Gelatinases, Neurofilaments and N-Acetyl aspartic Acid as Biomarkers in Different Pathologies

Background: Gelatinases (MMP-2 and MMP-9) are zinc and calcium dependent endopeptidases that degrade several components of the extracellular matrix.The role of gelatinases in inflammation seems to be opposite. MMP-9, which can be produced also by neutrophils, has been associated to inflammation in several pathologies; also, the environmental conditions (i.e.pH) might influence the release of MMP-9 from neutrophils. On the contrary, MMP-2 has been associated to both tissue repair and damage, although its role is not yet clear. Furthermore, the possible presence in body fluids of a 66 kDa active form of MMP-9, which might not be modulated by its inhibitor TIMP-1, could open new insights on MMP-9 as marker of inflammation. Neurofilaments (Nf) are normally contained into the axoplasm and are released in the extracellular fluid following axonal injury. N-Acetyl aspartic Acid (NAA) is one of the most abundant molecules in the CNS. Recently, the combination of neurofilaments and NAA was identified as promising marker of axonal damage in Multiple Sclerosis (MS) patients. Aims: In the present study, I aimed to evaluate the role of active MMP-9 and MMP-2 as biomakers in inflammatory conditions (Multiple Sclerosis (MS) and in Spontaneous Intra Cerebral Hemorrhage (SICH)), and the role of Nf light and heavy subunits and NAA as biomarkers of axonal damage in a relatively large cohort of PPMS. I also investigated the presence in serum of the 66 kDa MMP-9, and pH effect on the neutrophils response. Methods: The 66 kDa active MMP-9 form was identified by employing immunoprecipitation and affinity chromatography. Human neutrophils were isolated from buffy-coats and placed in medium at different pH in absence or presence of stimulus (LPS or IL-8). The MMP-9 released was assayed by ELISA. The levels of MMP-9, MMP-2, TIMP-1 and TIMP-2 were measured by activity assay systems and ELISA in serum of 28 SICH patients, whereas only MMP-2 and TIMP-2 were measured in Cerebrospinal fluid (CSF) and serum of 67 patients with RRMS and in 129 controls. NAA, Nf light and Nf heavy were assayed by GC-MS, Luminex and ELISA, respectively, in CSF of 28 Primary Progressive (PP) and 10 Transitional Progressive MS patients and 15 controls. Main Results: The separation of MMP-9 from MMP-2 by affinity chromatography and immunoprecipitation showed that the 66 kDa form of MMP-9 is present in serum of healthy donors. MMP-9 release increased when neutrophils were placed in medium at acid pH, whereas it was uneffected by alkaline conditions. CSF and serum values of active MMP-2/TIMP-2 ratios as well as intrathecal synthesis of active MMP-2 were higher in MS patients than in non-inflammatory conditions and in Magnetic Resonance Imaging (MRI) inactive than in MRI active patients. Serum levels of active MMP-9 and MMP-2 as well as Perihematomal Edema (PE) volume were significantly different over time in SICH patients: MMP-9 and PE volume increase, whereas MMP- 2 decreases. Nf heavy levels were two fold higher in progressive MS patients. NAA levels were negatively correlated with black hole volume. Combined biomarkers classified an higher number of patients with abnormal biomarker levels than single markers. Conclusions: The presence of the 66 kDa active MMP-9 in the serum of healthy donors opens the possibility to investigate the role of this form also in pathological conditions. The different neutrophil response to environmental pH changes strengthen the hypothesis of MMP- 9 as marker of inflammation. The results obtained in this study about MMP-9 and MMP-2 in patients affected by SICH and MS, suggest that the two gelatinases may have opposite roles in inflammation. While MMP-9 enhance the inflammation, MMP-2 seems associated to its resolution. CSF Nf isoforms and NAA may reflect various aspects of axonal damage in Multiple Sclerosis. In particular, Nf heavy can be considered as a specific marker of axonal damage accumulation in patients with essentially progressive MS course.