Biomedical subjects

Rupert Timpl

Publications and source records attributed to Rupert Timpl.

At least 37 records · Page 2Linked to original sources

PubMed2002

Role of heparan sulfate domain organization in endostatin inhibition of endothelial cell function.

The anti-angiogenic activity of endostatin (ES) depends on interactions with heparan sulfate (HS). In the present study, intact HS chains of >/=15 kDa bound quantitatively to ES whereas N-sulfated HS decasaccharides, with affinity for several fibroblast growth factor (FGF) species, failed to bind. Instead, ES-binding oligosaccharides composed of mixed N-sulfated and N-acetylated disaccharide units were isolated from pig intestinal HS. A 10/12mer ES-binding epitope was identified, with two N-sulfated regions separated by at least one N-acetylated glucosamine unit (SAS-domain). Cleavage at the N-acetylation site disrupted ES binding. These findings point to interaction between discontinuous sulfated domains in HS and arginine clusters at the ES surface. The inhibitory effect of ES on vascular endothelial growth factor-induced endothelial cell migration was blocked by the ES-binding SAS-domains and by heparin oligosaccharides (12mers) similar in length to the ES-binding SAS-domains, but not by 6mers capable of FGF binding. We propose that SAS-domains modulate the biological activities of ES and other protein ligands with extended HS-binding sites. The results provide a rational explanation for the preferential interaction of ES with certain HS proteoglycan species.

Animals↗

PubMed2002

Binding of mouse nidogen-2 to basement membrane components and cells and its expression in embryonic and adult tissues suggest complementary functions of the two nidogens.

Nidogen-1 binds several basement membrane components by well-defined, domain-specific interactions. Organ culture and gene targeting approaches suggest that a high-affinity nidogen-binding site of the laminin gamma1 chain (gamma1III4) is important for kidney development and for nerve guidance. Other proteins may also bind gamma1III4, although human nidogen-2 binds poorly to the mouse laminin gamma1 chain. We therefore characterized recombinant mouse nidogen-2 and its binding to basement membrane proteins and cells. Mouse nidogen-1 and -2 interacted at comparable levels with collagen IV, perlecan, and fibulin-2 and, most notably, also with laminin-1 fragments P1 and gamma1III3-5, which both contain the gamma1III4 module. In embryos, nidogen-2 mRNA was produced by mesenchyme at sites of epithelial-mesenchymal interactions, but the protein was deposited on epithelial basement membranes, as previously shown for nidogen-1. Hence, binding of both nidogens to the epithelial laminin gamma1 chain is dependent on epithelial-mesenchymal interactions. Epidermal growth factor stimulated expression of both nidogens in embryonic submandibular glands. Both nidogens were found in all studied embryonic and adult basement membranes. Nidogen-2 was more adhesive than nidogen-1 for some cell lines and was mainly mediated by alpha3beta1 and alpha6beta1 integrins as shown by antibody inhibition. These findings revealed extensive coregulation of nidogen-1 and -2 expression and much more complementary functions of the two nidogens than previously recognized.

Animals↗

PubMed2002

Biglycan organizes collagen VI into hexagonal-like networks resembling tissue structures.

The ability of the leucine-rich repeat (LRR) proteins biglycan, decorin, and chondroadherin to interact with collagen VI and influence its assembly to supramolecular structures was studied by electron microscopy and surface plasmon resonance measurements in the BIAcore 2000 system. Biglycan showed a unique ability to organize collagen VI into extensive hexagonal-like networks over a time period of only a few minutes. Only the intact molecule, substituted with two dermatan sulfate chains, had this capacity. Intact decorin, with one dermatan sulfate chain only, was considerably less efficient, and aggregates of organized collagen VI were found only after several hours. Chondroadherin without glycosaminoglycan substitutions did not induce any ordered collagen VI organization. However, all three related LRR proteins were shown to interact with collagen VI using electron microscopy and surface plasmon resonance. Biglycan and decorin were exclusively found close to the N-terminal parts of the collagen VI tetramers, whereas chondroadherin was shown to bind close to both the N- and C-terminal parts of collagen VI. In the formed hexagonal networks, biglycan was localized to the intra-network junctions of the collagen VI filaments. This was demonstrated by electron microscopy after negative staining of gold-labeled biglycan in aggregation experiments with collagen VI.

Animals↗

PubMed2002

Crystal structure of a C-terminal fragment of growth arrest-specific protein Gas6. Receptor tyrosine kinase activation by laminin G-like domains.

Receptor tyrosine kinases of the Axl family are activated by Gas6, the product of growth arrest-specific gene 6. Gas6-Axl signaling is implicated in cell survival, adhesion, and migration. The receptor-binding site of Gas6 is located within a C-terminal pair of laminin G-like (LG) domains that do not resemble any other receptor tyrosine kinase ligand. We report the crystal structure at 2.2-A resolution of a Gas6 fragment spanning both LG domains (Gas6-LG). The structure reveals a V-shaped arrangement of LG domains strengthened by an interdomain calcium-binding site. LG2 of Gas6-LG contains two unusual features: an alpha-helix cradled by one edge of the LG beta-sandwich and a conspicuous patch of surface-exposed hydrophobic residues. Mutagenesis of some residues in this patch reduces Gas6-LG binding to the extracellular domain of Axl as well as Axl activation in glioblastoma cells, identifying a component of the receptor-binding site of Gas6.

Amino Acid Sequence↗

PubMed2002

The 1.9-A crystal structure of the noncollagenous (NC1) domain of human placenta collagen IV shows stabilization via a novel type of covalent Met-Lys cross-link.

Triple-helical collagen IV protomers associate through their N- and C-termini forming a three-dimensional network, which provides basement membranes with an anchoring scaffold and mechanical strength. The noncollagenous (NC1) domain of the C-terminal junction between two adjacent collagen IV protomers from human placenta was crystallized and its 1.9-A structure was solved by multiple anomalous diffraction (MAD) phasing. This hexameric NC1 particle is composed of two trimeric caps, which interact through a large planar interface. Each cap is formed by two alpha 1 fragments and one alpha 2 fragment with a similar previously uncharacterized fold, segmentally arranged around an axial tunnel. Each monomer chain folds into two structurally very similar subdomains, which each contain a finger-like hairpin loop that inserts into a six-stranded beta-sheet of the neighboring subdomain of the same or the adjacent chain. Thus each trimer forms a quite regular, but nonclassical, sixfold propeller. The trimer-trimer interaction is further stabilized by a previously uncharacterized type of covalent cross-link between the side chains of a Met and a Lys residue of the alpha 1 and alpha 2 chains from opposite trimers, explaining previous findings of nonreducible cross-links in NC1. This structure provides insights into NC1-related diseases such as Goodpasture and Alport syndromes.

Amino Acid Sequence↗

PubMed2002

Expression and distribution of laminin alpha1 and alpha2 chains in embryonic and adult mouse tissues: an immunochemical approach.

Protein levels, mRNA expression, and localization of laminin alpha1 and alpha2 chains in development and in adult mice were examined. Recombinant fragments were used to obtain high-titer-specific polyclonal antibodies for establishing quantitative radioimmuno-inhibition assays. This often demonstrated an abundance of alpha2 chain, but also distinct amounts of alpha1 chain for adult tissues. The highest amounts of alpha1 were found in placenta, kidney, testis, and liver and exceeded those of alpha2. All other tissue extracts showed a higher content of alpha2, which was particularly high in heart and muscle when compared to alpha1. Content of gamma1 chain, shared by most laminins, was also analyzed. This demonstrated gamma1 chain levels being equal to or moderately exceeding the sum of alpha1 and alpha2 chains, indicating that these isoforms represent the major known laminin isoforms in most adult mouse tissues so far examined. Moreover, we found good correlation between radioimmuno-inhibition data and mRNA levels of adult tissues as measured by quantitative real-time reverse transcriptase-PCR. Embryonic tissues were also analyzed by radioimmuno-inhibition assays. This demonstrated for day 11 embryos comparable amounts of alpha1 and gamma1 and a more than 25-fold lower content of alpha2. This content increased to about 10% of alpha1 in day 13 embryos. The day 18 embryo showed in heart, kidney, and liver, but not yet in brain and lung, alpha1/alpha2 chain ratios comparable to those in adult tissues. Immunostaining demonstrated alpha1 in Reichert's membrane (day 7.5), while alpha2 could not be detected before day 11.5. These data were compared with immunohistochemical localization results on several more embryonic and adult tissue sections. Our results regarding localization are consistent with those of earlier work with some notable exceptions. This was in part due to epitope masking for monoclonal antibodies commonly used in previous studies in esophagus, intestine, stomach, liver, kidney, and spleen.

Animals↗

PubMed2002

Proteinases of the bone morphogenetic protein-1 family convert procollagen VII to mature anchoring fibril collagen.

Collagen VII is the major structural component of the anchoring fibrils at the dermal-epidermal junction in the skin. It is secreted by keratinocytes as a precursor, procollagen VII, and processed into mature collagen during polymerization of the anchoring fibrils. We show that bone morphogenetic protein-1 (BMP-1), which exhibits procollagen C-proteinase activity, cleaves the C-terminal propeptide from human procollagen VII. The cleavage occurs at the BMP-1 consensus cleavage site SYAA/DTAG within the NC-2 domain. Mammalian tolloid-like (mTLL)-1 and -2, two other proteases of the astacin enzyme family, were able to process procollagen VII at the same site in vitro. Immunohistochemical and genetic evidence supported the involvement of these enzymes in cleaving type VII procollagen in vivo. Both BMP-1 and mTLL-1 are expressed in the skin and in cultured cutaneous cells. A naturally occurring deletion in the human COL7A1 gene, 8523del14, which is associated with dystrophic epidermolysis bullosa and eliminates the BMP-1 consensus sequence, abolished processing of procollagen VII, and in mutant skin procollagen VII accumulated at the dermal-epidermal junction. On the other hand, deficiency of BMP-1 in the skin of knockout mouse embryos did not prevent processing of procollagen VII to mature collagen, suggesting that mTLL-1 and/or mTLL-2 can substitute for BMP-1 in the processing of procollagen VII in situ.

Animals↗

PubMed2002

The type XIII collagen ectodomain is a 150-nm rod and capable of binding to fibronectin, nidogen-2, perlecan, and heparin.

Type XIII collagen consists of a short N-terminal intracellular domain, a transmembrane domain, and a collagenous ectodomain, and it is found at many sites of cell adhesion. We report on the characterization of recombinant type XIII collagen. The shed ectodomain was purified from insect cell culture medium and shown to form 240-kDa trimers with a T(m) of 42 degrees C. Correct chain association into a triple-helical conformation was confirmed by limited pepsin digestion and CD spectroscopy. Rotary shadowing electron microscopy of the ectodomain revealed it to be a 150-nm rod with two flexible hinges separating 31-, 52-, and 68-nm portions. The rods represent the collagenous domains 1-3, and the hinges coincide with the non-collagenous domains 2 and 3. By using surface plasmon resonance analysis, the ectodomain showed interaction with immobilized fibronectin, nidogen-2, and perlecan with K(D) values in the nanomolar range. The binding sites of type XIII collagen for fibronectin were localized to the collagenous domains, whereas the binding activities for nidogen-2 and perlecan resided in the pepsin-sensitive portions of the ectodomain. Furthermore, the ectodomain bound significantly to heparin, which also inhibited shedding of the ectodomain in insect cell cultures. The results reveal that type XIII collagen is notably distinct in its structure compared with other cell-surface proteins, and the in vitro binding with fibronectin, heparin, and two basement membrane components is indicative of multiple cell-matrix interactions in which this ubiquitously expressed protein participates.

Amino Acid Sequence↗

PubMed2002

Endostatin regulates endothelial cell adhesion and cytoskeletal organization.

Endostatin, an endogenous angiogenesis inhibitor, attenuates endothelial cell migration through an unknown mechanism. We show that endostatin induced tyrosine phosphorylation of focal adhesion kinase and paxillin, and promoted formation of focal adhesions and actin stress fibers, similar to fibroblast growth factor-2 (FGF-2). In cells cotreated with endostatin and FGF-2, focal adhesions and actin stress fibers were decreased, indicating that endostatin disturbs cell-matrix adhesion. Reduced tyrosine phosphorylation and cytoplasmic relocalization of beta-catenin in cells treated with FGF-2 and endostatin indicates that loosening of cell-cell adhesion is also disturbed by endostatin. These data provide a molecular basis both for the lack of effect of endostatin on the normal, quiescent vasculature, and its antagonistic effects on stimulated tumor vessels.

Actins↗

PubMed2002

Functional studies on recombinant domains of Mac-2-binding protein.

Mac-2-binding protein (M2BP) is a secreted glycoprotein suggested to have a role in host defense. It forms linear and ring-shaped oligomers, with each ring segment being composed of two monomers. We have produced recombinant human M2BP fragments comprising domains 1 and 2 (M2BP-1,2) and domains 3 and 4 (M2BP-3,4) in 293 human kidney cells to characterize structural and functional properties of M2BP. Both fragments were obtained in a native and glycosylated form, as analyzed by CD spectroscopy, trypsin susceptibility, and enzymatic deglycosylation. These results strongly suggest that both fragments are autonomous folding units. All three potential N-glycosylation sites in M2BP-1,2 and all four in M2BP-3,4 were found to be occupied. M2BP-1,2 expressed in tunicamycin-treated cells contained no glycosyl residues, indicating that O-glycosylation is not occurring. Ultracentrifugation revealed that M2BP-1,2 is homogeneously dimeric in the nanomolar range reflecting the properties of intact M2BP. Domain 2 (BTB/POZ domain) is thus identified as the dimerization domain of M2BP, because it has been formerly shown that recombinant domain 1 is monomeric. M2BP-3,4 showed a concentration-dependent self-association, and aggregates of different size and shape were shown by electron microscopy. In contrast to this irregular aggregation of M2BP-3,4, it has been formerly shown that a fragment comprising domains 2-4 still has the ability to form ring-like structures, although the rings are protein-filled, and thus domain 2 appears to be indispensable for ring formation. Solid phase assays showed that M2BP-3,4 contains binding sites for galectin-3, nidogen, and collagens V and VI, whereas M2BP-1,2 is inactive in binding. Both fragments showed no cell adhesive activity in contrast to native M2BP, suggesting that a concerted binding action and/or multivalent interactions of rings are necessary for cell attachment.

Antigens, Neoplasm↗

PubMed2002

Complete sequence, recombinant analysis and binding to laminins and sulphated ligands of the N-terminal domains of laminin alpha3B and alpha5 chains.

The N-terminal sequences of mouse laminin alpha3B and alpha5 chains have been completed and demonstrate the presence of a signal peptide followed by a complete laminin N-terminal (LN) module (domain VI). These signal peptides were released after recombinant production of larger fragments comprising domains VI/V (45-65 kDa) from this region yielding properly folded proteins, which were secreted from HEK-293-EBNA cells. Pepsin digestion of these fragments yielded products of 25-35 kDa, which consisted only of domain V. The alphaVI/V fragments were able to inhibit self-assembly of laminin-1, with those from the alpha3B and alpha5 chains being more active than those from alpha1 and alpha2 chains. Domain V fragments, however, showed a reduced activity, indicating the major contribution of the LN module in inhibition. These interactions were confirmed by surface-plasmon-resonance assays demonstrating moderate affinities (K(d)=0.02 to >6 microM) for the binding to laminin-1. This indicated that laminins containing alpha3B or alpha5 chains should also be able to form non-covalent networks by polymerization. The LN modules also showed heparin binding in affinity chromatography, which was strongest for alpha1/alpha2, moderate for alpha3B, whereas no binding was observed for alpha5. They all bound to heparan sulphate chains of perlecan and to sulphatides, with a lower variability in binding activity. Specific antibodies were raised against alpha3BVI/V and alpha5VI/V and were shown to stain basement membrane zones in various mouse tissues. These antibodies also allowed the identification of a new laminin assembly form 5B consisting of alpha3B, beta3 and gamma2 chains.

Amino Acid Sequence↗

PubMed2002

The leucine-rich repeat protein PRELP binds perlecan and collagens and may function as a basement membrane anchor.

PRELP (proline arginine-rich end leucine-rich repeat protein) is a heparin-binding leucine-rich repeat protein in connective tissue extracellular matrix. In search of natural ligands and biological functions of this molecule, we found that PRELP binds the basement membrane heparan sulfate proteoglycan perlecan. Also, recombinant perlecan domains I and V carrying heparan sulfate bound PRELP, whereas other domains without glycosaminoglycan substitution did not. Heparin, but not chondroitin sulfate, inhibited the interactions. Glycosaminoglycan-free recombinant perlecan domain V and mutated domain I did not bind PRELP. The dissociation constants of the PRELP-perlecan interactions were in the range of 3-18 nm as determined by surface plasmon resonance. As expected, truncated PRELP, without the heparin-binding domain, did not bind perlecan. Confocal immunohistochemistry showed that PRELP outlines basement membranes with a location adjacent to perlecan. We also found that PRELP binds collagen type I and type II through its leucine-rich repeat domain. Electron microscopy visualized a complex with PRELP binding simultaneously to the triple helical region of procollagen I and the heparan sulfate chains of perlecan. Based on the location of PRELP and its interaction with perlecan heparan sulfate chains and collagen, we propose a function of PRELP as a molecule anchoring basement membranes to the underlying connective tissue.

Animals↗

PubMed2002

Recombinant human laminin-10 (alpha5beta1gamma1). Production, purification, and migration-promoting activity on vascular endothelial cells.

The laminin (LN) family of large heterotrimeric extracellular matrix glycoproteins has multiple functions: LNs take part in the regulation of processes such as cell migration, differentiation, and proliferation, in addition to contributing to the structure of basement membranes. LN-10, composed of alpha5, beta1, and gamma1 chains, is widely distributed in most basement membranes of both epithelia and endothelia. We determined the complete human cDNA sequence for the LN alpha5 chain and produced recombinant human LN-10 (rLN-10) in HEK293 cells by triple transfection of full-length cDNAs encoding the human LN alpha5, beta1, and gamma1 chains. The rLN-10 was purified using affinity chromatography and had an apparent molecular mass of approximately 800 kDa in SDS-PAGE and a native domain structure in rotary shadowing electron microscopy. By using function-blocking monoclonal antibodies, integrin alpha(3)beta(1) was found to be a major mediator of adhesion of HT-1080 and human saphenous vein endothelial cells. Human saphenous vein endothelial cells adhered more strongly to rLN-10 than to LN-1 and LN-8 and showed better migration on rLN-10, compared with several other matrices. Considering the cell adhesive and migration-promoting properties of rLN-10 on endothelial cells, this molecule could be useful in improving the biocompatibility and endothelialization of vascular grafts.

Base Sequence↗

PubMed2002

Defective laminin 5 processing in cylindroma cells.

Cylindromas are benign skin tumors occurring as multiple nodules characteristically well circumscribed by an excess of basement membrane-like material. To determine the molecular defects leading to extracellular matrix accumulation, the ultrastructural, immunological, and biochemical properties of cylindroma tissue and isolated cells were analyzed. In cylindromas, hemidesmosomes are reduced in number, heterogeneous and immature compared to the normal dermal-epidermal junction. Expression of the alpha6beta4 integrin in tumor cells is weaker than in basal keratinocytes of the epidermis. Moreover, although in the epidermis alpha2beta1-integrin expression is restricted to the basal cell layer, it is found in all neoplastic cells within the nodules. Laminin 5 is present throughout the whole thickness of the basement membrane-like zone whereas laminin 10 is restricted to the interface adjacent to the tumor cells. Furthermore, laminin 5 is not properly processed and most of the alpha3A and gamma2 laminin chains remain as 165-kd and 155-kd polypeptides, respectively. Mature laminin 5 is thought to be necessary for correct hemidesmosome and basement membrane formation and its abnormal processing, as well as the low expression of alpha6beta4 integrins, could explain the lack of mature hemidesmosomes. Together, the results show that multiple molecular defects, including alteration of laminin 5 and its integrin receptors, contribute to structural aberrations of the basement membrane and associated structures in cylindromas.

Antibodies↗

PubMed2002

Evidence of nidogen-2 compensation for nidogen-1 deficiency in transgenic mice.

Previous studies have shown that inhibition of nidogen-laminin binding interferes with basement membrane stabilization in various mouse organ cultures while no overt phenotype has been observed following inactivation of the nidogen-1 gene in mice. We have now used recombinant mouse nidogen-1 and nidogen-2 in order to evaluate a possible compensation between the two isoforms in the knock-out mice. Essentially, a comparable in vitro binding of nidogens-1 and -2 to the same laminin gamma1 chain structure and to several other basement membrane proteins has been revealed. Quantitative radioimmuno-assays have demonstrated high concentrations of nidogen-1 exceeding those of laminin gamma1 and nidogen-2 by factors of 5 and 20-50, respectively, in tissue extracts of wild-type mice. A three- to sevenfold increase in nidogen-2 was observed in heart and muscle of mice with nidogen-1 deficiency and confirmed by a similar increase in the intensity of immunogold staining of these tissues. However, a few of the tissues from mice with the gene knock-out still contained some nidogen-1-like immunoreactivity (1% of wild-type). Furthermore, both nidogen isoforms showed a similar distribution in various organs during embryonic development which, however, as shown previously, changed in some adult tissues. The data support the nidogen-2 compensation hypothesis to explain the limited phenotype observed following elimination of the nidogen-1 gene.

Adaptation, Physiological↗

PubMed2002

Domain IV of mouse laminin beta1 and beta2 chains.

Domain IV, consisting of about 230 residues, represents a particular protein module so far found only in laminin beta1 and beta2 chains. Both domains were obtained by recombinant production in mammalian cells. They showed a globular structure, as expected from electron microscopic examination of laminins. Fragment beta1IV was obtained as a monomer and a disulfide-bonded dimer, and both were modified to approximately 50% by a single chondroitin sulfate chain attached to Ser721 of an SGD consensus sequence. Dimerization is caused by an odd number of cysteines, with three of them having a partial thiol character. Whether both modifications also occur in tissue forms of laminin remains to be established. Fragment beta2IV was only obtained as a monomer, as it lacked one crucial cysteine and the SGD sequence. It required, however, the presence of two adjacent LE modules for proper folding. Polyclonal antibodies raised against both fragments showed no cross-reaction with each other and allowed establishment of beta chain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 5-25-fold lower content of beta2 compared with beta1 chains in various tissue extracts of adult mice. Tissues derived from beta2-deficient mice failed to react with the beta2-specific antibodies but showed a twofold higher content of beta1 than heterozygotes. The antibodies to beta2 showed broader tissue staining than reported previously, including in particular a distinct reaction with the extrasynaptic endomysium of skeletal muscle. Immunogold staining localized both beta chains primarily to basement membranes of kidney, muscle and various other tissues.

Amino Acid Sequence↗

PubMed2002

Structure and function of collagen-derived endostatin inhibitors of angiogenesis.

Endostatins are inhibitors of endothelial cell migration and angiogenesis and have been shown to reduce tumor growth in animal models. They are derived from the nontriplehelical C-terminal NC1 domains of collagens XV and XVIII, which are released proteolytically in trimeric form and further converted to monomeric endostatins of about 20 kDa. Both endostatin isoforms share a compact globular fold, but differ in certain binding properties for proteins and cells, as well as in tissue distribution. Differences in activity were found between NC1 domains and endostatins and are related to the oligomerization state. Endostatin effects are not restricted to endothelial cells, but also control renal epithelial cells and neuronal guidance in C. elegans. Cellular receptors are still insufficiently characterized and include for endostatin-XVIII heparan sulfate proteoglycans. Receptor engagement elicits various downstream effects including tyrosine kinase and gene activation. Much remains to be learned, however, about details of the signal transduction cascades and how they interfere with pro-angiogenic factors under physiological conditions and during therapeutic treatment.

Animals↗

PubMed2002

Endostatin reduces vascularization, blood flow, and growth in a rat gliosarcoma.

Endostatin, the 20-kDa C-terminal fragment of collagen XVIII, has previously been shown to inhibit growth and induce regression of different experimental tumors in rodents. In this study, we show that recombinant murine and human endostatin, produced in 293 EBNA cells and yeast, respectively, inhibit ectotopic as well as orthotopic growing BT4Cn gliosarcomas in BD-IX rats. In rats in which s.c. gliomas were grown for a total of 29 days, systemic treatment with recombinant murine endostatin induced about 50% reduction of intratumoral blood flow and tumor size after only 10 days of therapy. In contrast, the blood flow to irrelevant organs was unaffected by endostatin, indicating its specificity of action. Tumors were not observed to increase in size or regrow after cessation of therapy. Furthermore, endostatin-treated rats with i.c. tumors had significantly longer survival time than did untreated controls. In the treated rats, endostatin therapy resulted in a reduced tumor blood vessel volume and an increased tumor cell density with an increased apoptotic index within a given tumor volume, as verified by flow cytometry and by staining with deoxynucleotidyltransferase-mediated dUTP nick-end labeling. This work verifies the general anti-angiogenic and antitumor effects of endostatin and indicates that the protein may also be considered as a treatment strategy for malignant brain tumors.

Angiogenesis Inhibitors↗