Targeted Deletion of Collagen V in Tendons and Ligaments Results in a Classic Ehlers-Danlos Syndrome Joint Phenotype

Collagen V mutations underlie classic Ehlers-Danlos syndrome, and joint hypermobility is an important clinical manifestation. We define the function of collagen V in tendons and ligaments, as well as the role of alterations in collagen V expression in the pathobiology in classic Ehlers-Danlos syndrome. A conditional Col5a1^flox/flox mouse model was bred with Scleraxis-Cre mice to create a targeted tendon and ligament Col5a1-null mouse model, Col5a1^Δten/Δten. Targeting was specific, resulting in collagen V–null tendons and ligaments. Col5a1^Δten/Δten mice demonstrated decreased body size, grip weakness, abnormal gait, joint laxity, and early-onset osteoarthritis. These gross changes were associated with abnormal fiber organization, as well as altered collagen fibril structure with increased fibril diameters and decreased fibril number that was more severe in a major joint stabilizing ligament, the anterior cruciate ligament (ACL), than in the flexor digitorum longus tendon. The ACL also had a higher collagen V content than did the flexor digitorum longus tendon. The collagen V–null ACL and flexor digitorum longus tendon both had significant alterations in mechanical properties, with ACL exhibiting more severe changes. The data demonstrate critical differential regulatory roles for collagen V in tendon and ligament structure and function and suggest that collagen V regulatory dysfunction is associated with an abnormal joint phenotype, similar to the hypermobility phenotype in classic Ehlers-Danlos syndrome.Ehlers-Danlos syndrome (EDS) is a hereditary connective tissue disorder characterized by joint hypermobility, skin extensibility, and connective tissue fragility.^1–5 The combined prevalence of all types of EDS is approximately 1 in 5000. More than half of these cases are characterized by joint hypermobility,⁶ and hypermobility is one of the major diagnostic criteria for the classic subtype of EDS. Joint hypermobility can cause chronic joint and limb pain, recurring joint dislocation, and sports injuries with potential degenerative complications, including muscle weakness, precocious osteoarthritis, spondylosis, and lower bone mass.^6–9In more than 90% of patients with classic EDS, collagen V mutations have been identified,^3,10 and approximately half are null-allele mutations resulting in COL5A1 haploinsufficiency.^10–12 Besides the null-allele mutations, mutations scattered throughout COL5A1 and COL5A2 genes and some mRNA splicing mutations in COL5A1 also have been identified.¹⁰ This finding has led to the proposal that classic EDS is a collagen V disease resulting from altered collagen V expression.¹⁰Collagen V is a quantitatively minor, regulatory component in collagen I–rich connective tissues, including dermis, tendons and ligaments, bones, blood vessels, and cornea.¹³ Collagen V content relative to collagen I varies from a high of 10% to 20% in cornea to 2% to 5% of the total fibril-forming collagens in most other tissues.^14,15 Collagen V regulates collagen fibrillogenesis by nucleating fibril assembly in in vitro self-assembly assays, cell culture analyses, and mouse models.^14,16–19 The data support a model whereby the collagen V:I ratio in different tissues determines the initial diameter and number of fibrils assembled. By varying the number of collagen V nucleation sites for a given collagen I concentration, the fibroblast can regulate fibril number and diameter in a tissue-specific manner, ie, more sites result in increased fibrils assembled with smaller diameters.^13,17Several mouse models have been established to elucidate the function of collagen V tissue-specific fibril assembly. A traditional homozygous deletion in the Col5a1 gene is embryonic lethal because of a virtual lack of fibril formation at the beginning of organogenesis in the early embryo, although the Col5a1^−/− mice synthesize and secrete collagen I at a level comparable with that of wild-type controls.¹⁸ This model demonstrates that collagen V is essential for the assembly of collagen I–containing fibrils in the low-collagen-concentration environment of the embryo and is consistent with a critical role in nucleation of fibril assembly. The heterozygous Col5a1^+/− mice demonstrate haploinsufficiency, with approximately 50% of wild-type collagen V expression. Col5a1^+/− mice are excellent models of classic EDS.²⁰ There were two subpopulations of fibrils in the mutant dermis. One had increased diameters and normal fibril structure, and these were immunoreactive for collagen V; the second group had very large diameters with aberrant fibril structure and were negative for collagen V reactivity. This suggests relatively normal assembly when nucleated with collagen, but the expression level of collagen V was insufficient to nucleate all available collagen I and, therefore, dysfunctional fibril assembly in the high-collagen-content environment. This abnormal fibril growth recapitulated that seen clinically in the dermis of EDS patients. In addition, the Col5a1^+/− flexor digitorum longus tendon (FDL) also demonstrates decreased cross-sectional area and stiffness compared with that in the wild-type controls,²¹ consistent with the joint hypermobility and dislocations seen in EDS patients.To overcome the embryonic lethal phenotype and permit analyses of the roles of collagen V in the development and maturation of a tissue-specific extracellular matrix, our group created a conditional collagen V–null mouse model by using a Cre/loxP approach.²² When targeted to the corneal stroma, a severe dysfunctional regulation of fibrillogenesis and corneal opacity was observed in the targeted collagen V–null mice. Unlike cornea, the tendon has a low collagen V content. The mature tendon contains uniaxial fibrils with a very heterogeneous population of fibril diameters, and the mechanical properties of the tendon depend on the increases in fibril diameter seen with development.²³Our aim was to explore specific regulatory roles for collagen V in tendons and ligaments, as well as its roles in the pathophysiology associated with joint hypermobility in classic EDS. Our conditional Col5a1^flox/flox mouse model was targeted to tendons and ligaments using Cre driven by a Scleraxis (Scx) promoter (Scx-Cre) to produce the Col5a1^Δten/Δten mouse model. Scx-Cre targets the deletion to tendons and ligaments. The data demonstrate that the absence of collagen V results in a disruption in tendon and ligament structure and function. In addition, there were consistent differences between the FDL tendon and the anterior cruciate ligament (ACL), indicating tissue-specific regulatory properties. The absence of collagen V also resulted in alterations in the joint consistent with the hypermobility seen in classic EDS.