Matrix stiffness modulates the activity of MMP-9 and TIMP-1 in hepatic stellate cells to perpetuate fibrosis

Dariusz Lachowski,David Pinato,Krista Rombouts,Armando Del Rio Hernandez,Alistair Rice,Ernesto Cortes

doi:10.1038/s41598-019-43759-6

Abstract

Liver fibrosis is characterised by a dense and highly cross-linked extracellular matrix (ECM) which promotes progression of diseases such as hepatocellular carcinoma. The fibrotic microenvironment is characterised by an increased stiffness, with rigidity associated with disease progression. External stiffness is known to promote hepatic stellate cell (HSC) activation through mechanotransduction, leading to increased secretion of ECM components. HSCs are key effector cells which maintain the composition of the ECM in health and disease, not only by regulating secretion of ECM proteins such as collagen, but also ECM-degrading enzymes called matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). Uninhibited MMPs degrade ECM proteins to reduce external rigidity. Using fibronectin-coated polyacrylamide gels to alter substrate rigidity without altering ligand density, we show that fibrotic rigidities downregulate MMP-9 expression and secretion, and also upregulate secretion of TIMP-1, though not its expression. Using tissue immunofluorescence studies, we also report that the expression of MMP-9 is significantly decreased in activated HSCs in fibrotic tissues associated with hepatocellular carcinoma. This suggests the presence of a mechanical network that allows HSCs to maintain a fibrotic ECM, with external rigidity providing feedback which affects MMP-9 and TIMP-1 secretion, which may become dysregulated in fibrosis.

Highlights

Liver fibrosis in hepatocellular carcinoma (HCC) is characterised by a remarkable extracellular matrix (ECM) stiffness, with extensive deposition and cross-linking of extracellular proteins, including fibrillar and basement membrane collagens
The matrix remodelling proteins matrix metalloproteinase 2 (MMP-2) and 9 (MMP-9) are two key matrix metalloproteinases (MMPs) secreted from hepatic stellate cell (HSC) that degrade collagen[15], and a sensitivity of MMP and tissue inhibitors of matrix metalloproteinases (TIMPs) secretion by HSCs to external rigidity may underlie an important feedback loop that regulates the composition of the ECM in fibrosis
We hypothesized that substrate stiffness would affect MMP-2, MMP-9 and TIMP-1 gene expression

Summary

Introduction

Liver fibrosis in hepatocellular carcinoma (HCC) is characterised by a remarkable extracellular matrix (ECM) stiffness, with extensive deposition and cross-linking of extracellular proteins, including fibrillar and basement membrane collagens. Quiescent HSCs maintain ECM homeostasis by balancing the extracellular proteolytic activity of MMPs with the production of ECM proteins, but when HSCs are activated in disease or following injury, excess collagen production and altered matrix degradation leads to a stiff fibrotic state[6]. The pathological state of fibrosis is associated with an increased matrix stiffness due to the altered composition of the ECM, with stiffness dependent on both collagen abundance and cross-linking[10] This increased rigidity is detected by HSCs through cell surface receptors known as integrins, leading to HSC activation[11,12,13]. The matrix remodelling proteins matrix metalloproteinase 2 (MMP-2) and 9 (MMP-9) are two key MMPs secreted from HSCs that degrade collagen[15], and a sensitivity of MMP and TIMP secretion by HSCs to external rigidity may underlie an important feedback loop that regulates the composition of the ECM in fibrosis

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Results

Conclusion