Hemozoin Impairs MMPs/ TIMPs Balance and Promotes IL-8 Release in Human Microvascular Endothelial Cells. Possible Key Role of MMP-9 in Cerebral Malaria

Cerebral malaria (CM) is a life-threatening encephalopathy caused by the cytoadherence of malaria-infected erythrocytes to brain endothelial cells followed by blood-brain barrier (BBB) damage and neurological sequelae. In recent years, increased levels of several matrix metalloproteinases (MMPs) have been related to glial activation in either human or murine CM models; interestingly, malarial pigment hemozoin (HZ) was shown to enhance MMP-9 expression and activity in human monocytes. However, a detailed analysis of the effects of HZ on the production of MMPs and tissue inhibitors (TIMPs) by endothelial cells is still missing. In the present work, human microvascular endothelial cells (HMEC) were treated with different doses of native HZ for 48-72h. The first consequence of this treatment was a significant morphological modification of HZ-treated cells, which showed an elongated form, instead of the classical polygonal shape; notably, such a change is often related to enhanced invasion/migration ability and to the production of cytokine/chemokine. Therefore, several parameters, including total and specific gelatinolytic activity; protein expression of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1 and TIMP-2; and IL-8 release were studied. Analysis through in situ gelatin zymography assay revealed an HZ-dependent enhancement of total gelatinolytic activity, which according to additional direct gelatin zymography was due to ex novo induction of MMP-9 activity, while basal levels of MMP-2 activity were not altered. Further investigation by western blot of the expression of both gelatinases and their inhibitors showed that HZ enhanced both MMP-2 and MMP-9 proteins, as well as TIMP-2, the specific inhibitor of MMP-2, while TIMP-1 (inhibitor of MMP-9) expression was not altered. HZ also enhanced protein levels of MMP-1 and MMP-3, two enzymes involved in the cascade of proteolytic activation of MMP-9. Moreover, specific ELISA assay showed a HZ-dependent release of IL-8, a MMP-9-related chemokine involved in monocytes/neutrophils recruitment. Interestingly, blocking experiments with anti-IL-8 antibodies and synthetic MMP-9 inhibitors showed that HZ-dependent release of IL-8 was partially due to cleavage by MMP-9, while enhanced MMP-9 activity was not dependent on IL-8 levels. Taken together, the present data suggest that in HZ-treated HMEC the following cascade of events occurs: first, enhanced MMP-1, MMP-2, MMP-3, MMP-9 and TIMP-2 protein expression is promoted, while TIMP-1 protein levels are not modified; as a result of the impaired balance between MMPs and their inhibitors, as well as a consequence of the promoted activating cascade involving MMP-1 and MMP-3, ex novo activity of MMP-9 is induced, without affecting MMP-2 activity; consequently, total gelatinolytic activity is increased, and MMP-9-mediated cleavage of pro-IL-8 is favoured, resulting in higher IL-8 release, which might also be responsible for the change in shape. Collectively, all these events may concur to the damage of BBB during CM, the recruitment and extravasation of mononuclear cells and a major role for MMP-9 is proposed. Acknowledgements: 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