Collagen V haploinsufficiency in a murine model of classic Ehlers-Danlos syndrome is associated with deficient structural and mechanical healing in tendons.

Classic Ehlers-Danlos syndrome (EDS) patients suffer from connective tissue hyperelasticity, joint instability, skin hyperextensibility, tissue fragility, and poor wound healing due to heterozygous mutations in COL5a1 or COL5a2 genes. This study investigated the roles of collagen V in establishing structure and function in uninjured patellar tendons as well as in the injury response using a Col5a1+/- mouse, a model for classic EDS. These analyses were done comparing tendons from a classic EDS model (Col5a1+/- ) with wild-type controls. Tendons were subjected to mechanical testing, histological, and fibril analysis before injury as well as 3 and 6 weeks after injury. We found that Col5a1+/- tendons demonstrated diminished recovery of mechanical competency after injury as compared to normal wild-type tendons, which recovered their pre-injury values by 6 weeks post injury. Additionally, the Col5a1+/- tendons demonstrated altered fibril morphology and diameter distributions compared to the wild-type tendons. This study indicates that collagen V plays an important role in regulating collagen fibrillogenesis and the associated recovery of mechanical integrity in tendons after injury. In addition, the dysregulation with decreased collagen V expression in EDS is associated with a diminished injury response. The results presented herein have the potential to direct future targeted therapeutics for classic EDS patients. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2707-2715, 2017.

Abdominal wall hernias-A local manifestation of systemically impaired quality of the extracellular matrix.

BACKGROUND: Throughout life, inguinal hernia develops in approximately every fourth man, some of whom develop multiple hernias. If patients at risk of developing multiple hernias could be identified by a serologic biomarker, treatment might be able to be tailored and improved. Evidence suggests that abdominal wall hernia formation is associated with altered collagen metabolism. The aim of this study was to evaluate biomarkers for type IV and V collagen turnover in patients with multiple hernias and control subjects without hernia. METHODS: Venous blood was collected from 88 men (mean age, 62 years) with a history of more than 3 hernia repairs and 86, age-matched men without hernias. Biomarkers for synthesis of collagen type IV (P4NP) and type V (P5CP) as well as breakdown (C4M and C5M) were measured in serum by validated, solid-phase, competitive assays. Collagen turnover was indicated by the ratio between the biomarker for synthesis and breakdown. RESULTS: Type IV collagen turnover was 1.4-fold increased in patients with multiple hernias compared to control subjects (P < .001), whereas type V collagen turnover was 1.7-fold decreased (P < .001). Diagnostic power of P5CP was 0.83 (95%C.I.:0.77-0.89), P < .001. CONCLUSION: Patients with multiple hernias exhibit increased turnover of type IV collagen and a decreased turnover of type V collagen, demonstrating systemically altered collagen turnover. Biomarkers for type V collagen turnover may be used to identify patients at risk for or with multiple hernias.

Regulatory role of collagen V in establishing mechanical properties of tendons and ligaments is tissue dependent.

Patients with classic (type I) Ehlers-Danlos syndrome (EDS), characterized by heterozygous mutations in the Col5a1 and Col5a2 genes, exhibit connective tissue hyperelasticity and recurrent joint dislocations, indicating a potential regulatory role for collagen V in joint stabilizing soft tissues. This study asked whether the contribution of collagen V to the establishment of mechanical properties is tissue dependent. We mechanically tested four different tissues from wild type and targeted collagen V-null mice: the flexor digitorum longus (FDL) tendon, Achilles tendon (ACH), the anterior cruciate ligament (ACL), and the supraspinatus tendon (SST). Area was significantly reduced in the Col5a1(ΔTen/ΔTen) group in the FDL, ACH, and SST. Maximum load and stiffness were reduced in the Col5a1(ΔTen/ΔTen) group for all tissues. However, insertion site and midsubstance modulus were reduced only for the ACL and SST. This study provides evidence that the regulatory role of collagen V in extracellular matrix assembly is tissue dependent and that joint instability in classic EDS may be caused in part by insufficient mechanical properties of the tendons and ligaments surrounding each joint.

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.

Increased type I and V collagen expression in uterine leiomyomas during the menstrual cycle.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the ratios of type I and type V collagen expression were significantly increased in the leiomyoma tissues at the protein level, as compared with those in the normal myometrium tissues through the menstrual cycle. These results suggest that increased expression of type I and type V collagen might play a role in the pathogenesis of uterine leiomyoma.

Interleukin-17-dependent autoimmunity to collagen type V in atherosclerosis.

RATIONALE: Considerable evidence shows atherosclerosis to be a chronic inflammatory disease in which immunity to self-antigens contributes to disease progression. We recently identified the collagen type V [col(V)] α1(V) chain as a key autoantigen driving the Th17-dependent cellular immunity underlying another chronic inflammatory disease, obliterative bronchiolitis. Because specific induction of α1(V) chains has previously been reported in human atheromas, we postulated involvement of col(V) autoimmunity in atherosclerosis. OBJECTIVE: To determine whether col(V) autoimmunity may be involved in the pathogenesis of atherosclerosis. METHODS AND RESULTS: Here, we demonstrate Th17-dependent anti-col(V) immunity to be characteristic of atherosclerosis in human coronary artery disease (CAD) patients and in apolipoprotein E-null (ApoE(-/-)) atherosclerotic mice. Responses were α1(V)-specific in CAD with variable Th1 pathway involvement. In early atherosclerosis in ApoE(-/-) mice, anti-col(V) immunity was tempered by an interleukin (IL)-10-dependent mechanism. In support of a causal role for col(V) autoimmunity in the pathogenesis of atherosclerosis, col(V) sensitization of ApoE(-/-) mice on a regular chow diet overcame IL-10-mediated inhibition of col(V) autoimmunity, leading to increased atherosclerotic burden in these mice and local accumulation of IL-17-producing cells, particularly in the col(V)-rich adventitia subjacent to the atheromas. CONCLUSIONS: These findings establish col(V) as an autoantigen in human CAD and show col(V) autoimmunity to be a consistent feature in atherosclerosis in humans and mice. Furthermore, data are consistent with a causative role for col(V) in the pathogenesis of atherosclerosis.

Reflux-induced collagen type v sensitization: potential mediator of bronchiolitis obliterans syndrome.

BACKGROUND: Lung transplantation continues to have poor long-term survival partly because of the high incidence of bronchiolitis obliterans syndrome (BOS). Gastroesophageal reflux disease (GERD) has been implicated in BOS pathogenesis. We investigated the role of collagen type V [col(V)] sensitization in this process. METHODS: Only primary lung transplant recipients were included. Reflux status was assessed with pH monitoring, impedance plethysmography, and esophagogastroduodenoscopy. Sensitivity to col(V) was determined with trans vivo delayed-type hypersensitivity reaction (DTH). Kaplan-Meier analyses were performed. RESULTS: Of the 54 recipients, 26 had proven GERD. There were no significant between-group differences in diagnosis; donor and recipient age; sex; ischemic time; single vs bilateral; human leukocyte antigen A, B, and DR matching cytomegalovirus status; acute rejections; or mean follow-up period. The mean DTH response in the GERD group was 25.7 x 10(-4) inches vs 18.3 x 10(-4) inches in the non-GERD group (P = .023). There was a significant reduction in BOS-free survival in the GERD group for both BOS-I (GERD+, 28.3%; GERD-, 86.6%; P = .0001) and BOS-II/III (GERD+, 66.2%; GERD-, 91.7%; P = .0374). A second cohort of 53 patients awaiting lung transplantation also was assayed. The mean DTH response in the GERD group was 24.0 x 10(-4) inches vs 13.1 x 10(-4) inches in the non-GERD group (P = .003). There were no differences in age or sex. CONCLUSIONS: GERD is strongly associated with the development of BOS after primary lung transplantation. Col(V) sensitization is associated with reflux and BOS and may play an intermediary role in the pathogenesis of BOS. Trials using col(V) reactivity to assess the impact of antireflux procedures in patients with lung transplantation and idiopathic pulmonary fibrosis are warranted.

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.

[Functional histology of dermis]. / Histologie fonctionnelle du derme.

The skin is composed of epidermis, dermis and subcutaneous tissue that interconnect anatomically. The dermis is an integrated system of fibrous and amorphous connective tissue that accommodates nerve and vascular networks, epidermally derived appendages, fibroblasts, macrophages and mast cells. Elastic and collagen tissue are the main types of fibrous connective tissue. The elastic connective tissue is assembled in a continuous network including mature elastic fibers, immature elaunin fibers and oxytalan fibers. Mature elastic fibers and elaunin have microfibrillar and amorphous matrix components while oxytalan fibers only contain microfibrils. Several molecules have been identified as constituents of the elastic fibers. Among the most characterized of these molecules is elastin in amorphous matrix, fibrillins 1 and 2 and LTBP-2 (ligand of latent TGFbeta) in microfibrils and fibulins which interconnect elastin and fibrillins. Elastic fibers provides elasticity to the skin. Under electron microscope, collagen fibers appears as of bundles of periodically banded fibrils which are composed of collagens types I, III and V; type V collagen is believed to assist in regulating fibril diameter. They are associated with FACITs (fibril-associated collagen with interrupted triple helixes) collagens types XIV et XVI. Collagen fibers provide tensile strength to the skin. Non fibrous connective tissue molecules include finely filamentous glycoproteins, glycosaminoglycans and proteoglycans of "the ground substance" (hyaluronic acid and chondroitin sulphate, dermatan sulphate, versican, decorin). Fibroblasts, macrophages and mast cells are regular residents of the dermis. The main function of these cells are well known. Fibroblasts are responsible for the synthesis and the degradation of fibrous and non fibrous connective tissue matrix proteins. Macrophages are phagocytic; they process and present antigen to immunocompetent lymphoid cells. Mast cells are responsible for IgE mediated acute, subacute and chronic inflammation. All these cells have a long list of other functions, in particular they are involved in coagulation, wound healing and tissue remodeling.

Collagen type V enhances matrix contraction by human periodontal ligament fibroblasts seeded in three-dimensional collagen gels.

Extracellular matrix components play an important role in modulating cellular activity. To study such capacities of the matrix, fibroblasts are frequently cultured in a three-dimensional gel and contraction is assessed as a measure of cellular activity. Since a connective tissue contains several types of collagen, we investigated the effect of gels composed of collagen I alone or in combination with 10% collagen III and/or 5% collagen V on contraction by human periodontal ligament fibroblasts. Gels containing collagen V contracted much faster than those without this type of collagen. Blocking of the integrin beta1-subunit with an activity-blocking antibody delayed (gels with collagen V) or almost completely blocked (gels without collagen V) contraction. Use of an antibody directed against integrin alpha2beta1 resulted in delay of gel contraction for gels both with and without collagen V. Anti-integrin alpha v beta3 or RGD peptides partially blocked contraction of gels containing collagen V, but had no effect on gels consisting of collagen I alone. The beta1-containing integrins are involved in the basal contraction by fibroblasts that bind to collagens I and III. The enhanced contraction, stimulated by collagen V, appears to be mediated by integrin alpha v beta3. We conclude that collagen V may play an important modulating role in connective tissue contraction. Such a modulation may occur during the initial stages of wound healing and/or tissue regeneration.

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.

Glypican-1 and alpha4(V) collagen are required for Schwann cell myelination.

Schwann cell myelination requires interactions with the extracellular matrix (ECM) mediated by cell surface receptors. Previously, we identified a type V collagen family member, alpha4(V) collagen, which is expressed by Schwann cells during peripheral nerve differentiation. This collagen binds with high affinity to heparan sulfate through a unique binding motif in the noncollagenous N-terminal domain (NTD). The principal alpha4(V) collagen-binding protein on the Schwann cell surface is the heparan sulfate proteoglycan glypican-1. We investigated the role of alpha4(V) collagen and glypican-1 in Schwann cell terminal differentiation in cultures of Schwann cells and dorsal root ganglion neurons. Small interfering RNA-mediated suppression of glypican-1 expression decreased binding of alpha4(V)-NTD to Schwann cells, adhesion and spreading of Schwann cells on alpha4(V)-NTD, and incorporation of alpha4(V) collagen into Schwann cell ECM. In cocultures, alpha4(V) collagen coassembles with laminin on the surface of polarized Schwann cells to form tube-like ECM structures that are sites of myelination. Suppression of glypican-1 or alpha4(V) collagen expression significantly inhibited myelination. These results demonstrate an important role for these proteins in peripheral nerve terminal differentiation.

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.

Role of 12-lipoxygenase in the stimulation of p38 mitogen-activated protein kinase and collagen alpha5(IV) in experimental diabetic nephropathy and in glucose-stimulated podocytes.

The 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism is implicated in extracellular matrix (ECM) synthesis, but its role in podocytes has not been studied. This study tested whether 12-LO induction by diabetes or by high glucose (HG) in cultured podocytes alters glomerular basement membrane by activating signal transduction pathways culminating in ECM synthesis. Sprague-Dawley rats received an injection of diluent (control [C]) or streptozotocin 65 mg/kg (DM) and were killed at 1 or 4 mo. Glomerular 12-LO mRNA and protein levels were higher in DM than in C glomeruli at 1 and 4 mo, and 12-LO localized predominantly in podocytes. Glomerular p38 mRNA and protein were higher in DM at months 1 and 4, but phospho-p38 mitogen-activated protein (MAPK) was increased only at month 1. Glomerular collagen alpha5(IV)/glutaraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratio was increased in DM at month 1 but not at month 4, whereas collagen alpha5(IV) protein was higher at both 1 and 4 mo. Mouse podocytes were cultured in media with 25 mM glucose (HG) with or without the 12-LO inhibitor cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC) or with 5.5 mM glucose + 19.5 mM mannitol (low glucose [LG+M]) for 10 d at 37 degrees C. 12-LO mRNA and protein levels were higher in HG than in LG+M as was the p38 MAPK/GAPDH mRNA ratio. Phospho-p38 MAPK protein but not total p38 MAPK was higher in HG compared with LG+M. Collagen alpha5(IV)/GAPDH mRNA ratio and protein were higher in HG than in LG+M. 12-LO inhibition by CDC decreased HG-induced phospho-p38 MAPK and the phospho-p38/total p38 MAPK ratio, collagen alpha5(IV)/GAPDH mRNA ratio, and collagen alpha5(IV) protein expression. In summary, diabetes in vivo and exposure of podocytes to HG in vitro stimulated 12-LO, p38 MAPK, and collagen alpha5(IV) mRNA and (activated) protein. 12-LO inhibition by CDC diminished the expression of podocyte phospho-p38 MAPK and collagen alpha5(IV) mRNA and protein. These findings implicate 12-LO and the p38 MAPK signaling pathway in the mediation of ECM synthesis by podocytes in diabetes.

[Molecular basis of the classical type of Ehlers-Danlos syndrome]. / Molekularne podloze zespolu Ehlersa-Danlosa typu klasycznego.

Classical type of Ehlers-Danlos syndrome (EDS) is the most common variant of the disease, however, its molecular background is still unknown. The paper presents the latest findings concerning this type of EDS. In most cases, mutations in genes encoding type V collagen were found. In some cases, the mutations were excluded indicating the genetic heterogeneity of the disease.