Matrix MetalloProteinase In Multiple Sclerosis

In Multiple Sclerosis (MS), Matrix MetalloProteinase (MMP) activity in tissues is the result of a balance between MMPs and their Tissue Inhibitors (TIMPs).

MMP-9 predominates in acute MS lesions and is inhibited by TIMP-1, while MMP-2 may participate in the remodeling of the ExtraCellular Matrix (ECM) such as in chronic disease and is inhibited by TIMP-2.

These differences may be reflected in Serum and CerebroSpinal Fluid (CSF). We have tried to characterize MMP-2 and MMP-9 activities, in relation to their respective TIMPs, 2 and 1, as a factor of different types of the disease, as this information was not previously clearly stated.

We found the MMP-2/TIMP-2 ratio in Serum to show higher values in Secondary/Progressive (SP, p=0.02) and Primary/Progressive (PP, p=0.01) MS than short disease duration (SDD) Relapsing/Remitting (RR) MS, but not different from the healthy control (HC) group.

Whereas the MMP-9/TIMP-1 ratio in Serum showed higher (p=0.04) values in SDD RR MS than PP but also in active patients, evaluated either clinically (p=0.006).

Or, from the Magnetic Resonance Imaging (MRI, p < 0.05), compared to inactive disease. CSF MMP to TIMP ratios did not differ between MS subtypes, suggesting systemic rather CNS-restricted changes.

These results show that an increase in MMP-2/TIMP-2 ratio marks chronic progression in MS, but it is as high as in HC, and also confirm that high MMP-9 activity characterizes short duration relapsing and active forms of the disease.

Objective

1. Determine Serum levels of Matrix MetalloProteinase-2 (MMP-2), MMP-9, Tissue Inhibitor of MetalloProteinase-1 (TIMP-1), and TIMP-2 in patients with Secondary/Progressive (SP) MS
2. Determine the relationship between Serum levels and MRI activity
3. Evaluate the effect of Interferon (IFN-ß) therapy on these measures

Background
High Serum levels of MMP-9 and low levels of TIMP-1 predict the appearance of new Gadolinium-enhancing (Gd⁺) lesions in Relapsing/Remitting (RR) MS.

Methods
Monthly Gd⁺ Brain MRI and measures of serum MMP-2, MMP-9, TIMP-1, and TIMP-2 at 3-month intervals were performed for up to 3 years in 33 patients with SPMS participating in a Phase III study of IFN-ß-1b.

Results
Patients who developed new Gd⁺ lesions had higher levels of MMP-9 than patients who did not develop Gd⁺ lesions (median 351 vs 226 ng/mL, p = 0.049).

The ratio of MMP-9/TIMP-1 predicted new Gd⁺ lesion on the concurrent scan (OR = 2.23, 95% CI 0.99 to 4.99, p = 0.052) and on the following scan (OR = 2.16, 95% CI 1.01 to 4.63, p = 0.048), whereas levels of MMP-2/TIMP-2 did not.

Median levels of TIMP-1 were higher and MMP-9 trended lower for IFN-ß compared to placebo recipients (TIMP-1: 1,450 vs 1,185 ng/mL, p = 0.024; MMP-9: 225 vs 339 ng/mL, p = 0.081). IFN-ß did not influence levels of MMP-2 and TIMP-2.

Conclusion
The ratio of MMP-9/TIMP-1 may predict MRI activity in SPMS. The effect of IFN-ß-1b in MS, as measured by reduction in new Gd⁺ lesions, may be partly explained by altering MMP-9/TIMP-1 ratio.

Matrix MetalloProteinase-9 (MMP-9) was detected by Zymography and Enzyme-Linked Immunosorbent Assay (ELISA) in matched Serum and CerebroSpinal Fluid (CSF) samples from patients with Neurological Diseases.

Patients with Relapsing/Remitting Multiple Sclerosis (RR-MS) had Serum and CSF MMP-9 levels comparable to those from patients with Inflammatory Neurological Diseases (INDs).

But, higher than patients with Non-Inflammatory Neurological Diseases (NINDs) and healthy donors (HDs).

MMP-9 increased in active RR-MS in comparison with inactive RR-MS implying that MMP-9 in MS is related with clinical disease activity.

A correlation between the CSF/Serum Albumin (Q(AIb)) and CSF/Serum MMP-9 (Q(MMP-9)) was observed in IND and NIND but not in RR-MS patients.

Indicating that CSF MMP-9 levels in NIND and IND patents could be influenced by Serum MMP-9 and Blood-Brain Barrier (BBB) permeability properties.

MS patients had higher values of Q(MMP-9):Q(Alb)(MMP-9 index) than IND and NIND patients suggesting that in MS the increase in CSF MMP-9 could be due to Intrathecal synthesis of MMP-9.

A significant inverse correlation was found between MMP-9 and its endogenous inhibitor TIMP-1 in RR-MS indicating that in MS patients both the increase in MMP-9 and the decrease in TIMP-1 Serum levels could contribute to BBB disruption and T-Lymphocyte entry into the CNS.

In Multiple Sclerosis, Matrix MetalloProteinases (MMPs) are effectors of crucial pathogenetic steps, such as Blood-Brain Barrier breakdown, invasion of Brain Parenchyma by Immune Cells and DeMyelination.

However, only limited data are available on the types of MMPs induced in the course of Multiple Sclerosis, and on the role of their endogenous antagonists, the Tissue Inhibitors of MetalloProteinases (TIMPs).

We quantified the transcriptional expression of six MMPs and the four TIMPs in lesions and in Normal-Appearing White Matter (NAWM) from post-mortem Multiple Sclerosis Brain tissue by real-time polymerase chain reaction, and compared levels with those in Brain tissue from six control patients without Neurological Disease.

The mRNA expression of MMP-7 and -9, but not of other MetalloProteinases [MMP-2 and -3, and Tumour Necrosis Factor-alpha (TNF-)-converting-enzyme] was equally upregulated throughout all stages of lesion formation with active inflammation, and in most of matched NAWM tissue.

The transcription of Cytokines TNF-/ß and InterLeukin-2 (IL-2), known modulators of MMPs, was upregulated only in distinct stages of lesion formation, while their receptors were not induced at all.

Which suggests that additional signalling molecules participate in the sustained upregulation of MMP-7 and -9 in Multiple Sclerosis. None of the TIMPs showed a significant induction over baseline expression of controls.

We hypothesize that an imbalance between MMP and TIMP expression may cause a persistent ProteoLytic overactivity in Multiple Sclerosis, that may be a factor for continuous tissue destruction, and hence for Secondary disease progression.

Infection of the Central Nervous System (CNS) of susceptible mice with Mouse Hepatitis Virus (MHV), a positive-strand RNA Virus that is a member of the CoronaViridae family, reproducibly results in an acute Encephalomyelitis followed by a DeMyelinating Disease similar to the human DeMyelinating Disease Multiple Sclerosis (MS).

MHV infection triggers a robust cell-mediated response in which both CD4⁺ and CD8⁺ T-Cells are essential in controlling Viral replication and spread.

However, Viral clearance is incomplete and Viral RNA and protein can persist within White Matter Tracts (Axons).

Areas of Viral persistence are often associated with DeMyelinating lesions, and recent studies have indicated an important role for both T-Cells and Macrophages in contributing to Myelin destruction.

The molecular mechanisms governing Leukocyte trafficking and accumulation within the CNS of MHV-infected mice are just now being understood and recent studies indicate that Chemokines and Chemokine Receptors have an important role in this process.

This article will provide an overview on how these molecules regulate T-Cell and Macrophage trafficking into the CNS of MHV-infected mice.

And, illustrate the delicate balance that exists with regards to expression of Chemokines and their receptors, as it relates to both host defense and disease development.

Chemotactic Cytokines or Chemokines form a family of ProInflammatory Proteins that are functionally linked to various classes of Proteases, including Matrix MetalloProteinases (MMPs).

Both families of molecules are key players in the migration of Inflammatory Cells in AutoImmune Diseases and in invasive Cancers.

For example, the Chemokine InterLeukin-8 acts as a fast secretagogue of Gelatinase B in Granulocytes and is increased in the Synovial fluid of Arthritis patients and may locally recruit and activate Neutrophils.

The latter are the most abundant inflammatory cell type in the joints of patients with Rheumatoid Arthritis. In the case of the inflamed joint, the contribution of Matrix remodeling Enzymes in the breakdown of cartilage and bone is trivial.

Gelatinase B (MMP-9) was documented in AutoImmune Diseases and Cancer by ImmunoHistoChemistry with the use of MonoClonal AntiBodies.

Studies in Rheumatoid Arthritis and Multiple Sclerosis led us to postulate the "Remnant Epitopes Generate AutoImmunity" or REGA model for AutoImmunity.

This model is based on the PathoPhysiological role of three major classes of molecules involved in Aspecific Primary Immune Defense Mechanisms: the Cytokines, the Chemokines and the Proteases.

The REGA model has proven to be useful for the development of disease treatment strategies. Particular Cytokines are disease-limiting and may thus be used for the treatment of AutoImmune Disorders.

Cytokines and Chemokines that induce Enzymes promote disease and may be antagonized. Along this line of research, we have recently identified natural and biosynthetic Chemokine Antagonists.

Some of these have shown potent AntiViral activity against Human Immunodeficiency Virus. It is expected that these might also become useful in the treatment of AutoImmune Diseases and invasive Cancers.

A similar effect may be expected by the antagonization of damaging Proteases or with the use of recombinant or synthetic Enzyme Inhibitors.

Recent evidence has highlighted the fact that Axon injury is an important component of Multiple Sclerosis pathology. The issue of whether a CNS Antigen-specific Immune Response is required to produce Axon injury remains unresolved.

We investigated the extent and time course of Axon injury in a rodent model of a Delayed-Type HyperSensitivity (DTH) reaction directed against the Mycobacterium Bacille Calmette-Guerin (BCG).

Using MRI, we determined whether the ongoing Axon injury is restricted to the period during which the Blood-Brain Barrier is compromized.

DTH lesions were initiated in adult rats by IntraCerebral injection of heat-killed BCG followed by a peripheral challenge with BCG.

Our findings demonstrate that a DTH reaction to a non-CNS Antigen within a CNS White Matter Tract (Axons) leads to Axon injury.

Ongoing Axon injury persisted throughout the 3-month period studied and was not restricted to the period of Blood-Brain Barrier breakdown, as detected by MRI enhancing lesions.

We have previously demonstrated that Matrix MetalloProteinases (MMPs) are upregulated in Multiple Sclerosis Plaques and DTH lesions.

In this study we demonstrated that microinjection of activated MMPs into the Cortical White Matter results in Axon injury.

Our results show that Axon injury, possibly mediated by MMPs, is Immunologically non-specific and may continue behind an intact Blood-Brain Barrier.

Matrix MetalloProteinases (MMPs) are increasingly being implicated in the pathogenesis of several CNS diseases.

In Multiple Sclerosis, MMPs could be responsible for the influx of inflammatory MonoNuclear Cells into the CNS, contribute to Myelin destruction and disrupt the integrity of the Blood-Brain Barrier

In Alzheimer's disease, MMPs might mediate the deposition of Amyloid ß-Proteins; and MMPs are known to contribute to the invasiveness of malignant Glioma Cells and might regulate their angiogenic capacity. Nonetheless, MMPs could also have beneficial roles in recovery from CNS injury.

Therefore, both the identity of the MMP and its cellular origin could determine whether disease pathogenesis or regeneration occurs, and thus synthetic MMP Inhibitors might be valuable for treating some CNS diseases.

Dendritic Cells (DC) are Antigen-Presenting Cells (APC) that most efficiently initiate and control Immune responses. Migration processes of blood DC are crucial to exert their professional Antigen-presenting functions.

Matrix-degrading MetalloProteinases (MMP) are ProteoLytic Enzymes, which are considered to be key Enzymes in ExtraCellular Matrix (ECM) turnover and mediators of cell migration.

Tissue Inhibitors of MetalloProteinases (TIMP) are important regulators of MMP activity. Here we investigate whether blood Monocyte-derived immature DC (iDC) and mature DC (mDC) express, produce and secrete functionally active MMP-1, -2, -3 and -9 and their Inhibitors TIMP-1 and -2, and examine their involvement in Multiple Sclerosis (MS).

On mRNA level, we observed high numbers of MMP-2 and TIMP-2 mRNA expressing iDC in MS. On protein level, high percentages of MMP-1, -2 and -9 expressing iDC by flow cytometry.

And, high MMP-1 secretion by Western blot together with high MMP-2 and -9 activities in iDC supernatants as studied with Zymography were observed.

Similarly, MS is associated with high percentages of MMP-2 and -3 and of TIMP-1 expressing mDC by flow cytometry together with high MMP-3 secretion and high MMP-9 activity in culture supernatants.

Spontaneous migratory capacity of both iDC and mDC over ECM-coated filters was higher in MS compared to Healthy Controls (HC).

In conclusion, blood Monocyte-derived iDC and mDC express, produce and secrete several MMP and TIMP. Alterations in these molecules as observed in MS may be functionally important for DC functioning.

ExtraCellular Matrix (ECM) molecules play important roles in the PathoBiology of the major human Central Nervous System (CNS) Inflammatory/DeMyelinating disease Multiple Sclerosis (MS).

This mini-review highlights some recent work on CNS Endothelial Cell interactions with Vascular basement membrane ECM as part of the Cellular Immune Response, and roles for White Matter ECM molecules in DeMyelination and ReMyelination in MS lesions.

Recent basic and clinical investigations of MS emphasize Axonal injury, not only in chronic MS plaques, but also in acute lesions; progressive Axonal degeneration in Normal-Appearing White Matter also may contribute to Brain and Spinal Cord Atrophy in MS patients.

Remodeling of the InterStitial White Matter ECM molecules that affect Axon regeneration, however, is incompletely characterized.

Our ongoing ImmunoHistoChemical studies demonstrate enhanced ECM Versican, a Neurite and Axon growth-inhibiting White Matter ECM ProteoGlycan, and Dermatan Sulfate ProteoGlycans at the edges of inflammatory MS lesions.

This suggests that enhanced ProteoGlycan deposition in the ECM and Axonal growth inhibition may occur early and are involved in expansion of active lesions.

Decreased ECM ProteoGlycans and their Phagocytosis by Macrophages along with Myelin in plaque centers imply that there is "injury" to the ECM itself.

These results indicate that White Matter ECM ProteoGlycan alterations are integral to MS pathology at all disease stages and that they contribute to a CNS ECM that is inhospitable to Axon regrowth/regeneration.

Matrix MetalloProteinases (MMPs) are hypothesized to play an important role in the pathogenesis of several Central Nervous System Disorders.

Increased levels of expression of MMP-9 (Gelatinase B) and MMP-2 (Gelatinase A) have been observed in Alzheimer's Disease, Stroke, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis.

This suggests an aberrant regulation of MMPs that could lead to inappropriate expression of MMP activity.

To allow us to evaluate the effect of increased levels of active MMP-9 in the Central Nervous System, mutant forms of the Enzyme were designed to AutoCatalytically remove the pro domain, yielding active Enzyme.

This was accomplished by modifying residues in the Cysteine switch AutoInhibitor region of the ProPeptide.

Stable cell lines and transgenic mice that express G100L and D103N AutoActive forms of human MMP-9 were developed to study the role of dysregulation of MMP-9 in disease.

Matrix MetalloProteinases (MMPs) are proteases involved in ExtraCellular Matrix (ECM) remodeling, Leukocyte infiltration into lesions and Myelin degradation in the Central Nervous System (CNS) disease Multiple Sclerosis (MS).

We have investigated whether MMP-12 (Macrophage Metalloelastase) is expressed in MS lesions at various stages.

In control patient tissue and (p)reactive MS lesions, only occasional Microglial and Astrocyte staining was detected. In contrast, in active DeMyelinating lesions, Phagocytic Macrophages were MMP-12 positive.

A lower proportion of Phagocytes was positive for MMP-12 in chronic active DeMyelinating lesions and inactive lesions. This suggests a role for MMP-12 during DeMyelination in MS.