Exposure to oxLDL impairs TGF-β activity in human tendon cells

Abstract

Background: Previous studies have shown that patients with hypercholesterolemia experience elevated levels of oxidized LDL (oxLDL), a molecule which triggers inflammation and collagenase activity. In this study we discovered novel mechanistic effects of oxLDL on tendon cells and the mediators regulating matrix remodeling by analyzing the expression and activity of related proteins and enzymes. These effects may contribute to tendon damage in patients with high cholesterol.

Methods: Isolated human tendon cells (male and female donors age 28 ± 1.4 age 37 ± 5.7, respectively) were incubated in the presence or absence of oxLDL. The influence of oxLDL on the expression level of key mRNA and proteins was examined using real time quantitative PCR, ELISA and Western blots. The activities of enzymes relevant to collagen synthesis and breakdown (lysyl oxidase and matrix metalloproteinases) were quantified using fluorometry. Finally, the isolated human tendon cells in a 3D construct were exposed to combinations of oxLDL and TGF-β to examine their interacting effects on collagen matrix remodeling.

Results: The one-way ANOVA of gene expression indicates that key mRNAs including TGFB, COL1A1, DCN, and LOX were significantly reduced in human tendon cells by oxLDL while MMPs were increased. The oxLDL reduced the activity of LOX at 50 µg/ml, whereas conversely MMP activities were induced at 25 µg/ml (P ≤ 0.01). COL1A1 synthesis and TGF-β secretion were also inhibited (P ≤ 0.05). Adding recombinant TGF-β reversed the effects of oxLDL on the expression of collagens and LOX. OxLDL also impaired collagen matrix remodeling (P ≤ 0.01), and adding TGF-β restored the native phenotype.

Conclusion: Exposure to oxLDL in patients with hypercholesterolemia may adversely affect the mechanical and structural properties of tendon tissue through a direct action of oxLDL on tendon cells, including impairment of TGF-β expression. This impairment leads to disturbed matrix remodeling and synthesis, thereby potentially leading to increased risk of acute or chronic tendon injury. Our discovery may provide an opportunity for developing effective treatments for tendon injury in hypercholesterolemia patients by targeting the TGF-β pathway.

Keywords: Collagen matrix; Hyperlipidemia; TGF-β; Tendon; oxLDL.

Fig. 1

Graphic illustration of the experiment design and data collection methods

Fig. 2

oxLDL changes the expression of collagen and the factors that mediate matrix remodeling. Exposure of human tendon cells to progressively increasing doses of oxLDL resulted in reduction of key mRNAs involved in anabolic processes such as COL1A1, COL1A2, COL3A1, DCN, ILK, ITGB1, and LOX. Conversely, the expression of specific MMPs (MMP1, MMP3, and MMP14) were increased. Mean ± SE; * P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; n = 4 biological replicates

Fig. 3

oxLDL changes the activity of enzymes regulating matrix remodeling, and protein expression of collagen. OxLDL increases MMPs activity (a) and reduces LOX activity (b) in human tendon cells. Western blot shows reductions of COL1A1 protein in human tendon cells after incubation with oxLDL (c). The densitometry analysis of Col1A1/Vinculin ratios shows the effect is statically significant and responsive to the dose of oxLDL (d). The uncropped images of blots are included in Supplementary information. Mean ± SE; ns P > 0.05; * P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; n = 4 biological replicates

Fig. 4

oxLDL changes the expression of collagen and lox via TGF-β. oxLDL decreases TGF-β expression at the level of mRNA (a) and protein (b). Adding recombinant TGF-β to human tendon cells treated with oxLDL rescued the expression of collagen and LOX mRNA (c) but not MMPs (d). Mean ± SE; * P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; n = 4 biological replicates

Fig. 5

oxLDL disturbed matrix remodeling of BAT via TGF-β. The scanned micrograph of BATs shows impaired BATs remodeling in presence of oxLDL which was reversed by adding recombinant TGF-β (a). The impact of oxLDL on thickness of BAT was significant while adding TGF-β abrogated this effect (b). Mean ± SE; ns P > 0.05; **P ≤ 0.01; n = 4 biological replicates