Hemozoin (Malarial Pigment) Promotes MMP-9 Activation and IL-8 Release in Human Microvascular Endothelial Cells

INTRODUCTION: Increased levels of several matrix metalloproteinases (MMPs) have been recently related to glial activation and blood-brain barrier (BBB) damage in cerebral malaria (CM) models (1-2). Hemozoin (HZ), a ferriprotoporphirin IX crystal produced by Plasmodium parasite from the catabolysm of hemoglobin, was shown to enhance MMP-9 expression and activity in human monocytes (3). However, a complete profiling of the effects of HZ on the production of MMPs and tissue inhibitors (TIMPs) by endothelial cells was still missing. In the present work, human microvascular endothelial cells (HMEC) were treated with different doses of native HZ and the gelatinolytic activity, the MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1 and TIMP-2 protein expression, and the release of IL-8, a MMP-9-related chemokine, were studied. RESULTS: In situ gelatin zymography on HMEC supernatants revealed an HZ-dependent enhancement of total gelatinolytic activity, which appeared totally due to ex novo induction of MMP-9; basal MMP-2 activity was not altered. Further investigation by western blot analysis showed that HZ enhanced both MMP-2 and MMP-9 proteins, as well as TIMP-2 (inhibitor of MMP-2); on the other hand, TIMP-1 (inhibitor of MMP-9) was not altered. Additionally, protein expressions of MMP-1 and MMP-3, two enzymes involved in the proteolytic activating cascade of MMP-9, were also promoted. Results from specific ELISA assay showed higher IL-8 release from HMEC after HZ treatment compared to untreated controls. Crossed blocking experiments with anti-IL-8 antibodies and synthetic MMP-9 inhibitors indicated that HZ-dependent IL-8 release was partially due to the cleavage by MMP-9, while enhanced MMP-9 activity was not dependent on IL-8 levels. Taken altogether, the present data suggest that HZ stimulates MMP-1, MMP-2, MMP-3, MMP-9 and TIMP-2 (but not TIMP-1) protein expression by HMEC. As a result of impaired balance between MMPs and TIMPs, as well as a consequence of the promoted activating cascade involving MMP-1 and MMP-3, the activity of MMP-9 (but not of MMP-2) is induced ex novo, increasing total gelatinolytic activity and MMP-9-mediated shedding of pro-IL-8. Collectively, all these events may concur to the rupture of BBB, favouring the recruitment and extravasation of mononuclear cells during CM. ACKNOWLEDGMENTS: The financial support of the FP6-IP18835 ANTIMAL, Regione Piemonte, Ricerca Sanitaria Finalizzata and Compagnia di San Paolo in the context of the Italian Malaria Network is acknowledged. 1. Szklarczyk A. et al. (2007) J. Neurovirol. 13, 2-10 2. Van Den Steen P. E. et al. (2006) Lab. Invest 86, 873-888 3. Prato M. et al. (2005) J. Immunol. 175, 6436-64425.