Regulation of collagen fibril nucleation and initial fibril assembly involves coordinate interactions with collagens V and XI in developing tendon

Abstract

Collagens V and XI comprise a single regulatory type of fibril-forming collagen with multiple isoforms. Both co-assemble with collagen I or II to form heterotypic fibrils and have been implicated in regulation of fibril assembly. The objective of this study was to determine the roles of collagens V and XI in the regulation of tendon fibrillogenesis. Flexor digitorum longus tendons from a haplo-insufficient collagen V mouse model of classic Ehlers Danlos syndrome (EDS) had decreased biomechanical stiffness compared with controls consistent with joint laxity in EDS patients. However, fibril structure was relatively normal, an unexpected finding given the altered fibrils observed in dermis and cornea from this model. This suggested roles for other related molecules, i.e. collagen XI, and compound Col5a1(+/-),Col11a1(+/-) tendons had altered fibril structures, supporting a role for collagen XI. To further evaluate this, transcript expression was analyzed in wild type tendons. During development (E18-P10) both collagen V and XI were comparably expressed; however, collagen V predominated in mature (P30) tendons. The collagens had a similar expression pattern. Tendons with altered collagen V and/or XI expression (Col5a1(+/-); Col11a1(+/-); Col5a1(+/-),Col11a1(+/-); Col11a1(-/-); Col5a1(+/-),Col11a1(-/-)) were analyzed at E18. All genotypes demonstrated a reduced fibril number and altered structure. This phenotype was more severe with a reduction in collagen XI. However, the absence of collagen XI with a reduction in collagen V was associated with the most severe fibril phenotype. The data demonstrate coordinate roles for collagens V and XI in the regulation of fibril nucleation and assembly during tendon development.

FIGURE 1.

Altered biomechanical properties in Col5a1^+/− mouse tendons. Cross-sectional area, maximum load, maximum stress, stiffness, and modulus were measured in P60 FDL tendons from Col5a1^+/+ and Col5a1^+/− mice. Cross-sectional areas were significantly decreased in Col5a1^+/− tendons compared with wild type tendons. There was a significant reduction in stiffness in Col5a1^+/− tendons compared with control tendons, consistent with increased elasticity. The maximum load, stress, and modulus were comparable in both genotypes. Asterisk, p < 0.05.

FIGURE 2.

Mature Col5a1^+/− tendons demonstrate a fibril structure comparable with wild type controls, whereas Col5a1^+/−,Col11a1^+/− tendons have an EDS-like fibril phenotype. A and B, collagen fibril structure was analyzed in the tendons from Col5a1^+/− and wild type mice at P30 using transmission electron microscopy. Fibril structures in Col5a1^+/− tendons and wild type controls were comparable, with each showing a heterogeneous population of fibrils with normal, near circular fibril cross-sections (arrow). However, there was an increased number of small and of large diameter fibrils in the mutant mice. In addition, the fibril cross-sectional profiles were less regular in the Col5a1^+/− tendons (arrow). C, fibril structure in the compound heterozygotes appears comparable with the Col5a^+/− tendon, but fibril cross-sectional profiles are more irregular. In addition, there is a subpopulation of structurally aberrant fibrils (arrow). D–F, fibril diameter distributions of tendons at P30 had a broad, heterogeneous population of fibrils with a bimodal distribution in wild type, Col5a1^+/−, and Col5a1^+/−,Col11a1^+/− mice. Both sets of mutant tendons demonstrate an increased number of small diameter fibrils relative to the wild type controls. In addition, both mutant genotypes developed a shoulder composed of larger fibrils with a heterogeneous distribution of diameters. This shoulder was substantially better developed in the compound heterozygous versus Col5a1^+/− tendon.

FIGURE 3.

Differential expression of collagens V and XI during tendon development. A, qualitative transcript analysis of collagen V and XI α-chain gene expression from P10 to P90 indicates that a number of different isoforms are possible during tendon development. B, quantitative transcript analysis of collagen V and XI α-chain genes from E18 to P90 quantitatively depicts the different isoforms possible throughout development, showing a shift from early expression of both collagen V and XI α-chains to preferential expression of collagen V beginning at 1 month. All five α-chain genes decrease with development. The inset shows only the expression of the collagen XI chains with an expanded scale at 1–3 months. C, immunoblot analyses of α1(V) and α1(XI) chain expression during tendon development detected the α1 chains of collagens I, V, and XI. The position of cross-linked collagen I α-chains including β1,1 is indicated by the square bracket. Molecular weight markers are presented on the right. D, immunolocalization of collagens V and XI in early development shows reactivity for α1(V) and α1(XI) throughout the P4 tendon.

FIGURE 4.

Compound heterozygous (Col5a1^+/−,Col11a1^+/−) mice demonstrate a fibril phenotype in early tendon development. Col5a1^+/− and Col11a^+/− tendons show normal fibril morphology and a small increase in fibril diameter relative to wild type tendons (arrows in A–C). In contrast, compound heterozygous mice have larger diameter fibrils that are beginning to display irregular fibril cross-sections (arrows in D) compared with both the wild type and haplo-insufficient mice. Transmission electron micrographs are of cross-sections from E18 mouse FDL tendons.

FIGURE 5.

A severe collagen XI-null fibril phenotype is enhanced by reduction in collagen V, indicating synergistic regulatory roles. The loss of collagen XI in Col11a1^−/− tendons (B and C) is associated with abnormally large fibrils with aberrant structures (arrows) compared with wild type controls (A). A deficiency in collagen XI coupled with a reduction in collagen V results in an increased severity of the fibril phenotype, including a substantial decrease in the number of fibrils (C). The disruption in fibrillogenesis results in disrupted fiber assembly (D–F, asterisks) and overall tendon structure. Again, this is more severe in the compound mutant tendons. Transmission electron micrographs of E18 mouse tendons in cross-section are shown. D–F are low magnification micrographs of regions comparable with those in A–C, respectively.

FIGURE 6.

Decreases in collagens V and XI lead to dysfunctional regulation of fibrillogenesis and a decreased number of fibrils assembled in early tendon development. A, fibril diameter distributions from E18 tendons with altered collagen V and XI expression are shown. The reduction in collagens V and XI was associated with changes in the diameter distributions relative to wild type controls. All mutant tendons except Col5a1^+/− demonstrated the development of a right-hand shoulder with larger and more heterogeneous fibril diameters. This is illustrated by the increase in the range of the last quartile (Q4-Q3) that increases from 17 nm in wild type tendons to 76 nm in Col11a1^−/−,Col5a1^+/− tendons, with the other genotypes being intermediate. B, shown is analysis of fibril number in tendons with altered collagen V and XI expression. A decrease in fibril density of ∼20% is seen in Col5a1^+/− tendons. Col11a1^+/−, compound Col11a1^+/−,Col5a1^+/−, and Col11a1^−/− tendons demonstrated a significant decrease in fibril number relative to wild type tendons of ∼26–29%. In contrast, the Col11a1^−/−,Col5a1^+/− tendons showed an ∼44% decrease. These data are consistent with nucleation and assembly of fewer fibrils. Horizontal lines indicate pairs of data that were significantly different from each other (p < 0.001) in analysis of variance. The down arrow and percentage indicate the % decrease in fibril density for each genotype compared with wild type controls.