Modulation of matrix metalloproteinases and their tissue inhibitors in haemozoin-treated human microvascular endothelial cells

Introduction: Recently, the in vivo involvement of matrix metalloproteinases (MMPs) in blood–brain barrier (BBB) damage during cerebral malaria (CM) has been suggested. Moreover, MMP-9 is induced by malaria pigment (HZ, haemozoin) in human monocytes in vitro. Here, the effects of HZ on balances between MMPs and their tissue inhibitors (TIMPs) in a human microvascular endothelial cell line (HMEC-1) are described. Methods: Cells were treated for 72 hours with different doses (6–25 lM) of native (n) or lipid-free HZ. Therefore, total gelatinolytic activity by fluorogenic gelatin conversion assay; levels of gelatinases (MMP-2 and MMP-9) by gelatin zymography; MMP-1, MMP-3, TIMP-1, and TIMP-2 protein expression by Western blotting; and cell morphology and microtubule-like vessel formation by microscopy were studied. Results: nHZ enhanced total gelatinolytic activity by inducing de novo proMMP-9 and MMP-9 without affecting basal proMMP-2 levels. TIMP-1 (endogenous MMP-9 inhibitor) was not altered by nHZ, whereas MMP-2 inhibitor (TIMP-2) was enhanced. Additionally, nHZ induced MMP-1 and MMP-3, two enzymes sequentially involved in proMMP-9 proteolytic activation. Cells treated with nHZ appeared elongated and were able to form microtubule-like vessels on synthetic basement membrane surfaces. Lipid-free HZ did not reproduce the effects of nHZ. Conclusions: The present data suggest that the lipid moiety of HZ alters the MMP/TIMP balances, promotes proteolytic proMMP-9 activation, and enhances total gelatinolytic activity and cell invasiveness in HMEC-1. These findings might be useful to understand the mechanisms underlying BBB damage during CM. MMPs may be considered as candidate targets for adjuvant therapy. Acknowledgements: FP6-IP18834 ANTIMAL, Regione Piemonte (RSF2008bis), Compagnia di San Paolo, Universita` di Milano (PUR 2009), Fund for Scientific Research (FWOVlaanderen) and Research Fund of KULeuven (GOA/2007/15 and CREA/09/023) are acknowledged.