Fragility of reconstituted type V collagen fibrils with the chain composition of α1(V)α2(V)α3(V) respective of the D-periodic banding pattern.

The triple-helical domains of two subtypes of type V collagen were prepared from human placenta, one with the chain composition of [α1(V)](2)α2(V) (Vp112) and the other with the chain composition of α1(V)α2(V)α3(V) (Vp123) with limited pepsin treatment. In order to characterize the triple-helical domain of the type Vp123 collagen molecule, the reconstituted aggregate structure formed from the pepsin-treated collagen was compared by using transmission electron microscopy. The diameter of the fibrils reconstituted from types pepsin-treated type Vp123 collagen and type Vp112 collagen was highly uniform and less than the D-periodicity at all the temperatures examined, suggesting that the major triple-helical domain of both subtypes has a potency to limit their lateral growth. Both fibrils were approximately 45 nm in width and showed the D-periodic banding pattern along their axes at 34°C. In contrast to type Vp112, the reconstituted type Vp123 fibrils showed no banding pattern along their axes when they were reconstituted at 37°C. The banded fibrils once reconstituted from type Vp123 at 34°C tend to lose their characteristic pattern within 60 min when they were incubated at 37°C. One explanation is that a slightly higher content of hydrophobic residues of type Vp123 collagen than those of type V112p collagen augmented the intermolecular interaction that disturbs the D-periodicity governed essentially by electrostatic interactions. Taken together with recent data in Col5a3 gene-targeted mice, the results suggest that type V123 collagen exists not only as a periodic banded fibril but also as nonfibrillar meshwork structures.

Col V siRNA engineered tenocytes for tendon tissue engineering.

The presence of uniformly small collagen fibrils in tendon repair is believed to play a major role in suboptimal tendon healing. Collagen V is significantly elevated in healing tendons and plays an important role in fibrillogenesis. The objective of this study was to investigate the effect of a particular chain of collagen V on the fibrillogenesis of Sprague-Dawley rat tenocytes, as well as the efficacy of Col V siRNA engineered tenocytes for tendon tissue engineering. RNA interference gene therapy and a scaffold free tissue engineered tendon model were employed. The results showed that scaffold free tissue engineered tendon had tissue-specific tendon structure. Down regulation of collagen V α1 or α2 chains by siRNAs (Col5α1 siRNA, Col5α2 siRNA) had different effects on collagen I and decorin gene expressions. Col5α1 siRNA treated tenocytes had smaller collagen fibrils with abnormal morphology; while those Col5α2 siRNA treated tenocytes had the same morphology as normal tenocytes. Furthermore, it was found that tendons formed by coculture of Col5α1 siRNA treated tenocytes with normal tenocytes at a proper ratio had larger collagen fibrils and relative normal contour. Conclusively, it was demonstrated that Col V siRNA engineered tenocytes improved tendon tissue regeneration. And an optimal level of collagen V is vital in regulating collagen fibrillogenesis. This may provide a basis for future development of novel cellular- and molecular biology-based therapeutics for tendon diseases.

A novel splice variant in the N-propeptide of COL5A1 causes an EDS phenotype with severe kyphoscoliosis and eye involvement.

BACKGROUND: The Ehlers-Danlos Syndrome (EDS) is a heritable connective tissue disorder characterized by hyperextensible skin, joint hypermobility and soft tissue fragility. The classic subtype of EDS is caused by mutations in one of the type V collagen genes (COL5A1 and COL5A2). Most mutations affect the type V collagen helical domain and lead to a diminished or structurally abnormal type V collagen protein. Remarkably, only two mutations were reported to affect the extended, highly conserved N-propeptide domain, which plays an important role in the regulation of the heterotypic collagen fibril diameter. We identified a novel COL5A1 N-propeptide mutation, resulting in an unusual but severe classic EDS phenotype and a remarkable splicing outcome. METHODOLOGY/PRINCIPAL FINDINGS: We identified a novel COL5A1 N-propeptide acceptor-splice site mutation (IVS6-2A>G, NM_000093.3_c.925-2A>G) in a patient with cutaneous features of EDS, severe progressive scoliosis and eye involvement. Two mutant transcripts were identified, one with an exon 7 skip and one in which exon 7 and the upstream exon 6 are deleted. Both transcripts are expressed and secreted into the extracellular matrix, where they can participate in and perturb collagen fibrillogenesis, as illustrated by the presence of dermal collagen cauliflowers. Determination of the order of intron removal and computational analysis showed that simultaneous skipping of exons 6 and 7 is due to the combined effect of delayed splicing of intron 7, altered pre-mRNA secondary structure, low splice site strength and possibly disturbed binding of splicing factors. CONCLUSIONS/SIGNIFICANCE: We report a novel COL5A1 N-propeptide acceptor-splice site mutation in intron 6, which not only affects splicing of the adjacent exon 7, but also causes a splicing error of the upstream exon 6. Our findings add further insights into the COL5A1 splicing order and show for the first time that a single COL5A1 acceptor-splice site mutation can perturb splicing of the upstream exon.

Regulation of collagen fibril nucleation and initial fibril assembly involves coordinate interactions with collagens V and XI in developing tendon.

Collagens V and XI comprise a single regulatory type of fibril-forming collagen with multiple isoforms. Both co-assemble with collagen I or II to form heterotypic fibrils and have been implicated in regulation of fibril assembly. The objective of this study was to determine the roles of collagens V and XI in the regulation of tendon fibrillogenesis. Flexor digitorum longus tendons from a haplo-insufficient collagen V mouse model of classic Ehlers Danlos syndrome (EDS) had decreased biomechanical stiffness compared with controls consistent with joint laxity in EDS patients. However, fibril structure was relatively normal, an unexpected finding given the altered fibrils observed in dermis and cornea from this model. This suggested roles for other related molecules, i.e. collagen XI, and compound Col5a1(+/-),Col11a1(+/-) tendons had altered fibril structures, supporting a role for collagen XI. To further evaluate this, transcript expression was analyzed in wild type tendons. During development (E18-P10) both collagen V and XI were comparably expressed; however, collagen V predominated in mature (P30) tendons. The collagens had a similar expression pattern. Tendons with altered collagen V and/or XI expression (Col5a1(+/-); Col11a1(+/-); Col5a1(+/-),Col11a1(+/-); Col11a1(-/-); Col5a1(+/-),Col11a1(-/-)) were analyzed at E18. All genotypes demonstrated a reduced fibril number and altered structure. This phenotype was more severe with a reduction in collagen XI. However, the absence of collagen XI with a reduction in collagen V was associated with the most severe fibril phenotype. The data demonstrate coordinate roles for collagens V and XI in the regulation of fibril nucleation and assembly during tendon development.

Hemizygous deletion of COL3A1, COL5A2, and MSTN causes a complex phenotype with aortic dissection: a lesson for and from true haploinsufficiency.

Aortic dilatation/dissection (AD) can occur spontaneously or in association with genetic syndromes, such as Marfan syndrome (MFS; caused by FBN1 mutations), MFS type 2 and Loeys-Dietz syndrome (associated with TGFBR1/TGFBR2 mutations), and Ehlers-Danlos syndrome (EDS) vascular type (caused by COL3A1 mutations). Although mutations in FBN1 and TGFBR1/TGFBR2 account for the majority of AD cases referred to us for molecular genetic testing, we have obtained negative results for these genes in a large cohort of AD patients, suggesting the involvement of additional genes or acquired factors. In this study we assessed the effect of COL3A1 deletions/duplications in this cohort. Multiplex ligation-dependent probe amplification (MLPA) analysis of 100 unrelated patients identified one hemizygous deletion of the entire COL3A1 gene. Subsequent microarray analyses and sequencing of breakpoints revealed the deletion size of 3,408,306 bp at 2q32.1q32.3. This deletion affects not only COL3A1 but also 21 other known genes (GULP1, DIRC1, COL5A2, WDR75, SLC40A1, ASNSD1, ANKAR, OSGEPL1, ORMDL1, LOC100129592, PMS1, MSTN, C2orf88, HIBCH, INPP1, MFSD6, TMEM194B, NAB1, GLS, STAT1, and STAT4), mutations in three of which (COL5A2, SLC40A1, and MSTN) have also been associated with an autosomal dominant disorder (EDS classical type, hemochromatosis type 4, and muscle hypertrophy). Physical and laboratory examinations revealed that true haploinsufficiency of COL3A1, COL5A2, and MSTN, but not that of SLC40A1, leads to a clinical phenotype. Our data not only emphasize the impact/role of COL3A1 in AD patients but also extend the molecular etiology of several disorders by providing hitherto unreported evidence for true haploinsufficiency of the underlying gene.

The role of type V collagen fibril as an ECM that induces the motility of glomerular endothelial cells.

Although type V collagen (Col V) is present in developing and mature connective tissues of glomeruli, its primary function has not been elucidated yet. The purpose of this study was to elucidate the role of Col V fibrils in glomerular cells. We isolated primary cells from porcine kidney and cultured them on Col V fibrils reconstructed from purified Col V molecules extracted from porcine cornea. Time-lapse observation showed that Col V fibrils induce dynamic movement of glomerular endothelial cells (GEC) by stimulating them to extend long filopodial protrusions and wide lamellipodia. Col V signaling mediated through beta1 integrin activated phosphorylation of paxillin at tyrosine 118 (paxillin-pY118) and of focal adhesion kinase at tyrosine 861 (FAKpY861) at the cell periphery; a second Col V signal was mediated through neuroglycan 2 and activated FAKpY397. FAKpY861 was present in loose attachment points between Col V fibrils and GEC, allowing the cells to migrate easily. Activation of FAKpY397 induced incomplete focal adhesion at the centers of cells and caused cell movement. Therefore both signaling pathways facilitated cell motility, which was inhibited by the addition of antibodies to beta1 integrin, NG2, and Col V. We suggest that Col V fibrils activate 'outside-in' signaling in GEC and induce their dynamic motility.

Graded arrangement of collagen fibrils in the equine superficial digital flexor tendon.

By using ultramorphological and biochemical methods, we analyzed the regional differences between the three parts of the equine superficial digital flexor tendon (SDFT), namely, the myotendinous junction (MTJ), middle metacarpal (mM), and osteotendinous junction (OTJ). Cross-sectional images showed unique distributions of collagen fibrils of varying diameters in each region. Small collagen fibrils (diameter <100 nm) were distributed predominantly in the MTJ region, and the OTJ region was relatively rich in large collagen fibrils (diameter >200 nm). In the mM region, the collagen fibrils were intermediately distributed between the MTJ and OTJ. The results indicate a graded arrangement of collagen fibrils in the tendon. Type V collagen was detected preferentially in the MTJ region. Since type V collagen is believed to be one of the collagens regulating collagen fibril formation, its possible functionality in the MTJ region in terms of fibril formation and fibril arrangement in the tendon has been discussed here.

Morphologic characterization of organized extracellular matrix deposition by ascorbic acid-stimulated human corneal fibroblasts.

PURPOSE: To characterize the structure and morphology of extracellular matrix (ECM) synthesized by untransformed, cultured human corneal fibroblasts in long-term cultures. METHODS: Human corneal stromal keratocytes were expanded in transwell culture in the presence of fetal bovine serum and a stable derivative of vitamin C. The cells were allowed to synthesize a fibrillar ECM for up to 5 weeks. Constructs were assessed by light (phase-contrast and differential interference-contrast) and transmission (standard and quick freeze/deep etch) microscopy. RESULTS: Electron micrographs revealed stratified constructs with multiple parallel layers of cells and an extracellular matrix comprising parallel arrays of small, polydisperse fibrils (27-51 nm) that often alternate in direction. Differential interference contrast images demonstrated oriented ECM fibril arrays parallel to the plane of the construct, whereas quick-freeze, deep-etch micrographs showed the details of the matrix interaction with fibroblasts through arrays of membrane surface structures. CONCLUSIONS: Human keratocytes, cultured in a stable vitamin C derivative, are capable of assembling extracellular matrix, which comprises parallel arrays of ECM fibrils. The resultant constructs, which are highly cellular, are morphologically similar to the developing mammalian stroma, where organized matrix is derived. The appearance of arrays of structures on the cell membranes suggests a role in the local organization of synthesized ECM. This model could provide critical insight into the fundamental processes that govern the genesis of organized connective tissues such as the cornea and may provide a scaffolding suitable for tissue engineering a biomimetic stroma.

Collagen types I, III, and V constitute the thick collagen fibrils of the mouse decidua.

A mammal's endometrium is deeply remodeled while receiving and implanting an embryo. In addition to cell proliferation and growth, endometrial remodeling also comprises synthesis and degradation of several molecular components of the extracellular matrix. All of these events are orchestrated by a precise sequence of ovarian hormones and influenced by several types of cytokines. As we have previously reported, an intriguing and rapid increase in collagen fibril diameter occurs in the decidualized areas of the endometrium, surrounding the implantation crypt, whereas collagen fibrils situated far from the embryo remain unchanged. Collagen fibrilogenesis is a complex molecular process coordinated by a number of factors, such as the types and amounts of glycosaminoglycans and proteoglycans associated with collagen molecules. Collagen genetic type, mechanical stress, aging, and other factors not yet identified also contribute to this development. A recent study suggests that thick fibrils from mouse decidua are formed, at least in part, by aggregation of thin fibrils existing in the stroma before the onset of decidualization. In the present ultrastructural study using single and double immunogold localization, we showed that both thin and thick collagen fibrils present in the mouse pregnant endometrium endometrium are heterotypic structures formed at least by type I, type III, and type V collagens. However, type V collagen predominates in the thick collagen fibrils, whereas it is almost absent of the thin collagen fibrils. The putative role of type V homotrimer in the rapid increase of the diameter of collagen fibrils of the mouse decidua is discussed.

Procollagen C proteinase enhancer 1 genes are important determinants of the mechanical properties and geometry of bone and the ultrastructure of connective tissues.

Procollagen C proteinases (pCPs) cleave type I to III procollagen C propeptides as a necessary step in assembling the major fibrous components of vertebrate extracellular matrix. The protein PCOLCE1 (procollagen C proteinase enhancer 1) is not a proteinase but can enhance the activity of pCPs approximately 10-fold in vitro and has reported roles in inhibiting other proteinases and in growth control. Here we have generated mice with null alleles of the PCOLCE1 gene, Pcolce, to ascertain in vivo roles. Although Pcolce-/- mice are viable and fertile, Pcolce-/- male, but not female, long bones are more massive and have altered geometries that increase resistance to loading, compared to wild type. Mechanical testing indicated inferior material properties of Pcolce-/- male long bone, apparently compensated for by the adaptive changes in bone geometry. Male and female Pcolce-/- vertebrae both appeared to compensate for inferior material properties with thickened and more numerous trabeculae and had a uniquely altered morphology in deposited mineral. Ultrastructurally, Pcolce-/- mice had profoundly abnormal collagen fibrils in both mineralized and nonmineralized tissues. In Pcolce-/- tendon, 100% of collagen fibrils had deranged morphologies, indicating marked functional effects in this tissue. Thus, PCOLCE1 is an important determinant of bone mechanical properties and geometry and of collagen fibril morphology in mammals, and the human PCOLCE1 gene is identified as a candidate for phenotypes with defects in such attributes in humans.

Type V and VI collagen for cohesion of dermal fibrillar structures.

Type V and VI collagen were capable to joint each other and with type I and IV collagen, as well as mucopolysaccharides. This capability suggested that these collagens function for cohesion of fibrillar tissue components of dermis. This study demonstrated the locality of these types of collagen in dermis. Fresh specimens of normal skin were fixed in 2% paraformaldehyde in phosphate-buffered saline, overnight. Besides, in order to loosen the twist of collagen fibril, some pieces of the skin specimens were treated by citrate buffer pH 3.0, prior to fixation. The specimens were embedded in Technovit 4100 and the ultrathin sections were stained by antibody to type V collagen and followed by antibody to type I, III, IV and VI collagen. The immune reactant was visualized by gold particles for electron microscopic observation. Type V and VI collagen formed networks in dermis and jointed to collagen fibrils, elastic fibre and basal lamina. Type V collagen was found inside collagen fibrils, broad elastic fibres and junctions. Dermo-epidermal junction showed type V collagen on the dermal aspects of basal lamina and at the sites where anchoring filaments joint to basal lamina, while in junction of mesenchymal tissues, no precise structural components for type V collagen were identified. Type VI collagen wove with type V collagen in dermis and associated with mucopolysaccharides. In conclusion, type V collagen formed networks in dermal interfibrillar space and participated in assembling collagen fibrils and forming broad elastic fibres. Epithelial and mesenchymal cells cohered to the underlying dermal matrix in the junction by type V collagen. Type VI collagen interwove with type V collagen in the interfibrous space and associated with mucopolysaccharides. Types V and VI collagen preserved architecture of dermal matrix.

Collagenofibrotic glomerulopathy with a widespread expression of type-V collagen.

Collagenofibrotic glomerulopathy is considered as a form of glomerulopathy in which organized collagen type III progressively deposits. We report a case of this disease with widespread expression of collagen type V. A 65-year-old woman was admitted to our hospital for further evaluation of nephrotic-range proteinuria. The patient had had anemia and hypertension for 9 years, and proteinuria for 3 years. A renal biopsy specimen showed a remarkable mesangial expansion with Congo red-negative and periodic acid-Schiff-positive deposits. At the ultrastructural level, two forms of bundling fibers were found in the mesangium and subendothelial side of the glomerular basement membranes (GBM). The GBM itself appeared normal. Immunohistochemical investigation showed that the glomerular lesions were strongly reactive with both anti-collagen type-III and -V antibodies. Immunoelectron microscopy demonstrated collagen type V in both forms of bundling fibers. Despite therapy, her renal function declined. The clinical course and renal pathology of this case were in accordance with collagenofibrotic glomerulopathy except for the widespread expression of collagen type V. Collagen type V is a fibrillar collagen capable of forming banding fibrils. This report poses the question whether collagen type V accumulates only in this particular case or whether it is a normal component in collagenofibrotic glomerulopathy.

Order of intron removal influences multiple splice outcomes, including a two-exon skip, in a COL5A1 acceptor-site mutation that results in abnormal pro-alpha1(V) N-propeptides and Ehlers-Danlos syndrome type I.

Ehlers-Danlos syndrome (EDS) type I (the classical variety) is a dominantly inherited, genetically heterogeneous connective-tissue disorder. Mutations in the COL5A1 and COL5A2 genes, which encode type V collagen, have been identified in several individuals. Most mutations affect either the triple-helical domain of the protein or the expression of one COL5A1 allele. We identified a novel splice-acceptor mutation (IVS4-2A-->G) in the N-propeptide-encoding region of COL5A1, in one patient with EDS type I. The outcome of this mutation was complex: In the major product, both exons 5 and 6 were skipped; other products included a small amount in which only exon 5 was skipped and an even smaller amount in which cryptic acceptor sites within exon 5 were used. All products were in frame. Pro-alpha1(V) chains with abnormal N-propeptides were secreted and were incorporated into extracellular matrix, and the mutation resulted in dramatic alterations in collagen fibril structure. The two-exon skip occurred in transcripts in which intron 5 was removed rapidly relative to introns 4 and 6, leaving a large (270 nt) composite exon that can be skipped in its entirety. The transcripts in which only exon 5 was skipped were derived from those in which intron 6 was removed prior to intron 5. The use of cryptic acceptor sites in exon 5 occurred in transcripts in which intron 4 was removed subsequent to introns 5 and 6. These findings suggest that the order of intron removal plays an important role in the outcome of splice-site mutations and provide a model that explains why multiple products derive from a mutation at a single splice site.

Immunohistological and morphometric analysis of inflammatory cells in rapidly progressive periodontitis and adult periodontitis.

The purpose of this study was to localize, characterize, and quantify in situ the inflammatory cells in the gingival connective tissue prior and subsequent to the initial therapy of ten patients with rapidly progressive periodontitis (RPP) and five patients with adult periodontitis (AP). Using immunohistological techniques, the amount of T lymphocytes, alphabeta-T lymphocytes, gammadelta-T lymphocytes, B lymphocytes, and plasma cells was determined at the beginning of the periodontal therapy (baseline) and at the time of periodontal surgery. Furthermore, the distribution of collagen types I, III, V, and VI was investigated using transmission electron microscopy. At baseline, patients with RPP revealed much higher numbers of inflammatory cells than patients with AP. During initial therapy of patients with RPP, the amount of T cells, alphabeta-T cells, and gammadelta-T cells was reduced significantly (P<0.05). Biopsies of patients with AP revealed a statistically significant reduction of all cell types, except alphabeta-T cells and gammadelta-T cells in the deep connective tissue. The transmission electron microscopy of biopsies from patients with RPP and AP with severe inflammation taken at baseline revealed that collagen types I and III were destroyed nearly completely in areas with leukocyte infiltration, whereas collagen types V and VI revealed a more pronounced labeling reaction. The results revealed that, during initial therapy, the amount of inflammatory cells was reduced significantly more in biopsies of patients with AP than in patients with RPP. At baseline, the inflamed gingival tissue consists mainly of collagen types V and VI in areas with infiltrates of inflammatory cells.