Collagen Structure-Function Mapping Informs Applications for Regenerative Medicine.

Type I collagen, the predominant protein of vertebrates, assembles into fibrils that orchestrate the form and function of bone, tendon, skin, and other tissues. Collagen plays roles in hemostasis, wound healing, angiogenesis, and biomineralization, and its dysfunction contributes to fibrosis, atherosclerosis, cancer metastasis, and brittle bone disease. To elucidate the type I collagen structure-function relationship, we constructed a type I collagen fibril interactome, including its functional sites and disease-associated mutations. When projected onto an X-ray diffraction model of the native collagen microfibril, data revealed a matrix interaction domain that assumes structural roles including collagen assembly, crosslinking, proteoglycan (PG) binding, and mineralization, and the cell interaction domain supporting dynamic aspects of collagen biology such as hemostasis, tissue remodeling, and cell adhesion. Our type III collagen interactome corroborates this model. We propose that in quiescent tissues, the fibril projects a structural face; however, tissue injury releases blood into the collagenous stroma, triggering exposure of the fibrils\u27 cell and ligand binding sites crucial for tissue remodeling and regeneration. Applications of our research include discovery of anti-fibrotic antibodies and elucidating their interactions with collagen, and using insights from our angiogenesis studies and collagen structure-function model to inform the design of super-angiogenic collagens and collagen mimetics

Bone growth during rapamycin therapy in young rats

<p>Abstract</p> <p>Background</p> <p>Rapamycin is an effective immunosuppressant widely used to maintain the renal allograft in pediatric patients. Linear growth may be adversely affected in young children since rapamycin has potent anti-proliferative and anti-angiogenic properties.</p> <p>Methods</p> <p>Weanling three week old rats were given rapamycin at 2.5 mg/kg daily by gavage for 2 or 4 weeks and compared to a Control group given equivalent amount of saline. Morphometric measurements and biochemical determinations for serum calcium, phosphate, iPTH, urea nitrogen, creatinine and insulin-growth factor I (IGF-I) were obtained. Histomorphometric analysis of the growth plate cartilage, in-situ hybridization experiments and immunohistochemical studies for various proteins were performed to evaluate for chondrocyte proliferation, chondrocyte differentiation and chondro/osteoclastic resorption.</p> <p>Results</p> <p>At the end of the 2 weeks, body and tibia length measurements were shorter after rapamycin therapy associated with an enlargement of the hypertrophic zone in the growth plate cartilage. There was a decrease in chondrocyte proliferation assessed by <it>histone-4 </it>and <it>mammalian target of rapamycin </it>(<it>mTOR</it>) expression. A reduction in <it>parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP) </it>and an increase in <it>Indian hedgehog </it>(<it>Ihh</it>) expression may explain in part, the increase number of hypertrophic chondrocytes. The number of TRAP positive multinucleated chondro/osteoclasts declined in the chondro-osseous junction with a decrease in the <it>receptor activator of nuclear factor kappa β ligand </it>(<it>RANKL</it>) and <it>vascular endothelial growth factor </it>(<it>VEGF</it>) expression. Although body and tibial length remained short after 4 weeks of rapamycin, changes in the expression of chondrocyte proliferation, chondrocyte differentiation and chondro/osteoclastic resorption which were significant after 2 weeks of rapamycin improved at the end of 4 weeks.</p> <p>Conclusion</p> <p>When given to young rats, 2 weeks of rapamycin significantly decreased endochondral bone growth. No catch-up growth was demonstrated at the end of 4 weeks, although markers of chondrocyte proliferation and differentiation improved. Clinical studies need to be done to evaluate these changes in growing children.</p

Phenotypic and biochemical consequences of collagen X mutations in mice and humans

Skeletal biology has entered an exciting period with the technological advances in murine transgenesis and human genetics. This review focuses on how these two approaches are being used to address the role of collagen X, the major extracellular matrix component of the focal zone of endochondral ossification, the hypertrophic cartilage zone. The hypothesized role of this unique collagen in skeletal morphogenesis and the phenotypic and biochemical consequences resulting from the disruption of its function are discussed. Specifically, data from three murine models, including transgenic mice with a dominant interference phenotype for collagen X, and two sets of mice with an inactivated collagen X gene through gene targeting and homologous recombination, as well as the human disorder of Schmid metaphyseal chondrodysplasia resulting from mutations in collagen X, are summarized and compared. Several inconsistencies and unresolved issues regarding the murine and human phenotypes and the function of collagen X are discussed.link_to_subscribed_fulltex

Type I collagen molecular map lends insights into the domain structure of the fibril and the genotype-phenotype relationship for some collagen mutations

Our molecular map of type I collagen was previously correlated with the Orgel et al., 2006 x-ray fibril diffraction model to identify cell and matrix interaction domains. Here we used two strategies to analyze mutation patterns to pinpoint functionally significant regions. First, regions of the \u3b11(I) chains were identified having three or more consecutive glycines either associated with lethal or silent phenotypes. Many of these regions co-localized with sites for interactions with mineralization proteins such as phosphophoryn, cell surface receptors, and matrix metalloproteinases, or for intermolecular crosslinking. Five of the larger runs of silent glycines, although each on separate monomers in the D-period, clustered vertically within a narrow fibril region- herein called the major silent zone (MSZ). Second, the distribution of OI substitution mutations on the COL1A1 and COL1A2 genes were examined and found to be statistically different from that expected on the basis of base pair mutation rates, suggesting differential phenotypic consequences of mutations occurring on different collagen regions. For example, some glycines were predicted to have high mutation rates yet did not; notably, most localized within or near the MSZ or other runs of silent glycines. Together, these results pinpointed several regions of the collagen triple helix- most notably within the cell interaction domain, and a narrow cross-fibril zone just N-terminal to the major cell surface integrin binding site GFOGER- as being particularly sensitive to glycine mutations and likely having highly crucial biological functions. Thus for some collagen mutations, disease phenotypes may result, at least in part, from disruption of crucial protein functions such as mineralization or cell-fibril interactions