Dysfunctional tendon collagen fibrillogenesis in collagen VI null mice

Abstract

Tendons are composed of fibroblasts and collagen fibrils. The fibrils are organized uniaxially and grouped together into fibers. Collagen VI is a non-fibrillar collagen expressed in developing and adult tendons. Human collagen VI mutations result in muscular dystrophy, joint hyperlaxity and contractures. The purpose of this study is to determine the functional roles of collagen VI in tendon matrix assembly. During tendon development, collagen VI was expressed throughout the extracellular matrix, but enriched around fibroblasts and their processes. To analyze the functional roles of collagen VI a mouse model with a targeted inactivation of Col6a1 gene was utilized. Ultrastructural analysis of Col6a1-/- versus wild type tendons demonstrated disorganized extracellular micro-domains and associated collagen fibers in the Col6a1-/- tendon. In Col6a1-/- tendons, fibril structure and diameter distribution were abnormal compared to wild type controls. The diameter distributions were shifted significantly toward the smaller diameters in Col6a1-/- tendons compared to controls. An analysis of fibril density (number/μm(2)) demonstrated a ~2.5 fold increase in the Col6a1-/- versus wild type tendons. In addition, the fibril arrangement and structure were aberrant in the peri-cellular regions of Col6a1-/- tendons with frequent very large fibrils and twisted fibrils observed restricted to this region. The biomechanical properties were analyzed in mature tendons. A significant decrease in cross-sectional area was observed. The percent relaxation, maximum load, maximum stress, stiffness and modulus were analyzed and Col6a1-/- tendons demonstrated a significant reduction in maximum load and stiffness compared to wild type tendons. An increase in matrix metalloproteinase activity was suggested in the absence of collagen VI. This suggests alterations in tenocyte expression due to disruption of cell-matrix interactions. The changes in expression may result in alterations in the peri-cellular environment. In addition, the absence of collagen VI may alter the sequestering of regulatory molecules such as leucine rich proteoglycans. These changes would result in dysfunctional regulation of tendon fibrillogenesis indirectly mediated by collagen VI.

Fig. 1. Collagen VI is enriched in the peri-cellular region of tendon fibroblasts

Collagen VI immuno-reactivity was localized throughout the tendon extracellular matrix in P1 and P30 wild type mice (A,B). No reactivity was observed in the Col6a1−/− control mice (C). At higher magnifications (B) reactivity was enriched in the peri-cellular regions around tendon fibroblasts in both P1 and P30 mice (arrows). Nuclei were labeled with DAPI.

Fig. 2. Altered tendon fibroblast, extracellular micro-domains and fiber organization in collagen VI null tendons

Transmission electron micrographs of FDL tendons at P1 and P30 from Col6a1+/+ and Col6a1−/− mice. (A) At P1, wild type tendon fibroblasts showed a well organized arrangement with extended processes defining extracellular micro-domains containing small collagen fibers (fibril bundles). (B) In contrast, Col6a1−/− tendon fibroblasts had altered cell shape, and their processes had numerous thin branches resulting in disorganized micro-domains and fibers. (C) At P30, the wild type tendon fibroblasts defined well organized micro-domains and larger organized fibers. (D) In contrast, P30 Col6a1−/− tendon fibroblasts show altered shape, arrangement and organization with abnormal micro-domain organization and disrupted fiber formation. Fibers (F); Fibroblast processes (arrows).

Fig. 3. Dysfunctional tendon fibrillogenesis in collagen VI null mice

(A) Transmission electron micrographs of P30 FDL tendons form Col6a1+/+ and Col6a1−/− mice. Wild type fibrils have normal circular cross sectional profiles and a broad heterogeneous distribution of fibrils in both peri-cellular and central areas (labeled P and C). In contrast, Col6a1−/− tendons have smaller diameter fibrils. In the peri-cellular area, very large, structurally aberrant, twisted fibrils are frequently observed (arrows). In central area, structurally aberrant fibrils are not observed. (B,C) Histograms of fibril number (density) and fibril diameter distributions in the peri-cellular area and central area from P30 Col6a1+/+ and Col6a1−/− mice. Fibril number is significantly increased in Col6a1−/− tendons in both peri-cellular and central areas compared to wild type controls. The fibril diameter distributions were shifted toward smaller diameters in peri-cellular and central areas of Col6a1−/− tendons compared to wild type controls with a significant difference in mean fibril diameter in both regions (p<0.001).

Fig. 4. Altered biomechanical properties of tendon in collagen VI null mice

Cross-sectional area, maximum load, maximum stress, stiffness and modulus were measured in P30 FDL tendons from Col6a1+/+ and Col6a1−/− mice. Cross sectional areas are significantly decreased in Col6a1−/− tendons compared to wild type control tendons. There is a significant reduction in maximum load and stiffness in Col6a1−/− tendons compared to wild type control tendons. However, maximum stress and modulus are comparable in both genotypes. Asterisk; P<0.05.

Fig. 5. Increased MMP-2 in collagen VI null tendons

Collagen VI deficiency tends to increase MMP2 activation. Tendon extracts of P30 FDL tendons from Col6a1+/+ and Col6a1−/− mice were analyzed using zymography (A-C) and western blotting (D-F). Tendon extracts from both genotypes contained proMMP-2 and MMP-2 activity (A). The activity of proMMP-2 (B) and MMP-2 (C) tends to increase in Col6a1−/− tendons compared to wild type controls. Western blotting analysis of the same extracts used for zymography confirms the expression of proMMP-2 and MMP-2 (D). The expression of proMMP-2 (E) and MMP-2 (F) tends to increase in Col6a1−/− tendon extracts consistent with zymography data. The amount of extract loaded was normalized to β-actin.