Endostatin, a carboxyl-terminal fragment of collagen XVIII, has been shown to regress tumors in mice. In this study, we have analyzed the mechanism of endostatin action on endothelial cells and nonendothelial cells. Endostatin treatment of cow pulmonary artery endothelial cells caused apoptosis, as demonstrated by three methods, annexin V-fluorescein isothiocyanate staining, caspase 3, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling assay. Moreover, addition of endostatin led to a marked reduction of the Bcl-2 and Bcl-XL anti-apoptotic protein, whereas Bax protein levels were unaffected. These effects were not seen in several nonendothelial cells. Collectively, these findings provide important mechanistic insight into endostatin action. (+info)
Endostatin binds to blood vessels in situ independent of heparan sulfate and does not compete for fibroblast growth factor-2 binding.
Endostatin is a carboxyl-terminal proteolytic fragment of collagen XVIII and a potent inhibitor of angiogenesis. The mechanism of action is unknown, but the crystal structure of endostatin predicts a prominent heparan sulfate binding site, suggesting that endostatin competitively inhibits heparin-binding angiogenic factors, such as basic fibroblast growth factor (FGF-2). The goal of the study was to map endostatin binding sites in intact human tissues and to determine whether this binding is heparan sulfate dependent. In situ binding was performed with recombinant epitope-tagged murine endostatin. Endostatin predominantly binds to blood vessels of different calibers in a saturable fashion. In addition, binding to some epithelial basement membranes is seen. The localization pattern is similar to that reported for collagen XVIII, endostatin's parent molecule. In breast carcinomas, endostatin co-localizes largely with FGF-2. In a surprising contrast to FGF-2, endostatin binding is resistant to treatment with heparitinase, demonstrating that binding is not mediated by heparan sulfate proteoglycans. Furthermore, FGF-2 and heparin do not compete for endostatin binding, providing additional evidence for the discreteness of endostatin and FGF-binding sites. (+info)
Angiogenesis inhibitor endostatin is a distinct component of elastic fibers in vessel walls.
Theendothelial cell inhibitor endostatin (22 kDa) is part of the carboxyl-terminal globular domain of collagen XVIII and shows a widespread tissue distribution. Immunohistology of adult mouse tissues demonstrated a preferred localization in many vessel walls and some other basement membrane zones. A strong immunogold staining was observed across elastic fibers in the multiple elastic membranes of aorta and other large arteries. Staining was less strong along sparse elastic fibers of veins and almost none was observed in the walls of arterioles and capillaries. Strong evidence was also obtained for some intracellular and basement membrane associations. Immunogold double staining of elastic fibers showed a close colocalization of endostatin with fibulin-2, fibulin-1, and nidogen-2, but not with perlecan. Reasonable amounts of endostatin could be extracted from aorta and skin by EDTA, followed by detergents, with aorta being the richest source of the inhibitor identified so far. Solubilizations with collagenase and elastase were approximately fivefold less efficient. Immunoblots of aortic extracts detected major endostatin components of 22-25 kDa whereas skin extracts also contained some larger components. Solid-phase assays demonstrated distinct binding of recombinant mouse endostatin to the fibulins and nidogen-2, consistent with their tissue colocalization. Together, the data indicate several different ways for endostatin to be associated with the extracellular matrix, and its release may determine biological activation. This also defines a novel function for some elastic tissues. (+info)
The generation of endostatin is mediated by elastase.
Endostatin, a potent inhibitor of angiogenesis and tumor growth, is a COOH-terminal fragment of collagen XVIII derived through cleavage of an Ala-His linkage by an as yet unidentified endostatin-processing enzyme. Endostatin was originally isolated from the conditioned medium of hemangioendothelioma (EOMA) cells. By investigating the processing of collagen XVIII to endostatin by EOMA cells, we show here that the generation of endostatin can be mediated by an elastase activity. We also show that several members of the elastase family can act as an endostatin-processing enzyme by specifically cleaving the Ala-His linkage and releasing endostatin from a precursor molecule. We further suggest that the generation of endostatin from collagen XVIII is at least a two-step process, involving a metal-dependent early step and an elastase activity-dependent final step. (+info)
Purification and characterization of recombinant murine endostatin in E. coli.
Endostatin, a carboxyl-terminal fragment of collagen XVIII is known as an anti-angiogenic agent, that specifically inhibits the proliferation of endothelial cell and the growth of several primary tumor. We report here the purification and characterization of the recombinant murine endostatin (rmEndostatin) which was expressed in a prokaryotic expression system. This rmEndostatin has similar physiochemical properties of yeast-produced recombinant endostatin, and it also specifically inhibits the proliferation and migration of bovine capillary endothelial cells stimulated by basic fibroblast growth factor. The biological activity of rmEndostatin was also shown by its anti-angiogenic ability on the chorioallantoic membrane of chick embryo in vivo. In this article, we demonstrate the refolding and purification of rmEndostatin, expressed using E. coli system, to a biologically active and soluble form. In addition, these results confirm the activity of endostatin as a potent anti-angiogenic agent. (+info)
Reality testing in cancer treatment: the phase I trial of endostatin.
Shortly before his death in 1995, Kenneth B. Schwartz, a cancer patient at Massachusetts General Hospital (MGH), founded The Kenneth B. Schwartz Center at MGH. The Schwartz Center is a non-profit organization dedicated to supporting and advancing compassionate health care delivery which provides hope to the patient, support to caregivers, and encourages the healing process. The center sponsors the Schwartz Center Rounds, a monthly multidisciplinary forum where caregivers reflect on important psychosocial issues faced by patients, their families, and their caregivers, and gain insight and support from fellow staff members. The September 1999 Schwartz Center Rounds addressed the growing attention around the phase I trial of Endostatin. Endostatin represents a new treatment paradigm. It is an anti-angiogenic protein, an endogenous fragment of collagen XVIII. In an attempt to ensure a fair allocation of a very limited number of treatment slots in this classical phase I trial, a lottery was established. More than 1,400 patients enrolled within two days of the lottery, all vying for three places in the first cohort. Two contrasting cases are presented, each a potentially eligible patient. The discussion focuses on the dilemma presented by patients desperate for an unproven treatment and the responsibility of staff to explain and support without compounding the hype or suffocating the hope. (+info)
Secreted cathepsin L generates endostatin from collagen XVIII.
Endostatin, an inhibitor of angiogenesis and tumor growth, was identified originally in conditioned media of murine hemangioendothelioma (EOMA) cells. N-terminal amino acid sequencing demonstrated that it corresponds to a fragment of basement membrane collagen XVIII. Here we report that cathepsin L is secreted by EOMA cells and is responsible for the generation of endostatin with the predicted N-terminus, while metalloproteases produce larger fragments in a parallel processing pathway. Efficient endostatin generation requires a moderately acidic pH similar to the pericellular milieu of tumors. The secretion of cathepsin L by a tumor cell line of endothelial origin suggests that this cathepsin may play a role in angiogenesis. We propose that cleavage within collagen XVIII's protease-sensitive region evolved to regulate excessive proteolysis in conditions of induced angiogenesis. (+info)
Increased concentrations of the circulating angiogenesis inhibitor endostatin in patients with systemic sclerosis.
OBJECTIVE: Endostatin is an angiogenesis inhibitor derived from type XVIII collagen. The aim of this study was to determine the concentrations of circulating endostatin in patients with systemic sclerosis (SSc), and to assess the relationship between these concentrations, extension of tissular sclerosis, and presence of cutaneous scars or ulcers. METHODS: The study involved 50 patients with SSc and 30 healthy subjects. Cutaneous extension of sclerosis was graded according to Barnett's classification system: 33 patients had grade I SSc and 17 patients had grades II or III SSc. The results of pulmonary function tests were abnormal in 31 of 50 patients, 8 of whom also had abnormalities on chest radiograms. Cutaneous scars or ulcers were found in 22 of 50 patients. Endostatin concentrations were determined using a competitive enzyme immunoassay method. RESULTS: The mean circulating endostatin concentration was significantly higher in the SSc group than in the healthy subjects group (mean +/- SD 53.2 +/- 22.4 ng/ml versus 9.9 +/- 9.7 ng/ml; P < 10(-4)), in patients with grade II or grade III SSc than in patients with grade I SSc (63.2 +/- 20.2 ng/ml versus 45.1 +/- 15.6 ng/ml; P < 10(-2)), in patients with abnormal findings on chest radiograms than in patients with normal findings on chest radiograms (67.6 +/- 22.4 ng/ml versus 50.4 +/- 21.6 ng/ml; P < 0.05), and in patients with cutaneous scars or ulcers than in patients without these manifestations (60.9 +/- 25.9 ng/ml versus 47.2 +/- 13.3 ng/ml; P < 10(-2)). CONCLUSION: Circulating endostatin concentrations are significantly increased in patients with SSc. Production of endostatin may result from tissular sclerosis and could contribute to the development of ischemic manifestations. (+info)