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.

OBJECTIVETo test the hypothesis that polycystin-1 (PC1) is involved in orthodontic tooth movement as a mechanical sensor.MATERIALS AND METHODSThe 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.RESULTSIn 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.CONCLUSIONSThese 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.

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 p27Kip1 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 p27Kip1 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.

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. α5β1 and αvβ3 integrins form stable complexes with immobilized endostatin (KD = ∼1.8 × 10−8m, 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 αvβ3 integrin at the interface between the β-propeller domain of the αv subunit and the βA domain of the β3 subunit. In addition, we report that α5β1 and αvβ3 integrins bind to heparin/heparan sulfate. The ectodomain of the α5β1 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 α5β1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.

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 I 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-ER*Cre;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. (C) 2009 Elsevier Inc. All rights reserved.

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.

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.

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.

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.

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 [OD 595 ]=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 μm versus 64.2±4.4 μm), and exhibited increased [ 3 H]-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 (OD 595 =0.316±0.038), migration (79.5±5.8 μm), 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.

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. Magn Reson Med, 2006. © 2006 Wiley‐Liss, Inc.

Human MutationVolume 25, Issue 3 p. 316-316 Letter to the Editors A response to Suzuki et al. “How pathogenic is the p.D104N/endostatin polymorphic allele of COL18A1 in Knobloch syndrome?”† Stylianos E. Antonarakis, Corresponding Author Stylianos E. Antonarakis [email protected] Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, SwitzerlandUniversity of Geneva Medical School, Department of Genetic Medicine and Development, CMU - rue Michel-Servet 1, CH-1211 Geneva 4, SwitzerlandSearch for more papers by this authorAlexandre Reymond, Alexandre Reymond Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland Center for Integrative Genomics, University of Lausanne, Lausanne, SwitzerlandSearch for more papers by this authorOlivier Menzel, Olivier Menzel Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, SwitzerlandSearch for more papers by this authorReidunn CJ Bekkeheien, Reidunn CJ Bekkeheien Department of Cell Biology, Harvard Medical School, Boston, MassachusettsSearch for more papers by this authorNaomi Fukai, Naomi Fukai Department of Cell Biology, Harvard Medical School, Boston, MassachusettsSearch for more papers by this authorEileen Boye, Eileen Boye Department of Cell Biology, Harvard Medical School, Boston, MassachusettsSearch for more papers by this authorGyorgy Kosztolanyi, Gyorgy Kosztolanyi Department of Medical Genetics, University of Pécs, Pécs, HungarySearch for more papers by this authorSalim Aftimos, Salim Aftimos Department of Clinical Genetics, Starship Children's Hospital, Auckland, New ZealandSearch for more papers by this authorSamuel Deutsch, Samuel Deutsch Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, SwitzerlandSearch for more papers by this authorHamish S. Scott, Hamish S. Scott Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, SwitzerlandSearch for more papers by this authorBjorn R. Olsen, Bjorn R. Olsen Department of Cell Biology, Harvard Medical School, Boston, MassachusettsSearch for more papers by this authorMichel Guipponi, Michel Guipponi Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, SwitzerlandSearch for more papers by this author Stylianos E. Antonarakis, Corresponding Author Stylianos E. Antonarakis [email protected] Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, SwitzerlandUniversity of Geneva Medical School, Department of Genetic Medicine and Development, CMU - rue Michel-Servet 1, CH-1211 Geneva 4, SwitzerlandSearch for more papers by this authorAlexandre Reymond, Alexandre Reymond Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland Center for Integrative Genomics, University of Lausanne, Lausanne, SwitzerlandSearch for more papers by this authorOlivier Menzel, Olivier Menzel Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, SwitzerlandSearch for more papers by this authorReidunn CJ Bekkeheien, Reidunn CJ Bekkeheien Department of Cell Biology, Harvard Medical School, Boston, MassachusettsSearch for more papers by this authorNaomi Fukai, Naomi Fukai Department of Cell Biology, Harvard Medical School, Boston, MassachusettsSearch for more papers by this authorEileen Boye, Eileen Boye Department of Cell Biology, Harvard Medical School, Boston, MassachusettsSearch for more papers by this authorGyorgy Kosztolanyi, Gyorgy Kosztolanyi Department of Medical Genetics, University of Pécs, Pécs, HungarySearch for more papers by this authorSalim Aftimos, Salim Aftimos Department of Clinical Genetics, Starship Children's Hospital, Auckland, New ZealandSearch for more papers by this authorSamuel Deutsch, Samuel Deutsch Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, SwitzerlandSearch for more papers by this authorHamish S. Scott, Hamish S. Scott Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, SwitzerlandSearch for more papers by this authorBjorn R. Olsen, Bjorn R. Olsen Department of Cell Biology, Harvard Medical School, Boston, MassachusettsSearch for more papers by this authorMichel Guipponi, Michel Guipponi Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, SwitzerlandSearch for more papers by this author First published: 23 February 2005 https://doi.org/10.1002/humu.20140Citations: 1 † Communicated by Peter Byers AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. REFERENCES Hanson EH, Imperatore G, Burke W. 2001. HFE gene and hereditary hemochromatosis: a HuGE review. Human genome epidemiology. Am J Epidemiol 154: 193– 206. Marneros AG, Olsen BR. 2003. Age-dependent iris abnormalities in collagen XVIII/endostatin deficient mice with similarities to human pigment dispersion syndrome. Invest Ophthalmol Vis Sci 44: 2367– 2372. Menzel O, Bekkeheien RC, Reymond A, Fukai N, Boye E, Kosztolanyi G, Aftimos S, Deutsch S, Scott HS, Olsen BR, Antonarakis SE, Guipponi M. 2004. Knobloch syndrome: novel mutations in COL18A1, evidence for genetic heterogeneity, and a functionally impaired polymorphism in endostatin. Hum Mutat 23: 77– 84. Suzuki OT, Bagatini K, Sertié AL, Passos-Bueno MR. 2005. How pathogenic is the pD104N/endostatin polymorphic allele of COL18A1 in Knobloch syndrome? [Letter] Hum Mutat 25: 000– 000. Utriainen A, Sormunen R, Kettunen M, Carvalhaes LS, Sajanti E, Eklund L, Kauppinen R, Kitten GT, Pihlajaniemi T. 2004. Structurally altered basement membranes and hydrocephalus in a type XVIII collagen deficient mouse line. Hum Mol Genet 13: 2089– 2099. Citing Literature Volume25, Issue3March 2005Pages 316-316 ReferencesRelatedInformation

Human MutationVolume 25, Issue 3 p. 314-315 Letter to the Editors How pathogenic is the p.D104N/endostatin polymorphic allele of COL18A1 in Knobloch syndrome?† Oscar T. Suzuki, Oscar T. Suzuki Human Genome Center, Department of Biology, Institute of Biosciences, University of São Paulo, São Paulo, BrazilSearch for more papers by this authorKelly Bagatini, Kelly Bagatini Human Genome Center, Department of Biology, Institute of Biosciences, University of São Paulo, São Paulo, BrazilSearch for more papers by this authorAndréa L. Sertié, Andréa L. Sertié Human Genome Center, Department of Biology, Institute of Biosciences, University of São Paulo, São Paulo, BrazilSearch for more papers by this authorMaria Rita Passos-Bueno, Corresponding Author Maria Rita Passos-Bueno passos@ib.usp.br Human Genome Center, Department of Biology, Institute of Biosciences, University of São Paulo, São Paulo, BrazilRua do Matão 277, Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil 05508-900Search for more papers by this author Oscar T. Suzuki, Oscar T. Suzuki Human Genome Center, Department of Biology, Institute of Biosciences, University of São Paulo, São Paulo, BrazilSearch for more papers by this authorKelly Bagatini, Kelly Bagatini Human Genome Center, Department of Biology, Institute of Biosciences, University of São Paulo, São Paulo, BrazilSearch for more papers by this authorAndréa L. Sertié, Andréa L. Sertié Human Genome Center, Department of Biology, Institute of Biosciences, University of São Paulo, São Paulo, BrazilSearch for more papers by this authorMaria Rita Passos-Bueno, Corresponding Author Maria Rita Passos-Bueno passos@ib.usp.br Human Genome Center, Department of Biology, Institute of Biosciences, University of São Paulo, São Paulo, BrazilRua do Matão 277, Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil 05508-900Search for more papers by this author First published: 23 February 2005 https://doi.org/10.1002/humu.20139Citations: 3 † Communicated by Peter Byers AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume25, Issue3March 2005Pages 314-315 RelatedInformation