Abstract
IntroductionWe have previously found an increased mast cell density in tendon biopsies from patients with patellar tendinopathy compared to controls. This study examined the influence of mast cells on basic tenocyte functions, including production of the inflammatory mediator prostaglandin E2 (PGE2), extracellular matrix remodeling and matrix metalloproteinase (MMP) gene transcription, and collagen synthesis.MethodsPrimary human tenocytes were stimulated with an established human mast cell line (HMC-1). Extracellular matrix remodeling was studied by culturing tenocytes in a three-dimensional collagen lattice. Survival/proliferation was assessed with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay. Levels of mRNA for COX-2, COL1A1, MMP1, and MMP7 were determined by quantitative real-time polymerase chain reaction (qPCR). Cox-2 protein level was assessed by Western blot analysis and type I procollagen was detected by immunofluorescent staining. PGE2 levels were determined using an enzyme-linked immunosorbent assay (ELISA).ResultsMast cells stimulated tenocytes to produce increased levels of COX-2 and the pro-inflammatory mediator PGE2, which in turn decreased COL1A1 mRNA expression. Additionally, mast cells reduced the type I procollagen protein levels produced by tenocytes. Transforming growth factor beta 1 (TGF-β1) was responsible for the induction of Cox-2 and PGE2 by tenocytes. Mast cells increased MMP1 and MMP7 transcription and increased the contraction of a three-dimensional collagen lattice by tenocytes, a phenomenon which was blocked by a pan-MMP inhibitor (Batimastat).ConclusionOur data demonstrate that mast cell-derived PGE2 reduces collagen synthesis and enhances expression and activities of MMPs in human tenocytes.
Highlights
We have previously found an increased mast cell density in tendon biopsies from patients with patellar tendinopathy compared to controls
Tenocytes treated with Human mast cell line 1 (HMC-1) conditioned media for 24 or 48 hours showed a significant increase in cell survival/proliferation over untreated control samples (Figure 1)
We show that under conditions where production of prostaglandin E2 (PGE2) both in mast cells and tencytes were substantially blocked with indomethacin, Indo mast cell sonicate (MCS) did not have any effect on COL1A1 expression by tenocytes relative to control untreated cells
Summary
We have previously found an increased mast cell density in tendon biopsies from patients with patellar tendinopathy compared to controls. This study examined the influence of mast cells on basic tenocyte functions, including production of the inflammatory mediator prostaglandin E2 (PGE2), extracellular matrix remodeling and matrix metalloproteinase (MMP) gene transcription, and collagen synthesis. The primary cell type in tendons, tenocytes, may be influenced by the presence of mast cells in their microenvironment. We utilized an in vitro culture system using human primary tendon fibroblasts (tenocytes) and an established human mast cell line (HMC-1) to investigate potential effects of mast cells on tenocyte gene expression and function. Our data provide evidence for mechanisms by which mast cells could contribute to the development of tendinopathy, including a transforming growth factor beta (TGFβ)-dependent upregulation of cyclooxygenase (COX)-2, a reduction of COL1A1 mRNA and type I procollagen protein levels in tenocytes, and a matrix metalloproteinases (MMP)-dependent increase in collagen remodeling activity. The current studies support the hypothesis that inflammatory cells may be involved in the development of tendon injury or overuse pathology [7]
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