Targeted deletion of collagen V in tendons and ligaments results in a classic Ehlers-Danlos syndrome joint phenotype

Abstract

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.

Figure 1

Targeted deletion of collagen V in flexor digitorum longus (FDL) tendons. A: Cre excision was targeted to tendons by using a Scleraxis promoter (Scx-Cre) in a double reporter (mTmG) mouse. Analysis of cross sections from postnatal day 4 (P4) limbs shows ubiquitous expression of red fluorescence (mT) in control mTmG mice. In contrast, Scx-Cre/mTmG mice show green fluorescence (mG) in the tendon, indicating targeted Cre excision. Other tissues show no Cre recombinase activity. DAPI (blue), nuclear localization. B: Col5a1 mRNA expression in the postnatal day 10 Col5a1^Δten/Δten FDL is at background levels at real-time PCR. In contrast, expression in Col5a1^flox/flox (Col5a1^f/f) control mice is comparable with that in the wild-type mice. C: Postnatal day 10 FDLs are collagen V null in Col5a1^Δten/Δten mice. Immunoblot analysis using antibodies against the alpha 1 chain of collagen V [a1(V)] and against β-actin as a loading control. Control Col5a1^flox/flox and wild-type mice express comparable collagen V. D: Collagen V immunoreactivity (red) is present in postnatal day 30 control (Ctrl) FDLs but absent in Col5a1^Δten/Δten mice. DAPI (blue), nuclear localization. Scale bars = 20 μm (A).

Figure 2

Targeted deletion of collagen V in anterior cruciate ligament (ACL) driven by Scleraxis-Cre (Scx-Cre). A: Posterior view of the knee joint of Scx-Cre/mTmG mouse at postnatal day 10 shows the ACL and posterior cruciate ligament (PCL) with green fluorescent protein expression (left panel). No green fluorescent protein is expressed in the mTmG control mouse knee joint (right panel). Arrows indicate the position of the ACL and PCL. The image was obtained with a fluorescence dissecting microscope. B: Immunofluorescence microscopy showed the depletion of collagen V expression in the Col5a1^Δten/Δten ACL at postnatal day 10 (right panel) compared to the controls (left panel). Green, collagen V; red, phalloidin; blue, DAPI stained nucleus. C: Immunoblotting shows no expression of collagen V in Col5a1^Δten/Δten ACLs. The Col5a1^flox/flox (Col5a1^f/f) mouse ACL expressed the same amount of collagen V as did that of wild-type C57BL/6 mice. Ctrl, control; double reporter, mTmG.

Figure 3

Joint laxity and aberrant gait in Col5a1^Δten/Δten mice. A and B:Col5a1^Δten/Δten mice with a targeted deletion of collagen V in tendons and ligaments show smaller size and deformation of the limbs in representative 8-month-old female mice (arrows indicate joint dislocation). C: Decreased body weight in Col5a1^Δten/Δten mice. Postnatal day 60 male wild-type (n = 8) and mutant mice (n = 8) mice were used. D: Forelimb grip strength measurement from the same group of mice as in C show weakness of Col5a1^Δten/Δten mice relative to control mice. E: Abnormal gait in Col5a1^Δten/Δten mice. Representative hindlimb prints of control and Col5a1^Δten/Δten mice. The control mice walk in a straight line with regular, even steps and wide toe spread. The Col5a1^Δten/Δten mice show short stride lengths (vertical bars), with small toe spread. F:Col5a1^Δten/Δten mice exhibit excess joint laxity compared with that in the control mice when the joint is overextended passively (arrows, ankle joint). ^∗∗∗P < 0.001. Ctrl, control.

Figure 4

Early-onset osteoarthritis in Col5a1^Δten/Δten mice. Representative histologic sections of tibial condyles in control and Col5a1^Δten/Δten mice at postnatal day 30. Decalcified knee joint embedded in OCT. Frontal sections of the knee joint were stained with Safranin O and Fast green. A: The cartilage surface in the control mouse is smooth. B: The lateral surface of the tibia condyles in Col5a1^Δten/Δten knee joint is covered with fibrous tissue and chronic inflammation cells. The cartilage erosion, chondrocyte death, and proteoglycan depletion indicate the onset of osteoarthritis in Col5a1^Δten/Δten knee joint.

Figure 5

Altered biomechanical properties in Col5a1^Δten/Δten flexor digitorum longus. Cross-sectional area, stiffness, and modulus were measured in postnatal day 60 (P60) male flexor digitorum longus tendons from Col5a1^Δten/Δten and Scx-Cre control mice. The significant decrease in cross-sectional area and stiffness in Col5a1^Δten/Δten FDLs is consistent with the joint laxity phenotype. The modulus is comparable in both genotypes. ^∗P < 0.05, ^∗∗P < 0.01.

Figure 6

Aberrant fiber structure and organization in Col5a1^Δten/Δten tendons during early tendon development. A and B: Postnatal day 4 control (Ctrl) mouse flexor digitorum longus (FDL) contained organized fibers (F) that are relatively uniform in size. The fibers were present in microdomains that were defined by tenocyte cytoplasmic processes (curved arrows). B and D: Collagen protofibrils can be seen during deposition (arrowhead). C and D:Col5a1^Δten/Δten mouse FDLs have less-organized fibers (F). In Col5a1^Δten/Δten FDLs, there are fewer fibers than in the wild-type controls. The collagen V–null FDLs have larger fibers with more heterogeneity in size than in control tendons. In addition, in the absence of collagen V, the fibers are less organized, ie, less regularly packed, with more space separating them than in the controls. In addition, the tenocyte processes (curved arrows) defining the microdomains are less organized than in the controls.

Figure 7

Abnormal fibril structure in mature Col5a1^Δten/Δten tendons. A and B: The Col5a1^Δten/Δten flexor digitorum longus (FDL) has larger and more heterogeneous fibrils than do the wild-type controls. In addition, fibrils assembled in collagen V–null tendons show aberrant structures (arrow). C: Histograms represent the distribution of fibril diameters in the FDL tendon of control and Col5a1^Δten/Δten mice. The Col5a1^Δten/Δten mice have broader distribution of the fibril diameters, with increased fibril numbers of both small-diameter and large-diameter fibrils. The diameter was measured in three different mice of each group, with seven images from each mouse. D: The mean fibril diameter is increased significantly in the FDL of Col5a1^Δten/Δten mice. E: The fibril density is decreased significantly in the FDL of Col5a1^Δten/Δten mice. The means ± SD, n = 21, and Student's t-test (D and E). All FDLs were from male mice at postnatal day 30. ^∗∗∗P < 0.001. Ctrl, control; Q, quartile.

Figure 8

High collagen V content in anterior cruciate ligament (ACL) versus flexor digitorum longus (FDL). Immunoblot analysis shows different collagen V expression levels in different tissues of postnatal day 30 wild-type C57BL/6 mice. Cornea expresses the most collagen V, followed by ACL, which is comparable with that in the skin but much higher than that in FDL and bone. β-Actin was used as the protein loading control.

Figure 9

Abnormal anterior cruciate ligament (ACL) structure in the Col5a1^Δten/Δten mouse. A and B: Transmission electron microscopy shows a significant increase in fibril diameter and aberrant fibril structure in the Col5a1^Δten/Δten ACL. The alterations in the collagen V–null ACL were more severe. C: Histograms show the broader distribution of fibril diameters in the ACL of Col5a1^Δten/Δten mice compared with that in the control mice. D: The mean fibril diameter is increased significantly in the ACL of Col5a1^Δten/Δten mice ACL. E: The fibril density was decreased significantly in Col5a1^Δten/Δten mice. Fibril diameter and density were measured in three different mice of each group, with three to seven images from each mouse; the data are presented as means ± SD, and Student's t-test was used (C–E). All mice were postnatal day 30 males. ^∗∗∗P < 0.001. Ctrl, control; Q, quartile.

Figure 10

Altered biomechanical properties in anterior cruciate ligament (ACL) of Col5a1^Δten/Δten mice. The biomechanical properties are altered significantly in the absence of collagen V, with a significant decrease in stiffness and modulus in the Col5a1^Δten/Δten ACL compared with that in the controls (Ctrl). ^∗∗∗P < 0.001.

Figure 11

Targeted depletion of collagen V in tendon and ligament induces joint instability. A: Joint laxity and chronic fibrous inflammation presented in Col5a1^Δten/Δten mice knee joints at postnatal day 4. Sagittal sections of knee joints from postnatal day 4 mice show that the fibroblasts and fibers of the anterior cruciate ligament (ACL) align parallel in the ACL midsubstance in the control (Ctrl) mice. In the Col5a1^Δten/Δten ACL, the fibers were disorganized and wavy. The asterisks show synovial inflammation that contained fibrous connective tissue, blood vessels, and chronic inflammatory cells in the Col5a1^Δten/Δten ACL. Stain: H&E. B: Polarized light microscopy in control mice shows large, well-organized ACL fibers. In contrast, the fibers in Col5a1^Δten/Δten ACL are wavy and smaller. The specimens were the same as in A. Stain: picrosirius red.