Biomedical subjects

David E Birk

Publications and source records attributed to David E Birk.

At least 19 recordsLinked to original sources

PubMed2006

Decorin regulates assembly of collagen fibrils and acquisition of biomechanical properties during tendon development.

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.

Animals↗

PubMed2006

Murine model of the Ehlers-Danlos syndrome. col5a1 haploinsufficiency disrupts collagen fibril assembly at multiple stages.

The most commonly identified mutations causing Ehlers-Danlos syndrome (EDS) classic type result in haploinsufficiency of proalpha1(V) chains of type V collagen, a quantitatively minor collagen that co-assembles with type I collagen as heterotypic fibrils. To determine the role(s) of type I/V collagen interactions in fibrillogenesis and elucidate the mechanism whereby half-reduction of type V collagen causes abnormal connective tissue biogenesis observed in EDS, we analyzed mice heterozygous for a targeted inactivating mutation in col5a1 that caused 50% reduction in col5a1 mRNA and collagen V. Comparable with EDS patients, they had decreased aortic stiffness and tensile strength and hyperextensible skin with decreased tensile strength of both normal and wounded skin. In dermis, 50% fewer fibrils were assembled with two subpopulations: relatively normal fibrils with periodic immunoreactivity for collagen V where type I/V interactions regulate nucleation of fibril assembly and abnormal fibrils, lacking collagen V, generated by unregulated sequestration of type I collagen. The presence of the aberrant fibril subpopulation disrupts the normal linear and lateral growth mediated by fibril fusion. Therefore, abnormal fibril nucleation and dysfunctional fibril growth with potential disruption of cell-directed fibril organization leads to the connective tissue dysfunction associated with EDS.

Alleles↗

PubMed2006

Collagen fibril assembly during postnatal development and dysfunctional regulation in the lumican-deficient murine cornea.

The transparent cornea is the outer barrier of the eye and is its major refractive surface. Development of a functional cornea requires a postnatal maturation phase involving development, growth and organization of the stromal extracellular matrix. Lumican, a leucine-rich proteoglycan, is implicated in regulating assembly of collagen fibrils and the highly organized extracellular matrix essential for corneal transparency. We investigated the regulatory role(s) of lumican in fibril assembly during postnatal corneal development using wild type (Lum+/+) and lumican-null (Lum-/-) mice. In Lum+/+ mice, a regular architecture of small-diameter fibrils is achieved in the anterior stroma by postnatal day 10 (P10), while the posterior stroma takes longer to reach this developmental maturity. Thus, the anterior and the posterior stroma follow distinct developmental timelines and may be under different regulatory mechanisms. In Lum-/- mice, it is the posterior stroma where abnormal lateral associations of fibrils and thicker fibrils with irregular contours are evident as early as P10. In contrast, the anterior stroma is minimally perturbed by the absence of lumican. In Lum+/+ mice, lumican is expressed throughout the developing stroma at P10, with strong expression limited to the posterior stroma in the adult. Therefore, the posterior stroma, which is most vulnerable to lumican-deficiency, demonstrates an early developmental defect in fibril structure and architecture in the Lum-/- mouse. These defects underlie the reported increased light scattering and opacity detectable in the adult. Our findings emphasize the early regulation of collagen structure by lumican during postnatal development of the cornea.

Animals↗

PubMed2006

Collagen dysregulation in the dermis of the Sagg/+ mouse: a loose skin model.

The Sagg/+ mouse is an ethylnitrosourea-derived mutant with a dermal phenotype similar to some of the subtypes of Ehlers-Danlos syndrome (EDS) and cutis laxa. The dermis of the Sagg/+ mouse has less dense and more disorganized collagen fibers compared to controls. The size of extracted Type I dermal collagen was the same as that observed in normal skin; however, more collagen could be extracted from Sagg/+ skin, which also showed decreased collagen content and decreased steady-state levels of alpha1(I), alpha2(I), alpha1(V), and alpha2(V) procollagen mRNAs. The biomechanical properties of Sagg/+ skin were significantly decreased relative to normal skin. However, there were no significant differences in the quantities of the major collagen cross-links, that is, dehydrohydroxylysinonorleucine and dehydrohistidinohydroxymerodesmosine between Sagg/+ and normal skin. Electron microscopic evaluation of Sagg/+ skin indicated that the mutation interferes with the proper formation of collagen fibrils and the data are consistent with a mutation in Type V collagen leading to haploinsufficiency with the formation of two sub-populations of collagen fibrils, one normal and one with irregular shape and a larger diameter. Further study of this novel mutation will allow the identification of new mechanisms involved in the regulation of normal and pathologic collagen gene expression.

Animals↗

PubMed2006

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.

Alleles↗

PubMed2006

Analysis of expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human ciliary body after latanoprost.

PURPOSE: To determine the effect of latanoprost on the expression of human matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in the ciliary body. METHODS: Total RNA was isolated, and qualitative RT-PCR was performed to detect the mRNA of MMPs and TIMPs in human ciliary body tissue and explant cultures of ciliary body smooth muscle (CBSM) cells. CBSM cell cultures were treated with vehicle control or latanoprost acid for 24 hours. Quantitative RT-PCR of cell cultures from five different donors was performed to determine relative changes in expression. GAPDH served as an endogenous control. RESULTS: The mRNA of MMP-1, -2, -3, -11, -12, -14, -15, -16, -17, -19, and -24 as well as TIMP-1 to -4 were found in ciliary body tissue and CBSM cells. MMP-9 was present after latanoprost treatment. In control CBSM cells, the relative expression of MMP mRNA was MMP-2 and -14 > MMP-24 > MMP-1, -11, -15, -16, and -19 > MMP-3 and 17, > MMP-12. The relative expression of TIMP mRNA was TIMP-2 > TIMP-1 > TIMP-3 > TIMP-4. Latanoprost increased MMP-3 (in three of five cultures), MMP-17 (in four of five cultures), and TIMP-3 (in all five cultures); MMP-1, -2, -12, -14, -15, and -16 and TIMP-4 were downregulated. CONCLUSIONS: The transcription of the genes for MMP-3 and -17 is increased by latanoprost treatment. MMP-9 is present after latanoprost treatment and may also mediate ECM changes. TIMP-3 is upregulated and may compensate for the increase in MMPs. These coordinated changes could be expected to mediate the latanoprost-induced alteration of ECM in the ciliary body.

Adult↗

PubMed2006

Structural abnormalities of the cornea and lid resulting from collagen V mutations.

PURPOSE: Type V collagen forms heterotypic fibrils with type I collagen and accounts for 10% to 20% of corneal collagen. The purpose of this study was to define the ocular phenotype resulting from mutations in the type V collagen genes COL5A1 and COL5A2 and to study the pathogenesis of anomalies in a Col5a1-deficient mouse. METHODS: Seven patients with classic Ehlers-Danlos syndrome (EDS) due to COL5A1 haploinsufficiency and one with an exon-skipping mutation in COL5A2 underwent an ocular examination, corneal topography, pachymetry, and specular microscopy. A Col5a1-haploinsufficient mouse model of classic EDS was used for biochemical and immunochemical analyses of corneas. Light and electron microscopy were used to quantify stromal thickness, fibril density, fibril structure, and diameter. RESULTS: Five males and three females (mean age, 26 +/- 13.57 years; range, 11-52) were studied. All patients had "floppy eyelids." The corneas of all eyes were thinner (mean corneal thickness: 435.75 +/- 12.51 microm) when compared with control corneas (568.89 +/- 28.46 microm; P < 0.0001). In the Col5a1+/- mouse cornea, type V collagen content was reduced by approximately 49%, and stromal thickness was reduced by approximately 26%. Total collagen deposition in Col5a1(+/-) corneas also was reduced. Collagen fibril diameters were increased, but fibril density was decreased throughout the stroma at all developmental stages. CONCLUSIONS: In the eye, COL5A1 and COL5A2 mutations manifest as abnormally thin and steep corneas with floppy eyelids. Mechanisms involved in producing the latter anomalies probably involve altered regulation of collagen fibrillogenesis due to abnormalities in heterotypic type I/V collagen interactions similar to those observed in the Col5a1+/- mouse cornea.

Adolescent↗

PubMed2006

Effect of latanoprost on the expression of matrix metalloproteinases and their tissue inhibitors in human trabecular meshwork cells.

PURPOSE: To determine the effect of latanoprost on the expression of human matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in the trabecular meshwork (TM). METHODS: Total RNA was isolated, and qualitative RT-PCR was performed to detect the mRNA of MMPs and TIMPs in human TM tissue and explant cultures of TM endothelial cells. Cultures of TM cells were treated with vehicle control or latanoprost acid for 24 hours. Real-time RT-PCR of cell cultures from five different donors was performed to determine relative changes in expression. GAPDH served as an endogenous control. RESULTS: The mRNA of MMP-1, -2, -3, -11, -12, -14, -15, -16, -17, -19, and -24 and of TIMP-1 to -4 was present in TM tissue and cultures of TM cells. MMP-9 was not found. In control TM endothelial cells, the relative expression of MMP mRNA were MMP-2 and -14 > MMP-16, -19, and -24 > MMP-15 > MMP-11 and -17 > MMP-1 and -3 > MMP-12. The relative expressions of TIMP mRNA were TIMP-1 > TIMP-2 and -3 > TIMP-4. Latanoprost increased MMP-1 (in four of five cultures), MMP-3 (in four of five cultures), MMP-17 (in three of five cultures), MMP-24 (in all five cultures), TIMP-2, -3, and -4 expression (in three of five cultures); MMP-11 and -15 were downregulated. CONCLUSIONS: Contrary to the expected result, latanoprost seems to have a significant effect on TM cells. The transcription of the genes for MMP-1, -3, -17, and -24 is increased by latanoprost treatment. TIMP-2, -3, and -4 are also upregulated. The upregulation of these TIMPs may compensate for the increase of those MMPs. The absence of MMP-9 and concurrent upregulation of a greater number of TIMPs may explain the limited effect of latanoprost on TM outflow.

Adult↗

PubMed2006

Regulation of procollagen amino-propeptide processing during mouse embryogenesis by specialization of homologous ADAMTS proteases: insights on collagen biosynthesis and dermatosparaxis.

Mutations in ADAMTS2, a procollagen amino-propeptidase, cause severe skin fragility, designated as dermatosparaxis in animals, and a subtype of the Ehlers-Danlos syndrome (dermatosparactic type or VIIC) in humans. Not all collagen-rich tissues are affected to the same degree, which suggests compensation by the ADAMTS2 homologs ADAMTS3 and ADAMTS14. In situ hybridization of Adamts2, Adamts3 and Adamts14, and of the genes encoding the major fibrillar collagens, Col1a1, Col2a1 and Col3a1, during mouse embryogenesis, demonstrated distinct tissue-specific, overlapping expression patterns of the protease and substrate genes. Adamts3, but not Adamts2 or Adamts14, was co-expressed with Col2a1 in cartilage throughout development, and with Col1a1 in bone and musculotendinous tissues. ADAMTS3 induced procollagen I processing in dermatosparactic fibroblasts, suggesting a role in procollagen I processing during musculoskeletal development. Adamts2, but not Adamts3 or Adamts14, was co-expressed with Col3a1 in many tissues including the lungs and aorta, and Adamts2(-/-) mice showed widespread defects in procollagen III processing. Adamts2(-/-) mice had abnormal lungs, characterized by a decreased parenchymal density. However, the aorta and collagen fibrils in the aortic wall appeared normal. Although Adamts14 lacked developmental tissue-specific expression, it was co-expressed with Adamts2 in mature dermis, which possibly explains the presence of some processed skin procollagen in dermatosparaxis. The data show how evolutionarily related proteases with similar substrate preferences may have distinct biological roles owing to tissue-specific gene expression, and provide insights into collagen biosynthesis and the pathobiology of dermatosparaxis.

ADAM Proteins↗

PubMed2006

Functional knockout of the matrilin-3 gene causes premature chondrocyte maturation to hypertrophy and increases bone mineral density and osteoarthritis.

Mutations in the gene encoding matrilin-3 (MATN3), a noncollagenous extracellular matrix protein, have been reported in a variety of skeletal diseases, including multiple epiphyseal dysplasia, which is characterized by irregular ossification of the epiphyses and early-onset osteoarthritis, spondylo-epimetaphyseal dysplasia, and idiopathic hand osteoarthritis. To assess the role of matrilin-3 in the pathogenesis of these diseases, we generated Matn3 functional knockout mice using embryonic stem cell technology. In the embryonic growth plate of the developing long bones, Matn3 null chondrocytes prematurely became prehypertrophic and hypertrophic, forming an expanded zone of hypertrophy. This expansion was attenuated during the perinatal period, and Matn3 homozygous null mice were viable and showed no gross skeletal malformations at birth. However, by 18 weeks of age, Matn3 null mice had a significantly higher total body bone mineral density than Matn1 null mice or wild-type littermates. Aged Matn3 null mice were much more predisposed to develop severe osteoarthritis than their wild-type littermates. Here, we show that matrilin-3 plays a role in modulating chondrocyte differentiation during embryonic development, in controlling bone mineral density in adulthood, and in preventing osteoarthritis during aging. The lack of Matn3 does not lead to postnatal chondrodysplasia but accounts for higher incidence of osteoarthritis.

Aging↗

PubMed2005

Keratocan, a cornea-specific keratan sulfate proteoglycan, is regulated by lumican.

Lumican is an extracellular matrix glycoprotein widely distributed in mammalian connective tissues. Corneal lumican modified with keratan sulfate constitutes one of the major proteoglycans of the stroma. Lumican-null mice exhibit altered collagen fibril organization and loss of corneal transparency. A closely related protein, keratocan, carries the remaining keratan sulfate of the cornea, but keratocan-null mice exhibit a less severe corneal phenotype. In the current study, we examined the effect of lumican overexpression in corneas of wild type mice. These mice showed no alteration in collagen organization or transparency but had increased keratocan expression at both protein and mRNA levels. Corneas of lumican-null mice showed decreased keratocan. This coupling of keratocan expression with lumican also was observed after intrastromal injection of a lumican expression minigene into the corneal stroma of Lum-/- mice. Small interfering RNA knockdown of lumican in vitro reduced keratocan expression, whereas co-injection of a lumican-expressing minigene with a beta-galactosidase reporter driven by the keratocan promoter demonstrated an increase of keratocan transcriptional activity in response to lumican expression in Lum-/- corneas in vivo. These observations demonstrate that lumican has a novel regulatory role in keratocan expression at the transcriptional level. Such results help provide an explanation for the differences in severity of corneal manifestation found in Lum-/- and Kera-/- mice. The results also suggest a critical level of small proteoglycans to be essential for collagen organization but that overabundance is not detrimental to extracellular matrix morphogenesis.

Animals↗

PubMed2005

Influence of decorin and biglycan on mechanical properties of multiple tendons in knockout mice.

Evaluations of tendon mechanical behavior based on biochemical and structural arrangement have implications for designing tendon specific treatment modalities or replacement strategies. In addition to the well studied type I collagen, other important constituents of tendon are the small proteoglycans (PGs). PGs have been shown to vary in concentration within differently loaded areas of tendon, implicating them in specific tendon function. This study measured the mechanical properties of multiple tendon tissues from normal mice and from mice with knock-outs of the PGs decorin or biglycan. Tail tendon fascicles, patellar tendons (PT), and flexor digitorum longus tendons (FDL), three tissues representing different in vivo loading environments, were characterized from the three groups of mice. It was hypothesized that the absence of decorin or biglycan would have individual effects on each type of tendon tissue. Surprisingly, no change in mechanical properties was observed for the tail tendon fascicles due to the PG knockouts. The loss of decorin affected the PT causing an increase in modulus and stress relaxation, but had little effect on the FDL. Conversely, the loss of biglycan did not significantly affect the PT, but caused a reduction in both the maximum stress and modulus of the FDL. These results give mechanical support to previous biochemical data that tendons likely are uniquely tailored to their specific location and function. Variances such as those presented here need to be further characterized and taken into account when designing therapies or replacements for any one particular tendon.

Animals↗

PubMed2005

Targeted ablation of the abcc6 gene results in ectopic mineralization of connective tissues.

Pseudoxanthoma elasticum (PXE), characterized by connective tissue mineralization of the skin, eyes, and cardiovascular system, is caused by mutations in the ABCC6 gene. ABCC6 encodes multidrug resistance-associated protein 6 (MRP6), which is expressed primarily in the liver and kidneys. Mechanisms producing ectopic mineralization as a result of these mutations remain unclear. To elucidate this complex disease, a transgenic mouse was generated by targeted ablation of the mouse Abcc6 gene. Abcc6 null mice were negative for Mrp6 expression in the liver, and complete necropsies revealed profound mineralization of several tissues, including skin, arterial blood vessels, and retina, while heterozygous animals were indistinguishable from the wild-type mice. Particularly striking was the mineralization of vibrissae, as confirmed by von Kossa and alizarin red stains. Electron microscopy revealed mineralization affecting both elastic structures and collagen fibers. Mineralization of vibrissae was noted as early as 5 weeks of age and was progressive with age in Abcc6(-/-) mice but was not observed in Abcc6(+/-) or Abcc6(+/+) mice up to 2 years of age. A total body computerized tomography scan of Abcc6(-/-) mice revealed mineralization in skin and subcutaneous tissue as well as in the kidneys. These data demonstrate aberrant mineralization of soft tissues in PXE-affected organs, and, consequently, these mice recapitulate features of this complex disease.

ATP-Binding Cassette Transporters↗

PubMed2004

Single amino acid substitutions in procollagen VII affect early stages of assembly of anchoring fibrils.

Procollagen VII is a homotrimer of 350-kDa pro-alpha1(VII) chains, each consisting of a central collagenous domain flanked by the noncollagenous N-terminal NC1 domain and the C-terminal NC2 domain. After secretion from cells, procollagen VII molecules form anti-parallel dimers with a C-terminal 60-nm overlap. Characteristic alignment of procollagen VII monomers forming a dimer depends on site-specific binding between the NC2 domain and the triple-helical region adjacent to Cys-2634 of the interacting procollagen VII molecules. Formation of the intermolecular disulfide bonds between Cys-2634 and either Cys-2802 or Cys-2804 is promoted by the cleavage of the NC2 domain by procollagen C-proteinase. By employing recombinant procollagen VII variants harboring G2575R, R2622Q, or G2623C substitutions previously disclosed in patients with dystrophic epidermolysis bullosa, we studied how these amino acid substitutions affect intermolecular interactions. Binding assays utilizing an optical biosensor demonstrated that the G2575R substitution increased affinity between mutant molecules. In contrast, homotypic binding between the R2622Q or G2623C molecules was not detected. In addition, kinetics of heterotypic binding of all analyzed mutants to wild type collagen VII were different from those for binding between wild type molecules. Moreover, solid-state binding assays demonstrated that R2622Q and G2623C substitutions prevent formation of stable assemblies of procollagen C-proteinase-processed mutants. These results indicate that single amino acid substitutions in procollagen VII alter its self-assembly and provide a basis for understanding the pathomechanisms leading from mutations in the COL7A1 gene to fragility of the dermal-epidermal junction seen in patients with dystrophic forms of epidermolysis bullosa.

Amino Acid Substitution↗

PubMed2004

The fibril-associated collagen IX provides a novel mechanism for cell adhesion to cartilaginous matrix.

Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely alpha1beta1, alpha2beta1, alpha10beta1, and alpha11beta1, are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express alpha1beta1, alpha2beta1, or alpha10beta1 integrin as their only collagen receptor, showed fast attachment and spreading on human recombinant collagen IX indicating that it is an effective cell adhesion protein. To further study the recognition of collagen IX we produced recombinant alphaI domains in Escherichia coli. For each of the four alphaI domains, collagen IX was among the best collagenous ligands, making collagen IX exceptional compared with all other collagen subtypes tested so far. Rotary shadowing electron microscopy images of both alpha1I- and alpha2I-collagen IX complexes unveiled only one binding site located in the COL3 domain close to the kink between it and the COL2 domain. The recognition of collagen IX by alpha2I was considered to represent a novel mechanism for two reasons. First, collagen IX has no GFOGER motif, and the identified binding region lacks any similar sequences. Second, the alpha2I domain mutations D219R and H258V, which both decreased binding to collagen I and GFOGER, had very different effects on its binding to collagen IX. D219R had no effect, and H258V prevented type IX binding. Thus, our results indicate that collagen IX has unique cell adhesion properties when compared with other collagens, and it provides a novel mechanism for cell adhesion to cartilaginous matrix.

Amino Acid Sequence↗

PubMed2004

Type V collagen controls the initiation of collagen fibril assembly.

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-alpha1(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.

Animals↗

PubMed2004

Reduced type I collagen utilization: a pathogenic mechanism in COL5A1 haplo-insufficient Ehlers-Danlos syndrome.

To examine mechanisms by which reduced type V collagen causes weakened connective tissues in the Ehlers-Danlos syndrome (EDS), we examined matrix deposition and collagen fibril morphology in long-term dermal fibroblast cultures. EDS cells with COL5A1 haplo-insufficiency deposited less than one-half of hydroxyproline as collagen compared to control fibroblasts, though total collagen synthesis rates are near-normal because type V collagen represents a small fraction of collagen synthesized. Cells from patients with osteogenesis imperfecta (OI) and haplo-insufficiency for proalpha1(I) chains of type I collagen also incorporated about one-half the collagen as controls, but this amount was proportional to their reduced rates of total collagen synthesis. Collagen fibril diameter was inversely proportional to type V/type I collagen ratios (EDS > control > OI). However, a reduction of type V collagen, in the EDS derived cells, was associated with the assembly of significantly fewer fibrils compared to control and OI cells. These data indicate that in cell culture, the quantity of collagen fibrils deposited in matrix is highly sensitive to reduction in type V collagen, far out of proportion to type V collagen's contribution to collagen mass.

Cell Culture Techniques↗

PubMed2004

Position of single amino acid substitutions in the collagen triple helix determines their effect on structure of collagen fibrils.

Collagen II fibrils are a critical structural component of the extracellular matrix of cartilage providing the tissue with its unique biomechanical properties. The self-assembly of collagen molecules into fibrils is a spontaneous process that depends on site-specific binding between specific domains belonging to interacting molecules. These interactions can be altered by mutations in the COL2A1 gene found in patients with a variety of heritable cartilage disorders known as chondrodysplasias. Employing recombinant procollagen II, we studied the effects of R75C or R789C mutations on fibril formation. We determined that both R75C and R789C mutants were incorporated into collagen assemblies. The effects of the R75C and R789C substitutions on fibril formation differed significantly. The R75C substitution located in the thermolabile region of collagen II had no major effect on the fibril formation process or the morphology of fibrils. In contrast, the R789C substitution located in the thermostable region of collagen II caused profound changes in the morphology of collagen assemblies. These results provide a basis for identifying pathways leading from single amino acid substitutions in collagen II to changes in the structure of individual fibrils and in the organization of collagenous matrices.

Binding Sites↗