNSH supernatants, endostatin was detected that increased predominantly after hypoxia in supernatants and cellular lysates. Abeta(1-40) (80 microg/ml) supplementation to SKNSH neurons for 24 hr completely abolished the release of endostatin. These data show that endostatin is released by neurons to accumulate in amyloid plaques in Alzheimer's disease. Induction by hypoxia and complete abrogation of endostatin release after Abeta(1-40) challenge reveals intricate interactions between the two proteins and opens new avenues for the development of novel treatment strategies of AD patients. (+info)
Expression of type XVIII collagen during healing of corneal incisions and keratectomy wounds.
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PURPOSE: To determine the distribution of type XVIII collagen in mouse ocular tissues and to investigate the expression of type XVIII collagen during healing of corneal incisions and keratectomy wounds. METHODS: Immunohistochemical analysis of type XVIII collagen was performed in mouse ocular tissue, with polyclonal antibodies to the hinge domain. For wound-healing experiments, excimer laser keratectomy and single linear incisions were performed on mouse corneas. The corneas were harvested at various time points after wounding and processed for immunohistochemistry, in situ hybridization, competitive reverse transcription-polymerase chain reaction (RT-PCR), and Western blot analysis. RESULTS: In the unwounded mouse cornea, type XVIII collagen was expressed by the corneal epithelial cells. Type XVIII collagen was immunolocalized to the mouse corneal epithelium, epithelial basement membrane, Descemet's membrane, ciliary epithelium, lens capsule, retinal inner limiting membrane, and Bruch's membrane. In the early stages of wound healing after excimer laser keratectomy (days 3 and 7), type XVIII collagen staining of the epithelial basement membrane was absent, whereas its localization to Descemet's membrane was unchanged. After linear corneal incisions, however, type XVIII collagen was clearly seen in the stroma and in the epithelial basement membrane. Type XVIII collagen immunolocalization to the subepithelial stromal wound region peaked at 1 week after wounding, and its mRNA showed a corresponding temporal increase in expression within the same region after linear corneal incisions. CONCLUSIONS: The results suggest that type XVIII collagen is broadly expressed in ocular tissues and that it may have a role in wound healing, especially after incisional corneal wounds. (+info)
Collagen XVIII, a basement membrane heparan sulfate proteoglycan, interacts with L-selectin and monocyte chemoattractant protein-1.
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Leukocyte infiltration during inflammation is mediated by the sequential actions of adhesion molecules and chemokines. By using a rat ureteral obstruction model, we showed previously that L-selectin plays an important role in leukocyte infiltration into the kidney. Here we report the purification, identification, and characterization of an L-selectin-binding heparan sulfate proteoglycan (HSPG) expressed in the rat kidney. Partial amino acid sequencing and Western blotting analyses showed that the L-selectin-binding HSPG is collagen XVIII, a basement membrane HSPG. The binding of L-selectin to isolated collagen XVIII was specifically inhibited by an anti-L-selectin monoclonal antibody, EDTA, treatment of the collagen XVIII with heparitinase or heparin but not by chemically desulfated heparin. A cell binding assay showed that the L-selectin-collagen XVIII interaction mediates cell adhesion. Interestingly, collagen XVIII also interacted with a chemokine, monocyte chemoattractant protein-1, and presented it to a monocytic cell line, THP-1, which enhanced the alpha(4)beta(1) integrin-mediated binding of the THP-1 cells to vascular cell adhesion molecule-1. Thus, collagen XVIII may provide a link between selectin-mediated cell adhesion and chemokine-induced cellular activation and accelerate the progression of leukocyte infiltration in renal inflammation. (+info)
Recombinant endostatin forms amyloid fibrils that bind and are cytotoxic to murine neuroblastoma cells in vitro.
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Endostatin is a fragment of collagen XVIII that acts as an endogenous inhibitor of tumor angiogenesis and tumor growth. Anti-tumor effects have been described using both soluble and insoluble recombinant endostatin. However, differences in endostatin structure are likely to cause differences in bioactivity. In the present study we have investigated the structure and cellular effects of insoluble endostatin. We found that insoluble endostatin shows all the hallmarks of amyloid aggregates. Firstly, it binds Congo red and shows the characteristic apple-green birefringe when examined under polarized light. Secondly, electron microscopy shows that endostatin forms short unbranched fibrils. Thirdly, X-ray analysis shows the abundant presence of cross-beta sheets, the tertiary structure that underlies fibrillogenesis. None of these properties was observed when examining soluble endostatin. Soluble endostatin can be triggered to form cross-beta sheets following denaturation, indicating that endostatin is a protein fragment with an inherent propensity to form amyloid deposits. Like beta-amyloid, found in the brains of patients with Alzheimer's disease, amyloid endostatin binds to and is toxic to neuronal cells, whereas soluble endostatin has no effect on cell viability. Our results demonstrate a previously unrecognized functional difference between soluble and insoluble endostatin, only the latter acting as a cytotoxic amyloid substance. (+info)
Delivery of endostatin in experimental cancer therapy.
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Endostatin, the 20 kDa C-terminal fragment of collagen XVIII, has been shown to be an effective inhibitor of tumour angiogenesis and growth in different experimental systems and is currently in Phase II/III clinical trials. One challenging aspect of anti-angiogenic treatment is the mode of delivery of the active compound. In this paper we review some of the basic knowledge of endostatin and look specifically into the different possible ways in which endostatin may be administered. (+info)
Intraocular expression of endostatin reduces VEGF-induced retinal vascular permeability, neovascularization, and retinal detachment.
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Endostatin, a proteolytic fragment of collagen XVIII, is an endogenous inhibitor of tumor angiogenesis that also inhibits choroidal neovascularization. In this study, we assessed the effects of increased intraocular expression of endostatin on vascular endothelial growth factor (VEGF)-induced changes in the retina. After subretinal injection of a pair of gutless adenoviral vectors (AGV) designed to provide tamoxifen-inducible expression of endostatin, diffuse endostatin immunoreactivity was induced thoroughout the retina by administration of tamoxifen. Induction of endostatin in double transgenic mice with doxycycline-induced expression of VEGF in the retina resulted in significant suppression of leakage of intravascular [3H]mannitol into the retina. The ability of endostatin to reduce VEGF-induced retinal vascular permeability was confirmed by using [3H]mannitol leakage and two other parameters, fluorescein leakage and retinal thickness, after subretinal injection of a bovine immunodeficiency lentiviral vector coding for endostatin (BIV-vectored endostatin, or BIVendostatin). Subretinal injection of BIVendostatin resulted in more discrete, less intense staining for endostatin in the retina than that seen with the inducible AGV system, which suggested lower levels and allowed visualization of sites where endostatin was concentrated. Endostatin staining outlined retinal blood vessels, which suggested endostatin binding to a component of vessel walls. More prolonged or higher level expression of VEGF in the retina resulted in neovascularization and retinal detachment, both of which were also significantly reduced by BIVendostatin. These data suggest that endostatin may be an endogenous inhibitor of vasopermeability as well as neovascularization. In patients with diabetic retinopathy, endostatin gene transfer may provide a way to decrease the risk of three causes of visual loss: macular edema, neovascularization, and retinal detachment. (+info)
Endostatin expression by MDA-MB-435 breast cancer cells effectively inhibits tumor growth.
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Tumors must induce the formation of new blood vessels in order to grow and metastasize. Endostatin, a cleaved product of collagen XVIII, inhibits endothelial cell proliferation and suppresses tumor growth and metastases. Several recent reports have questioned the efficacy of endostatin as a tumor suppressor in experimental animals. Our objective was to determine whether endostatin expression in breast cancer cells inhibits neovascularization and tumor growth in nude mice. MDA-MB-435 cells were transfected with an endostatin expression vector while control cells were transfected with an empty vector. Endostatin expression and secretion were confirmed by RT-PCR and a dot blot assay. No differences were observed in the growth rates of the endostatin-expressing and control clones in vitro. When injected into male and female nude mice, tumors from the control clones increased in size 10-15 fold over 8-10 weeks. In contrast, the endostatin clones formed small tumors which did not increase in size after the first 3 weeks. The endostatinderived tumors had a significantly higher apoptotic index (5.6%) compared to controls (2.0%) and showed a marked reduction in vascularization. In conclusion, expression of endostatin in MDA-MB-435 breast cancer cells effectively suppressed breast tumor growth by inhibiting angiogenesis and increasing apoptosis. (+info)
Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha v beta 3 and alpha 5 beta 1 integrins.
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Tumstatin and endostatin are two inhibitors of angiogenesis derived from precursor human collagen molecules known as alpha 3 chain of type IV collagen and alpha1 chain of type XVIII collagen, respectively. Although both these inhibitors are noncollagenous (NC1) domain fragments of collagens, they only share a 14% amino acid homology. In the present study we evaluated the functional receptors, mechanism of action, and intracellular signaling induced by these two collagen-derived inhibitors. Human tumstatin prevents angiogenesis via inhibition of endothelial cell proliferation and promotion of apoptosis with no effect on migration, whereas human endostatin prevents endothelial cell migration with no effect on proliferation. We demonstrate that human tumstatin binds to alpha v beta 3 integrin in a vitronectin/fibronectin/RGD cyclic peptide independent manner, whereas human endostatin competes with fibronectin/RGD cyclic peptide to bind alpha 5 beta 1 integrin. The activity of human tumstatin is mediated by alpha v beta 3 integrin, whereas the activity of human endostatin is mediated by alpha 5 beta 1 integrin. Additionally, although human tumstatin binding to alpha v beta 3 integrin leads to the inhibition of Cap-dependent translation (protein synthesis) mediated by focal adhesion kinase/phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 pathway, human endostatin binding to alpha 5 beta 1 integrin leads to the inhibition of focal adhesion kinase/c-Raf/MEK1/2/p38/ERK1 mitogen-activated protein kinase pathway, with no effect on phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 and Cap-dependent translation. Collectively, such distinct properties of human tumstatin and human endostatin provide the first insight into their diverse antiangiogenic actions and argue for combining them for targeting tumor angiogenesis. (+info)