Vertebrate collagen fibrils are heterotypically composed of a quantitatively major and minor fibril collagen. In non-cartilaginous tissues, type I collagen accounts for the majority of the collagen mass, and collagen type V, the functions of which are poorly understood, is a minor component. Type V collagen has been implicated in the regulation of fibril diameter, and we reported recently preliminary evidence that type V collagen is required for collagen fibril nucleation (Wenstrup, R. J., Florer, J. B., Cole, W. G., Willing, M. C., and Birk, D. E. (2004) J. Cell. Biochem. 92, 113-124). The purpose of this study was to define the roles of type V collagen in the regulation of collagen fibrillogenesis and matrix assembly. Mouse embryos completely deficient in pro-␣1(V) chains were created by homologous recombination. The col5a1؊/؊ animals die in early embryogenesis, at approximately embryonic day 10. The type V collagen-deficient mice demonstrate a virtual lack of collagen fibril formation. In contrast, the col5a1؉/؊ animals are viable. The reduced type V collagen content is associated with a 50% reduction in fibril number and dermal collagen content. In addition, relatively normal, cylindrical fibrils are assembled with a second population of large, structurally abnormal collagen fibrils. The structural properties of the abnormal matrix are decreased relative to the wild type control animals. These data indicate a central role for the evolutionary, ancient type V collagen in the regulation of fibrillogenesis. The complete dependence of fibril formation on type V collagen is indicative of the critical role of the latter in early fibril initiation. In addition, this fibril collagen is important in the determination of fibril structure and matrix organization.Type V collagen is a member of the fibril subclass of collagens, which have in common a triple helical domain composed of an uninterrupted series of Gly-X-Y triplets. Type V collagen is a quantitatively minor component of predominantly type I collagen fibrils in most non-cartilaginous tissues. Several isoforms of type V collagen exist, which differ in the type and ratio of constituent chains, including heterotypic molecules containing type XI collagen chains. The most abundant and widely distributed isoform is ␣1(V) 2 ␣2(V), which forms heterotypic fibrils with type I collagen (1). The role of type V collagen in the organization and biological properties of collagenous extracellular matrix is poorly understood. Observations of an inverse correlation between type V collagen:type I collagen ratios and collagen fibril diameter in in vitro fibril assembly experiments (2), cell cultures (3, 4), and in various tissues (5) have led to the hypothesis that type V collagen serves as a negative regulator of collagen fibril diameter (3-5). That function may be mediated by retention of the non-collagenous amino-terminal propeptide after type V collagen molecules are incorporated into fibrils (2, 6 -9). This non-collagenous domain projects outward through the gap between adj...

Tendon function involves the development of an organized hierarchy of collagen fibrils. Small leucine-rich proteoglycans have been implicated in the regulation of fibrillogenesis and decorin is the prototypic member of this family. Decorin-deficient mice demonstrate altered fibril structure and mechanical function in mature skin and tail tendons. However, the developmental role(s) of decorin needs to be elucidated. To define these role(s) during tendon development, tendons (flexor digitorum longus) were analyzed ultrastructurally from postnatal day 10 to 90. Decorin-deficient tendons developed abnormal, irregularly contoured fibrils. Finite mixture modeling estimated that the mature tendon was a three-subpopulation mixture of fibrils with characteristic diameter ranges. During development, in each subpopulation the mean diameter was consistently larger in mutant mice. Also, diameter distributions and the percentage of fibrils in each subpopulation were altered. Biomechanical analyses demonstrated that mature decorin-deficient tendons had significantly reduced strength and stiffness; however, there was no reduction in immature tendons. Expression of decorin and biglycan, a closely related family member, was analyzed during development. Decorin increased with development while biglycan decreased. Spatially, both had a comparable localization throughout the tendon. Biglycan expression increased substantially in decorin-deficient tendons suggesting a potential functional compensation. The accumulation of structural defects during fibril growth, a period associated with decorin expression and low biglycan expression, may be the cause of compromised mechanical function in the absence of decorin. Our findings indicate that decorin is a key regulatory molecule and that the temporal switch from biglycan to decorin is an important event in the coordinate regulation of fibrillogenesis and tendon development.

Collagen fibrillogenesis is finely regulated during development of tissue-specific extracellular matrices. The role(s) of a leucine-rich repeat protein subfamily in the regulation of fibrillogenesis during tendon development were defined. Lumican-, fibromodulin-, and double-deficient mice demonstrated disruptions in fibrillogenesis. With development, the amount of lumican decreases to barely detectable levels while fibromodulin increases significantly, and these changing patterns may regulate this process. Electron microscopic analysis demonstrated structural abnormalities in the fibrils and alterations in the progression through different assembly steps. In lumican-deficient tendons, alterations were observed early and the mature tendon was nearly normal. Fibromodulin-deficient tendons were comparable with the lumican-null in early developmental periods and acquired a severe phenotype by maturation. The double-deficient mice had a phenotype that was additive early and comparable with the fibromodulin-deficient mice at maturation. Therefore, lumican and fibromodulin both influence initial assembly of intermediates and the entry into fibril growth, while fibromodulin facilitates the progression through growth steps leading to mature fibrils. The observed increased ratio of fibromodulin to lumican and a competition for the same binding site could mediate these transitions. These studies indicate that lumican and fibromodulin have different developmental stage and leucine-rich repeat protein specific functions in the regulation of fibrillogenesis.

The HLA-G gene is primarily expressed in placental cells that invade the maternal decidua during pregnancy. This gene encodes multiple isoforms that fulfill a variety of functions at the maternal-fetal interface throughout gestation. Recently, a null allele for the most abundant HLA-G isoform was associated with recurrent miscarriage in two independent studies, suggesting that reduced levels of the HLA-G1 protein may compromise successful pregnancy. We initiated the present study to determine whether other polymorphisms that could affect expression levels of HLA-G were associated with fetal loss in women participating in a 15-year prospective study of pregnancy outcome. We genotyped these subjects for 18 single-nucleotide polymorphisms in the 1,300 bp upstream of exon 1, 13 of which were identified as part of this study, as well as for an insertion/deletion (in/del) polymorphism in the 3' untranslated region. The 18 SNPs defined eight unique haplotypes. One polymorphism, -725C/G, was associated with fetal loss, with an increased risk for miscarriage in couples in which both partners carried the -725G allele, compared with couples not carrying this allele (odds ratio 2.76, 95% confidence interval 1.08-7.09; P=.035). Further, the G at nucleotide -725 creates a CpG dinucleotide, and we demonstrate that this CpG site is methylated on -725G alleles. Overall, this study identified extraordinary levels of variation in the 5'-upstream regulatory region of HLA-G and provides evidence for an association between a promoter-region SNP and fetal loss rates, further attesting to the novel features and critical role of this gene in pregnancy.

Switching epilepsy patients from the enzyme-inducers carbamazepine or phenytoin to the noninducing drugs levetiracetam or lamotrigine produces rapid and clinically significant amelioration in several serological markers of vascular risk. These findings suggest that phenytoin and carbamazepine may substantially increase the risk for cardiovascular and cerebrovascular disease.