Spheroidal Organoids Reproduce Characteristics of Longitudinal Depth Zones in Bovine Articular Cartilage.

Articular cartilage has multiple histologically distinct longitudinal depth zones. Development and pathogenesis occur throughout these zones. Cartilage explants, monolayer cell culture and reconstituted 3-dimensional cell constructs have been used for investigating mechanisms of pathophysiology in articular cartilage. Such models have been insufficient to reproduce zone-dependent cellular characteristics and extracellular matrix (ECM) upon investigation into cartilage development and pathogenesis. Therefore, we defined a chondrocyte spheroid model consistently formed with isolated chondrocytes from longitudinal depth zones without extrinsic materials. This spheroid showed zone-dependent characteristics of size, cartilage-specific ECM (collagen types I and II, aggrecan and keratan sulfate) and gene expressions of anabolic and catabolic molecules (matrix molecules and matrix metalloproteinase-13). In addition, the spheroid model is small enough to maintain the viability of cells and point symmetry to analyze the gradient of diffusive molecules. This spheroid organoid model will be useful to elucidate the mechanism of histogenesis and pathogenesis in articular cartilage.

Role of polycystin-1 in bone remodeling: orthodontic tooth movement study in mutant mice.

OBJECTIVE: To test the hypothesis that polycystin-1 (PC1) is involved in orthodontic tooth movement as a mechanical sensor. MATERIALS AND METHODS: The response to force application was compared between three mutant and four wild-type 7-week-old mice. The mutant mice were PC1/Wnt1-cre, lacking PC1 in the craniofacial region. An orthodontic closed coil spring was bonded between the incisor and the left first molar, applying 20 g of force for 4 days. Micro-computed tomography, hematoxylin and eosin staining, and tartrate-resistent acid phosphatase (TRAP) staining were used to study the differences in tooth movement among the groups. RESULTS: In the wild-type mice the bonded molar moved mesially, and the periodontal ligament (PDL) was compressed in the compression side. The compression side showed a hyalinized zone, and osteoclasts were identified there using TRAP staining. In the mutant mice, the molar did not move, the incisor tipped palatally, and there was slight widening of the PDL in the tension area. Osteoclasts were not seen on the bone surface or on the compression side. Osteoclasts were only observed on the other side of the bone-in the bone marrow. CONCLUSIONS: These results suggest a difference in tooth movement and osteoclast activity between PC1 mutant mice and wild-type mice in response to orthodontic force. The impaired tooth movement and the lack of osteoclasts on the bone surface in the mutant working side may be related to lack of signal from the PDL due to PC1 deficiency.

Molecular interplay between endostatin, integrins, and heparan sulfate.

Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. alpha5beta1 and alphavbeta3 integrins form stable complexes with immobilized endostatin (KD=approximately 1.8x10(-8) M, two-state model). Two arginine residues (Arg27 and Arg139) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the alphavbeta3 integrin at the interface between the beta-propeller domain of the alphav subunit and the betaA domain of the beta3 subunit. In addition, we report that alpha5beta1 and alphavbeta3 integrins bind to heparin/heparan sulfate. The ectodomain of the alpha5beta1 integrin binds to haparin with high affinity (KD=15.5 nM). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and alpha5beta1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.

Lack of type VIII collagen in mice ameliorates diabetic nephropathy.

OBJECTIVE: Key features of diabetic nephropathy include the accumulation of extracellular matrix proteins. In recent studies, increased expression of type VIII collagen in the glomeruli and tubulointerstitium of diabetic kidneys has been noted. The objectives of this study were to assess whether type VIII collagen affects the development of diabetic nephropathy and to determine type VIII collagen-dependent pathways in diabetic nephropathy in the mouse model of streptozotocin (STZ)-induced diabetes. RESEARCH DESIGN AND METHODS: Diabetes was induced by STZ injections in collagen VIII-deficient or wild-type mice. Functional and histological analyses were performed 40 days after induction of diabetes. Type VIII collagen expression was assessed by Northern blots, immunohistochemistry, and real-time PCR. Proliferation of primary mesangial cells was measured by thymidine incorporation and direct cell counting. Expression of phosphorylated extracellular signal-regulated kinase (ERK1/2) and p27(Kip1) was assessed by Western blots. Finally, Col8a1 was stably overexpressed in mesangial cells. RESULTS: Diabetic wild-type mice showed a strong renal induction of type VIII collagen. Diabetic Col8a1(-)/Col8a2(-) animals revealed reduced mesangial expansion and cellularity and extracellular matrix expansion compared with the wild type. These were associated with less albuminuria. High-glucose medium as well as various cytokines induced Col8a1 in cultured mesangial cells. Col8a1(-)/Col8a2(-) mesangial cells revealed decreased proliferation, less phosphorylation of Erk1/2, and increased p27(Kip1) expression. Overexpression of Col8a1 in mesangial cells induced proliferation. CONCLUSIONS: Lack of type VIII collagen confers renoprotection in diabetic nephropathy. One possible mechanism is that type VIII collagen permits and/or fosters mesangial cell proliferation in early diabetic nephropathy.

The polycystic kidney disease 1 (Pkd1) gene is required for the responses of osteochondroprogenitor cells to midpalatal suture expansion in mice.

Mechanical stress is known to modulate postnatal skeletal growth and development. However, the mechanisms underlying the mechanotransduction are not fully understood. Polycystin-1 (PC1) is a promising candidate among proteins that may play a role in the process as it has been shown to function as a flow sensor in renal epithelium and it is known to be important for skeletal development. To investigate whether PC1 is involved in mechanotransduction in skeletal tissues, mice with a conditional deficiency for PC1 in neural crest cells, osteoblasts or chondrocytes were subjected to midpalatal suture expansion. Dynamic bone labeling revealed that new bone formation in response to expansion was significantly reduced in Wnt1Cre;Pkd1 mice, as the suture area containing new bone was 14.0+/-3.4% in mutant mice versus 65.0+/-3.8% in control mice at 2 weeks (p<0.001). In contrast, stress-induced new bone formation was not affected in OsxCre;Pkd1 mice. The increase in cell proliferation and differentiation into osteoblasts, seen in wild-type mice 1 day after force delivery, was not observed until 14 days in Wnt1Cre;Pkd1 mice. TUNEL labeling showed a significant increase in apoptotic suture cells at days 1 and 3 (from 7.0+/-0.5% to 13.5+/-1.4% at day 1 and from 4.6+/-1.1% to 10.5+/-1.7% at day 3, p<0.05). Abnormal ossification of nasal cartilage of Wnt1Cre;Pkd1 mice was accelerated upon suture expansion. Such ossification was also observed, but to a lesser extent in Col2a1-ERCre;Pkd1 mice. Transcript levels of Runx2 and MMP13 were significantly increased in the nasal cartilage of Wnt1Cre;Pkd1 mice compared to controls (p<0.05 and p<0.001, respectively), and in mutant mice with expansion versus without expansion (p<0.05 and p<0.001, respectively). Lack of PC1 in chondroprogenitor cells also resulted in increased cell apoptosis and an altered arrangement of chondrocytes in nasal cartilage. These results indicate that PC1 plays a critical role in the response of osteochondroprogenitor cells to the mechanical tissue stress induced by midpalatal suture expansion. They also suggest that the combination of an in vivo mechanical model, such as midpalatal suture expansion, with conditional deficiency for proteins that play a role in mechanotransduction, represents a powerful experimental strategy to explore underlying mechanisms.

Collagen XVIII and corneal reinnervation following keratectomy.

The significance of collagen XVIII in the regulation of corneal reinnervation remains largely unknown. We used whole-mount immunoconfocal microscopy to localize collagen XVIII to the nerve basement membrane of wild-type (WT) mouse corneas. Transmission electron microscopy showed corneal nerve disorganization in collagen XVIII knockout mice (col18a1(-/-)). Antibody 2H3-specific neurofilament colocalized with collagens XVIII and IV and laminin-2 in WT mouse corneas, but did not colocalize with collagen IV and laminin-2 in col18a1(-/-) mouse corneas. Following keratectomy, col18a1(-/-) mice displayed decreased corneal neurite extension compared to WT mice. Our data indicate that collagen XVIII may play an important role in corneal reinnervation after wounding.

Growth of cranial synchondroses and sutures requires polycystin-1.

In vertebrates, coordinated embryonic and postnatal growth of the craniofacial bones and the skull base is essential during the expansion of the rostrum and the brain. Identification of molecules that regulate skull growth is important for understanding the nature of craniofacial defects and for development of non-invasive biologically based diagnostics and therapies. Here we report on spatially restricted growth defects at the skull base and in craniofacial sutures of mice deficient for polycystin-1 (Pkd1). Mutant animals reveal a premature closure of both presphenoid and sphenooccipital synchondroses at the cranial base. Furthermore, knockout mice lacking Pkd1 in neural crest cells are characterized by impaired postnatal growth at the osteogenic fronts in craniofacial sutures that are subjected to tensile forces. Our data suggest that polycystin-1 is required for proliferation of subpopulations of cranial osteochondroprogenitor cells of both mesodermal and neural crest origin during skull growth. However, the Erk1/2 signalling pathway is up-regulated in the Pkd1-deficient skeletal tissue, similarly to that previously reported for polycystic kidney.

Kinesin-2 controls development and patterning of the vertebrate skeleton by Hedgehog- and Gli3-dependent mechanisms.

Hedgehog signaling plays an essential role in patterning of the vertebrate skeleton. Here we demonstrate that conditional inactivation of the Kif3a subunit of the kinesin-2 intraflagellar transport motor in mesenchymal skeletal progenitor cells results in severe patterning defects in the craniofacial area, the formation of split sternum and the development of polydactyly. These deformities are reminiscent of those previously described in mice with deregulated hedgehog signaling. We show that in Kif3a-deficient mesenchymal tissues both the repressor function of Gli3 transcription factor and the activation of the Shh transcriptional targets Ptch and Gli1 are compromised. Quantitative analysis of gene expression demonstrates that the Gli1 transcript level is dramatically reduced, whereas Gli3 expression is not significantly affected by kinesin-2 depletion. However, the motor appears to be required for the efficient cleavage of the full-length Gli3 transcription factor into a repressor form.

Mechanical force-induced midpalatal suture remodeling in mice.

Mechanical stress is an important epigenetic factor for regulating skeletal remodeling, and application of force can lead to remodeling of both bone and cartilage. Chondrocytes, osteoblasts and osteoclasts all participate and interact with each other in this remodeling process. To study cellular responses to mechanical stimuli in a system that can be genetically manipulated, we used mouse midpalatal suture expansion in vivo. Six-week-old male C57BL/6 mice were subjected to palatal suture expansion by opening loops with an initial force of 0.56 N for the periods of 1, 3, 5, 7, 14 or 28 days. Periosteal cells in expanding sutures showed increased proliferation, with Ki67-positive cells representing 1.8+/-0.1% to 4.5+/-0.4% of total suture cells in control groups and 12.0+/-2.6% to 19.9+/-1.2% in experimental/expansion groups (p<0.05). Starting at day 1, cells expressing alkaline phosphatase and type I collagen were seen. New cartilage and bone formation was observed at the oral edges of the palatal bones at day 7; at the nasal edges only bone formation without cartilage appeared to occur. An increase in osteoclast numbers suggested increased bone remodeling, ranging from 60 to 160% throughout the experimental period. Decreased Saffranin O staining after day 3 suggested decreased proteoglycan content in the secondary cartilage. Micro-CT showed a significant increase in maxillary width at days 14 and 28 (from 2334+/-4 microm to 2485+/-3 microm at day 14 and from 2383+/-5 microm to 2574+/-7 microm at day 28, p<0.001). The suture width was increased at days 14 and 28, except in the oral third region at day 28 (from 48+/-5 microm to 36+/-4 microm, p<0.05). Bone volume/total volume was significantly reduced at days 14 and 28 (50.2+/-0.7% vs. 68.0+/-3.7% and 56.5+/-1.0% vs. 60.9+/-1.3%, respectively, p<0.05), indicative of increased bone marrow space. These findings demonstrate that expansion forces across the midpalatal suture promote bone resorption through activation of osteoclasts and bone and cartilage formation via increased proliferation and differentiation of periosteal cells. Mouse midpalatal suture expansion would be useful in further studies of the ability of mineralized tissues to respond to mechanical stimulation.

Effect of in ovo immobilization on development of chick hind-limb articular cartilage: an evaluation using micro-MRI measurement of delayed gadolinium uptake.

To examine the effect of immobilization on the development of articular cartilage, we assessed glycosaminoglycan (GAG) content in the chick articular surface by delayed gadolinium-enhanced MRI of cartilage (dGEMRIC). Chick embryos were paralyzed by decamethonium bromide (DMB) from day 10 to either day 13 or day 16. The GAG content of the chick knee was compared with that of nonparalyzed chick embryos. Histologic analysis was unable to quantify GAG content; however, dGEMRIC demonstrated that GAG content was higher in the femoral condyles of the nonparalyzed embryos on day 13, and on day 16 the GAG content was lower in both the femoral condyles and the tibial plateaus of the nonparalyzed embryos. These results suggest that paralysis delays embryonic hind-limb development. Osteoblastic activity at the cartilage canal, as demonstrated by staining for alkaline phosphatase (ALP), was present only in the nonparalyzed chick embryos on day 16. The GAG content of the cartilage decreased when the cartilage canals began to form on day 16. The effect of immobilization on hind-limb development was indicated by the differences in the GAG content of the cartilage anlage measured by dGEMRIC in the developing knee joint of paralyzed and nonparalyzed embryonic chicks.

Migration and growth are attenuated in vascular smooth muscle cells with type VIII collagen-null alleles.

OBJECTIVE: Type VIII collagen is upregulated after vascular injury and in atherosclerosis. However, the role of type VIII collagen endogenously expressed by smooth muscle cells (SMCs) and in the context of the vascular matrix microenvironment, which is rich in type I collagen, is not known. To address this, we have compared aortic SMCs from wild-type (WT) mice to SMCs from type VIII collagen-deficient (KO) mice when plated on type I collagen. METHODS AND RESULTS: Type VIII collagen was upregulated after wounding of WT SMCs. KO SMCs exhibited greater adhesion to type I collagen than WT SMCs (optical density [OD595]=0.458+/-0.044 versus 0.193+/-0.071). By contrast, the WT SMCs spread more (389+/-75% versus 108+/-14% increase in cell area), migrated further (total distance 80.6+/-6.2 microm versus 64.2+/-4.4 microm), and exhibited increased [3H]-thymidine uptake (160,000+/-22,300 versus 63,100+/-12,100 counts per minute) when compared with KO SMCs. Gelatin zymograms showed that WT SMCs expressed latent matrix metalloproteinase 2, whereas KO SMCs did not. Addition of exogenous type VIII collagen returned levels of KO SMC adhesion (OD595=0.316+/-0.038), migration (79.5+/-5.8 microm), and latent matrix metalloproteinase 2 expression to levels comparable to WT SMCs. CONCLUSIONS: This study suggests that SMCs can modify the matrix microenvironment by producing type VIII collagen, using it to overlay type I collagen, and generating a substrate favorable for migration.

Targeted disruption of Col8a1 and Col8a2 genes in mice leads to anterior segment abnormalities in the eye.

Collagen VIII is localized in subendothelial and subepithelial extracellular matrices. It is a major component of Descemet's membrane, a thick basement membrane under the corneal endothelium, where it forms a hexagonal lattice structure; a similar structure, albeit less extensive, may be formed in other basement membranes. We have examined the function of collagen VIII in mice by targeted inactivation of the genes encoding the two polypeptide subunits, Col8a1 and Col8a2. Analysis of these mice reveals no major structural defects in most organs, but demonstrates that type VIII collagen is required for normal anterior eye development, particularly the formation of a corneal stroma with the appropriate number of fibroblastic cell layers and Descemet's membrane of appropriate thickness. Complete lack of type VIII collagen leads to dysgenesis of the anterior segment of the eye: a globoid, keratoglobus-like protrusion of the anterior chamber with a thin corneal stroma. Descemet's membrane is markedly thinned. The corneal endothelial cells are enlarged and reduced in number, and show a decreased ability to proliferate in response to different growth factors in vitro. An important function of collagen VIII may therefore be to generate a peri- or subcellular matrix environment that permits or stimulates cell proliferation.

Loss of collagen XVIII enhances neovascularization and vascular permeability in atherosclerosis.

BACKGROUND: Plaque neovascularization is thought to promote atherosclerosis; however, the mechanisms of its regulation are not understood. Collagen XVIII and its proteolytically released endostatin fragment are abundant proteoglycans in vascular basement membranes and the walls of major blood vessels. We hypothesized that collagen XVIII in the aortic wall inhibits the proliferation and intimal extension of vasa vasorum. METHODS AND RESULTS: To test our hypothesis, we bred collagen XVIII-knockout (Col18a1(-/-)) mice into the atherosclerosis-prone apolipoprotein E-deficient (ApoE(-/-)) strain. After 6 months on a cholesterol diet, aortas from ApoE(-/-);Col18a1(-/-) and ApoE(-/-);Col18a1(+/-) heterozygote mice showed increased atheroma coverage and enhanced lipid accumulation compared with wild-type littermates. We observed more extensive vasa vasorum and intimal neovascularization in knockout but not heterozygote aortas. Endothelial cells sprouting from Col18a1(-/-) aortas were increased compared with heterozygote and wild-type aortas. In contrast, vascular permeability of large and small blood vessels was enhanced with even heterozygous loss of collagen XVIII but was not suppressed by increasing serum endostatin to wild-type levels. CONCLUSIONS: Our results identify a previously unrecognized function for collagen XVIII that maintains vascular permeability. Loss of this basement membrane proteoglycan enhances angiogenesis and vascular permeability during atherosclerosis by distinct gene-dose-dependent mechanisms.

Groucho homologue Grg5 interacts with the transcription factor Runx2-Cbfa1 and modulates its activity during postnatal growth in mice.

Runx2-Cbfa1, a Runt transcription factor, plays important roles during skeletal development. It is required for differentiation and function of osteoblasts. In its absence, chondrocyte hypertrophy is severely impaired and there is no vascularization of cartilage templates during skeletal development. These tissue-specific functions of Runx2 are likely to be dependent on its interaction with other proteins. We have therefore searched for proteins that may modulate the activity of Runx2. The yeast two-hybrid system was used to identify a groucho homologue, Grg5, as a Runx2-interacting protein. Grg5 enhances Runx2 activity in a cell culture-based assay and by analyses of postnatal growth in mice we demonstrate that Grg5 and Runx2 interact genetically. We also show that Runx2 haploinsufficiency in the absence of Grg5 results in a more severe delay in ossification of cranial sutures and fontanels than occurs with Runx2 haploinsufficiency on a wild-type background. Finally, we find shortening of the proliferative and hypertrophic zones, and expansion of the resting zone in the growth plates of Runx2(+/-) Grg5(-/-) mice that are associated with reduced Ihh expression and Indian hedgehog (Ihh) signaling. We therefore conclude that Grg5 enhances Runx2 activity in vivo.

Localization of collagen XVIII and the endostatin portion of collagen XVIII in aged human control eyes and eyes with age-related macular degeneration.

PURPOSE: Endostatin, a C-terminal fragment of collagen XVIII (coll XVIII) formed by proteolysis, specifically inhibits endothelial cell migration and proliferation in vitro and potently inhibits angiogenesis and tumor growth in vivo. The purpose of this study was to examine the immunolocalization of endostatin and coll XVIII in the retina and choroid of human donor tissue sections from aged control donor eyes and to determine whether the localization or relative levels are changed in age-related macular degeneration (AMD). METHODS: Ocular tissues were obtained from six aged control donors (age range, 75-86 years; mean age, 80.5 years) without evidence or history of chorioretinal disease and from nine donors with AMD (age range, 74-105 years; mean age, 88.6 years). Tissues were cryopreserved, and streptavidin alkaline phosphatase immunohistochemistry was performed with goat anti-human and mouse anti-human endostatin antibodies and rabbit anti-mouse coll XVIII. Blood vessels were identified with mouse anti-human CD-34 antibody in adjacent sections. Pigment in RPE and choroidal melanocytes was bleached. Three independent observers scored the immunohistochemical reaction product. RESULTS: In aged control eyes, coll XVIII and endostatin (the endostatin portion of coll XVIII) immunoreactivity was observed in large retinal blood vessels and in capillaries in some individuals, but the internal limiting membrane (ILM) had the most intense retinal immunostaining. There was no significant difference in immunoreactivity to both antibodies in retinal blood vessels in aged control eyes. In the choroid, endostatin and coll XVIII were localized to blood vessels, Bruch's membrane, and RPE basal lamina. AMD retina and choroid had a similar pattern and intensity of coll XVIII immunostaining, as observed in control eyes but reaction product was more diffuse in the choroid. Endostatin immunoreactivity was significantly higher in ILM (P = 0.037) in AMD retina and significantly lower in the choriocapillaris, Bruch's membrane, and RPE basal lamina of AMD choroids (P < 0.05) and completely negative in some areas of AMD choroids. CONCLUSIONS: These data suggest that reduced levels of the endostatin portion of coll XVIII in Bruch's membrane, RPE basal lamina, intercapillary septa, and choriocapillaris in eyes with AMD may be permissive for choroidal neovascularization.

Collagen XVIII/endostatin is essential for vision and retinal pigment epithelial function.

Age-related macular degeneration (ARMD) with abnormal deposit formation under the retinal pigment epithelium (RPE) is the major cause of blindness in the Western world. basal laminar deposits are found in early ARMD and are composed of excess basement membrane material produced by the RPE. Here, we demonstrate that mice lacking the basement membrane component collagen XVIII/endostatin have massive accumulation of sub-RPE deposits with striking similarities to basal laminar deposits, abnormal RPE, and age-dependent loss of vision. The progressive attenuation of visual function results from decreased retinal rhodopsin content as a consequence of abnormal vitamin A metabolism in the RPE. In addition, aged mutant mice show photoreceptor abnormalities and increased expression of glial fibrillary acidic protein in the neural retina. Our data demonstrate that collagen XVIII/endostatin is essential for RPE function, and suggest an important role of this collagen in Bruch's membrane. Consistent with such a role, the ultrastructural organization of collagen XVIII/endostatin in basement membranes, including Bruch's membrane, shows that it is part of basement membrane molecular networks.

Knobloch syndrome: novel mutations in COL18A1, evidence for genetic heterogeneity, and a functionally impaired polymorphism in endostatin.

Knobloch syndrome (KNO) is an autosomal recessive disorder characterized by high myopia, vitreoretinal degeneration with retinal detachment, and congenital encephalocele. Pathogenic mutations in the COL18A1 gene on 21q22.3 were recently identified in KNO families. Analysis of two unrelated KNO families from Hungary and New Zealand allowed us to confirm the involvement of COL18A1 in the pathogenesis of KNO and to demonstrate the existence of genetic heterogeneity. Two COL18A1 mutations were identified in the Hungarian family: a 1-bp insertion causing a frameshift and a premature in-frame stop codon and an amino acid substitution. This missense variant is located in a conserved amino acid of endostatin, a cleavage product of the carboxy-terminal domain of collagen alpha 1 XVIII. D1437N (D104N in endostatin) likely represents a pathogenic mutation, as we show that the endostatin N104 mutant is impaired in its affinity towards laminin. Linkage to the COL18A1 locus was excluded in the New Zealand family, providing evidence for the existence of a second KNO locus. We named the second unmapped locus for Knobloch syndrome KNO2. Mutation analysis excluded COL15A1, a member of the multiplexin collagen subfamily similar to COL18A1, as being responsible for KNO2.

Characterization of endostatin binding to heparin and heparan sulfate by surface plasmon resonance and molecular modeling: role of divalent cations.

Endostatin (20 kDa) is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds zinc, heparin/heparan sulfate, laminin, and sulfatides and inhibits angiogenesis and tumor growth. Here we determined the kinetics and affinity of the interaction of endostatin with heparin/heparan sulfate and investigated the effects of divalent cations on these interactions and on the biological activities of endostatin. The binding of human recombinant endostatin to heparin and heparan sulfate was studied by surface plasmon resonance using BIAcore technology and further characterized by docking and molecular dynamics simulations. Kinetic data, evaluated using a 1:1 interaction model, showed that heparan sulfate bound to and dissociated from endostatin faster than heparin and that endostatin bound to heparin and heparan sulfate with a moderate affinity (K(D) approximately 2 microm). Molecular modeling of the complex between endostatin and heparin oligosaccharides predicted that, compared with mutagenesis studies, two further arginine residues, Arg(47) and Arg(66), participated in the binding. The binding of endostatin to heparin and heparan sulfate required the presence of divalent cations. The addition of ZnCl(2) to endostatin enhanced its binding to heparan sulfate by approximately 40% as well as its antiproliferative effect on endothelial cells stimulated by fibroblast growth factor-2, suggesting that this activity is mediated by the binding of endostatin to heparan sulfate. In contrast, no increase in the antiangiogenic and anti-proliferative activities of endostatin promoted by vascular endothelial growth factor was observed upon the addition of zinc.

Double knockout mice reveal a lack of major functional compensation between collagens XV and XVIII.

Generation of double knockout mice for collagen types XV and XVIII indicated surprisingly that the mice are viable and do not suffer from any new major defects. Although the two collagens are closely related molecules sharing similarities in tissue expression, we conclude that their biological roles are essentially separate, that of type XV in muscle and type XVIII in the eye. Detailed comparisons of the null mice eyes indicated that type XV collagen seems to be involved in the tunica vasculosa lentis regression process, whereas type XVIII is in the regression of vasa hyaloidea propria, and only minor compensatory effects could be detected. Furthermore, the essential role of type XVIII collagen in the eye is highlighted by the occurrence of this collagen in the epithelial basement membranes of the iris and the ciliary body and in the inner limiting membrane of the retina, sites lacking type XV.