Shriners Hospitals for Children - Tampa

The biosynthetic response of calf articular cartilage explants to dynamic compression was examined over a wide range of amplitudes, waveforms, and frequencies. Glycosaminoglycan synthesis was assessed by 35S-sulfate incorporation, and amino acid uptake and protein synthesis were assessed by 3H-proline incorporation. Two culture chambers were designed to allow uniaxial radially unconfined compression and mechanical testing of cartilage disks: one chamber was used inside a standard incubator; the other was used with a mechanical spectrometer and allowed load and displacement to be monitored during compression. Dynamic stiffness measurements of 3-mm diameter disks identified a characteristic frequency [0.001 Hz (cycles/sec)] that separated low- and high-frequency regimes in which different flow and deformation phenomena predominated; e.g., at 0.0001-0.0001 Hz, significant fluid was exuded from cartilage disks, whereas at 0.01-1 Hz, hydrostratic pressure increased within disks. At the higher frequencies, oscillatory strains of only approximately 1-5% stimulated 3H-proline and 35S-sulfate incorporation by approximately 20-40%. In contrast, at the lower frequencies (a) compressions of less than 5% had no effect, consistent with the dosimetry of biosynthetic inhibition by static compression (approximately 25% compression caused a approximately 20% inhibition of radiolabel incorporation), and (b) higher amplitudes (cycling between disk thicknesses of 1.25 and 0.88-1.00 mm) stimulated 3S-sulfate incorporation by approximately 20-40%, consistent with the kinetics of response to a single 2-h compression and release. None of the compression protocols was associated with detectable alterations in (e.g., compression-induced depletion of) total glycosaminoglycan content. This study provides a framework for identifying both the physical and biological mechanisms by which dynamic compression can modulate chondrocyte biosynthesis. In addition, the culture and compression methodology potentially allows in vitro evaluation of clinical strategies of continuous passive motion therapy to stimulate cartilage remodeling.

STUDY DESIGN: An outcome questionnaire was constructed to evaluate patient satisfaction and performance and to discriminate among patients with adolescent idiopathic scoliosis. OBJECTIVES: To determine reliability and validity in a new quality-of-life instrument for measuring progress among scoliosis patients. SUMMARY OF BACKGROUND DATA: Meta-analysis of the surgical treatment of adolescent idiopathic scoliosis determined that a uniform assessment of outcome did not exist. In addition, patient measures of well-being as opposed to process measures (e.g., radiographs) were not consistently reported. This established the need for a standardized questionnaire to assess patient measures in conjunction with process measures. METHODS: The instrument consists of 24 questions divided into seven equally weighted domains as determined by factor analysis: pain, general self-image, postoperative self-image, general function, overall level of activity, postoperative function, and satisfaction. The questionnaire takes approximately 5 minutes to complete and is taken at predetermined time intervals. A total of 244 of patients from three different sites responded to the questionnaire. RESULTS: The reliability based on internal consistency was confirmed with a Cronbach's alpha coefficient greater than 0.6 for each domain. In addition, acceptable correlation coefficient values greater than 0.68 were obtained for each domain by the test-retest method on normal controls. Similarly; to establish validity of the questionnaire, responses of normal high school students were compared with that of the patients. Consistent differences were noted in the domains between the two groups with P < 0.003. The largest differences were in pain (control, 29.96 +/- 0.20; patient, 13.23 +/- 5.55) and general level of activity (control, 14.96 +/- 0.20; patient, 12.16 +/- 3.23). Examination of the relationship between the domains and patient satisfaction showed that pain correlates with satisfaction to the greatest degree (Pearson's correlation co-efficient, r = -0.511; P < 0.001), followed by self-image (r = 0.412; P < 0.001). CONCLUSIONS: This questionnaire addresses patient measures for evaluation of outcome in adolescent idiopathic scoliosis surgery by examining several domains. It also allows for dynamic monitoring of scoliosis patients as they become adults. This is a validated instrument with good reliability measures.

Mature human aorta contains a 70-kDa versican fragment, which reacts with a neoepitope antiserum to the C-terminal peptide sequence DPEAAE. This protein therefore appears to represent the G1 domain of versican V1 (G1-DPEAAE441), which has been generated in vivo by proteolytic cleavage at the Glu441-Ala442 bond, within the sequence DPEAAE441-A442RRGQ. Because the equivalent aggrecan product (G1-NITEGE341) and brevican product (G1-EAVESE395) are generated by ADAMTS-mediated cleavage of the respective proteoglycans, we tested the capacity of recombinant ADAMTS-1 and ADAMTS-4 to cleave versican at Glu441-Ala442. Both enzymes cleaved a recombinant versican substrate and native human versican at the Glu441-Ala442 bond and the mature form of ADAMTS-4 was detected by Western analysis of extracts of aortic intima. We conclude that versican V1 proteolysis in vivocan be catalyzed by one or more members of the ADAMTS family of metalloproteinases. Mature human aorta contains a 70-kDa versican fragment, which reacts with a neoepitope antiserum to the C-terminal peptide sequence DPEAAE. This protein therefore appears to represent the G1 domain of versican V1 (G1-DPEAAE441), which has been generated in vivo by proteolytic cleavage at the Glu441-Ala442 bond, within the sequence DPEAAE441-A442RRGQ. Because the equivalent aggrecan product (G1-NITEGE341) and brevican product (G1-EAVESE395) are generated by ADAMTS-mediated cleavage of the respective proteoglycans, we tested the capacity of recombinant ADAMTS-1 and ADAMTS-4 to cleave versican at Glu441-Ala442. Both enzymes cleaved a recombinant versican substrate and native human versican at the Glu441-Ala442 bond and the mature form of ADAMTS-4 was detected by Western analysis of extracts of aortic intima. We conclude that versican V1 proteolysis in vivocan be catalyzed by one or more members of the ADAMTS family of metalloproteinases. Versican is a member of the family of large aggregating proteoglycans, which also includes aggrecan, neurocan, and brevican. Although aggrecan is most abundant in cartilages (1Hascall V.C. Calabro A. Midura R.J. Yanagishita M. Methods Enzymol... 1994; 230: 390-417Google Scholar) and both neurocan and brevican are largely restricted to nervous tissues (2Yamada H. Watanabe K. Shimonaka M. Yamaguchi Y. J. Biol. Chem... 1994; 269: 10119-10126Google Scholar), versican has a rather wide tissue distribution (3Bode-Lesniewska B. Dours-Zimmermann M.T. Odermatt B.F. Briner J. Heitz P.U. Zimmermann D.R. J. Histochem. Cytochem... 1996; 44: 303-312Google Scholar). It has been identified in loose connective tissues and in fibrous, articular, and elastic cartilages. It is also detectable in the central and peripheral nervous system, in the epidermis, and in all three wall layers of veins and elastic arteries. Furthermore, versican can exist in a number of isoforms, namely V0, V1, V2, V3, and Vint (4Ito K. Shinomura T. Zako M. Ujita M. Kimata K. J. Biol. Chem... 1995; 270: 958-965Google Scholar, 5Zako M. Shinomura T. Ujita M. Ito K. Kimata K. J. Biol. Chem... 1995; 270: 3914-3918Google Scholar, 6Dours-Zimmermann M.T. Zimmermann D.R. J. Biol. Chem... 1994; 269: 32992-32998Google Scholar, 7Lemire J.M. Braun K.R. Maurel P. Kaplan E.D. Schwartz S.M. Wight T.N. Arterioscler. Thromb. Vasc. Biol... 1999; 19: 1630-1639Google Scholar). The V1 isoform is composed of a G1 domain, chondroitin sulfate (CS)1 attachment domain (GAG-beta) and the G3 domain. The V0 and V1 isoforms differ only by the presence of the GAG-alpha domain in the V0 form, which adds 987 amino acids and about five putative CS-attachment sites, adjacent to the hyaluronan binding domain (4Ito K. Shinomura T. Zako M. Ujita M. Kimata K. J. Biol. Chem... 1995; 270: 958-965Google Scholar, 6Dours-Zimmermann M.T. Zimmermann D.R. J. Biol. Chem... 1994; 269: 32992-32998Google Scholar). The V2 isoform, which is a predominant brain proteoglycan (8Schmalfeldt M. Dours-Zimmermann M.T. Winterhalter K.H. Zimmermann D.R. J. Biol. Chem... 1998; 273: 15758-15764Google Scholar), is composed of the G1 domain, GAG-alpha domain, and the G3 domain. Both V0 and V1 are detected at the mRNA level in the human aorta, and versican is also detected in the aorta by immunohistochemistry with antibodies recognizing both variants (3Bode-Lesniewska B. Dours-Zimmermann M.T. Odermatt B.F. Briner J. Heitz P.U. Zimmermann D.R. J. Histochem. Cytochem... 1996; 44: 303-312Google Scholar, 9Yao L.Y. Moody C. Schonherr E. Wight T.N. Sandell L.J. Matrix Biol.. 1994; 14: 213-225Google Scholar). Furthermore, recent data show that cultured smooth muscle cells contain mRNA for V0, V1, and V3 isoforms (7Lemire J.M. Braun K.R. Maurel P. Kaplan E.D. Schwartz S.M. Wight T.N. Arterioscler. Thromb. Vasc. Biol... 1999; 19: 1630-1639Google Scholar). In contrast to aggrecan, for which the degradative pathways have been described in detail (10Sandy J.D. Thompson V. Doege K. Verscharen C. Biochem. J... 2000; 351: 161-166Google Scholar), very little is known regarding versican turnover. A 66-kDa protein, which is immunologically related to versican, has been described in fetal human skin (11Sorrell J.M. Carrino D.A. Baber M.A. Caplan A.I. Anat. Embryol... 1999; 199: 45-56Google Scholar). However, the metabolic origin of this protein was not described. On the other hand, the findings (2Yamada H. Watanabe K. Shimonaka M. Yamaguchi Y. J. Biol. Chem... 1994; 269: 10119-10126Google Scholar, 12Sandy J.D. Boynton R.E. Flannery C.R. J. Biol. Chem... 1991; 266: 8198-8205Google Scholar) that both aggrecan and brevican are degradedin vivo by glutamyl endopeptidases, which appear to belong to the ADAMTS family of metalloproteinases (13Kuno K. Kanada N. Nakashima E. Fujiki F. Ichimura F. Matsushima K. J. Biol. Chem... 1997; 272: 556-562Google Scholar, 14Tortorella M.D. Burn T.C. Pratta M.A. Abbaszade I. Hollis J.M. Liu R. Rosenfeld S.A. Copeland R.A. Decicco C.P. Wynn R. Rockwell A. Yang F. Duke J.L. Solomon K. George H. Bruckner R. Nagase H. Itoh Y. Ellis D.M. Ross H. Wiswall B.H. Murphy K. Hillman Jr., M.C. Hollis G.F. Arner E.C. et al.Science.. 1999; 284: 1664-1666Google Scholar, 15Abbaszade I. Liu R.Q. Yang F. Rosenfeld S.A. Ross O.H. Link J.R. Ellis D.M. Tortorella M.D. Pratta M.A. Hollis J.M. Wynn R. Duke J.L. George H.J. Hillman Jr., M.C. Murphy K. Wiswall B.H. Copeland R.A. Decicco C.P. Bruckner R. Nagase H. Itoh Y. Newton R.C. Magolda R.L. Trzaskos J.M. Hollis G.F. Arner E.C. Burn T.C. J. Biol. Chem... 1999; 274: 23443-23450Google Scholar), suggested to us that versican might also be degraded in this manner. In this paper we provide the first evidence that versican is indeed processed in vivo by a glutamyl endopeptidase that appears to belong to the ADAMTS family. Abdominal aorta was anonymously obtained from an organ donor with approval from the University of Washington human subjects review committee. During preparation of the organs for transplantation, excess aortic tissue was trimmed from around the entry point of the celiac artery. This tissue was kept in University of Wisconsin solution (16Wahlberg J.A. Southard J.H. Belzer F.O. Cryobiology.. 1986; 23: 477-482Google Scholar) at 4 °C until the tissue was either frozen intact in liquid nitrogen or dissected on ice in Ca2+- and Mg2+-free phosphate-buffered saline with proteinase inhibitors (10 mm EDTA, 0.1 mm AEBSF, 1 μg/ml pepstatin) into the intimal, medial, and adventitial layers. Tissue samples (100–500 mg of wet weight) were frozen for storage and immediately after thawing were extracted twice for 24 h in 1 ml of 4 m guanidine-HCl, 10 mm MES, 50 mmsodium acetate, 5 mm EDTA, 0.1 mm AEBSF, 5 mm iodoacetamide, 0.3 m aminohexanoic acid, 15 mm benzamidine, 1 μg/ml pepstatin, pH 6.8, at 4 °C. The samples were then centrifuged for 10 min at 12,000 ×g at 4 °C. The clear supernatants were combined (1.5–2.0 ml), and three volumes of ice-cold ethanol/5 mm sodium acetate were added. After 16 h at −20 °C, the precipitate was collected by centrifugation at 13,000 × g for 20 min at 4 °C. The pellet was dried, dissolved in 50 mm Tris, 50 mm sodium acetate, 10 mm EDTA, pH 7.6, and deglycosylated by digestion for 1.5 h at 37 °C with chondroitinase ABC (25 milliunits/100 μg of GAG, protease-free, Seikagaku). Versican was prepared from human smooth muscle cell-conditioned medium after labeling with [35S]methionine for 24 h as described previously (17Olin K.L. Potter-Perigo S. Barrett P.H. Wight T.N. Chait A. J. Biol. Chem... 1999; 274: 34629-34636Google Scholar). Antiserum DPEAAE (abbreviated to anti-DP in the text below) was raised in rabbits against the synthetic peptide CGGDPEAAE conjugated to ovalbumin (by Research Genetics, Huntsville, AL), and the antibodies were affinity-purified on a peptide-substituted Sulfolink column from Pierce and Co. Anti-CDAGWLADQTVRYPI (also called HAL) was obtained from Dr. Steve Carlson; this antiserum is known to a which with and is in the proteoglycan of the G1 domain of aggrecan, versican, and also in protein J.D. 1995; 266: Scholar). was raised in rabbits against aggrecan G1 domain by Dr. to the peptide sequence of human was from Dr. 1995; 266: Scholar), and the antiserum to recombinant human versican V1 in was from Dr. J.R. J. 1995; Scholar). for the GAG-alpha domain and the domain of human versican have been previously described M.T. Zimmermann D.R. J. Biol. Chem... 1994; 269: 32992-32998Google Scholar, D.R. Dours-Zimmermann M.T. M. J. Biol... 1994; Scholar). The was from Research The antiserum was raised in rabbits by of the synthetic peptide conjugated to ovalbumin (by Research this peptide of human Western analysis was on with antibodies at and by as previously described J.D. 1995; 266: Scholar, J.D. Thompson V. Verscharen C. Biochem. J... 1998; Scholar). of the V0 and V1 isoforms of versican, was on × of human aorta from organ were in at 4 °C. After in were and for by the with as a The affinity-purified anti-DP was at 10 of the versican antiserum was at and the was at were with The and for the cleavage were generated F. Scholar) with a of of and of and The cleavage were identified with a level of human ADAMTS-1 was and as previously described R. J. Biol. Chem... 2000; Scholar). was a from of the substrate recombinant A by of the of the domain of versican V1 in has been described D.R. Dours-Zimmermann M.T. M. J. Biol... 1994; Scholar). The substrate below) includes a an and cleavage to the versican The ADAMTS at Glu441-Ala442 is as ADAMTS the substrate was first against for 4 h to phosphate-buffered was with ADAMTS μg of proteinase 20 μg of in of 50 mm Tris, 10 mm pH at 37 °C for 16 h and (10 μg for Western analysis or 20 μg for were on digestion of native 10 μg of protein as from human aortic from at about μg of versican or μg of from Dr. was with ADAMTS-1 or ADAMTS-4 in 50 mm Tris, mm 10 mm pH at 37 °C for 16 The samples were then as dried, and on for Western of the five known cleavage of human aggrecan (10Sandy J.D. Thompson V. Doege K. Verscharen C. Biochem. J... 2000; 351: 161-166Google Scholar, 14Tortorella M.D. Burn T.C. Pratta M.A. Abbaszade I. Hollis J.M. Liu R. Rosenfeld S.A. Copeland R.A. Decicco C.P. Wynn R. Rockwell A. Yang F. Duke J.L. Solomon K. George H. Bruckner R. Nagase H. Itoh Y. Ellis D.M. Ross H. Wiswall B.H. Murphy K. Hillman Jr., M.C. Hollis G.F. Arner E.C. et al.Science.. 1999; 284: 1664-1666Google Scholar, 15Abbaszade I. Liu R.Q. Yang F. Rosenfeld S.A. Ross O.H. Link J.R. Ellis D.M. Tortorella M.D. Pratta M.A. Hollis J.M. Wynn R. Duke J.L. George H.J. Hillman Jr., M.C. Murphy K. Wiswall B.H. Copeland R.A. Decicco C.P. Bruckner R. Nagase H. Itoh Y. Newton R.C. Magolda R.L. Trzaskos J.M. Hollis G.F. Arner E.C. Burn T.C. J. Biol. Chem... 1999; 274: 23443-23450Google Scholar, J.D. Flannery C.R. J. Scholar, J.D. Scholar) with the known cleavage of brevican by ADAMTS-4 (2Yamada H. Watanabe K. Shimonaka M. Yamaguchi Y. J. Biol. Chem... 1994; 269: 10119-10126Google Scholar, C. Pratta M. Solomon K. Arner E.C. S. J. Biol. Chem... 2000; Scholar) is in in I. The substrate for the was on the cleavage within the the of chondroitin sulfate in the domain of aggrecan and the that represent J. Biochem. 1997; Scholar). this that the of the ADAMTS aggregating is by an in the on the of the bond with a on the of the A rather loose sequence for cleavage is as the the are are or more and a of on this the of the known isoforms of human versican were for evidence of cleavage and sequence of the known ADAMTS cleavage for aggrecan and brevican and the versican cleavage described in this cleavage A A A A for A A A A A A 1 1 or or cleavage versican cleavage A A A A A A A A for to for or or cleavage in a for to This suggested the presence of a very cleavage in the at the Glu441-Ala442 bond in V1 versican and at the equivalent in V0 versican The of a cleavage at in V1 versican was by the that the of this to the of the protein is to the of the known cleavage in aggrecan at and brevican at The also suggested a cleavage at in the GAG-alpha of both the V0 and V2 isoforms of versican and on vivo of the product of this cleavage be described Because the neoepitope antiserum has been previously J.D. 1995; 266: Scholar, J.R. J. J.H. R.A. Flannery C.R. M. J.D. J. Biol. Chem... 1995; 270: Scholar, K. R.A. J. 1996; Scholar) to the that an equivalent antiserum to the putative versican neoepitope sequence DPEAAE be of the equivalent versican We therefore generated a antiserum to the peptide were for 24 h with and the were from the medium by (17Olin K.L. Potter-Perigo S. Barrett P.H. Wight T.N. Chait A. J. Biol. Chem... 1999; 274: 34629-34636Google Scholar) and with chondroitinase ABC for analysis on a × the presence of very 1 and which in the to On Western analysis both of were with and the antiserum only 1 with the GAG-alpha antiserum with the of M.T. Zimmermann D.R. J. Biol. Chem... 1994; 269: 32992-32998Google Scholar, D.R. Dours-Zimmermann M.T. M. J. Biol... 1994; Scholar), 1 as the V0 isoform and as the V1 isoform of of a proteoglycan preparation from human smooth muscle cells L.Y. Moody C. Schonherr E. Wight T.N. Sandell L.J. Matrix Biol.. 1994; 14: 213-225Google Scholar) were on a for Western analysis with namely and 1 A as was and this was with with and very with and was also evidence for the presence of a product as which more It 1 and represent versican isoforms V0 and V1, in is also that V0 and V1 not on this In to versican, the antiserum also detected abundant in the to and the antiserum 1 detected at about and of were The antiserum 1 with in the smooth muscle versican that the not with the DPEAAE sequence is in intact versican Furthermore, the antiserum not with of the that and that are generated by proteolysis of versican at other the the of versican in human aorta we extracts of aorta and of the and on the The of with of the and was in of aorta, only the with are 1 versican the V1 was the only detectable very This was with and also with The of detectable of with in this is to the of this antiserum to and The antiserum also detected abundant in the to which to in the smooth muscle cell-conditioned medium have not been was the that a 70-kDa as 4 on 1 was detected with and and this very with the neoepitope antiserum The and of this product suggested that the cleavage product of human versican V1 from the cleavage In a product at about as was also in and are with the generated by cleavage of the versican V0 isoform at the in the domain. The of to 4 is with the very of V0 to V1 versican in The of of and 4 was by that the of both was by of the antiserum with the peptide CGGDPEAAE at 10 for h 1 The at about in to be Because of the of with affinity-purified C-terminal neoepitope J.D. 1995; 266: Scholar), we tested antiserum against a wide of aggrecan with the C-terminal and (10Sandy J.D. Thompson V. Doege K. Verscharen C. Biochem. J... 2000; 351: 161-166Google Scholar, J.D. 1995; 266: Scholar, J.D. Thompson V. Verscharen C. Biochem. J... 1998; Scholar) not with the affinity-purified Western in which with not with and of prepared from the human aorta samples for the Western a distribution for the In this both the versican and the to be most abundant in the detectable in in the and to in the The analysis of human aorta samples from with to a with that the of both the and were in the and Because the human aorta extracts versican with and to the we the that the C-terminal at can be generated by digestion of versican with ADAMTS this we recombinant human ADAMTS-1 and ADAMTS-4 with a recombinant human versican domain substrate and the by on The substrate preparation as a of about 50 which the substrate with ADAMTS-1 of the substrate and generated a product of about which as a and a product at about the the ADAMTS-4 more of the substrate the ADAMTS-1 digestion generated the product digestion of were for Western analysis with the antiserum and the neoepitope antiserum The substrate with the antiserum and with the antiserum After digestion with either ADAMTS-1 or ADAMTS-4 the substrate was and were obtained at about and which in to the as and were with the antiserum 4 and only the was this as the with a C-terminal at In the of the generated by ADAMTS-1 was by of the antiserum with the peptide was also with the and an On the that the of the with both anti-DP and the the of the with of data show that both ADAMTS-1 and ADAMTS-4 are of the versican substrate at the Glu441-Ala442 bond to an of which at about and a C-terminal of which at about The for the of the and with to is the of the of ADAMTS digestion of the recombinant of with both ADAMTS-1 and ADAMTS-4 were by and generated from both and ovalbumin and The for the substrate is and the from cleavage at the Glu441-Ala442 bond are and The with ADAMTS-1 a at which to This was not in the ADAMTS-4 with of more digestion with The with both enzymes that were very to the for the C-terminal the ADAMTS-1 product was at and the ADAMTS-4 product at were in the of both the of the to the This appears to be to very proteolysis of the product from the this product reacts with which the C-terminal and also the which 5 10 at the the ADAMTS can also cleave native versican at the Glu441-Ala442 bond, we versican from human aortic with and The were by Western with and anti-DP The 1 and versican abundant versican in the to as of the 70-kDa The of the in this to that in 1 is to the very with the anti-DP antiserum in the digestion with ADAMTS-1 not the of a in the of 70-kDa with ADAMTS-4 all of the versican and also most of the versican and generated a at which was with anti-DP show that the 70-kDa can be generated by of aortic versican with ADAMTS-1 and human aortic contains ADAMTS proteinase we extracts by Western with an antiserum to human ADAMTS-4 The a of protein at about which with the mature form of recombinant of the presence of in extracts was obtained by that was and by of the antiserum with the peptide at 10 for 1 h at of the recombinant versican substrate or native versican with recombinant enzymes and that the ADAMTS-4 was more the ADAMTS-1 μg of this native versican was with and μg of for 16 and the of product was by Western This that the ADAMTS-4 was about to more an equivalent of the ADAMTS-1 this ADAMTS-4 was also to be more aggrecan was as substrate that the of ADAMTS-4 was not to versican as a also that both enzymes degraded aggrecan more versican the aggrecan as by of substrate was obtained with 0.1 μg of or μg of versican digestion about μg of and not be obtained with 5 μg of the (also This paper that the mature human aorta contains a of V1 versican (G1-DPEAAE441), which can be generated by ADAMTS-1 or ADAMTS-4 digestion of intact human This product has been by Western with three versican and and a antiserum raised to an C-terminal neoepitope The appears to in is in extracts in the presence of proteinase and the can be detected by immunohistochemistry of the extracts that contain the versican also contain the mature form of human ADAMTS-4 is that this member of the ADAMTS family is for in The of the 70-kDa to other versican in the aorta be from of that about of the protein in the product appears to be in human skin the 66-kDa versican described by and (11Sorrell J.M. Carrino D.A. Baber M.A. Caplan A.I. Anat. Embryol... 1999; 199: 45-56Google Scholar) reacts with the antiserum in this A. Carrino and A. I. This 66-kDa protein is more abundant in fetal skin with an for this protein in the of extracts also a 1 that appears to represent the product generated by ADAMTS cleavage at of the V0 isoform of versican, and this product is also in human skin The data also the of a for ADAMTS cleavage of in aggregating The of and to cleave versican at a clear to versican I. J.D. Potter-Perigo S. Wight T.N. S.A. S. 1996; Scholar), also the of this ADAMTS is to from this sequence ADAMTS cleavage in versican at that is in versican isoforms V0 and V2 In this versican V2 is the most abundant isoform in brain (8Schmalfeldt M. Dours-Zimmermann M.T. Winterhalter K.H. Zimmermann D.R. J. Biol. Chem... 1998; 273: 15758-15764Google Scholar), we have human brain extracts for the presence of the This versican is indeed abundant in this and P. In the the and generated of neurocan F. M. Y. S. Watanabe E. A. 1998; Scholar) from cleavage at an ADAMTS which is from to be in human neurocan at the sequence suggested a in the of to that of proteoglycan by the ADAMTS family of an binding domain Jr., J. 1995; Scholar) adjacent the binding for the of the aggrecan domain with ADAMTS-4 has also been by the of in R.A. J. K. J. Biol. Chem... 2000; Scholar, C. C. E. Matrix Biol... 2000; 19: Scholar). of might in as ADAMTS appears to be In the ADAMTS of has one or more in to the and domain of all family It has been suggested that the sulfate binding on this which other is not to cells by a K. Matsushima K. J. Biol. Chem... 1998; 273: Scholar). In recent data M. Pratta M. Liu R.Q. Abbaszade I. Ross H. Burn T. Arner E. J. Biol. Chem... 2000; M.A. Tortorella M.D. Arner E.C. J. Biol. Chem... 2000; Scholar) has suggested that the aggrecan and ADAMTS-4 is the sulfate of the substrate that of ADAMTS-mediated proteolysis by appears to In this is that of the or of an ADAMTS proteinase by K. Matsushima K. J. Biol. Chem... 1998; 273: Scholar) might provide an for and of smooth muscle cells R.A. J. Scholar, Jr., T.C. Thromb. Scholar). an of versican might the of versican G1 and G3 domain which have been to and Y. Yang B. C. V. K. Yang J. 1999; Scholar, C. V. Y. Yang J. Biol. Chem... 2000; Scholar). The data on ADAMTS-1 and ADAMTS-4 cleavage of versican, with the on and of aggrecan I. Liu R.Q. Yang F. Rosenfeld S.A. Ross O.H. Link J.R. Ellis D.M. Tortorella M.D. Pratta M.A. Hollis J.M. Wynn R. Duke J.L. George H.J. Hillman Jr., M.C. Murphy K. Wiswall B.H. Copeland R.A. Decicco C.P. Bruckner R. Nagase H. Itoh Y. Newton R.C. Magolda R.L. Trzaskos J.M. Hollis G.F. Arner E.C. Burn T.C. J. Biol. Chem... 1999; 274: 23443-23450Google Scholar, K. Y. H. H. M. H. K. 2000; Scholar, M.D. Pratta M. Liu R.Q. J. Ross O.H. Abbaszade I. Burn T. Arner E. J. Biol. Chem... 2000; Scholar) and ADAMTS-4 cleavage of brevican C. Pratta M. Solomon K. Arner E.C. S. J. Biol. Chem... 2000; Scholar) that three ADAMTS family members not are all to cleavage of one or more aggregating In this the of ADAMTS family members to for as in the of ADAMTS-4 as M.D. Burn T.C. Pratta M.A. Abbaszade I. Hollis J.M. Liu R. Rosenfeld S.A. Copeland R.A. Decicco C.P. Wynn R. Rockwell A. Yang F. Duke J.L. Solomon K. George H. Bruckner R. Nagase H. Itoh Y. Ellis D.M. Ross H. Wiswall B.H. Murphy K. Hillman Jr., M.C. Hollis G.F. Arner E.C. et al.Science.. 1999; 284: 1664-1666Google Scholar), It be to the of and which are for this for cleavage of in the of the proteoglycans, and also to which family members are for the of proteoglycan substrate in In this be that we that both ADAMTS-1 and appear to aggrecan more versican and that for both ADAMTS-4 was the most of this be in that the against aggrecan might be to the substrate for aggrecan to versican 50 or the of M.A. Tortorella M.D. Arner E.C. J. Biol. Chem... 2000; Scholar) on the proteoglycan in In the of the to the might be related to a of the recombinant enzymes to or prepared and enzymes with more or be to this in the with to the of is that the T. H. K. H. T. Y. T. Y. M. H. N. Y. H. T. R. Y. Matsushima K. J. 2000; Scholar) a with tissue in the and a of in the this from an to as aggrecan versican or other to be We Dr. and for the human aortic The of Thompson and the at the University of is chondroitin sulfate a and with 1 antiserum DPEAAE antiserum to human versican V1

OBJECTIVE: To determine the proteolytic fragmentation patterns and N-terminal sequence of aggrecan fragments in human synovial fluid from patients with inflammatory arthritides, joint injury, or osteoarthritis (OA). METHODS: Knee synovial fluid was obtained from patients with joint injury, OA, acute pyrophosphate arthritis (pseudogout), reactive arthritis, psoriatic arthritis, or juvenile rheumatoid arthritis. Chondroitin sulfate-substituted aggrecan fragments present in the fluid were purified by cesium chloride gradient centrifugation and enzymatically deglycosylated. Core protein species were determined by N-terminal analysis and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with electroblotting and detection with monoclonal antibody 3B3. RESULTS: Samples from patients with joint injury, OA, and inflammatory joint disease all showed a similar 3-band pattern, with core sizes of approximately 200 kd, 170 kd, and 135 kd. In all samples, diffuse immunoreactive products were also seen, with an apparent size of > 250 kd. N-terminal analysis of core preparations of all samples showed a consistent single predominant sequence, beginning at alanine 374 of the human aggrecan core protein. CONCLUSION: The aggrecan fragments present in joint fluids from patients with various inflammatory arthritides, joint injury, or OA result from a predominant cleavage of the human aggrecan core protein at the glutamate 373-alanine 374 bond within the interglobular domain, between the G1 and G2 domains. The consistent pattern of fragments seen on SDS-PAGE and the single predominant N-terminal sequence suggest a common degradative mechanism of aggrecan in these different joint conditions. The identity of the proteolytic agent (aggrecanase), however, remains unknown. These results appear to have important implications with regard to the development of therapies to protect cartilage from degradation in patients with joint disease.

Synovial fluid was collected from patients with recent knee injury and from patients with early or late stage osteoarthritis. Chondroitin sulfate-substituted aggrecan fragments present in these fluids, and in normal bovine synovial fluid, were purified by cesium chloride gradient centrifugation, enzymically deglycosylated and fractionated by gel filtration on Superose-12. Each sample contained two major aggrecan core protein populations with apparent molecular masses of approximately 90 kD and 150 kD. For all samples, NH2-terminal analysis of both populations gave a single major sequence beginning ARGSV. This NH2 terminus results from cleavage of the human aggrecan core protein at the Glu 373-Ala 374 bond within the interglobular domain between the G1 and G2 domains. Cleavage at this site also occurs during control and interleukin-1 stimulated aggrecan catabolism in bovine cartilage explant cultures (Sandy, J., P. Neame, R. Boynton, and C. Flannery. 1991. J. Biol. Chem. 266:8683-8685). These results indicate that the major aggrecan fragments present in both osteoarthritic human synovial fluid and in normal bovine synovial fluid are large, being composed of a short NH2-terminal stretch of the interglobular domain, the G2 domain, the keratan sulfate domain, and variable lengths of the chondroitin sulfate domain(s). We conclude that the release of aggrecan fragments from articular cartilage into the synovial fluid seen at all stages of human osteoarthritis (Lohmander, L. S. 1991. Acta Orthop. Scand. 62:623-632) is promoted by the action of a normal cartilage proteinase which cleaves the Glu 373-Ala 374 bond of the interglobular domain.

Degradation of the large cartilage proteoglycan aggrecan in arthritis involves an unidentified enzyme aggrecanase, and at least one of the matrix metalloproteinases. Proteinase-sensitive cleavage sites in the aggrecan interglobular domain (IGD) have been identified for many of the humman MMPs, as well as for aggrecanase and other proteinases. The major MMP expressed by chondrocytes stimulated with retinoic acid to degrade their matrix is collagenase-3 or MMP-13. Because of its potential role in aggrecan degradation we examined the specificity of MMP-13 for an aggrecan substrate. The results show that MMP-13 cleaves aggrecan in the IGD at the same site (..PEN314-FFG..) identified for other members of the MMP family, and also at a novel site ..VKP384-VFE.. not previously observed for other proteinases.

BACKGROUND: Diabetes is a major threat to public health in the United States and worldwide. Understanding the role of environmental chemicals in the development or progression of diabetes is an emerging issue in environmental health. OBJECTIVE: We assessed the epidemiologic literature for evidence of associations between persistent organic pollutants (POPs) and type 2 diabetes. METHODS: Using a PubMed search and reference lists from relevant studies or review articles, we identified 72 epidemiological studies that investigated associations of persistent organic pollutants (POPs) with diabetes. We evaluated these studies for consistency, strengths and weaknesses of study design (including power and statistical methods), clinical diagnosis, exposure assessment, study population characteristics, and identification of data gaps and areas for future research. CONCLUSIONS: Heterogeneity of the studies precluded conducting a meta-analysis, but the overall evidence is sufficient for a positive association of some organochlorine POPs with type 2 diabetes. Collectively, these data are not sufficient to establish causality. Initial data mining revealed that the strongest positive correlation of diabetes with POPs occurred with organochlorine compounds, such as trans-nonachlor, dichlorodiphenyldichloroethylene (DDE), polychlorinated biphenyls (PCBs), and dioxins and dioxin-like chemicals. There is less indication of an association between other nonorganochlorine POPs, such as perfluoroalkyl acids and brominated compounds, and type 2 diabetes. Experimental data are needed to confirm the causality of these POPs, which will shed new light on the pathogenesis of diabetes. This new information should be considered by governmental bodies involved in the regulation of environmental contaminants.

The catabolism of aggrecan has been studied in calf articular cartilage explant cultures. The chondroitin sulfate-rich, high buoyant density products that accumulate in culture medium have been purified, and NH2-terminal sequence data have been obtained. Aggrecan released from the tissue in the presence or absence of interleukin-1 alpha, whether analyzed before or after reduction and alkylation, exhibited only one major and one minor NH2-terminal sequence. The major sequence, ARGXVILXAKPDF, shows very high similarity to a region of the interglobular domain (between the G1 and G2 domains) of both human and rat aggrecan. The minor sequence, VEVS, was that previously described for the NH2 terminus of the intact core protein. These results indicate that catabolism of aggrecan in cartilage explants involves proteolytic cleavage within a conserved region of the interglobular domain and that this results in the separation of the G1 domain from the remainder of the molecule. A major product of this process is a large nonaggregating species that consists of an NH2-terminal sequence beginning with ARG (and composed of about 100 residues of the interglobular domain) that is attached to an intact G2 domain followed by an extended section of the chondroitin sulfate-bearing domain toward the COOH terminus.

The objectives of this study were to determine the viscoelastic shear properties of articular cartilage and to investigate the effects of the alteration of proteoglycan structure on these shear properties. Glycosidase treatments (chondroitinase ABC and Streptomyces hyaluronidase) were used to alter the proteoglycan structure and content of the tissue. The dynamic viscoelastic shear properties of control and treated tissues were measured and statistically compared. Specifically, cylindrical bovine cartilage specimens were subjected to oscillatory shear deformation of small amplitude (gamma degrees = 0.001 radian) over a physiological range of frequencies (0.01-20 Hz) and at various compressive strains (5, 9, 12, and 16%). The dynamic complex shear modulus was calculated from the measurements. The experimental results show that the solid matrix of normal articular cartilage exhibits intrinsic viscoelastic properties in shear over the range of frequencies tested. These viscoelastic shear properties were found to be dependent on compressive strains. Our data also provide significant insights into the structure-function relationships for articular cartilage. Significant correlations were found between the material properties (the magnitude of dynamic shear modulus, the phase shift angle, and the equilibrium compressive modulus), and the biochemical compositions of the cartilage (collagen, proteoglycan, and water contents). The shear modulus was greatly reduced when the proteoglycans were degraded by either chondroitinase ABC or Streptomyces hyaluronidase. The results suggest that the ability of collagen to resist tension elastically provides the stiffness of the cartilage matrix in shear and its elastic energy storage capability. Proteoglycans enmeshed in the collagen matrix inflate the collagen network and induce a tensile prestress in the collagen fibrils. This interaction of the collagen and proteoglycan within the cartilage matrix provides the complex mechanism that allows the tissue to resist shear deformation.

Products generated by the digestion of human aggrecan with recombinant human stromelysin have been purified and analyzed by N-terminal sequencing and C-terminal peptide isolation. N-terminal analysis of chondroitin sulfate-bearing fragments revealed a clearly identifiable sequence initiating at residue Phe342 of human aggrecan, providing evidence for a cleavage site at the Asn341-Phe342 bond located within the interglobular domain. This cleavage site, which separates the G1 domain from the remainder of the molecule, was confirmed by isolation from the liberated G1 domain of a C-terminal tryptic peptide with the sequence YDAICYTGEDFVDIPEN (in which the C-terminal residue is Asn341). This peptide was also isolated from tryptic digests of hyaluronan-binding proteins (A1D4 samples) prepared by CsCl gradient centrifugation of extracts of mature human articular cartilages. Since these A1D4 samples contain G1 domain which accumulates as a result of aggrecan catabolism in vivo, these results clearly indicate that stromelysin cleaves the Asn341-Phe342 bond of human aggrecan in situ.

ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs-1) is a member of the ADAMTS family of metalloproteases which, together with ADAMTS-4 and ADAMTS-5, has been shown to degrade members of the lectican family of proteoglycans. ADAMTS-1 mRNA is induced in granulosa cells of periovulatory follicles by the luteinizing hormone surge through a progesterone receptor-dependent mechanism. Female progesterone receptor knockout (PRKO) mice are infertile primarily due to ovulatory failure and lack the normal periovulatory induction of ADAMTS-1 mRNA. We therefore investigated the protein localization and function of ADAMTS-1 in ovulating ovaries. Antibodies against two specific peptide regions, the pro-domain and the metalloprotease domain of ADAMTS-1, were generated. Pro-ADAMTS-1 of 110 kDa was identified in mural granulosa cells and appears localized to cytoplasmic secretory vesicles. The mature (85-kDa pro-domain truncated) form accumulated in the extracellular matrix of the cumulus oocyte complex (COC) during the process of matrix expansion. Each form of ADAMTS-1 protein increased >10-fold after the ovulatory luteinizing hormone surge in wild-type but not PRKO mice. Versican is also localized selectively to the ovulating COC matrix and was found to be cleaved yielding a 70-kDa N-terminal fragment immunopositive for the neoepitope DPEAAE generated by ADAMTS-1 and ADAMTS-4 protease activity. This extracellular processing of versican was reduced in ADAMTS-1-deficient PRKO mouse ovaries. These observations suggest that one function of ADAMTS-1 in ovulation is to cleave versican in the expanded COC matrix and that the anovulatory phenotype of PRKO mice is at least partially due to loss of this function.

PURPOSE: To determine the effect of serum on morphology, growth, and proteoglycan synthesis by primary cultures of collagenase-isolated bovine keratocytes. METHODS: Keratocytes were isolated from bovine corneas using sequential collagenase digestion and cultured in Dulbecco's modified Eagle's medium (DMEM), with and without fetal bovine serum (FBS). Proteoglycans synthesized by the cells in culture and by keratocytes in intact cornea culture were metabolically radiolabeled with 35SO4. The proteoglycans were characterized by their sensitivity to keratanase, chondroitinase ABC, and heparatinase and by their size on Superose 6 HR. Cell number was determined by measuring DNA content of the culture dishes. RESULTS: Keratocytes cultured in 10% FBS proliferated, appeared fibroblastic, and synthesized only 9% of the total glycosaminoglycan as keratan sulfate (KS), whereas cells in serum-free media were quiescent, appeared dendritic, and synthesized 47% KS, a value similar to the 45% KS for corneas radiolabeled overnight in organ culture. This increased proportion of KS synthesis in serum-free media was caused by a moderate increase in KS synthesis combined with a substantial decrease in chondroitin sulfate (CS) synthesis. Fractionation on Superose 6 High Resolution showed the size and relative amounts of the CS- and KS-containing proteoglycans synthesized by keratocytes in serum-free media also more closely resembled that of keratocytes in corneas in organ culture than keratocytes in media containing serum. CONCLUSIONS: A comparison of proteoglycan synthesis and cell morphology between keratocytes in corneas in organ culture and in cell culture indicates that keratocytes maintain a more native biosynthetic phenotype and appearance when cultured in serum-free media. These results also suggest that culturing in the presence of serum fundamentally alters the keratocyte phenotype to an activated cell, mimicking certain changes observed during wound healing.

Two forms of dermatan sulfate proteoglycans, called DS-PGI and DS-PGII, have been isolated from both bovine fetal skin and calf articular cartilage and characterized. The proteoglycans were isolated using either (a) molecular sieve chromatography under conditions where DS-PGI selectively self-associates or (b) chromatography on octyl-Sepharose, which separates DS-PGI from DS-PGII based on differences in the hydrophobic properties of their core proteins. The NH2-terminal amino acid sequence of DS-PGI from skin and cartilage is identical. The NH2-terminal amino acid sequence of DS-PGII from skin and cartilage is identical. However, the amino acid sequence data and tryptic peptide maps demonstrate that the core proteins of DS-PGI and DS-PGII differ in primary structure. In DS-PGI from bovine fetal skin, 81-84% of the glycosaminoglycan was composed of IdoA-GalNAc(SO4) disaccharide repeating units. In DS-PGI from calf articular cartilage, only 25-29% of the glycosaminoglycan was composed of IdoA-GalNAc(SO4). In DS-PGII from bovine fetal skin, 85-93% of the glycosaminoglycan was IdoA-GalNAc(SO4), whereas in DS-PGII from calf articular cartilage, only 40-44% of the glycosaminoglycan was IdoA-GalNAc(SO4). Thus, analogous proteoglycans from two different tissues, such as DS-PGI from skin and cartilage, possess a core protein with the same primary structure, yet contain glycosaminoglycan chains which differ greatly in iduronic acid content. These differences in the composition of the glycosaminoglycan chains must be determined by tissue-specific mechanisms which regulate the degree of epimerization of GlcA-GalNAc(SO4) into IdoA-GalNAc(SO4) and not by the primary structure of the core protein.

CONTEXT: Information on the use of oral bisphosphonate agents to treat pediatric osteogenesis imperfecta (OI) is limited. OBJECTIVE: The objective of the investigation was to study the efficacy and safety of daily oral alendronate (ALN) in children with OI. DESIGN AND PARTICIPANTS: We conducted a multicenter, double-blind, randomized, placebo-controlled study. One hundred thirty-nine children (aged 4-19 yr) with type I, III, or IV OI were randomized to either placebo (n = 30) or ALN (n = 109) for 2 yr. ALN doses were 5 mg/d in children less than 40 kg and 10 mg/d for those 40 kg and greater. MAIN OUTCOME MEASURES: Spine areal bone mineral density (BMD) z-score, urinary N-telopeptide of collagen type I, extremity fracture incidence, vertebral area, iliac cortical width, bone pain, physical activity, and safety parameters were measured. RESULTS: ALN increased spine areal BMD by 51% vs. a 12% increase with placebo (P < 0.001); the mean spine areal BMD z-score increased significantly from -4.6 to -3.3 (P < 0.001) with ALN, whereas the change in the placebo group (from -4.6 to -4.5) was insignificant. Urinary N-telopeptide of collagen type I decreased by 62% in the ALN-treated group, compared with 32% with placebo (P < 0.001). Long-bone fracture incidence, average midline vertebral height, iliac cortical width, bone pain, and physical activity were similar between groups. The incidences of clinical and laboratory adverse experiences were also similar between the treatment and placebo groups. CONCLUSIONS: Oral ALN for 2 yr in pediatric patients with OI significantly decreased bone turnover and increased spine areal BMD but was not associated with improved fracture outcomes.

Asporin, a novel member of the leucine-rich repeat family of proteins, was partially purified from human articular cartilage and meniscus. Cloning of human and mouse asporin cDNAs revealed that the protein is closely related to decorin and biglycan. It contains a putative propeptide, 4 amino-terminal cysteines, 10 leucine-rich repeats, and 2 C-terminal cysteines. In contrast to decorin and biglycan, asporin is not a proteoglycan. Instead, asporin contains a unique stretch of aspartic acid residues in its amino-terminal region. A polymorphism was identified in that the number of consecutive aspartate residues varied from 11 to 15. The 8 exons of the human asporin gene span 26 kilobases on chromosome 9q31.1–32, and the putative promoter region lacks TATA consensus sequences. The asporin mRNA is expressed in a variety of human tissues with higher levels in osteoarthritic articular cartilage, aorta, uterus, heart, and liver. The deduced amino acid sequence of asporin was confirmed by mass spectrometry of the isolated protein resulting in 84% sequence coverage. The protein contains anN-glycosylation site at Asn281 with a heterogeneous oligosaccharide structure and a potentialO-glycosylation site at Ser54. The name asporin reflects the aspartate-rich amino terminus and the overall similarity to decorin. Asporin, a novel member of the leucine-rich repeat family of proteins, was partially purified from human articular cartilage and meniscus. Cloning of human and mouse asporin cDNAs revealed that the protein is closely related to decorin and biglycan. It contains a putative propeptide, 4 amino-terminal cysteines, 10 leucine-rich repeats, and 2 C-terminal cysteines. In contrast to decorin and biglycan, asporin is not a proteoglycan. Instead, asporin contains a unique stretch of aspartic acid residues in its amino-terminal region. A polymorphism was identified in that the number of consecutive aspartate residues varied from 11 to 15. The 8 exons of the human asporin gene span 26 kilobases on chromosome 9q31.1–32, and the putative promoter region lacks TATA consensus sequences. The asporin mRNA is expressed in a variety of human tissues with higher levels in osteoarthritic articular cartilage, aorta, uterus, heart, and liver. The deduced amino acid sequence of asporin was confirmed by mass spectrometry of the isolated protein resulting in 84% sequence coverage. The protein contains anN-glycosylation site at Asn281 with a heterogeneous oligosaccharide structure and a potentialO-glycosylation site at Ser54. The name asporin reflects the aspartate-rich amino terminus and the overall similarity to decorin. Cartilage matrix consists of fibrillar networks, primarily of collagen II and highly negatively charged molecules of aggrecan. There are also a number of noncollagenous glycoproteins that apparently contribute to the regulation of tissue assembly and properties. Among them is the family of the leucine-rich repeat (LRR) 1The abbreviations used are: LRRleucine-rich repeatGdnHClguanidinium hydrochlorideHPLChigh-pressure liquid chromatographyMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flightPAGEpolyacrylamide gel electrophoresisPCRpolymerase chain reactionproteins, which contains several members found in the extracellular matrix. There are currently 11 known members of this family. These molecules share a common structure with a central stretch of LRRs. This LRR domain is flanked by disulfide bridged loops, with 4 cysteine residues preceding the LRR domain and 2 on its C-terminal side. Apart from chondroadherin, these proteins also contain divergent amino-terminal extensions with features unique for the different proteins. Based on amino acid sequence and gene organization the family can be divided into four distinct groups. leucine-rich repeat guanidinium hydrochloride high-pressure liquid chromatography matrix-assisted laser desorption/ionization time-of-flight polyacrylamide gel electrophoresis polymerase chain reaction Decorin (1Krusius T. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7683-7687Crossref PubMed Scopus (415) Google Scholar) and biglycan (2Fisher L.W. Termine J.D. Young M.F. J. Biol. Chem. 1989; 264: 4571-4576Abstract Full Text PDF PubMed Google Scholar) constitute the first group (class I). These proteins have 10 LRRs and carry one and two chondroitin or dermatan sulfate chains, respectively. The glycosaminoglycan chains are linked to serine residues in the amino terminus. The molecules in this group are secreted with a propeptide. The second group (class II) consists of fibromodulin (3Oldberg Å. Antonsson P. Lindblom K. Heinegård D. EMBO J. 1989; 8: 2601-2604Crossref PubMed Scopus (228) Google Scholar), lumican (4Blochberger T.C. Vergnes J.P. Hempel J. Hassell J.R. J. Biol. Chem. 1992; 267: 347-352Abstract Full Text PDF PubMed Google Scholar), keratocan (5Corpuz L.M. Funderburgh J.L. Funderburgh M.L. Bottomley G.S. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 9759-9763Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), PRELP (6Bengtsson E. Neame P.J. Heinegård D. Sommarin Y. J. Biol. Chem. 1995; 270: 25639-25644Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), and osteoadherin (7Sommarin Y. Wendel M. Shen Z. Hellman U. Heinegård D. J. Biol. Chem. 1998; 273: 16723-16729Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Like the class I proteins they consist of 10 LRRs. With the exception of PRELP, they all carry polylactosamine or keratan sulfate chains linked to the LRR region and sulfated tyrosine residues in the amino-terminal extension. In contrast, the amino terminus of PRELP has a cluster of positively charged amino acid residues that mediates binding to heparan sulfate (8Bengtsson E. Aspberg A. Heinegård D. Sommarin Y. Spillmann D. J. Biol. Chem. 2000; 275: 40695-40702Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Unlike all other family members, osteoadherin contains a COOH-terminal extension (7Sommarin Y. Wendel M. Shen Z. Hellman U. Heinegård D. J. Biol. Chem. 1998; 273: 16723-16729Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Epiphycan/PG-Lb/DSPG3 (9Johnson H.J. Rosenberg L. Choi H.U. Garza S. Hook M. Neame P.J. J. Biol. Chem. 1997; 272: 18709-18717Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 10Shinomura T. Kimata K. J. Biol. Chem. 1992; 267: 1265-1270Abstract Full Text PDF PubMed Google Scholar, 11Deere M. Johnson J. Garza S. Harrison W.R. Yoon S.J. Elder F.F.B. Kucherlapati R. Hook M. Hecht J.T. Genomics. 1996; 38: 399-404Crossref PubMed Scopus (22) Google Scholar), mimecan/osteoglycin (12Madisen L. Neubauer M. Plowman G. Rosen D. Segarini P. Dasch J. Thompson A. Ziman J. Bentz H. Purchio A.F. DNA Cell Biol. 1990; 9: 303-309Crossref PubMed Scopus (75) Google Scholar, 13Funderburgh J.L. Corpuz L.M. Roth M.R. Funderburgh M.L. Tasheva E.S. Conrad G.W. J. Biol. Chem. 1997; 272: 28089-28095Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), and opticin/oculoglycan (14Reardon A.J. Le Goff M. Briggs M.D. McLeod D. Sheehan J.K. Thornton D.J. Bishop P.N. J. Biol. Chem. 2000; 275: 2123-2129Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 15Friedman J.S. Ducharme R. Raymond V. Walter M.A. Invest. Ophthalmol. Vis. Sci. 2000; 41: 2059-2066PubMed Google Scholar, 16Hobby P. Wyatt M.K. Gan W. Bernstein S. Tomarev S. Slingsby C. Wistow G. Mol. Vis. 2000; 6: 72-78PubMed Google Scholar) form the third group (class III). These are smaller molecules with only 6 LRRs and all contain sulfated tyrosine residues in the amino-terminal extension. In addition, epiphycan carries chondroitin sulfate, other O-linked oligosaccharides, and a cluster of glutamate residues in this region (9Johnson H.J. Rosenberg L. Choi H.U. Garza S. Hook M. Neame P.J. J. Biol. Chem. 1997; 272: 18709-18717Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The amino-terminal extension of opticin carriesO-linked oligosaccharides (14Reardon A.J. Le Goff M. Briggs M.D. McLeod D. Sheehan J.K. Thornton D.J. Bishop P.N. J. Biol. Chem. 2000; 275: 2123-2129Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and contains a heparin-binding consensus sequence (17Cardin A.D. Weintraub H.J. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar). Chondroadherin (18Neame P.J. Sommarin Y. Boynton R.E. Heinegård D. J. Biol. Chem. 1994; 269: 21547-21554Abstract Full Text PDF PubMed Google Scholar) forms the fourth branch on the extracellular matrix LRR protein family tree (class IV). This protein contains 10 LRRs, but lacks both amino- and COOH-terminal extensions outside the cysteine motifs. Nyctalopin, a recently published glycosylphosphatidylinositol-anchored LRR protein may also be a member of this subfamily (19Bech-Hansen N.T. Naylor M.J. Maybaum T.A. Sparkes R.L. Koop B. A. M.A. Young 2000; PubMed Scopus Google Scholar, C. K. H. S. C. J. A. S. A. B. W. A. 2000; PubMed Scopus Google Scholar). is from the the of LRR proteins into on sequence not the of the biglycan, and epiphycan are chondroitin or dermatan sulfate and may be related the different class II LRR proteins. A that is of the class and LRR proteins is a to to collagen the LRR This is a binding in the The different extensions a variety of for with other matrix other of and of the fibrillar of these molecules to have in the assembly of collagen is by in PubMed Scopus Google Scholar, M. Heinegård D. J. PubMed Scopus Google Scholar, E. Heinegård D. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar, E. Heinegård D. J. Biol. Chem. Full Text PDF PubMed Google Scholar) by gene H. H. J. Cell Biol. 1997; PubMed Scopus Google Scholar, S. T. C. H. J. Cell Biol. 1998; PubMed Scopus Google Scholar, L. A. R. Heinegård D. Å. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these collagen the of the LRR protein is The with a of of proteins in human found a number of proteins to be one a with of This related to fibromodulin in a variety of the structure a putative oligosaccharide chain and tissue of the It a novel member of the LRR protein family to the group (class I). The protein is asporin on the of a stretch in the amino-terminal region and the similarity with decorin. human cartilage of tissue to and of tissue at The tissues into and a in of for at 4 the was by at at 4 for in the cartilage from by with a of in D. Sommarin Y. PubMed Scopus Google Scholar). The was divided into 4 a and the was used for The was by by 4 and to a 6 in of for protein by at and by M. Sommarin Y. Heinegård D. J. PubMed Scopus Google Scholar). The proteins from the into two a proteins of to and a smaller the smaller proteins. The to and by by The was a of in the the was with of the and with a of to in the at a of of 10 for protein by at and by The asporin by by 10 and on a of in The was with and the proteins at a of with the of 2 for protein by at and by The asporin and by to 10 and to a The proteins with a from to at a of of 2 for and by of the a of proteoglycan. the was to of the to by and by chromatography that the was at the from the chromatography asporin and by by 4 This was on two of 6 and and at with of for protein by at by with was at to of to the by on a with a of in acid at a of The was at on with of on the gel and DNA to J. E. T. A Scholar). The amino acid from asporin used to the with the W. W. D.J. J. Mol. Biol. 1990; PubMed Scopus Google Scholar). The identified from this and The resulting sequence was used for and osteoarthritic articular cartilage was at in and in liquid and mRNA purified P. Neame P. Sommarin Y. Heinegård D. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). was with and with II of the mRNA with the asporin was the polymerase chain reaction with and and DNA at for the DNA was for of at at and 2 at The resulting was isolated from purified the and into the The and several of the resulting the and on a DNA In to and two and The resulting sequence the II of the A first mouse asporin sequence was from a of the mouse of with the human asporin this sequence the and first was and asporin from this by and The was into and all four and The human and mouse asporin in with the and respectively. 10 of isolated from human osteoarthritic articular cartilage on and to a of and from The with a of the human with by the DNA and of the to the The to or by the to be with in and in a for of was of of and of the and was with and at for 4 and on the polyacrylamide by with on and in the gel in a and and 10 and at and at respectively. and at of or at in The was by the of 10 of which also the of the a at purified from the acid used to the sequence coverage. and that the to a smaller the was used M. C. H. M. H. E. Chem. 2000; PubMed Scopus Google Scholar). The mass was of protein from the gel H. S. A. B. 1997; PubMed Scopus Google Scholar) by to the acid the matrix. was used for a matrix-assisted laser desorption/ionization time-of-flight mass The was used in the with and of 26 the and for of of protein the was with of The used to the and oligosaccharide and structure W. Chem. 2000; PubMed Scopus Google Scholar) and C. E. PubMed Scopus Google Scholar), respectively. of human articular cartilage with 4 by the matrix proteins from the of the in the of the by gel on 6 in two one proteins and the other with proteins of The proteins in this by asporin was in the also These on a at asporin and fibromodulin they from other proteins in the to asporin from fibromodulin on a at with a the two proteins with fibromodulin the to asporin and fibromodulin by gel with in the and chromatography A to the collagen of fibromodulin E. Heinegård D. J. Biol. Chem. Full Text PDF PubMed Google Scholar) to the protein with collagen the that also asporin was with the collagen not The of a of human by electrophoresis a of not The was on a in was in a identified by These and by gel on two of and 6 in a with a of the of the protein from by and two but by of the of the amino at was to both with a of the protein was not and isolated that with the that of these was from to but the of these are in the sequence of the identified by are the and the The the amino acid of the in a the identified by are the and the The the amino acid of the is sequence in a putative extracellular matrix The deduced sequence contains several leucine-rich and the two COOH-terminal cysteine residues of the extracellular matrix protein family. was used the in a number of other of these a that a and the amino-terminal of the extracellular matrix LRR proteins. The of the novel LRR protein was from human osteoarthritic cartilage, to the and of the consensus The mouse was identified of the mouse with the human sequence and from mouse on the sequences. The human and mouse asporin are in The amino acid of the two proteins are The four amino-terminal the of decorin and biglycan Mol. Biol. 1997; PubMed Scopus Google Scholar), which asporin a member of the class I branch of the LRR proteins. decorin and biglycan, asporin contains a highly putative sequence acid residues The putative site to the site in biglycan Y. A.D. P.J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). of the LRRs of asporin to decorin and biglycan a in amino acid sequence repeat of a tree of the extracellular matrix LRR proteins confirmed that asporin to the I the decorin and biglycan branch Unlike decorin and biglycan, asporin contains consensus glycosaminoglycan in its amino terminus. There one consensus site for and in the human and mouse asporin In contrast to all identified extracellular matrix LRR proteins, asporin has a stretch of aspartic acid residues in its amino-terminal region. found that the number of consecutive aspartic acid residues is of the human asporin the sequence the first residues in the human sequence revealed that at this several with of residues M. Johnson J. Garza S. Harrison W.R. Yoon S.J. Elder F.F.B. Kucherlapati R. Hook M. Hecht J.T. Genomics. 1996; 38: 399-404Crossref PubMed Scopus (22) Google Scholar, L. Neubauer M. Plowman G. Rosen D. Segarini P. Dasch J. Thompson A. Ziman J. Bentz H. Purchio A.F. DNA Cell Biol. 1990; 9: 303-309Crossref PubMed Scopus (75) Google Scholar, 13Funderburgh J.L. Corpuz L.M. Roth M.R. Funderburgh M.L. Tasheva E.S. Conrad G.W. J. Biol. Chem. 1997; 272: 28089-28095Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, A.J. Le Goff M. Briggs M.D. McLeod D. Sheehan J.K. Thornton D.J. Bishop P.N. J. Biol. Chem. 2000; 275: 2123-2129Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 15Friedman J.S. Ducharme R. Raymond V. Walter M.A. Invest. Ophthalmol. Vis. Sci. 2000; 41: 2059-2066PubMed Google Scholar) identified in the human In addition, the sequence of human asporin for the used in the human asporin was from tissue from several that this a found in the stretch of the mouse which and tree of extracellular matrix LRR proteins. The tree was with W. the of the extracellular matrix LRR protein A of with and for the and mouse respectively. The human asporin gene is on chromosome 9q31.1–32, the This also contains the for the LRR proteins and mimecan/osteoglycin A asporin was from sequence and a number of to the and of the sequence of a from chromosome that the asporin sequence number The first is also in the in the asporin gene 26 kilobases and consists of 8 the the and the the the in decorin and biglycan the are in the in decorin and biglycan It is exons are in the of the is the in decorin. Like biglycan consensus TATA is found of the first of the asporin A number of binding identified of the asporin in the human asporin sequence is from number exons number are in the of this The of of the sequence is in sequence in and in to of the first or the second of the The sequence is from number exons number are in the of this The of of the in a sequence is in sequence in and in to of the first or the second of the that the asporin gene for a of kilobases a human tissue found that the of was in the by the the was in the other articular cartilage is not on the was not to asporin in cartilage with that in other the of human osteoarthritic cartilage a that 10 of cartilage of was with 2 of from the other and that the the of asporin may be higher in articular cartilage in the other tissues A for the and of asporin was by of a mRNA which and tissues The asporin with a of human tissues with the levels in and levels found in heart, and in the and asporin was in and There was a of in the central in and A asporin was in mass spectrometry was used to the protein with the with the from the novel The identified of asporin are in The identified 84% of the protein The only in the sequence is the amino-terminal the consecutive This is to the of this which and mass spectrometry from asporin identified by mass acid with a mass of identified only are in in a with a mass of identified only are in The protein has one site at of asporin with confirmed that the protein contains oligosaccharides by the in its on This in the of the Asn281 by mass spectrometry this was not in protein A of mass was which can be by of to a H. T. G. C. 2000; PubMed Scopus Google Scholar). be both in in A mass of was of the oligosaccharide and putative of the linked to Asn281 in asporin from cartilage and are in the of the The of oligosaccharides at Asn281 was confirmed This has a different resulting in mass This mass with the and putative of on human mass at in human asporin was with mass and structure not in a mass at in human asporin was with mass and structure not The amino-terminal of the protein contains a potentialO-glycosylation site at which may be with oligosaccharide not the time-of-flight mass two with and that to the amino-terminal linked oligosaccharide The mass the two which be of linked oligosaccharide This is by the of in these the of a in the amino-terminal sequence that have consecutive residues in the a of and is for the with and respectively. to that the protein contains the sequence is to its this not be from the partially purified asporin the of the protein from the gel the of by mass of was for the protein from the The mass was to in the by the of with the that of the gel with This in with a higher The mass of the protein with consecutive residues in the amino-terminal is by a mass for oligosaccharide of and for O-linked oligosaccharide of to the the mass that the amino-terminal is in the is a member of the LRR protein family closely related to decorin and biglycan. The four amino-terminal the of the class I LRR proteins. decorin and biglycan asporin contains a putative with a site to the sequence for the to Y. A.D. P.J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). the sequence and of the LRR of asporin are to of decorin and biglycan to other members of the proteins. This is also from the tree of the LRR proteins. the decorin A. Genomics. PubMed Scopus Google Scholar) and biglycan L.W. U. W. W. Termine J.D. Young M.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar) the human asporin gene is divided into 8 The are in the sequence at the to of decorin and biglycan. The exons of the human asporin gene span 26 kilobases on chromosome It is not the asporin gene also contains the decorin gene A. Genomics. PubMed Scopus Google Scholar). Like in the biglycan gene L.W. U. W. W. Termine J.D. Young M.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar), TATA was found in the region of of a number of for in the of the deduced The extracellular matrix LRR protein to be in of and epiphycan (class and to chromosome Asporin, and (class and are found on chromosome a gene a LRR protein amino-terminal has asporin and osteoadherin J. T. Y. Genomics. 1998; PubMed Scopus Google Scholar). PRELP, and opticin (class and to chromosome (class is unique in not of a cluster but found in on chromosome It that several have resulting in the organization of the LRR The biglycan gene may have to chromosome four LRR protein to be one on chromosome and on chromosome The asporin amino-terminal extension is in stretch of aspartate to asporin recently identified in the and the S. A. S. C. J. J. Mol. 2000; PubMed Scopus Google Scholar). These proteins to by the to the class I LRR proteins on the amino-terminal cysteine and the amino acid sequence of the LRRs. of aspartic acid residues in the amino-terminal extensions these proteins of Unlike in human the mouse and the two are by other amino acid the of a number of and in this region in the amino terminus for this negatively charged amino acid In contrast to decorin and biglycan, asporin is not a proteoglycan. It contains consensus for glycosaminoglycan the and the amino-terminal cysteine decorin (1Krusius T. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7683-7687Crossref PubMed Scopus (415) Google Scholar) and biglycan (2Fisher L.W. Termine J.D. Young M.F. J. Biol. Chem. 1989; 264: 4571-4576Abstract Full Text PDF PubMed Google Scholar) have one and two respectively. There a consensus sequence for in the human This was confirmed by which in a on and of the by mass The linked oligosaccharide in all the from different tissue or all are at a in the tissue is not The that the protein was from a of tissue from several can of also contribute to the on in to oligosaccharide may on the of The protein contains putative site in the human that to be with linked has in the amino-terminal extension of epiphycan and It to be oligosaccharide in this region may the of this The of the of class I LRR proteins is It has that this sequence the glycosaminoglycan structure of decorin Å. Antonsson P. J. 1996; PubMed Scopus Google Scholar) and biglycan P. D.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The of a in which not contain glycosaminoglycan consensus that the may have other mRNA is expressed in a number of different articular cartilage, the levels found in and uterus, by levels in other tissues with of The of the protein are not in to the protein from fibromodulin in a collagen both proteins to to This be in with of other LRR proteins of the members have to to collagen with in the of aspartic acid residues in asporin are a of the extracellular matrix of and has a acid sequence in the of the protein Å. A. Heinegård D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: PubMed Scopus Google Scholar). This protein to and may have a in J. 1994; PubMed Scopus Google Scholar). asporin also has a in is not In this the of the protein in is of in of the of in this in S. C. D. K. M. L. Scholar). A may be the found in epiphycan (9Johnson H.J. Rosenberg L. Choi H.U. Garza S. Hook M. Neame P.J. J. Biol. Chem. 1997; 272: 18709-18717Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) and in Å. A. Heinegård D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The stretch of the protein has to Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. 1994; PubMed Scopus Google Scholar). putative for asporin be to the stretch in with other matrix in with fibromodulin and lumican and also of the glycosaminoglycan chains of decorin and biglycan, the family This for the collagen in the The other two members of the family decorin and biglycan have to A. M. L.M. Heinegård D. Ruoslahti E. J. 1994; PubMed Scopus Google Scholar). also asporin has this to be are to and at the of for with time-of-flight mass

Keratocan (Kera) is a cornea-specific keratan sulfate proteoglycan (KSPG) in the adult vertebrate eye. It belongs to the small leucine-rich proteoglycan (SLRP) gene family and is one of the major components of extracellular KSPG in the vertebrate corneal stroma. The Kera gene is expressed in ocular surface tissues including cornea and eyelids during morphogenesis. Corneal KSPGs play a pivotal role in matrix assembly, which is accountable for corneal transparency. In humans, mutations of the KERA gene are associated with cornea plana (CNA2) that manifests decreases in vision acuity due to the flattened forward convex curvature of cornea. To investigate the biological role of the Kera gene and to establish an animal model for corneal plana, we generated Kera knockout mice via gene targeting. Northern and Western blotting and immunohistochemical analysis showed that no Kera mRNA or keratocan protein was detected in the Kera–/– cornea. The expression levels of other SLRP members including lumican, decorin, and fibromodulin were not altered in the Kera–/– cornea as compared with that of the wild-type littermates. Mice lacking keratocan have normal corneal transparency at the age of 12 months. However, they have a thinner corneal stroma and a narrower cornea-iris angle of the anterior segment in comparison to the wild-type littermates. As demonstrated by transmission electron microscopy, Kera–/– mice have larger stromal fibril diameters and less organized packing of collagen fibrils in stroma than those of wild type. Taken together, our results showed that ablation of the Kera gene resulted in subtle structural alterations of collagenous matrix and did not perturb the expression of other SLRPs in cornea. Keratocan thus plays a unique role in maintaining the appropriate corneal shape to ensure normal vision. Keratocan (Kera) is a cornea-specific keratan sulfate proteoglycan (KSPG) in the adult vertebrate eye. It belongs to the small leucine-rich proteoglycan (SLRP) gene family and is one of the major components of extracellular KSPG in the vertebrate corneal stroma. The Kera gene is expressed in ocular surface tissues including cornea and eyelids during morphogenesis. Corneal KSPGs play a pivotal role in matrix assembly, which is accountable for corneal transparency. In humans, mutations of the KERA gene are associated with cornea plana (CNA2) that manifests decreases in vision acuity due to the flattened forward convex curvature of cornea. To investigate the biological role of the Kera gene and to establish an animal model for corneal plana, we generated Kera knockout mice via gene targeting. Northern and Western blotting and immunohistochemical analysis showed that no Kera mRNA or keratocan protein was detected in the Kera–/– cornea. The expression levels of other SLRP members including lumican, decorin, and fibromodulin were not altered in the Kera–/– cornea as compared with that of the wild-type littermates. Mice lacking keratocan have normal corneal transparency at the age of 12 months. However, they have a thinner corneal stroma and a narrower cornea-iris angle of the anterior segment in comparison to the wild-type littermates. As demonstrated by transmission electron microscopy, Kera–/– mice have larger stromal fibril diameters and less organized packing of collagen fibrils in stroma than those of wild type. Taken together, our results showed that ablation of the Kera gene resulted in subtle structural alterations of collagenous matrix and did not perturb the expression of other SLRPs in cornea. Keratocan thus plays a unique role in maintaining the appropriate corneal shape to ensure normal vision. The vertebrate cornea is a tough and transparent tissue that provides greater than 60% of refractive power to the incoming light to cast a focused image on retina. To exact its proper functions, the cornea has to maintain transparent, appropriate curvature for refraction and toughness for protection. Genetic and epigenetic factors that cause the change of corneal curvature, reduction of transparency, and weakness in structure can lead to the impairment of visual acuity. The corneal stroma consists of uniformly small collagen fibrils with an average diameter of 25 nm that are arranged in orthogonal lamellae. Stromal keratocytes are responsible for the formation and maintenance of a unique collagenous matrix that is essential for proper corneal curvature and transparency (1Linsenmayer T.F. Fitch J.M. Birk D.E. Ann. N. Y. Acad. Sci. 1990; 580: 114-160Crossref Scopus (73) Google Scholar, 2Hay E.D. Int. Rev. Cytol. 1980; 63: 263-322Crossref PubMed Scopus (297) Google Scholar). The mechanisms that govern the assembly of different levels of stromal architecture are not well understood; however, proteoglycan-collagen and collagen-collagen interactions have been implicated. It has been suggested that the stoichiometry and interaction of different collagen types play an important role in modulating collagen fibril diameter (1Linsenmayer T.F. Fitch J.M. Birk D.E. Ann. N. Y. Acad. Sci. 1990; 580: 114-160Crossref Scopus (73) Google Scholar, 3Doane K.J. Babiarz J.P. Fitch J.M. Linsenmayer T.F. Birk D.E. Exp. Cell Res. 1992; 202: 82-95Crossref Scopus (48) Google Scholar, 4Hahn R.A. Birk D.E. Development. 1992; 84: 383-393Google Scholar, 5Rada J.A. Cornuet P.K. Hassell J.R. Exp. Eye Res. 1993; 56: 635-648Crossref PubMed Scopus (291) Google Scholar). The proteoglycans in the stroma are members of the small leucine-rich proteoglycan (SLRP) 1The abbreviations used are: SLRP, small leucine-rich proteoglycan; KO, knockout; KS, keratan sulfate; E, embryonic day. gene family and are thought to regulate collagenous matrix assembly in connective tissue because of their bifunctional character: the protein moiety that binds collagen fibrils and the highly charged hydrophilic glycosaminoglycans that regulate interfibrillar spacing (1Linsenmayer T.F. Fitch J.M. Birk D.E. Ann. N. Y. Acad. Sci. 1990; 580: 114-160Crossref Scopus (73) Google Scholar, 2Hay E.D. Int. Rev. Cytol. 1980; 63: 263-322Crossref PubMed Scopus (297) Google Scholar, 6Weber I.T. Harrison R.W. Iozzo R.V. J. Biol. Chem. 1996; 271: 31767-31770Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 7Iozzo R.V. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 112-174Crossref Scopus (454) Google Scholar, 8Scott J.E. Biochemistry. 1996; 35: 8795-8799Crossref PubMed Scopus (215) Google Scholar, 9Bettelheim F.A. Plessy B. Biochim. Biophys. Acta. 1975; 381: 203-214Crossref PubMed Scopus (94) Google Scholar, 10Rawe I.M. Tuft S.J. Meek K.M. Histochem. J. 1992; 24: 311-318Crossref PubMed Scopus (31) Google Scholar, 11Hassell J.R. Cintron C. Kublin C. Newsome D.A. Arch. Biochem. Biophys. 1983; 222: 362-369Crossref PubMed Scopus (179) Google Scholar). In addition to interactions with collagen fibrils, corneal stromal proteoglycans also play a role in corneal hydration due to the highly negative charge of their sulfated carbohydrate moieties and the glycosaminoglycan chains. Keratocan, lumican, and mimecan/osteoglycin are the major keratan sulfate-containing proteoglycans in vertebrate corneal stroma. Their core proteins consist of 6–10 tandem repeats of 24 amino acids with hydrophobic residues in conserved positions. We and others have recently shown that mice lacking lumican revealed an age-dependent corneal opacity and a high proportion of abnormally thick collagen fibrils in the corneal stroma (12Charkravarti S. Magnuson T. Lass J.H. Jepsen K.J. LaMantia C. Carroll H. J. Cell Biol. 1998; 112: 987-996Google Scholar, 13Chakravarti S. Petroll W.M. Hassell J.R. Jester J.V. Lass J.H. Paul J. Birk D.E. Investig. Ophthalmol. Vis. Sci. 2000; 11: 3365-3373Google Scholar, 14Saika S. Shiraishi A. Liu C.-Y. Funderburgh J.L. Kao C.W. Converse R.L. Kao W.W.-Y. J. Biol. Chem. 2000; 275: 2607-2612Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). In humans, the first SLRP gene that is directly linked to a disease is the keratocan gene (KERA). Mutations of the human KERA gene are associated with cornea plana (CNA2) in which the forward convex curvature is flattened, leading to a decrease in light refraction (15Pellegata N.S. Dieguez-Lucena J.L. Joensuu T. Lau S. Montgomery K.T. Krahe R. Kivela T. Kucherlapati R. Forsius H. de la Chapelle A. Nat. Genet. 2000; 25: 91-95Crossref PubMed Scopus (124) Google Scholar). The clinical synopsis of CNA2 includes reduced visual activity, extreme hyperopia, hazy corneal limbus, corneal parenchymal opacities, thin corneal stroma, and indistinct sclerocorneal boundary. Moreover CNA2 with extremely flat cornea is often associated with angle closure glaucoma. We have found previously that, unlike lumican or mimecan/osteoglycin, which is ubiquitously expressed by most interstitial connective tissues, keratocan expression is specific to the adult corneal stroma in the mouse. During embryonic development, Kera gene expression tracks ocular surface tissues morphogenesis including cornea and eyelids (16Liu C.-Y. Shiraishi A Kao C.W. Converse R.L. Funderburgh J.L. Corpuz L.M. Conrad G.W. Kao W.W.-Y. J. Biol. Chem. 1998; 273: 22584-22588Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). To investigate the biological role of the Kera in vivo and to generate an animal model for corneal plana, we created Kera knockout mice via gene targeting. Western Blotting—To characterize keratocan in wild-type mice, two corneas were isolated from both eyes of normal FVB/N postnatal day 1, 7, and 14 mice. The corneas were minced and extracted in 1 ml of 4 m guanidine-HCl containing 10 mm sodium acetate, 10 mm sodium EDTA, 5 mmp-aminobenzamidine, and 0.1 m [epsis ]-amino-n-caproic acid at 4 °C overnight. The extracts were dialyzed exhaustively in distilled water at room temperature, and the water-insoluble fraction was dissolved in 0.1 m Tris acetate solution (pH 6.0) containing 6 m urea. The protein concentration was measured by spectrophotometer at A280 nm. Aliquots (100 μg of protein) were incubated with or without endo-β-galactosidase (0.1 unit/ml) at 37 °C overnight. An equal volume of 2× SDS sample buffer was added into digested and undigested samples, boiled for 5 min, electrophoresed on an SDS-PAGE gradient (4–15%) or regular 10% gel, and transferred to polyvinylidene difluoride membrane. Affinity-purified antibodies against mouse keratocan peptide (VRQAYEIQDPEDWDVHDDFYC) were raised in rabbit and used to detect the keratocan in corneal samples. Alkaline phosphatase-conjugated goat anti-rabbit IgG (heavy and light) was used as secondary antibody, and the immunocomplex was visualized with a Western Blue™ kit (Promega, Madison, WI). To detect decorin and fibromodulin of the mouse corneas, affinity-purified antibodies against mouse decorin peptide (IIPYDPDNPLISMC (17Fisher L.W. Stubbs III, J.T. Young M.F. Acta Orthop. Scand. Suppl. 1995; 266: 61-65Crossref PubMed Scopus (420) Google Scholar)) or mouse fibromodulin (CDKVGRKVFSKLRHLER and CDPYDPYPYEPSEPYPYGVEE) (a generous gift from Dr. Larry Fisher, National Institutes of Health) 2See csdb.nidcr.nih.gov/csdb/frame_reagents.htm on the World Wide Web. were raised in rabbit and used as primary antibody. Horseradish peroxidase-conjugated goat anti-rabbit IgG (heavy and light) was used as secondary antibody, and the immunocomplex was visualized with ECL™ Western blotting detection substrate (Pierce). Generation of Kera Knockout Mice by Gene Targeting—The mouse keratocan genomic 6.6-kb BamHI-EcoRI fragment, which contains 3.1-kb promoter, exon 1, intron 1, exon 2, and 1.1-kb intron 2, was first cloned into pBluescript SK vector (Stratagene, La Jolla, CA), and the resulting plasmid was named pKera-BR6.6 (16Liu C.-Y. Shiraishi A Kao C.W. Converse R.L. Funderburgh J.L. Corpuz L.M. Conrad G.W. Kao W.W.-Y. J. Biol. Chem. 1998; 273: 22584-22588Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). A 0.6-kb XbaI fragment was then deleted from the 5′ end of the pKera-BR6.6, and the plasmid was self-ligated. Next a 2.6-kb XhoI fragment containing 1.0-kb intron 1, entire exon 2, and 1.1-kb intron 2 was deleted from the 3′ end and replaced with a positive selection marker, neomycin resistance gene (pgkpr-neopA) cassette in the antisense orientation with respect to the Kera gene. The resulting plasmid was designated as pKera5′-pgk-Neo. A 1.6-kb KpnI fragment, which contains 0.6-kb exon 2 and 1.0-kb intron 2, was excised from the pKera-BR6.6 and inserted into pKera5′-pgkNeo digested with KpnI. Finally a negative selection marker gene, diphtheria toxin A fragment (pgkpr-Dta) cassette, was placed on the 3′ end of the targeting vector. Therefore, the Kera gene targeting vector, pKera-TV, contains a 3.1- and a 1.6-kb homologous sequence on 5′ and 3′ ends, respectively. The pKera-TV (80 μg) was linearized and transfected into an embryonic stem cell line (Duffy) via electroporation (250 microfarads and 800 V/cm). After 12 days of selection with Geneticin® (G418, 500 μg/ml), the G418-resistant colonies were individually picked and expanded. The homologous recombinant embryonic stem clones were identified by PCR-based analysis. PCR was carried out with the Expand Long Template PCR System (Roche Diagnostics). The primers used for 5′ PCR were as follows: kera-1, 5′-gatggcctagtcggccatcactgcaaagag-3′; kera-2, 5′-cacccagagtattaaacagtttggggttgc-3′; neo(+), 5′-cgcttcctcgtgctttacggtatcgccgctc-3′. The primers used for 3′ PCR were as follows: of the 5′ PCR was by of °C for 1 min, °C for 1 min, and °C for of 3′ PCR was the at °C was The and mice were by the gene targeting core at the of keratocan and lumican were and for rabbit antibodies against mouse lumican S. Shiraishi A. Liu C.-Y. Funderburgh J.L. Kao C.W. Converse R.L. Kao W.W.-Y. J. Biol. Chem. 2000; 275: 2607-2612Abstract Full Text Full Text PDF PubMed Scopus (209) Google and mouse keratocan peptide as primary and peroxidase-conjugated goat anti-rabbit IgG antibodies were used as secondary antibodies as previously S. Shiraishi A. Liu C.-Y. Funderburgh J.L. Kao C.W. Converse R.L. Kao W.W.-Y. J. Biol. Chem. 2000; 275: 2607-2612Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). was visualized with The were with a in and by light In as well as detection were from The mouse keratocan (16Liu C.-Y. Shiraishi A Kao C.W. Converse R.L. Funderburgh J.L. Corpuz L.M. Conrad G.W. Kao W.W.-Y. J. Biol. Chem. 1998; 273: 22584-22588Abstract Full Text Full Text PDF PubMed Scopus (84) Google and lumican S. Shiraishi A. Kao C.W. Converse R.L. Funderburgh J.L. J. Conrad G.W. Kao W.W.-Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google in pBluescript plasmid vector were linearized at end of the with or antisense and of keratocan mRNA were generated by in appropriate for antisense and for keratocan at 37 °C for 2 The were by at 37 °C for The in were then into by the in m at °C for Finally the were by and 5 was used for the The mouse eyes and were with at 4 °C and in The in was on on as previously A. S. J.P. Kao W.W.-Y. Liu C.-Y. of the and Scholar). To were to a with at °C and with at room for 1 by with at The were visualized with by from and wild-type were in 0.1 m sodium with mm for 2 on The corneas were and with for 1 After in a by the corneas were and in a of and were and with for and selection of specific for analysis. were a and a was with acetate by were and at a transmission electron The was a line cornea stroma was into two for anterior and stroma. The anterior stroma was as the 10 to the and the stroma was as the 10 to of appropriate were at from were in a The were and diameters were measured an image analysis the of fibril the fibril diameter from two were and no were the Therefore, in the from different were The and from the were for by a 2 of 24 the of normal was appropriate of the We the model with and without the two in and and Therefore, the analysis on the is The of fibrils measured was and for the anterior and and for the were of in during the and characterize keratocan protein expression in the wild-type mice, corneas were from day 1, 7, and 14 mice. The keratocan core protein was detected by SDS-PAGE Western blotting with antibodies against an peptide of mouse keratocan the peptide Keratocan as in undigested 1, of the corneal with endo-β-galactosidase to the keratan sulfate reduced keratocan to its core protein of 1, is in cornea of mice at postnatal day 1 and a in both the and of in corneas of mice postnatal day and day a in which the in mice. Generation of Keratocan in the biological of keratocan in we generated keratocan knockout mice via gene targeting. To ensure mice we deleted the of exon 2, which contains the and replaced with cassette in the antisense orientation with respect to the Kera gene 2, The of the mice were by PCR with specific for the wild-type from the at both 5′ and 3′ 2, G418-resistant embryonic stem cell embryonic stem cell clones were identified and then into line transmission from was and no were different of Keratocan Knockout mice are and without as hazy cornea at the age of 4 In showed that keratocan mRNA was in the the lumican mRNA expression showed no the wild-type and the corneas the keratocan protein was as by an immunohistochemical not As shown by the light the cornea-iris angle of the anterior segment was and the corneal stroma was thinner in the Kera–/– as compared with the wild-type littermates. Western blotting of the corneal extracts from also demonstrated that no keratocan protein detected in the mice revealed a The expression of lumican, and decorin in the cornea as by Western blotting showed no alterations the wild-type and the corneas and fibromodulin expression have not been altered in the Kera–/– cornea. 10 μg of 4 m guanidine-HCl protein was to SDS-PAGE Western blotting were with affinity-purified and against decorin or fibromodulin It was that, in the Kera–/– decorin fibromodulin was undigested image corneal from and wild-type mice were transmission electron the mouse a in the corneal stroma S. Petroll W.M. Hassell J.R. Jester J.V. Lass J.H. Paul J. Birk D.E. Investig. Ophthalmol. Vis. Sci. 2000; 11: 3365-3373Google the anterior and were were the and wild-type the collagen fibrils were larger in was less regular fibril packing in the was the stroma with no anterior and stroma. The collagen fibrils in the of normal fibrils was for fibrils in both the anterior and stroma. However, fibril diameters in both were larger in the of the of collagen fibril diameters from wild-type and mice that mice have larger diameters in both the anterior and of the cornea than the wild-type mice the wild-type and a normal of fibril were no of fibrils that were different from the wild-type in anterior or of the However, the in both anterior and of the showed a to larger diameters compared with the wild-type The anterior fibrils a diameter of nm in the wild-type nm in the of nm was in the stroma, fibrils a diameter of nm in the wild-type nm in the of nm also was was no anterior and in the normal however, in the the anterior and nm is that the to a greater by the keratocan analysis of corneal collagen The of collagen fibril diameters were in the anterior and and and cornea of wild-type and and Kera–/– and littermates. The diameter for the Kera–/– and the wild-type are The fibril diameter and the are in image In mice, eyelids are at and in the of postnatal We that the of protein of keratocan during the postnatal of the mouse with the of the at However, the of keratocan protein in the cornea not with the of keratocan mRNA (16Liu C.-Y. Shiraishi A Kao C.W. Converse R.L. Funderburgh J.L. Corpuz L.M. Conrad G.W. Kao W.W.-Y. J. Biol. Chem. 1998; 273: 22584-22588Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). It is that the of keratocan at has also been found in lumican S. Shiraishi A. Kao C.W. Converse R.L. Funderburgh J.L. J. Conrad G.W. Kao W.W.-Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google and collagen W.W.-Y. J. J. Biol. Chem. 1983; Full Text PDF PubMed Google Scholar). are important to the of tissues during embryonic In ocular tissues, to corneal stroma, and We have showed previously that keratocan expression is to the keratocytes embryonic day is also expressed by different of in tissues as and during Keratocan knockout mice are and that keratocan is in tissues that Kera during embryonic The expression levels of other SLRP members including lumican, decorin, and fibromodulin were not altered in the Kera–/– cornea as compared with that of the wild-type that the in the Kera–/– mice resulted from the of of Genetic has shown that mice lacking a decrease keratocan mRNA and corneal collagen at H. R.L. 2000; PubMed Scopus Google Scholar). However, we did not in collagenous matrix of the Kera–/– cornea at not The of our from the knockout mice is not is that ablation of perturb the expression of other than Kera that are in collagen The corneas lumican, and mimecan/osteoglycin knockout mice different knockout mice have the most with corneal collagen fibrils, and thinner corneal stroma (12Charkravarti S. Magnuson T. Lass J.H. Jepsen K.J. LaMantia C. Carroll H. J. Cell Biol. 1998; 112: 987-996Google Scholar, 13Chakravarti S. Petroll W.M. Hassell J.R. Jester J.V. Lass J.H. Paul J. Birk D.E. Investig. Ophthalmol. Vis. Sci. 2000; 11: 3365-3373Google Scholar, 14Saika S. Shiraishi A. Liu C.-Y. Funderburgh J.L. Kao C.W. Converse R.L. Kao W.W.-Y. J. Biol. Chem. 2000; 275: 2607-2612Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). knockout mice have larger collagen fibrils with transparent cornea A. N. Conrad G.W. Mol. Vis. Google Scholar). Keratocan knockout mice also larger diameters of corneal collagen less organized However, mice, mice have a thinner corneal stroma and a narrower cornea-iris the keratocan expression is to lumican in the corneal stroma, keratocan not lumican and the corneal in the mice. suggested that keratocan and lumican to different of collagen The reduced of corneal stroma in the mice was due to reduced hydration by a decrease of keratan sulfate proteoglycan in Kera–/– cornea. expression is the in the cornea as compared with keratocan and lumican, and mimecan/osteoglycin have been to corneal in including and has been shown that, unlike lumican and has no in mouse cornea. the subtle in the cornea of knockout mice. corneal plana (CNA2) is an disease that is directly linked to the of at mutations have been are for a to at an amino acid change from to amino acid the in the tandem leucine-rich (15Pellegata N.S. Dieguez-Lucena J.L. Joensuu T. Lau S. Montgomery K.T. Krahe R. Kivela T. Kucherlapati R. Forsius H. de la Chapelle A. Nat. Genet. 2000; 25: 91-95Crossref PubMed Scopus (124) Google Scholar). are of sequence change from to at which results in an amino acid change from to in the the and tandem leucine-rich M.F. T. Investig. Ophthalmol. Vis. Sci. 2000; Scholar). In an CNA2 of a to was found in exon 2 that a change of to a a protein of amino acids (15Pellegata N.S. Dieguez-Lucena J.L. Joensuu T. Lau S. Montgomery K.T. Krahe R. Kivela T. Kucherlapati R. Forsius H. de la Chapelle A. Nat. Genet. 2000; 25: 91-95Crossref PubMed Scopus (124) Google Scholar). It is that mutations a keratocan gene that in the cornea because they not a negative in a To mice recombinant human KERA or into a mouse with an model to investigate the of keratocan in We for with the electron and of the of of at for the of fibril

A rat chondrosarcoma cell line and primary bovine chondrocytes have been used to study cell-mediated aggrecan catabolism. Addition of 1 microM retinoic acid to chondrosarcoma cultures resulted in aggrecan proteolysis with the release of greater than 90% of the cell layer aggrecan into the medium within 4 days. NH2-terminal sequencing of chondroitin sulfate-substituted catabolic products gave a single major NH2-terminal sequence of ARGNVILTXK, initiating at Ala374. This showed that the proteinase, commonly referred to as "aggrecanase," which cleaves the Glu373-Ala374 bond of the interglobular domain of aggrecan (Sandy, J. D., Neame, P. J., Boynton, R. E., and Flannery, C. R. (1990) J. Biol. Chem. 266, 8683-8685), is active in this cell system. Aggrecan G1 domain, generated by cleavage of the interglobular domain, was also liberated during catabolism and this was characterized with three antipeptide antisera. Anti-CDAGWL was used as a general probe for G1 domain. Anti-FVDIPEN was used to specifically detect G1 domain with COOH terminus of Asn341, the form which is readily generated by cleavage of aggrecan by a wide range of matrix metalloproteinases. Anti-NITEGE antiserum was used to specifically detect G1 domain with COOH terminus of Gln373, the form which is the expected product of "aggrecanase"-mediated cleavage of aggrecan. Western blot analysis indicated that a single form of G1 domain of about 60 kDa was formed. G1 domain of this size reacted with both anti-CDAGWL and anti-NITEGE but not with anti-FVDIPEN. Similar experiments with primary bovine chondrocyte cultures, treated with either retinoic acid or interleukin 1, showed that two forms of catabolic G1 domain, of about 62 and 66 kDa, were formed. Both of these forms reacted on Western blots with anti-CDAGWL and also with anti-NITEGE. It is suggested that cell-mediated catabolism of the aggrecan interglobular domain in these culture systems, whether promoted by retinoic acid or interleukin 1, primarily involves cleavage of the Glu373-Ala374 bond by aggrecanase. The accumulation of G1 domain with a COOH-terminal of Glu373 shows that such aggrecanase-mediated cleavage can occur independent of the cleavage of the Asn341-Phe342 bond by matrix metalloproteinases.

Studies of aggrecan proteolysis in human joints have implicated both the aggrecanase [ADAMTS, a disintegrin-like and metalloprotease (reprolysin-type) with thrombospondin type 1 motif] and matrix metalloproteinase (MMP) families. We have analysed the aggrecan core protein species present in vivo in both articular cartilage and synovial fluids from normal, acutely injured and osteoarthritic joints. Normal cartilage contains at least seven major G1 domain (the N-terminal globular domain of aggrecan)-bearing species, of which three (full-length core, G1-NITEGE(373) and G1-VDIPEN(341)) have been identified. The C-terminals of the others are unknown but digestion of fetal human aggrecan with MMP-3 and crude aggrecanase suggests that they are products of MMP-like activity in vivo. Normal synovial fluids contain at least 10 species, of which nine result from ADAMTS-dependent cleavage, and this cleavage occurs at all of the five known aggrecanase sites. Aggrecan fragments in the cartilage and synovial fluids of acutely injured joints are generally similar to normal, but all contain a markedly increased ratio of G1-NITEGE to G1-VDIPEN. Aggrecan from the cartilage of late-stage osteoarthritis patients is remarkably similar to normal, whereas the synovial fluid aggrecan is more fragmented than that from normal or injured knees. The analyses suggest that the role of the ADAMTS and these MMP-like activities in human cartilage are distinctly different. Excessive ADAMTS activity in vivo is destructive to cartilage matrix, since the bulk of the glycosaminoglycan (GAG)-bearing products are released from the tissue into the synovial fluid following cleavage of the Glu(373)-Ala(374) bond. In contrast, the MMP-like activity appears to be essentially non-destructive, since much of the GAG-bearing product is retained in the tissue following cleavages that are in the more C-terminal regions of the molecule.

Transforming growth factor-β (TGF-β1, -β2, and -β3) has been implicated in the ontogenetic transition from scarless fetal repair to adult repair with scar. Generally, TGF-β exerts its effects through type I and II receptors; however, TGF-β modulators such as latent TGF-β binding protein-1 (LTBP-1), decorin, biglycan, and fibromodulin can bind and potentially inhibit TGF-β activity. To more fully explore the role of TGF-β ligands, receptors, and potential modulators during skin development and wound healing, we have used a rat model that transitions from scarless fetal-type repair to adult-type repair with scar between days 16 and 18 of gestation. We showed that TGF-β ligand and receptor mRNA levels did not increase during the transition to adult-type repair in fetal skin, whereas LTBP-1 and fibromodulin expression decreased. In addition, TGF-β1 and -β3; type I, II, and III receptors; as well as LTBP-1, decorin, and biglycan were up-regulated during adult wound healing. In marked contrast, fibromodulin expression was initially down-regulated in adult repair. Immunostaining demonstrated significant fibromodulin induction 36 hours after injury in gestation day 16, but not day 19, fetal wounds. This inverse relationship between fibromodulin expression and scarring in both fetal and adult rat wound repair suggests that fibromodulin may be a biologically relevant modulator of TGF-β activity during scar formation. Transforming growth factor-β (TGF-β1, -β2, and -β3) has been implicated in the ontogenetic transition from scarless fetal repair to adult repair with scar. Generally, TGF-β exerts its effects through type I and II receptors; however, TGF-β modulators such as latent TGF-β binding protein-1 (LTBP-1), decorin, biglycan, and fibromodulin can bind and potentially inhibit TGF-β activity. To more fully explore the role of TGF-β ligands, receptors, and potential modulators during skin development and wound healing, we have used a rat model that transitions from scarless fetal-type repair to adult-type repair with scar between days 16 and 18 of gestation. We showed that TGF-β ligand and receptor mRNA levels did not increase during the transition to adult-type repair in fetal skin, whereas LTBP-1 and fibromodulin expression decreased. In addition, TGF-β1 and -β3; type I, II, and III receptors; as well as LTBP-1, decorin, and biglycan were up-regulated during adult wound healing. In marked contrast, fibromodulin expression was initially down-regulated in adult repair. Immunostaining demonstrated significant fibromodulin induction 36 hours after injury in gestation day 16, but not day 19, fetal wounds. This inverse relationship between fibromodulin expression and scarring in both fetal and adult rat wound repair suggests that fibromodulin may be a biologically relevant modulator of TGF-β activity during scar formation. Fetal repair is fundamentally different from adult repair. Adult skin wounds heal by scar formation, whereas fetal skin wounds heal by regeneration with restoration of normal skin architecture. This transition from scarless fetal repair to adult-type healing with scar occurs at specific times during gestation.1Adzick NS Longaker MT Characteristics of fetal tissue repair.in: Adzick NS Longaker MT Fetal Wound Healing. Elsevier, New York1992: 53-70Google Scholar The mechanism for scarless fetal repair is unknown, but it does not require systemic factors such as the fetal immune system, fetal serum, or amniotic fluid.2Bleacher JC Adolph VR Dillon PW Krummel TM Isolated fetal mouse limbs: gestational effects on tissue repair in an unperfused system.J Pediatr Surg. 1993; 28: 1312-1315Abstract Full Text PDF PubMed Scopus (15) Google Scholar, 3Ihara S Motobayashi Y Wound closure in foetal rat skin.Development. 1992; 114: 573-582PubMed Google Scholar, 4Ihara S Motobayashi Y Nagao E Kistler A Ontogenetic transition of wound healing pattern in rat skin occurring at the fetal stage.Development. 1990; 110: 671-680PubMed Google Scholar Isolated human fetal skin transplanted into adult athymic mice can heal without scar.5Adzick NS Lorenz HP Cells, matrix, growth factors, and the surgeon. The biology of scarless fetal wound repair.Ann Surg. 1994; 220: 10-18Crossref PubMed Scopus (233) Google Scholar Thus the capability for scarless repair is inherent to fetal skin itself. Fetal skin contains fetal fibroblasts and fetal extracellular matrix (ECM) that is distinct from adult fibroblasts and ECM.5Adzick NS Lorenz HP Cells, matrix, growth factors, and the surgeon. The biology of scarless fetal wound repair.Ann Surg. 1994; 220: 10-18Crossref PubMed Scopus (233) Google Scholar For instance, the fetal ECM has a higher ratio of type III to type I collagen,6Hallock GG Rice DC Merkel JR DiPaolo BR Analysis of collagen content in the fetal wound.Ann Plast Surg. 1988; 21: 310-315Crossref PubMed Scopus (50) Google Scholar, 7Merkel JR DiPaolo BR Hallock GG Rice DC Type I and type III collagen content of healing wounds in fetal and adult rats.Proc Soc Exp Biol Med. 1988; 187: 493-497Crossref PubMed Scopus (171) Google Scholar as well as a different profile of proteoglycan and glycosaminoglycan synthesis.8Mast BA Flood LC Haynes JH DePalma RL Cohen IK Diegelmann RF Krummel TM Hyaluronic acid is a major component of the matrix of fetal rabbit skin and wounds: implications for healing by regeneration.Matrix. 1991; 11: 63-68Crossref PubMed Scopus (93) Google Scholar It is probable that both the fetal skin cells and the ECM they synthesize are critical in scarless repair. In addition, fetal cells and fetal ECM likely have a dynamic and reciprocal relationship whereby fetal cells secrete a fetal ECM that can, in turn, modulate fetal cell migration, proliferation, and collagen synthesis.9Raghow R The role of extracellular matrix in postinflammatory wound healing and fibrosis.FASEB J. 1994; 8: 823-831Crossref PubMed Scopus (369) Google Scholar These results suggest that cellular gene regulation rather than the external (amniotic) environment is the critical factor in scarless repair. We hypothesize that fundamental differences in gene expression govern the transition to adult-type repair in fetal skin, whereby profibrotic molecules will be up-regulated and antifibrotic molecules will be down-regulated during the onset of adult-type repair. The transforming growth factor-β (TGF-β1, -β2, and -β3) ligands have been implicated in the ontogenetic transition from scarless fetal repair to adult repair with scar. Specifically, TGF-β1 and TGF-β2 may promote scar formation, whereas TGF-β3 may reduce scarring.10Shah M Foreman DM Ferguson MW Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring.J Cell Sci. 1995; 108: 985-1002Crossref PubMed Google Scholar The addition of exogenous TGF-β1 to normally scarless fetal wounds results in scar,11Krummel TM Michna BA Thomas BL Sporn MB Nelson JM Salzberg AM Cohen IK Diegelmann RF Transforming growth factor beta (TGF-beta) induces fibrosis in a fetal wound model.J Pediatr Surg. 1988; 23: 647-652Abstract Full Text PDF PubMed Scopus (197) Google Scholar, 12Lin RY Sullivan KM Argenta PA Meuli M Lorenz HP Adzick NS Exogenous transforming growth factor-beta amplifies its own expression and induces scar formation in a model of human fetal skin repair.Ann Surg. 1995; 222: 146-154Crossref PubMed Scopus (149) Google Scholar while neutralizing antibodies against TGF-β1 and TGF-β2 in adult wounds, decreases scarring.10Shah M Foreman DM Ferguson MW Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring.J Cell Sci. 1995; 108: 985-1002Crossref PubMed Google Scholar Although the addition of TGF-β alone is enough to induce scarring in the fetus, TGF-β neutralizing antibodies alone do not entirely prevent scarring in the adult, suggesting that factors other than TGF-β may also be important for scarless fetal repair. TGF-β ligands have a wide range of effects on cellular motility, proliferation, matrix production, and differentiation.13Bonewald LF Regulation and regulatory activities of transforming growth factor beta.Crit Rev Eukaryot Gene Expr. 1999; 9: 33-44PubMed Google Scholar Most cells secrete TGF-β in an inactive latent form that undergoes proteolytic cleavage for activation or associates with one of several latent TGF-β-binding proteins (LTBP) that may function in ECM storage, secretion, and activation of TGF-β.13Bonewald LF Regulation and regulatory activities of transforming growth factor beta.Crit Rev Eukaryot Gene Expr. 1999; 9: 33-44PubMed Google Scholar, 14Massague J TGF-beta signal transduction.Annu Rev Biochem. 1998; 67: 753-791Crossref PubMed Scopus (4041) Google Scholar Once activated, TGF-β exerts its biological effects through binding to type II and type I receptors.13Bonewald LF Regulation and regulatory activities of transforming growth factor beta.Crit Rev Eukaryot Gene Expr. 1999; 9: 33-44PubMed Google Scholar Type III receptor (also known as betaglycan) is a membrane-anchored proteoglycan that facilitates TGF ligand binding to the type II J TGF-beta signal transduction.Annu Rev Biochem. 1998; 67: 753-791Crossref PubMed Scopus (4041) Google Scholar III several ECM of the biglycan and can bind TGF-β and TGF-β A M E of the biglycan, and fibromodulin with transforming growth factor J. 1994; PubMed Scopus Google Scholar The addition of to a rat model of matrix and JR Y E of transforming growth factor-beta against scarring in 1992; PubMed Scopus Google Scholar both and in addition to TGF-β can collagen formation and S and with 1994; PubMed Scopus Google Scholar, E of fibromodulin and to on Biol 1993; Full Text PDF PubMed Google Scholar, for fibromodulin binding to collagen and the of type I collagen for and the J Biochem. 1998; PubMed Scopus Google Scholar In the we have used a rat model that the transition from scarless fetal-type repair to adult-type repair with scar between days 16 and 18 of gestation S Motobayashi Y Nagao E Kistler A Ontogenetic transition of wound healing pattern in rat skin occurring at the fetal stage.Development. 1990; 110: 671-680PubMed Google Scholar To we the of the transition through and of and fetal wounds. we the expression of potential profibrotic molecules TGF-β1 and and type I, II, and III and potential antifibrotic molecules LTBP-1, decorin, biglycan, and during rat fetal skin we that fetal skin levels of TGF-β ligand and receptor levels did not increase during the transition we showed that levels of LTBP-1 and modulators of with the onset of scar formation in the fetal expression of LTBP-1 and fibromodulin during the transition to adult-type repair with scar suggests that LTBP-1 and fibromodulin may have antifibrotic in fetal wound repair. To the role of LTBP-1 and fibromodulin in scar formation, we expression to -β2, and -β3; TGF-β type I, II, and III receptors; and and biglycan in an adult rat model of wound repair. We demonstrated that TGF-β1 and -β3; type I, II, and III receptors; as well as LTBP-1, decorin, and biglycan, were up-regulated during adult wound healing. The of LTBP-1 in an adult model of repair with scar is with its role as a antifibrotic however, in marked to that of the other was initially down-regulated in adult repair with its role as a antifibrotic we fibromodulin expression in and fetal wounds. We showed significant induction of fibromodulin expression in fetal wounds and induction in wounds. Thus a inverse relationship between fibromodulin expression and scar formation in both fetal and adult rat of wound repair. This suggests that fibromodulin may be a biologically relevant modulator of TGF-β activity during scar formation. of fibromodulin with TGF-β in gestation wounds may scarless fetal repair through TGF-β In addition, fibromodulin induction in fetal wounds a of fetal wound collagen is in a more than collagen in adult wounds. for the of adult wounds into more may the addition of fibromodulin to modulate both TGF-β activity and ECM were in a at the of and and were The of of a as of was day of gestation. To cutaneous gene expression during were on days 16, 19, and of gestation. the were and a was to the were and fetal skin from between the of the and the of the was The tissue was in and at For fetal wound were on days 16 and of gestation. The were A was were used on the of to wounds. were with for and the amniotic was with normal were and hours and in wounds skin from and as well as from wound was used as To gene expression during cutaneous wound were and To and differences in wound healing, such as the and as well as the were used to wound R differences in the growth of normal and PubMed Scopus Google Scholar skin wounds, the were on the of 16 wounds were by at 2 to effects from wounds. at a were and wounds on were in the at hours and and days after injury 16 wounds of skin from in To wound healing to with an skin 1 of for was in and at was a of and and with at 1 of I to of mRNA by in and New Scholar To specific was and a that is known to an for and were by 2 of in a and more J of after with and A Scholar was 1 of with of and 1 of II in a at for The was by at for To differences in gene expression during was is on the in the the of molecules the of mRNA R for the of Google Scholar For we the likely to the range by the of to was the of the 2 of and and of in a were the were for 1 by the different for 1 for 1 and for for a of was also in for as a The were by on a and a by for of TGF-β ligands, receptors, and modulators during adult cutaneous wound repair. was on from to wounds and 16 wounds To in mRNA levels after for were to expression at and by the to The results are as the was to significant differences in gene expression between and adult wounds at different are A is for TGF-β ligand receptor or modulator with the range of on the of the significant in gene expression to for the fibromodulin expression levels to the The range of is and is marked with an A adult is or more from at of were for and are in were the from the Analysis and and to to I receptor II receptor III receptor to in a was by the with a specific for a The of the are in were with was in and at in a for 2 were at with specific were in for by in at were from the To in mRNA expression for TGF-β ligands, and were and on a the for TGF-β TGF-β and TGF-β modulator were to expression at by the to and as the the was to significant differences in gene expression between and fetal skin, as well as significant differences in gene expression between and adult wounds at different A of was fetal skin and skin were in The were in and into for and or To fibromodulin expression in fetal wounds, was on and wounds 36 hours after as well as on 36 and 36 fetal and of the tissue activity was with for by for and the were with To reduce were at in a with a of were with S for the addition of to Biol 1993; Full Text PDF PubMed Google Scholar for 1 at times with and with a of for 1 the were and was for was for the were with and with were with of was to the collagen pattern in wounds, hours after tissue were the were and with acid for 1 and with and in acid for were in for 2 and and were on a with both an and a of the wound by were in the a with the of the The was with a after through a and The of the was by the of the in the The for was were and in for the of To scarless healing in we the effects of wounds on and fetal and hours after injury and was at hours in hours was healing, with restoration of normal and development The was by the of the of day 16 fetal wounds hours after injury restoration of normal skin collagen with between wounds hours and skin and To with the of the transition day wounds were on fetal was to the gestation day 16 wounds after hours In addition, fetal wounds with collagen and of regeneration in the wound at hours of day wounds at hours and of collagen the scar to skin of the day hours and To significant differences in gene expression are and after the transition was on gestation day 16, 19, and fetal for -β2, and -β3; type I, II, and III receptors; and decorin, biglycan, and We that molecules scar will be up-regulated and that molecules scar will be down-regulated during the onset of adult-type repair. was to significant differences in mRNA levels between and fetal A of was results that TGF-β1 levels did not between gestation days 16 and 18 whereas TGF-β2 and levels were higher at day 16 to day 18 and A and Type I and III receptor on the other did not during the transition and type II receptor was by at day 16 as to day 18 and In of TGF-β modulator LTBP-1 mRNA was and fibromodulin was higher at day 16 day 18 and however, were by in to and biglycan expression was not during the transition and we that potential profibrotic molecules TGF-β1 and and type I, II, and III were not up-regulated during the transition potential antifibrotic LTBP-1, and with the onset of adult-type repair in the To the role of LTBP-1 and fibromodulin in scar formation, we expression to -β2, and -β3; type I, II, and III receptors; and and biglycan during adult rat wound repair. was on from and wounds. We that potential antifibrotic molecules be or down-regulated during adult repair. was to significant differences in mRNA levels between and adult wounds at different A of was from S M S Transforming growth factors and and are during normal and wound Biol PubMed Scopus Google Scholar TGF-β1 and as well as type I and II were up-regulated to in adult we did not a significant in TGF-β2 expression and In addition, we also showed of the type III receptor and of the potential TGF-β LTBP-1, decorin, and biglycan were up-regulated in adult for was initially down-regulated hours after injury and levels to by hours with a at days 3 and and and fibromodulin is down-regulated during both fetal skin transition to adult-type repair and in adult we that the of a potential antifibrotic may promote scar formation. To we fibromodulin expression in and fetal wounds, 36 hours after higher fibromodulin to the and in gestation day 36 to day 36 fetal skin A and however, 36 hours after injury in skin, in the the wound In marked contrast, skin fibromodulin to the wound 36 hours after injury are with effects on cell motility, proliferation, and ECM Transforming growth factor-beta Biol 1995; PubMed Scopus Google Scholar In of wound TGF-β1 and are known to promote NS Lorenz HP Cells, matrix, growth factors, and the surgeon. The biology of scarless fetal wound repair.Ann Surg. 1994; 220: 10-18Crossref PubMed Scopus (233) Google Scholar, M Foreman DM Ferguson MW Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring.J Cell Sci. 1995; 108: 985-1002Crossref PubMed Google Scholar, TM Michna BA Thomas BL Sporn MB Nelson JM Salzberg AM Cohen IK Diegelmann RF Transforming growth factor beta (TGF-beta) induces fibrosis in a fetal wound model.J Pediatr Surg. 1988; 23: 647-652Abstract Full Text PDF PubMed Scopus (197) Google Scholar TGF-β3 has been to scarring in a rat wound model by M Foreman DM Ferguson MW Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring.J Cell Sci. 1995; 108: 985-1002Crossref PubMed Google Scholar results demonstrated that profibrotic molecules such as TGF-β1 and were in skin from and that levels did not increase during the transition from fetal to repair. TGF-β2 levels in were in to Thus we did not a between TGF-β1 expression and the onset of adult-type repair in the we between TGF-β3 expression and scarless fetal-type repair. Although we showed significant TGF-β3 mRNA in and transition with its role as a antifibrotic was not and TGF-β3 was up-regulated from role in and may its expression in Transforming growth factor-beta Biol 1995; PubMed Scopus Google however, is with the role of TGF-β3 as a antifibrotic also did not TGF-β3 to have antifibrotic effects to a rabbit A Transforming growth factor beta 3 beta wound healing without of scar and Surg. PubMed Scopus Google Scholar This with adult rat TGF-β3 expression after injury and with other of TGF-β3 in adult S M S Transforming growth factors and and are during normal and wound Biol PubMed Scopus Google Scholar and JH and of transforming growth factor beta beta and beta 3 during wound J 1993; Google Scholar wound that repair with scar. TGF-β3 in adult wounds against a antifibrotic role for TGF-β ligand levels did not increase during the fetal skin transition to adult-type repair with we that the of TGF-β induction after injury may be more relevant than TGF-β a of TGF-β induction in fetal Ferguson MW of growth factors in fetal wound 1991; PubMed Scopus Google Scholar and in RY Sullivan KM Argenta PA Meuli M Lorenz HP Adzick NS Exogenous transforming growth factor-beta amplifies its own expression and induces scar formation in a model of human fetal skin repair.Ann Surg. 1995; 222: 146-154Crossref PubMed Scopus (149) Google Scholar, KM Lorenz HP Meuli M RY Adzick NS A model of scarless human fetal wound repair is in transforming growth factor Pediatr Surg. 1995; Full Text PDF PubMed Scopus Google Scholar and rabbit wound M The expression of transforming growth factor type beta in fetal and adult rabbit skin Pediatr Surg. 1994; Full Text PDF PubMed Scopus Google Scholar demonstrated TGF-β1 mRNA induction hours after and to levels by 18 hours in a mouse model of induction and of TGF beta 1 is an to in the mouse 1993; PubMed Scopus Google Scholar We have by that TGF-β1 and are in rat skin wounds and that the of expression after injury with gestational Specifically, TGF-β1 and whereas TGF-β1 and induction in Although induction and of TGF-β may be one mechanism scarless fetal TGF-β is in fetal wounds. To TGF-β receptor in to may for scarless fetal repair in the of we the expression of TGF-β type I, II, and III during the transition Type I and II are that are for TGF-β activity in J TGF-beta signal transduction.Annu Rev Biochem. 1998; 67: 753-791Crossref PubMed Scopus (4041) Google In contrast, the type III receptor is a proteoglycan that has function but it may modulate TGF-β to the J TGF-beta signal transduction.Annu Rev Biochem. 1998; 67: 753-791Crossref PubMed Scopus (4041) Google Scholar that type II receptor levels during the fetal transition to adult-type whereas type I and III receptor levels did not Thus we demonstrated that TGF-β ligand receptor levels with scar formation during the transition from scarless fetal repair to adult-type repair with scar profibrotic molecules were not up-regulated in the transition to adult-type we potential antifibrotic molecules such as LTBP-1, decorin, biglycan, and fibromodulin be are a of molecules that may function to and extracellular TGF-β activity through to bind both ECM and AM and of latent transforming growth factor-beta binding a 1999; PubMed Scopus Google Scholar We a significant in LTBP-1 mRNA in as to This in LTBP-1 expression in gestation may increase TGF-β and potentially for scar formation in To LTBP-1 will be down-regulated in a model that through we LTBP-1 expression in adult rat wounds. we of LTBP-1 mRNA to between hours and 3 days after The of LTBP-1 suggests that TGF-β expression may be with LTBP-1 in adult repair. have TGF-β1 and mRNA expression in human fibroblasts in S LF and of latent transforming growth factor beta in cell of a latent the latent TGF Biol 1994; Full Text PDF PubMed Google Scholar LTBP-1 levels in adult-type LTBP-1 levels in adult wounds against a fundamental antifibrotic role for results that the onset of adult-type repair in the does not to TGF-β or LTBP-1 Although the in TGF-β induction in may be a major of scar formation, it does not entirely fetal wounds heal without scar the of the addition of antibodies against to scar in adult M Foreman DM Ferguson MW Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring.J Cell Sci. 1995; 108: 985-1002Crossref PubMed Google Scholar This suggests that other molecules TGF-β ligands, receptors, and binding proteins may fetal scarless repair. to the more is known the proteoglycan biglycan, and have in ECM cellular and growth factor E in cell Biol Full Text PDF PubMed Google Scholar and and to a biglycan, can bind I, II, and to and for fibromodulin binding to collagen and the of type I collagen for and the J Biochem. 1998; PubMed Scopus Google Scholar, The of on the of collagen in PubMed Scopus Google Scholar, E of a tissue matrix with collagen I and collagen Biol Full Text PDF PubMed Google Scholar collagen decorin, biglycan, and fibromodulin can bind -β2, and with fibromodulin the for as well as the latent TGF-β1 A M E of the biglycan, and fibromodulin with transforming growth factor J. 1994; PubMed Scopus Google Scholar to an model of has injury with TGF-β JR Y E of transforming growth factor-beta against scarring in 1992; PubMed Scopus Google Scholar Thus the is to both ECM and TGF-β for the regulation of scarless fetal repair. showed that mRNA levels in to whereas biglycan levels did not from mRNA on the other by in to of adult wounds demonstrated and biglycan expression by to whereas fibromodulin was initially down-regulated at hours and up-regulated by between days 3 and in and in adult with biglycan in adult and biglycan for scarless fetal repair. In contrast, fibromodulin expression with scar formation during both fetal skin development and adult wound repair. To the role of fibromodulin in fetal we for fibromodulin in and fetal wounds 36 hours after We showed that fibromodulin expression 36 hours after injury in but not in wounds. Thus fibromodulin levels are with scarless fetal whereas adult-type fetal repair with scar and adult repair are in scarless fetal the of induction and of in with and fibromodulin may TGF-β TGF-β may in ECM and scar formation more prevent the protein-1 that TGF-β1 expression in adult and cell biology of 1998; PubMed Scopus Google Scholar fibromodulin in fetal wounds may collagen and as by on wounds. fibromodulin in fetal wounds not scarless repair may the of it also fetal collagen may the other the of fibromodulin induction in adult wounds may increase TGF-β activity and to scar and The increase in adult wound fibromodulin expression at days 3 and in the repair and is to a significant antifibrotic the of fibromodulin in adult wounds also potentially of TGF-β reduces but does not entirely scarring in adult as adult wounds are in fibromodulin for the regulation of ECM to adult wounds into more may the addition of fibromodulin or fibromodulin into wounds for the of both TGF-β activity and ECM In as the of fetal skin during development from a of cell to a with and the for growth factors, receptors, and ECM biological modulators also ontogenetic This suggests that the biology the transition from scarless fetal repair to adult-type repair with scar in gestation may be by dynamic in levels of at the

OBJECTIVE: To identify characteristic changes in large aggregating (aggrecan) and small proteoglycan (PG) populations in articular cartilages during osteoarthritis (OA) and rheumatoid arthritis (RA). METHODS: Aggrecan populations in guanidine extracts of femoral condylar cartilages of 46 OA and 8 RA patients who underwent total knee arthroplasty, as well as of 2 fetuses and 6 normal adults, were separated in agarose-polyacrylamide composite gels. Small PGs (biglycan, decorin, and fibromodulin) in the same extracts were analyzed in 12% polyacrylamide gels. Gels were stained or electrophoretically transferred and probed with antibodies to aggrecan epitopes and to small PGs. Epitope contents of the samples were also compared by inhibition radioimmunoassay. RESULTS: There were significant differences found among normal and diseased samples in their electrophoretic mobilities, band distributions, and antibody staining. OA and especially RA samples were heavily degraded, lacked certain aggrecan populations, and contained fewer keratan sulfate and chondroitin-6-sulfate epitopes compared with normal samples. Levels of chondroitin-4-sulfate and "fetal-type" epitopes were elevated in the OA samples compared with the normal ones. More core proteins of small PGs were found in diseased than in normal cartilages, but they were more heterogeneous in size and glycosaminoglycan substitution. CONCLUSION: There is extensive degradation of both large and small PGs in diseased cartilages, but a repair process does exist, especially in OA cartilages. Chondrocytes of diseased cartilages are able to synthesize fetal-type aggrecans. Small PGs are glycosylated differently in diseased cartilages than in normal ones.