Matrix metalloproteinase-7 and matrix metalloproteinase-9 in pediatric multiple sclerosis

Matrix metalloproteinases and their tissue inhibitors play a key role in the pathogenesis of adult-onset multiple sclerosis, and were suggested as biomarkers of response to interferon-β, an established treatment in multiple sclerosis. However, data regarding pediatric population are scarce. We determined serum levels of matrix metalloproteinase-7, matrix metalloproteinase-9, and tissue inhibitor of matrix metalloproteinase-1 in children, and evaluated effects of interferon-β therapy on these measures. Serum samples from 14 children with relapsing, remitting multiple sclerosis at baseline and at month 12, and from 15 controls, were collected. Interferon-β treatment was initiated in eight patients. Mean serum matrix metalloproteinase-9 levels and matrix metalloproteinase-9/tissue inhibitor of matrix metalloproteinase-1 ratio were higher in patients compared with controls, and were reduced significantly in treated patients at month 12, but did not change in untreated patients. Mean matrix metalloproteinase-7 levels were lower in patients compared with controls, and increased significantly in the treated group, but did not change significantly in the untreated group. In pediatric multiple sclerosis, a shift in matrix metalloproteinase-9/tissue inhibitor of matrix metalloproteinase-1 balance toward proteolytic activity is evident, and interferon-β therapy demonstrates a beneficial effect on this disturbed balance.     

Matrix metalloproteinase-9 and matrix metalloproteinase-2 gene polymorphisms in multiple sclerosis

We investigated the association of matrix metalloproteinase-9 (-1562C/T, +279R/Q) and matrix metalloproteinase-2 (-1575G/A, -1306C/T) gene polymorphisms with multiple sclerosis (MS) susceptibility, gender differences and disability in 244 patients and 132 healthy subjects. A significant decrease of the -1562T allele carriers in MS patients compared to controls (Pa=0.01, Pacorr=0.05) in -1562C/T MMP-9 gene polymorphism was found, (odds ratio (OR) -0.58, 95% confidence interval (CI):0.38-0.89). Significant differences were also demonstrated between female patients and healthy females (Pa=0.01, Pacorr=0.05), (OR-0.53, 95% CI:0.32-0.86). Other polymorphisms were not associated either with MS susceptibility or with phenotype of the disease. No association with disability was found.     

CCR5 delta32, matrix metalloproteinase-9 and disease activity in multiple sclerosis

Chemokines and matrix metalloproteinases (MMPs) appear to be crucial in leukocyte recruitment to the central nervous system in multiple sclerosis (MS). CCR5 delta32, a truncated allele of the CC chemokine receptor CCR5 gene encoding a non-functional receptor, did not confer protection from MS. CCR5 delta32 was, however, associated with a lower risk of recurrent clinical disease activity. High CSF levels of MMP-9 activity were also associated with recurrent disease activity. These results directly link intrathecal inflammation to disease activity in patients with MS, suggesting that treatments targeting CCR5 or treatment with MMP inhibitors may attenuate disease activity in MS.     

Intrathecal synthesis of matrix metalloproteinase-9 in patients with multiple sclerosis: implication for pathogenesis

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.     

Brevican-containing perineuronal nets of extracellular matrix in dissociated hippocampal primary cultures

Perineuronal nets (PNN) are specialized extracellular matrix structures enwrapping CNS neurons, which are important regulators for neuronal and synaptic functions. Brevican, a chondroitin sulfate proteoglycan, is an integral component of PNN. Here, we have investigated the appearance of these structures in hippocampal primary cultures. The expression profile of brevican in mixed cultures resembles the in vivo pattern with a strong upregulation of all isoforms during the second and 3rd weeks in culture. Brevican is primarily synthesized by co-cultured glial fibrillary acidic protein (GFAP-)-positive astrocytes and co-assembles with its interaction partners in PNN-like structures on neuronal somata and neurites as identified by counterstaining with the PNN marker Vicia villosa lectin. Both excitatory and inhibitory synapses are embedded into PNN. Furthermore, axon initial segments are strongly covered by a dense brevican coat. Altogether, we show that mature primary cultures can form PNN, and that basic features of these extracellular matrix structures may be studied in vitro.     

Increase of matrix metalloproteinase-9 in peripheral blood of multiple sclerosis patients treated with high doses of methylprednisolone

Matrix metalloproteinases (MMPs) are believed to play a role in the pathogenesis of multiple sclerosis (MS). As methylprednisolone is the treatment of choice for a relapse, we investigated the effect of methylprednisolone on blood levels of MMPs. Baseline TIMP-1 and MMP-2 levels were lower in MS patients than in healthy controls. MMP-9 levels tended to be elevated. During therapy, MMP-9 levels demonstrate a dose-dependent increase. No effect was noted on TIMP-1 and MMP-2 levels. The short-lived increase of MMP-9 plasma levels may be at least in part due to an activation and an increase of granulocytes and monocytes by methylprednisolone.     

Role of matrix metalloproteinase-9 in a mouse model for amyotrophic lateral sclerosis

The pathogenesis of amyotrophic lateral sclerosis remains poorly understood, but microglial and astroglial activation are thought to contribute to motor neuron death. Evidence suggests that matrix metalloproteinase-9 (MMP-9) is a mediator of this deleterious effect. In this study, we evaluated the effect of MMP-9 on the pathogenesis of amyotrophic lateral sclerosis. Although marked microglial and astroglial proliferation was seen in the spinal cord and in-vitro studies proved MMP-9 to be produced by these cells, deletion of the MMP-9 gene in SOD1(G93A) mice accelerated rather than delayed the motor neuron disease and significantly reduced survival. Our results suggest that the effect of MMP-9 on mutant superoxide dismutase-1 (SOD1)-induced motor neuron disease is protective rather than hazardous. Therefore, the effect of pharmacological inhibition of MMP-9 activity is unlikely to be of therapeutical benefit in amyotrophic lateral sclerosis.     

Serum matrix metalloproteinase-3 levels correlate with disease activity in relapsing-remitting multiple sclerosis

BACKGROUND: Adhesion molecules and matrix metalloproteinases (MMPs) are known to be relevant to the ongoing development and disappearance of areas of demyelination in the white matter of the CNS of multiple sclerosis (MS) patients. This study examined whether serum matrix metalloproteinase-3 (MMP-3) levels correlate with disease activity in MS. METHODS: Serum MMP-3 levels in 47 consecutive patients with relapsing-remitting MS were measured by immunoassay every 4 weeks over a 15 month period. RESULTS: During the study period, 48 clinical relapses occurred. Serum MMP-3 levels within 1 month of relapse were significantly higher than during the remission phase. Sequential analysis showed that serum MMP-3 levels had increased transiently at the time of clinical relapse but returned to the normal range within a month. CONCLUSIONS: Circulatory MMP-3 levels are correlated with disease activity in relapsing-remitting MS. This may contribute to the breakdown of the blood-brain barrier at the time of relapse.     

Axonal regeneration in old multiple sclerosis plaques

Summary Cryostat sections of two old plaques removed at autopsy from the spinal cord of a 62-year-old man with multiple sclerosis of 24-year duration were studied by indirect immunofluorescence with antibodies to neurofilament proteins, glial fibrillary acidic protein (GFAP), glial hyaluronate-binding protein (GHAP), vimentin and laminin. The neurofilament monoclonal antibodies used in this study reacted with phosphorylated epitopes of the two large polypeptides of the neurofilament triplet (NF 150K, NF 200K). As previously reported [Dahl D, Labkovsky B, Bignami A (1989) Brain Res Bull 22:225–232], the neurofilament antibodies either stained axons in the distal stump of transected sciatic nerve in the early stages of regeneration or late in the process, i.e., after regenerating axons had reached the distal stump of the transected sciatic nerve. Both multiple sclerosis plaques were positive for GFAP and vimentin, but negative for GHAP, while astrocytes in myelinated spinal cord white matter stained with both GFAP and GHAP antibodies. Laminin immunoreactivity in the plaques and normal spinal cord was confined to blood vessels. One plaque was almost devoid of axons as evidenced by indirect immunofluorescence with neurofilament antibodies. Another plaque was packed with bundles of thin axons running an irregular course in the densely gliosed tissue. Axons in the plaque only stained with neurofilament antibodies reacting with sciatic nerve in the early stages of regeneration while axons in the surrounding myelinated white matter were decorated by all neurofilament antibodies, regardless of the time of appearance of immunoreactivity in crushed sciatic nerve. It is concluded that reactive astrocytes forming glial scars do not constitute a non-permissible substrate for axonal growth.Supported by NIH grant NS 13034 and by the Veterans Administration     

Novel astrocytic protein in multiple sclerosis plaques

Monoclonal antibody J1-31 (MAb J1-31, isotype IgG 2b) was raised against crude homogenate of brain tissue from a multiple sclerosis (MS) patient (autopsy sample; Malhotra et al.: Microbios Letters 26:151-157, 1984). In human brain, MAb J1-31 recognizes an intracellular protein antigen (J1-31 antigen), which bands at approximately 30,000 daltons under reducing conditions for sodium dodecyl sulfate gel electrophoresis (Singh et al.: Bioscience Reports 6:73-79, 1986). By immunofluorescence microscopy, MAb J1-31 stains those cells that are also stained by antiserum to glial fibrillary acidic protein (GFAP), namely astrocytes, retinal Müller cells, and tanycytes in the ependyma (Predy et al.: Bioscience Reports 7:491-502, 1987). In addition, MAb J1-31 stains ciliated ependymal cells that do not express GFAP. Using a model system for gliosis (laceration-type injury of rat spinal cord), we were able to show that astrocytes responding to central nervous system injury exhibit greatly enhanced staining for J1-31 antigen (Predy et al.: Journal of Neuroscience Research 19:397-404, 1988; Predy and Malhotra: Brain Research Bulletin in press, 1989). In this article, we demonstrate that immunofluorescence staining owing to MAb J1-31 is greatly enhanced in MS plaques, as compared to adjacent "apparently normal" white matter. (This is consistent with previous results as MS plaques characteristically show an astroglial response [reactive gliosis] leading to the formation of a glial scar [McKhann: Annual Review of Neuroscience 5:219-239, 1982].) In addition, we present further evidence that J1-31 antigen is distinct from GFAP, although these two proteins may be associated spatially with one another.     

Changes of matrix metalloproteinase-9 and its tissue inhibitor (TIMP-1) after autologous hematopoietic stem cell transplantation in multiple sclerosis

Elevated levels of matrix metalloproteinase-9 (MMP-9) associates with predictors of multiple sclerosis (MS) activity. We analysed serum levels and mRNA expression of MMP-9 and its inhibitor TIMP-1 in peripheral mononuclear cells of 14 MS patients after autologous hematopoietic stem cell transplantation (AHSCT). All but 2 patients stabilized after AHSCT. A significant decrease of MMP-9 levels was seen up to 36 months after AHSCT. TIMP-1 levels did not change. MMP-9 mRNA levels correlated with the CD4+ T cell count (p<0.0001). The significant and persistent change in MMP-9 activity after AHSCT may be caused in part by the effect of AHSCT in the CD4+ T cell count.     

Increased matrix metalloproteinase-9 activity in unstable carotid plaques. A potential role in acute plaque disruption

BACKGROUND AND PURPOSE: Acute disruption of atherosclerotic plaques precedes the onset of clinical syndromes, and studies have implicated a role for matrix metalloproteinases (MMPs) in this process. The aim of this study was to establish the character, level, and expression of MMPs in carotid plaques and to correlate this with clinical status, cerebral embolization, and histology. METHODS: Plaques were obtained from 75 consecutive patients undergoing carotid endarterectomy and divided into 4 groups according to symptomatology (group 1, asymptomatic; group 2, symptomatic >6 months before surgery; group 3, symptomatic within 1 to 6 months; group 4, symptomatic within 1 month). All patients underwent preoperative and intraoperative transcranial Doppler monitoring. Plaques were subjected to histological examination and quantification of MMPs by zymography and ELISA. RESULTS: The level of MMP-9 was significantly higher in group 4 (median 125.7 ng/mL for group 4, median <32 ng/mL for all other groups; P=0.003), with no difference in the levels of MMPs 1, 2, or 3. Furthermore, the MMP-9 concentration was significantly higher in plaques undergoing spontaneous embolization (P=0.019) and those with histological evidence of plaque instability (P<0.03). In situ hybridization demonstrated increased MMP-9 expression in highly symptomatic plaques in areas of intense inflammatory infiltrate. CONCLUSIONS: The concentration, production, and expression of MMP-9 is significantly higher in unstable carotid plaques. If this proves to be a causal relationship, MMP-9 may be a strong candidate for pharmacotherapy aimed at stabilizing plaques and preventing stroke.     

Perisynaptic aggrecan-based extracellular matrix coats in the human lateral geniculate body devoid of perineuronal nets

The extracellular matrix surrounds different neuronal compartments in the mature nervous system. In a variety of vertebrates, most brain regions are loaded with a distinct type of extracellular matrix around the somatodendritic part of neurons, termed perineuronal nets. The present study reports that chondrotin sulfate proteoglycan-based matrix is structured differently in the human lateral geniculate body. Using various chondrotin sulfate proteoglycan-based extracellular matrix antibodies, we show that perisomatic matrix labeling is rather weak or absent, whereas dendrites are contacted by axonal coats appearing as small, oval structures. Confocal laser scanning microscopy and electron microscopy demonstrated that these typical structures are associated with synaptic loci on dendrites. Using multiple labelings, we show that different chondrotin sulfate proteoglycan components of the extracellular matrix do not associate exclusively with neuronal structures but possibly associate with glial structures as well. Finally, we confirm and extend previous findings in primates that intensity differences of various extracellular matrix markers between magno- and parvocellular layers reflect functional segregation between these layers in the human lateral geniculate body.     

Decomposition and long-lasting downregulation of extracellular matrix in perineuronal nets induced by focal cerebral ischemia in rats

The upregulation of extracellular matrix components, especially chondroitin sulfate proteoglycans, after brain injury and stroke is known to accompany the glial reaction, forming repellent scars that hinder axonal growth and the reorganization of the injured neuronal networks. The extracellular matrix associated with perineuronal nets (PNs) in the primarily injured and remote regions has not yet been systematically analyzed. We use the model of permanent middle cerebral artery occlusion (MCAO) to investigate the acute and long-lasting consequences of ischemia for PNs, related to the damage of neurons and reactions of glial cells, in spontaneously hypertensive rats. Extracellular matrix components associated with PNs around cortical interneurons and neurons in thalamic nuclei were characterized 1, 7, 14, and 35 days after MCAO, using Wisteria floribunda agglutinin (WFA) staining and immunocytochemistry. The degradation of PNs in the infarct core was initiated by loss of WFA-binding matrix components, indicating the cleavage of glycosaminoglycan chains of chondroitin sulfate proteoglycans. Immunostaining showed the subsequent removal of proteoglycan core proteins within the extending microglia/macrophage invasion zone lasting for 2 weeks after MCAO. In the cortical periinfarct region, delineated by an astrocytic scar against the infarct core, the number of WFA-stained and proteoglycan core protein-immunoreactive PNs was permanently reduced. In the homolateral ventroposterior thalamus, the delayed decrease in perineuronal matrix was related to the distribution pattern of activated microglia and massive neuronal degeneration. It can be concluded from these results that complementary to the known upregulation of matrix components in the glial scar, deficits in the expression of the neuron-associated extracellular matrix develop in the periinfarct and remote regions. These deficits may contribute to the long-lasting functional impairments after stroke.     

Expression of annexin-1 in multiple sclerosis plaques

This study describes the distribution and identity of annexin-1 positive cells in the central nervous system in patients with multiple sclerosis (MS). Glucocorticoid-inducible, anti-inflammatory properties have been ascribed to annexin-1, a member of a family of calcium-binding proteins that are referred to collectively as annexins. We have found annexin-1 to be spatially associated with active MS lesions and demonstrated a stage-dependent expression of annexin-1 in MS plaques. All of the most important pathogenetically involved cells of MS lesions showed a strong annexin-1 reactivity. Both correlation analysis and double staining procedures suggested annexin-1 expression in macrophages and perivascular lymphocytes, where a cytoplasmic reactivity was displayed, whereas in activated, gemistocytic astrocytes it was also concentrated close to the plasma membrane. Although the exact roles of annexin-1 in this setting are still to be determined, a possible contribution to anti-inflammatory processes might be suggested.