Cellular and Molecular Effects of High-Molecular-Weight Heparin on Matrix Metalloproteinase 9 Expression.

René Huber,Lara Hauke,Ralf Lichtinghagen,Bernadette Lüns,Korbinian Brand,Karin Weissenborn,Rozan Attili/Abedalkhader,Daniela Küper

doi:10.3390/ijms20071595

René Huber, Lara Hauke + Show 6 more

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https://doi.org/10.3390/ijms20071595

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Abstract

Blood sampling with different anticoagulants alters matrix metalloproteinase (MMP-) 9 expression, thus influencing its concentration and diagnostic validity. Here, we aimed to evaluate the effects of different anticoagulants on MMP-9 regulation. MMP-9 expression was assessed in response to ethylenediaminetetraacetic acid, citrate, and high-/low-molecular-weight heparin (HMWH, LMWH) in co-culture experiments using THP-1, Jurkat, and HT cells (representing monocytes, T, and B cells). Triple and double cell line co-culture experiments revealed that HMWH treatment of THP-1 and Jurkat led to a significant MMP-9 induction, whereas other anticoagulants and cell type combinations had no effect. Supernatant of HMWH-treated Jurkat cells also induced MMP-9 in THP-1 suggesting monocytes as MMP-9 producers. HMWH-induced cytokine/chemokine secretion was assessed in co-culture supernatant, and the influence of cytokines/chemokines on MMP-9 production was analyzed. These experiments revealed that Jurkat-derived IL-16 and soluble intercellular adhesion molecule (sICAM-) 1 are able to induce MMP-9 and IL-8 production by THP-1. As a consequence, the increased MMP-9 expression found in HMWH blood samples may be influenced by HMWH-dependent secretion of IL-16 and sICAM-1 by T cells resulting in an increased production of MMP-9 and IL-8 by monocytes. IL-8, in turn, may support MMP-9 and its own expression in a positive autocrine feedback loop.

Highlights

Matrix metalloproteinases (MMPs) are a major group of endopeptidases degrading extracellular matrix (ECM) and basement membrane components [1] involved in the regulation of cellular events such as proliferation, differentiation, and migration [2]
Stimulation of individual cell line cultures (THP-1, Jurkat, HT cells) with 3.2 mg/well ethylenediaminetetraacetic acid (EDTA), 220 μL/well citrate, or 50 international units (IU)/well High-molecular-weight heparin (HMWH) revealed that matrix metalloproteinase (MMP-)9 expression and secretion are not influenced directly by any of these anticoagulants in monocytic cells (Figure 1a), T cells (Figure 1b), or B cells (Figure 1c) since no significant induction of MMP-9 mRNA could be shown by Quantitative polymerase chain reaction (qPCR)
The analysis of MMP-9 mRNA expression in a co-culture of THP-1, Jurkat, and HT cells demonstrated that MMP-9 expression increased significantly over time after addition of HMWH

Summary

Introduction

Matrix metalloproteinases (MMPs) are a major group of endopeptidases degrading extracellular matrix (ECM) and basement membrane components [1] involved in the regulation of cellular events such as proliferation, differentiation, and migration [2]. Stroke is regarded as the one of the leading causes of death in the world [6] and MMP-9 levels in the blood have been suggested to represent a suitable biomarker supporting its prognosis [7]. It was previously shown by our group and others that the way of blood sampling may affect subsequent analytical tests and that the kind of anticoagulant used, especially heparin, may alter the amount of MMP-9 (and other proteases or protease inhibitors) measured [8,9,10,11]. The clinical chemist who has to evaluate a patient’s blood MMP-9 concentration usually does not know about the patient’s medical treatment, and an impact of concurrent treatment upon measured MMP-9 levels has not been considered in practice, so far

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