Ehlers-Danlos syndrome type VI: collagen type specificity of defective lysyl hydroxylation in various tissues

The Ehlers-Danlos syndrome type VI is an inherited disorder of collagen metabolism characterized by a defective lysyl hydroxylase. The resulting lack of hydroxylysine has been found in several connective tissues, all of which show varying degrees of clinical symptoms. In the present study, collagen was isolated from different connective tissues and the degree of hydroxylation of lysyl residues was determined. Subsequently, collagen types I, II, III, IV, and V have been prepared from a number of tissues. Insufficient hydroxylation of lysyl residues was found in type I and type III collagen, whereas types II, IV, and V showed normal amounts of hydroxylysine. The expression of the defect, even for type I and type III collagen, varied widely from one tissue to another. A complete lack of hydroxylysine was observed in skin, while it was less pronounced in tissues such as bone, tendon, lung, or kidney. The data suggest the presence of several isoenzymes having varying affinities to the different collagen types.     

Contraction of collagen lattices by fibroblasts from patients and animals with heritable disorders of connective tissue

Fibroblasts derived from patients and animals presenting various heritable connective tissue disorders were investigated for the ability to retract a reconstituted collagen matrix. When seeded into gels, dermatosparactic calf and sheep fibroblasts did not exhibit the elongated shape of normal fibroblasts and did not contract the collagen lattice to the same extent as control fibroblasts. In contrast, several cell strains obtained from patients with Ehlers-Danlos syndrome type VII displayed contractile properties for collagen gels similar to controls. Delayed contraction was noted by two strains of fibroblasts from patients with Ehlers-Danlos syndrome type IV, whereas fibroblasts from patients with osteogenesis imperfecta, Marfan syndrome and cutis laxa had normal retraction properties.     

Effects of minoxidil on human skin fibroblasts in culture

Collagens are a family of extracellular proteins that have evolved to serve many specialized biologic functions. Fifteen collagen types with at least 18 genetically distinct polypeptide chains have been identified.¹ In skin, eight of these collagens make up a major portion of the extracellular matrix including specialized structures such as basement membrane (Type IV collagen) and anchoring fibrils (Type VII collagen). All collagens are synthesized as procollagen precursor molecules. In addition to synthesis and precise association and alignment of the three polypeptide chains, biosynthesis involves many posttranslational modifications.^2,3 These include synthesis of hydroxyproline catalyzed by prolyl hydroxylase and synthesis of hydroxylysine catalyzed by lysyl hydroxylase. Hydroxyproline is essential for maximum helical stability, and hydroxylysine participates in collagen cross-link formation as well as synthesis of specific glycosides catalyzed by collagen glycosyl transferases. Other enzymes incorporate into the procollagen molecule complex carbohydrate residues typical of glycoproteins. Following secretion from the cell, amino and carboxyterminal propeptides of the procollagen molecule are cleaved by separate enzymes. Certain lysyl and hydroxylysyl residues are then modified by lysyl oxidase to corresponding aldehydes, which interact with certain hydroxylysine residues to form intermodular cross-links.     

Collagen in current connective tissue research. Application of molecular biological methods to medical problems

Collagen plays an important role in connective tissue diseases. It is the most abundant protein of the human body and serves as a model protein in the study of a variety of diseases. At present, at least ten different genetically distinct collagen types are known and characterized to at least some extent. In patients with Ehlers-Danlos syndrome, Marfan syndrome and osteogenesis imperfecta, molecular defects in the type I collagen genes are already known. It is very likely that in the near future vast amounts of data about the collagens, collagen defects and their involvement in human pathophysiology will become available.     

Adenoviral gene transfer restores lysyl hydroxylase activity in type VI Ehlers-Danlos syndrome

Type VI Ehlers-Danlos syndrome is a disease characterized by disturbed lysine hydroxylation of collagen. The disease is caused by mutations in lysyl hydroxylase 1 gene and it affects several organs including the cardiovascular system, the joint and musculoskeletal system, and the skin. The skin of type VI Ehlers-Danlos syndrome patients is hyperelastic, scars easily, and heals slowly and poorly. We hypothesized that providing functional lysyl hydroxylase 1 gene to the fibroblasts in and around wounds in these patients would improve healing. In this study we tested the feasibility of transfer of the lysyl hydroxylase 1 gene into fibroblasts derived from rats and a type VI Ehlers-Danlos syndrome patient (in vitro) and into rat skin (in vivo). We first cloned human lysyl hydroxylase 1 cDNA into a recombinant adenoviral vector (Ad5RSV-LH). Transfection of human type VI Ehlers-Danlos syndrome fibroblasts (about 20% of normal lysyl hydroxylase 1 activity) with the vector increased lysyl hydroxylase 1 activity in these cells to near or greater levels than that of wild type, unaffected fibroblasts. The adenoviral vector successfully transfected rat fibroblasts producing both beta-galactosidase and lysyl hydroxylase 1 gene activity. We next expanded our studies to a rodent model. Intradermal injections of the vector to the abdominal skin of rats produced lysyl hydroxylase 1 mRNA and elevated lysyl hydroxylase 1 activity, in vivo. These data suggest the feasibility of gene replacement therapy to modify skin wound healing in type VI Ehlers-Danlos syndrome patients.     

Detection of type III collagen in skin fibroblasts from patients with Ehlers-Danlos syndrome type IV by immunofluorescence

Immunofluorescence showed that cultured skin fibroblasts from 15 out of 17 patients with Ehlers-Danlos syndrome type IV retained abnormal amounts of type III collagen within the cytoplasm. This was not shown by fibroblasts from normal subjects or from patients with other inherited connective tissue diseases. The diagnosis of Ehlers-Danlos syndrome type IV may be facilitated by this finding.     

Leopard syndrome associated with hyperelastic skin: analysis of collagen metabolism in cultured skin fibroblasts.

We present a patient with Leopard syndrome and hyperelastic skin. Biochemical analysis using cultured skin fibroblasts showed normal type III and V collagen synthesis, lysyl hydroxylation level of type I procollagen and processing of pro-alpha(1) and alpha(2)(I). Our results suggest that molecular defects of hyperelasticity in Leopard syndrome are not related to abnormal collagen metabolism, although not all steps of collagen synthesis have been investigated.     

Ehlers-Danlos-Syndrom

The Ehlers-Danlos syndrome (EDS) comprises a heterogenous group of nine hereditary connective tissue disorders, characterized by hyperelasticity of skin and hypermobility of joints to differing extents. The skin is easily injured and wound healing is delayed. The majority of EDS patients belong to EDS-types I-III. The pathogenesis in these cases is not known, although recent data suggest a role for collagen V. In contrast, the etiology of EDS-types IV, VI and VII has been found. While EDS IV is caused by a mutation in the collagen III gene, in EDS VI a mutation in the lysyl hydroxylase gene is present. In EDS VII, the underlying defect is a mutation in the collagen I gene. The EDS-types V, VII and X are very rare; their symptoms resemble those of EDS-type II.     

Overhydroxylation of lysyl residues is the initial step for altered collagen cross-links and fibril architecture in fibrotic skin.

In fibrotic skin of lipodermatosclerosis a substantial increase of the cross-link hydroxylysylpyridinoline is observed. Hydroxylysylpyridinoline is a typical cross-link of skeletal tissue and is thought to play a major part in the hardening of sclerotic tissue. We investigated whether the increase in hydroxylysylpyridinoline is due to overhydroxylation of lysyl residues in the collagen molecule, which may also be associated with an increase of glycosylated hydroxylysine residues. Furthermore, we determined whether the collagen fibrils in lipodermatosclerosis showed a decrease of the diameter in the tissue as well as in vitro after fibrillogenesis of pepsin-solubilized collagens. Isolated alpha-chains of pepsin solubilized collagen I showed an increase in lysyl hydroxylation (hyl/(hyl + lys)) as compared with normal control [alpha1(I): lipodermatosclerosis 0.18 +/- 0.01; control 0.12 +/- 0.01; alpha2(I): lipodermatosclerosis 0.36 +/- 0.02; control 0. 25 +/- 0.03, p < 0.001]. Furthermore, the content of enzymatic glycosylated hydroxlysine residues increased. This increase is associated with a decrease of fibril diameter of both tissue and fibrils formed in vitro of pepsin-solubilized collagens. In the same pool of collagens an increase in collagen III content was observed as compared with controls (lipodermatosclerosis 14.5% +/- 1.6, control 10.3% +/- 1.6, p < 0.001). Our results showed that the overhydroxylation of lysyl residues, which is required for the generation of hydroxylysylpyridinoline, is not only restricted to the telopeptides but also affects the helical part of the molecule. This process is further associated with an increase of glycosylated hydroxylysyl residues. These changes along with the increase in collagen III content seem to be responsible for the observed alteration in the architecture of collagen fibrils in sclerotic skin.     

In vivo evidence for a bridging role of a collagen V subtype at the epidermis-dermis interface

Collagen V is the defective product in most cases of classical Ehlers-Danlos syndrome (EDS), a connective tissue disorder typically characterized by skin fragility and abnormal wound healing. Collagen V assembles into diverse molecular forms. The predominant α1(V)(2)α2(V) heterotrimer controls fibrillogenesis in skin and other tissues. The α1(V)(3) minor form is thought to occur in skin, but its function is unknown. To elucidate its role, we generated transgenic mice that overexpress the human α1(V)(3) homotrimer in the epidermis. The transgene-derived product is deposited as thin unstriated fibrillar material in the basement membrane zone of embryonic and perinatal epidermis and hair follicles. Accumulation of α1(V)(3)-containing fibrils leads to ultrastructural modifications at the epidermis-dermis interface and provokes changes in biomechanical properties, although not statistically significant. Using superparamagnetic immunobeads to isolate authentic suprastructures and protein-binding assays, we demonstrate that the homotrimer is part of a protein network containing collagen IV, laminin-111, and the dermal collagen VI. Our data show that the homotrimer serves as a bridging molecule that contributes to the stabilization of the epidermal-dermal interface. This finding strongly suggests that collagen V may be expressed in skin as different subtypes with important but distinct roles in matrix organization and stability.     

Ultrastructural alterations of elastic fibers and other dermal components in ehlers-danlos syndrome of the hypermobile type

Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective tissue disorders with varied molecular abnormalities. Some morphologic alterations are described in collagen and elastic fibers. We report on previously undescribed ultrastructural changes in the elastic fibers found in the skin of 22 of 29 patients suffering from the hypermobile type of EDS. They existed in variable combinations of a fragmented appearance with frayed contours, internal microcavities, some elastotic-like changes, and discrete calcified foci. The collagen fibrils and their bundles were also altered with morphologic aspects reminiscent of those found in the classical type of EDS. Large globules of hyaluronic acid were also dispersed in the amorphous matrix.     

Collagen biosynthesis and isomorphism in a case of Ehlers-Danlos syndrome Type VI

Summary Collagen metabolism was studied in fibroblasts grown from a skin biopsy specimen of a patient who presented the striking clinical features of Ehlers-Danlos syndrome and, in particular, hyperextensibility of the skin, hypermobility of the joints, and kyphoscoliosis. A reduction in lysine hydroxylation, characteristic of Ehlers-Danlos Type VI, was observed after labelling of the collagen with ¹⁴C-proline and ³H-lysine. Other modifications in the collagen metabolism of fibroblast cultures were noted, including an increase in collagen and total protein synthesis, and an increase in both the Type I and Type III collagen. The percentage of Type III collagen was, however, lower than in the control fibroblasts. The results point out the complexity of collagen disturbances in Ehlers-Danlos Type VI.     

Analysis of the age-related composition of human skin collagen and collagens synthesized by fibroblast culture

Age-related differences in the composition and the post-translational modifications of human skin collagens were examined in the present study. The data were compared with results of collagen synthesis from in vivo-aged fibroblasts in culture. Skin extracts and newly synthesized collagen from fibroblast cultures derived from both old and young donor groups showed the same ratio of collagen III to collagen I. Furthermore, no difference was noted in the degree of prolyl and lysyl hydroxylation of collagen I and collagen III. Young and old fibroblasts synthesized a similar quantity of collagen in vitro. The data suggest that fibroblasts maintain a uniform level of collagen production, composition and modification independent of the age of the donor.     

Cutaneous metaplastic synovial cyst in Ehlers-Danlos syndrome: report of a second case

A cutaneous metaplastic synovial cyst is a rare entity that is probably caused by trauma or surgery. We report the second case of cutaneous metaplastic synovial cyst in a child with Ehlers-Danlos syndrome. His father is also affected with Ehlers-Danlos syndrome, and his diagnosis is substantiated by the demonstration of reduced synthesis of collagen type V.     

Prolyl and lysyl hydroxylases in collagen synthesis

Collagens are the most abundant proteins in the extracellular matrix. They provide a framework to build organs and tissues and give structural support to make them resistant to mechanical load and forces. Several intra‐ and extracellular modifications are needed to make functional collagen molecules, intracellular post‐translational modifications of proline and lysine residues having key roles in this. In this article, we provide a review on the enzymes responsible for the proline and lysine modifications, that is collagen prolyl 4‐hydroxylases, 3‐hydroxylases and lysyl hydroxylases, and discuss their biological functions and involvement in diseases.     

Painful Piezogenic Pedal Papules on a Child with Ehlers-Danlos Syndrome

A 5-year-old girl with Ehlers-Danlos syndrome developed painful piezogenic pedal papules. She had suffered from pain in her heels for the previous two years and had undergone extensive orthopedic examinations that revealed no abnormalities. On admission, she had typical signs associated with piezogenic pedal papules (PPP), with pain induced by standing and disappearance of the lesions and the pain on relief of pressure. These papules were due to herniation of subcutaneous fat into the dermis, possibly because of structural defects of the connective tissue. To the best of our knowledge, this is the first reported case of painful PPP occurring in childhood. We suggest that the herniation of subcutaneous fat in our patient was most probably due to the connective tissue defect that occurs in the Ehlers-Danlos syndrome.     

Phenytoin modulates connective tissue metabolism and cell proliferation in human skin fibroblast cultures

Phenytoin has been proposed for the treatment of certain dermatologic conditions involving connective tissue abnormalities. To understand the biochemical basis of connective tissue changes, we incubated human skin fibroblasts in culture with varying concentrations of phenytoin. The results indicated that fibroblast proliferation, detected by tritiated thymidine incorporation into cells, was slightly stimulated when short incubation periods and low concentrations of phenytoin were employed. However, with longer incubation times and higher phenytoin concentrations, a significant reduction in fibroblast proliferation was observed. Further studies demonstrated that incubation of cells with phenytoin did not affect the production of procollagen, measured as synthesis of radioactive hydroxyproline in the cultures. However, assay of prolyl hydroxylase, an enzyme participating in the post-translational synthesis of hydroxyproline during collagen biosynthesis, was significantly reduced in the fibroblast cultures. The activity of collagenase, an enzyme participating in degradation of collagen, was markedly decreased in cultures treated with phenytoin. Thus, phenytoin may modulate collagen metabolism primarily by affecting the degradation of collagen. The results support previous suggestions that phenytoin may be useful for treatment of patients with increased levels of collagenase, such as in recessive dystrophic epidermolysis bullosa.     

A case of Ehlers-Danlos syndrome occurring with Marfan's syndrome

The Ehlers-Danlos syndrome and the Marfan syndrome are well recognized inherited disorders of connective tissue. A case is reported of a young woman who showed the features of both Ehlers-Danlos and Marfan's syndrome; such a combination has previously been reported but is very rare.     

Biochemical and immunological studies of fibroblasts derived from a patient with Ehlers-Danlos Syndrome Type IV

Summary Fibroblasts derived from a skin biopsy of a patient with the Ehlers-Danlos syndrome (EDS) type IV were cultured in monolayer. The amount of collagen synthesized during a 24-h pulse was not different from that found with normal fibroblasts. Chromatographic procedures and immunofluorescence staining showed a normal synthesis of type I procollagen and collagen but a deficiency in synthesis of type III procollagen and collagen. This could be corroborated by radioimmuno assays showing a reduction in type III procollagen by about 90%. The secretion and degradation of collagens was not altered. The results demonstrate that the molecular defect in this particular patient is due to an impairment of the mechanism controlling the gene expression for type III procollagen.

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Abbreviations BAPN -aminopropionitrile - CM carboxy-methyl - DEAE diethylaminoethyl - EDS Ehlers-Danlos syndrome - EDTA ethylendiaminetetracetate - S.C. subcutaneous