Cell density-dependent stimulation of PAI-1 and hyaluronan synthesis by TGF-β in orbital fibroblasts

Abstract

During the course of Graves’ orbitopathy (GO), orbital fibroblasts are exposed to factors that lead to proliferation and extracellular matrix (ECM) overproduction. Increased levels of tissue plasminogen activator inhibitor type 1 (PAI-1 (SERPINE1)) might promote the accumulation of ECM components. PAI-1 expression is regulated by cell density and various cytokines and growth factors including transforming growth factorβ(TGF-β). We examined the effects of increasing cell densities and TGF-β on orbital fibroblasts obtained from GO patients and controls. Responses were evaluated by the measurement of proliferation, PAI-1 expression, and ECM production. There was an inverse correlation between cell density and the per cell production of PAI-1. GO orbital, normal orbital, and dermal fibroblasts behaved similarly in this respect. Proliferation rate also declined with increasing cell densities. Hyaluronan (HA) production was constant throughout the cell densities tested in all cell lines. In both GO and normal orbital fibroblasts, but not in dermal fibroblasts, TGF-β stimulated PAI-1 production in a cell density-dependent manner, reaching up to a five-fold increase above baseline. This has been accompanied by increased HA secretion and pericellular HA levels at high cell densities. Increasing cell density is a negative regulator of proliferation and PAI-1 secretion both in normal and GO orbital fibroblasts; these negative regulatory effects are partially reversed in the presence of TGF-β. Cell density-dependent regulation of PAI-1 expression in the orbit, together with the local cytokine environment, may have a regulatory role in the turnover of the orbital ECM and may contribute to the expansion of orbital soft tissue in GO.

Characteristics of Hyaluronan Synthesis Inhibition by 4-Methylumbelliferone in Orbital Fibroblasts

“…18 We assume that the effect of 4-MU on transcription of HAS genes with very low expression levels is less consistent; HAS2 expression was 2 orders of magnitude higher than HAS1 and HAS3 in the studied cells, as we described earlier. 11 This theory is supported by our observation that significant downregulating effect of 4-MU on the expression of HAS1 was observed in TGF-β stimulated cells where HAS1 transcription is substantially higher (Fig. 6B).…”

“…Human fibroblasts were cultured as described previously in detail. 11 The cells were studied between passages 2 and 8. For experiments, orbital and dermal fibroblasts (OFs and DFs) were plated in 24-well or 96-well plates, in confluent cell density (1.56 × 10 4 cells/cm 2 ) in M199 supplemented with 10% (v/v) FBS.…”

“…All experiments were performed at least 3 times and carried out in triplicate. Secreted HA levels in cell culture supernatants and levels of pericellular HA 11 were measured using the DuoSet Hyaluronan Kit (R&D Systems, Minneapolis, MN, USA). In each case, results were adjusted for the HA content of FBS.…”

“…10 We recently demonstrated that TGF-β enhances HA synthesis of OFs via stimulating HAS1 expression. 11 The breakdown of HA is carried out by 2 isoforms of hyaluronidases, HYAL1 and HYAL2. 12 4-methylumbelliferone (4-MU) is a coumarin derivative with no anticoagulant property and is available as a spasmolytic over-the-counter drug in several European countries.…”

Gypenosides Protect Orbital Fibroblasts in Graves Ophthalmopathy via Anti-Inflammation and Anti-Fibrosis Effects

“…We found that the expression and secretion of inflammatory cytokines (IL-6, IL-8, TNF-α, and MCP-1) were increased after IL-1β stimulation. Additionally, the expression and secretion of fibrotic factors (HA, α-SMA, FN, and Col-1, and extracellular matrix 51 ) were increased after TGF-β1 stimulation in OFs. Although all of these mediators were decreased after treatment with Gyps.…”