Molecular characteristics of periodontal health: Collagens: Defining the healthy human gingival collagen transcriptome: Defining the healthy human gingival collagen transcriptome.

BACKGROUND: Defining periodontal health has been an ambitious and complex goal. The numerous and varied definitions of what constitutes periodontal health have resulted in a collection of subjective and unreliable clinical findings to diagnose and classify periodontal health and disease. The aim of this study was to fundamentally delineate the molecular characteristics of healthy periodontal tissues in men and women as they age, using the most abundant connective tissue component: Collagens. METHODS: Healthy gingival biopsies were separated into "young" (aged 18-35 years, five men/five women) and "old" (≥60 years, five men/four women) age groups depending on biological sex. RNA was extracted and next-generation RNA sequencing was performed using Unique Molecular Identifiers. Collagen gene expression was determined and quantified for young and old, male and female individuals. RESULTS: Twenty-six human collagens were identified in healthy gingival tissues. In general, age and biological sex affected expression of collagen α-chain transcripts. Ten of the 26 human gingival collagen genes formed a unique pattern for gingival health. More specifically, the expression of fibrillary (types I and III), fibril-associated collagens with interrupted triple-helices (FACIT) and FACIT-like (types XII, XIV, and XX), network-forming (types IV and VI), transmembrane (type XVII), and multiplexin (types XV and XVIII) collagens, taken together, exhibited a distinct pattern of characteristics for gingival health that was independent of age or biological sex. CONCLUSIONS: Although specific α-chains of the collagen transcriptome were affected by age and biological sex, the compilation of various collagen transcripts can be used to define gingival health that is independent of age and biological sex.

Regeneration of collagen fibrils at the papillary dermis by reconstructing basement membrane at the dermal-epidermal junction.

The epidermal basement membrane deteriorates with aging. We previously reported that basement membrane reconstruction not only serves to maintain epidermal stem/progenitor cells in the epidermis, but also increases collagen fibrils in the papillary dermis. Here, we investigated the mechanism of the latter action. Collagen fibrils in the papillary dermis were increased in organotypic human skin culture treated with matrix metalloproteinase and heparinase inhibitors. The expression levels of COL5A1 and COL1A1 genes (encoding collagen type V α 1 chain and collagen type I α 1 chain, respectively) were increased in fibroblasts cultured with conditioned medium from a skin equivalent model cultured with the inhibitors and in keratinocytes cultured on laminin-511 E8 fragment-coated plates. We then examined cytokine expression, and found that the inhibitors increased the expression of PDGF-BB (platelet-derived growth factor consisting of two B subunits) in epidermis. Expression of COL5A1 and COL1A1 genes was increased in cultured fibroblasts stimulated with PDGF-BB. Further, the bifunctional inhibitor hydroxyethyl imidazolidinone (HEI) increased skin elasticity and the thickness of the papillary dermis in the skin equivalent. Taken together, our data suggests that reconstructing the basement membrane promotes secretion of PDGF-BB by epidermal keratinocytes, leading to increased collagen expression at the papillary dermis.

Abnormalities in esophageal smooth muscle induced by mutations in collagen XIX.

Collagen XIX is a nonfibrillar collagen that localizes in restricted tissues at very low amounts. A previous study on Col19a1 null mice revealed that collagen XIX is involved in esophageal muscle physiology and morphogenesis. Here, we use histological analysis to show that mice with a Col19a1 mutant lacking the NC3 domain and seven collagen triplets display abnormal transition of smooth to striated muscle in the abdominal segment of esophagus, and a widened esophagus with age. With two newly prepared antibodies, we analyzed the expression of collagen XIX in the mouse esophagus and show that collagen XIX colocalizes with α-smooth muscle actin. By immunoelectron microscopy, we confirmed the localization of collagen XIX in esophageal smooth muscle cells. Col19a1 mutant mice contained reduced levels of mutated Col19a1 mRNA. Interestingly, hepatocyte growth factor, which has an important role in esophageal striated muscle development, was reduced in the esophagus of the Col19a1 mutant mice. These findings suggest that collagen XIX may be critical for the function of esophageal smooth muscle cells as a scaffold for anteroposterior migration of esophagus-striated muscle cells.

Vascular endothelial growth factor (VEGF) polymorphism and increased risk of epithelial ovarian cancer.

INTRODUCTION: Angiogenesis (AG) is essential for epithelial ovarian cancer (EOC) development. Vascular endothelial growth factor (VEGF), encoded by the VEGF gene, and endostatin, the product of the COL18A1 gene, act as a potent promoter and an inhibitor of AG, respectively. In the present study, we tested whether VEGF C936T and COL18A1 D104N polymorphisms alter the risk of EOC. METHODS: Genomic DNA from 131 EOC patients and 137 controls were analyzed by polymerase chain reaction and restriction fragment length polymorphism methods. The differences between groups were analyzed by χ (2) or Fisher's exact test and logistic regression analysis. RESULTS: The frequency of the VEGF 936CC genotype was higher in patients than in controls (84.8% vs. 75.3%, P = 0.03). Individuals with respective genotypes had a 1.98 (95% CI 1.04-3.78)-fold increased risk of EOC than those with the remaining genotypes. An excess of VEGF 936CC plus COL18A1 DN genotype was seen in patients when compared to controls (48.6% vs. 30.5%); however, only a tendency toward a statistically significant difference in genotype frequencies was found in the study (P = 0.06), reflecting a trend toward an increased risk of 2.44 for EOC in carriers of the combined genotype. CONCLUSION: Our data present, for the first time, preliminary evidence that VEGF C936T alone or combined with the COL18A1 D104N polymorphism of AG constitutes an important inherited EOC determinant.

Collagen XII: Protecting bone and muscle integrity by organizing collagen fibrils.

Collagen XII, largest member of the fibril-associated collagens with interrupted triple helix (FACIT) family, assembles from three identical α-chains encoded by the COL12A1 gene. The molecule consists of three threadlike N-terminal noncollagenous NC3 domains, joined by disulfide bonds and a short interrupted collagen triple helix toward the C-terminus. Splice variants differ considerably in size and properties: "small" collagen XIIB (220 kDa subunit) is similar to collagen XIV, whereas collagen XIIA (350 kDa) has a much larger NC3 domain carrying glycosaminoglycan chains. Collagen XII binds to collagen I-containing fibrils via its collagenous domain, whereas its large noncollagenous arms interact with other matrix proteins such as tenascin-X. In dense connective tissues and bone, collagen XII is thought to regulate organization and mechanical properties of collagen fibril bundles. Accordingly, recent findings show that collagen XII mutations cause Ehlers-Danlos/myopathy overlap syndrome associated with skeletal abnormalities and muscle weakness in mice and humans.

Collagen XXII binds to collagen-binding integrins via the novel motifs GLQGER and GFKGER.

Collagen XXII, a FACIT (fibril-associated collagen with interrupted triple helices), is expressed at the myotendinous junction and the articular surface of joint cartilage. Cellular receptors like collagen-binding integrins are known to bind collagens with distinct binding motifs following the sequence GXOGER. In the present study, we demonstrate the sequences GLQGER and GFKGER as novel binding motifs between collagen XXII and collagen-binding integrins, especially α2ß1 integrin. Solid-phase assays and surface plasmon resonance spectroscopy revealed a direct interaction between α2ß1 integrin and the motif GFKGER. In addition, immunohistochemical analysis demonstrated partial co-localization of collagen XXII, α2ß1 integrin and α11ß1 integrin at the myotendinous junction. Furthermore, computational modelling of the motifs GLQGER and GFKGER showed perfect fitting of the sequences into the binding pocket of collagen-binding integrins. Taken together, we demonstrated that collagen XXII interacts with collagen-binding integrins via the new motifs GLQGER and GFKGER.

Mouse models in tendon and ligament research.

Mutant mouse models are valuable resources for the study of tendon and ligament biology. Many mutant mouse models are used because their manifested phenotypes mimic clinical pathobiology for several heritable disorders, such as Ehlers-Danlos Syndrome and Osteogenesis Imperfecta. Moreover, these models are helpful for discerning roles of specific genes in the development, maturation, and repair of musculoskeletal tissues. There are several categories of genes with essential roles in the synthesis and maintenance of tendon and ligament structures. The form and function of these tissues depend highly upon fibril-forming collagens, the primary extracellular macromolecules of tendons and ligaments. Models for these fibril-forming collagens, as well as for regulatory molecules like FACITs and SLRPs, are important for studying fibril assembly, growth, and maturation. Additionally, mouse models for growth factors and transcription factors are useful for defining regulation of cell proliferation, cell differentiation, and cues that stimulate matrix synthesis. Models for membrane-bound proteins assess the roles of cell-cell communication and cell-matrix interaction. In some cases, special considerations need to be given to spatio-temporal control of a gene in a model. Thus, conditional and inducible mouse models allow for specific regulation of genes of interest. Advances in mouse models have provided valuable tools for gaining insight into the form and function of tendons and ligaments.

[A current viewpoint on structure and evolution of collagens. II. The fibril-associated collagens with interrupted triple helices].

Fibril-associated collagens with interrupted triple helices (FACITs) form one of the subfamilies of collagen family. Being minor components of connective tissues in multicellular animals, FACITs play an important role in structurization of extracellular matrix whose peculiarities determine differences among tissues. FACITs take part in regulation of the sizes of banded collagen fibrils and are also a link between diverse components of extracellular matrix and cells in different tissues. The functional characteristics of FACIT molecules are determined by peculiarities of α-chain structure (interruptions in collagenous domains and module structure of N-terminal noncollagenous regions), trimeric molecules (trimerization domains), and supramolecular assemblies (mainly, association with banded fibrils and inability to form homopolymeric suprastructural aggregates). The evolution of FACITs is also discussed. A hypothetical model of structural changes leading to formation of FACIT subfamily is propounded.

Common genetic variants in the endothelial system predict blood pressure response to sodium intake: the GenSalt study.

BACKGROUND We examined the association between 14 endothelial system genes and salt-sensitivity of blood pressure (BP). METHODS After a 3-day baseline examination, during which time the usual diet was consumed, 1,906 Chinese participants received a 7-day low-sodium diet (51.3 mmol of sodium/day) followed by a 7-day high-sodium diet (307.8 mmol of sodium/day). BP measurements were obtained at baseline and at the end of each intervention using a random-zero sphygmomanometer. RESULTS The DDAH1 rs11161637 variant was associated with reduced BP salt sensitivity, conferring attenuated systolic BP (SBP) and mean arterial pressure (MAP) decreases from baseline to the low-sodium intervention (both P = 2×10(-4)). Examination of genotype-sex interactions revealed that this relation was driven by the strong associations observed in men (P for interactions = 1.10×10(-4) and 0.008, respectively). When switching from the low- to high-sodium intervention, increases in diastolic BP (DBP) and MAP were attenuated by the COL18A1 rs2838944 minor A allele (P = 1.41×10(-4) and 1.55×10(-4), respectively). Conversely, the VWF rs2239153 C variant was associated with increased salt sensitivity, conferring larger DBP and MAP reductions during low-sodium intervention (P = 1.22×10(-4) and 4.44×10(-5), respectively). Ten variants from 3 independent SELE loci displayed significant genotype-sex interactions on DBP and MAP responses to low-sodium (P for interaction = 1.56×10(-3) to 1.00×10(-4)). Among men, minor alleles of 4 correlated markers attenuated BP responses to low-sodium intake, whereas minor alleles of another 4 correlated markers increased BP responses. No associations were observed in women for these variants. Further, qualitative interactions were shown for 2 correlated SELE markers. CONCLUSIONS These data support a role for the endothelial system genes in salt sensitivity.

Type VII collagen: the anchoring fibril protein at fault in dystrophic epidermolysis bullosa.

Type VII collagen is a major component of the anchoring fibrils of the dermal-epidermal adhesion on the dermal side at the lamina densa/papillary dermis interface. Dystrophic epidermolysis bullosa (DEB) emerged as a candidate for type VII collagen mutations becausing anchoring fibrils were shown to be morphologically altered, reduced in number, or completely absent in patients with different forms of DEB. Circulating autoantibodies recognize type VII collagen epitopes in epidermolysis bullosa acquisita. The suggestion that type VII collagen is required for human epidermal tumorigenesis relates to the increasing numbers of life-threatening complications associated with developing squamous cell carcinomas because of the extended life span of affected individuals with recessive DEB.

Collagen XVI expression is upregulated in glioblastomas and promotes tumor cell adhesion.

The poor prognosis of glioblastoma patients is related to diffuse brain invasion and interaction of tumor cells with extracellular matrices (ECM). We describe expression and function of the FACIT-collagen XVI in glioblastomas. We found upregulation of collagen XVI mRNA as well as protein in glioblastomas as compared to normal cortex. SiRNA knockdown resulted in decreased cell adhesion whereas increased adhesion was observed on surfaces coated with collagen XVI. The migration of glioblastoma cells on this substrate remained unchanged. Our results demonstrate de-novo expression of collagen XVI in glioblastomas as part of the tumor specific remodeling of the ECM.

Is there an evolutionary relationship between WARP (von Willebrand factor A-domain-related protein) and the FACIT and FACIT-like collagens?

We suggest that there is an evolutionary relationship between von Willebrand factor A-domain-related protein (WARP), and the fibril-associated collagen with interrupted triple helix (FACIT) and FACIT-like subfamilies of collagens. Data from a comparison of amino acid sequences, domain organisation and chromosomal location are consistent with the hypothesis that WARP and these collagens share a common collagen ancestor. In support of this is the observation that the WARP 3' coding region is GC-rich suggesting that this may represent the remnant of a triple helix protein domain which WARP has 'lost' during evolution.

Collagen messenger RNA expression in the human amniochorion in premature rupture of membranes.

OBJECTIVE: It has been suggested that there is a decrease in the collagen content of the fetal membranes when there is premature rupture of the membranes before the onset of labor. This study was designed to determine whether decreased amniochorion collagen production (as measured by reduced amounts of messenger RNA) or alterations in relative production of different fibrillar and nonfibrillar collagens are associated with premature rupture of the membranes. STUDY DESIGN: Fetal membranes were collected after preterm (24-36 weeks of gestation) and term (> or =37 weeks of gestation) deliveries both with and without premature rupture of the membranes. Specimens with evidence of histologic chorioamnionitis were excluded. The messenger RNA levels for fibrillar collagen types I, III, and V and fibril-associated collagens with interrupted triple-helices types XII and XIV were measured with relative quantitative reverse transcriptase-polymerase chain reaction. RESULTS: The messenger RNA levels for fibrillar collagens decreased with advancing gestational age. Preterm premature rupture of membranes was associated with increased messenger RNA levels for fibrillar collagens and fibril-associated collagens with interrupted triple-helices collagen XII, but not type XIV. The greatest change in relative amounts of collagen messsenger RNA was demonstrated by an increased type I/XIV ratio, which was due to the up-regulation of type I levels, but not type XIV levels. CONCLUSION: A rise in fibrillar collagen production (messenger RNA) for types I, III, and V and fibril-associated collagens with interrupted triple-helices collagen type XII is observed with preterm premature rupture of the membranes. There is no evidence for a similar up-regulation of messenger RNA for fibril-associated collagens with interrupted triple-helices collagen type XIV. The rise in the collagen I/XIV messenger RNA ratio in preterm premature rupture of the membranes may result in collagen fibrils without enough stabilizing fibril-associated collagens with interrupted triple-helices type XIV on the fibril surface to maintain structural integrity.

Genomic organization and characterization of the human type XXI collagen (COL21A1) gene.

We cloned a 4.1-kb full-length cDNA based on a reported human genomic clone containing a partial open reading frame (ORF) coding for a novel collagen-like protein. Sequence analysis indicated that the ORF codes for the alpha(1)-chain of type XXI collagen. Assembly of the genomic data reveals a complete sequence of the human gene COL21A1. COL21A1 is localized to chromosome 6p11.2-12.3, spanning 337 kb in size. The gene contains 31 exons, in which the 5'-untranslated exons 1 and 1a are alternatively spliced. The exon/domain organization of COL21A1 resembles that of the reported FACIT collagen genes, including COL9A1, COL9A2, COL9A3, and COL19A1, suggesting that these genes may have derived from the same ancestor FACIT gene by duplication. The expression of COL21A1 in human tissues is developmentally regulated, with a higher level at fetal stages. Type XXI collagen is an extracellular matrix component of the blood vessel walls, secreted by smooth-muscle cells. Platelet-derived growth factor (PDGF) has a pronounced effect on the stimulation of COL21A1 expression in cultured aortic smooth-muscle cells, suggesting that alpha1(XXI) collagen may contribute to the extracellular matrix assembly of the vascular network during blood vessel formation.

Identification and analysis of collagen alpha 1(XXI), a novel member of the FACIT collagen family.

The FACIT collagens bind to the surface of collagen fibrils linking them with other matrix molecules. Bioinformatics analysis of cDNA clone DKFZp564B052 showed that it resembled the FACIT collagens and was therefore designated collagen alpha 1(XXI). Phylogenetic analyses of the N-terminal NC3 domains of alpha 1(XXI) and other FACIT collagens showed that (i) alpha 1(XXI) clustered with the FACIT collagens; (ii) collagen alpha 1(XXI) arose before the divergence of alpha 1(XII), alpha 1(XIV) and alpha 1(XX); (iii) collagen alpha 1(XIV) derived from the C-terminal region of the NC3 domain of a collagen alpha 1(XII)-like molecule; and (iv) collagen alpha 1(XX) derived from a collagen alpha 1(XIV)-like molecule. This study provides a framework for the evolution of the FACIT collagens which will be of value in linking NC3 domains with their functions.