Grazia Maria Liuzzi

Serum MMP-2 and MMP-9 are elevated in different multiple sclerosis subtypes.

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.

Inhibition of aquaporin-4 expression in astrocytes by RNAi determines alteration in cell morphology, growth, and water transport and induces changes in ischemia-related genes

Recent studies indicate a key role of aquaporin (AQP) 4 in astrocyte swelling and brain edema and suggest that AQP4 inhibition may be a new therapeutic way for reducing cerebral water accumulation. To understand the physiological role of AQP4‐mediated astroglial swelling, we used 21‐nucleotide small interfering RNA duplexes (siRNA) to specifically suppress AQP4 expression in astrocyte primary cultures. Semiquantitative RT‐PCR experiments and Western blot analysis showed that AQP4 silencing determined a progressive and parallel reduction in AQP4 mRNA and protein. AQP4 gene suppression determined the appearance of a new morphological cell phenotype associated with a strong reduction in cell growth. Water transport measurements showed that the rate of shrinkage of AQP4 knockdown astrocytes was one‐half of that of controls. Finally, cDNA microarray analysis revealed that the gene expression pattern perturbed by AQP4 gene silencing concerned ischemia‐related genes, such as GLUT1 and hexokinase. Taken together, these results indicate that 1) AQP4 seems to be the major factor responsible for the fast water transport of cultured astrocytes; 2) as in skeletal muscle, AQP4 is a protein involved in cell plasticity; 3) AQP4 alteration may be a primary factor in ischemia‐induced cerebral edema; and 4) RNA interference could be a new potent tool for studying AQP pathophysiology in those organs and tissues where they are expressed.

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 (QAlb) and CSF/serum MMP-9 (QMMP-9) was observed in IND and NIND but not in RR-MS patients, indicating that CSF MMP-9 levels in NIND and IND patients could be influenced by serum MMP-9 and blood-brain barrier (BBB) permeability properties. MS patients had higher values of QMMP-9:QAlb (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.

Aquaporin-4-containing astrocytes sustain a temperature- and mercury-insensitive swelling in vitro.

In order to understand the molecular mechanism underlying astroglial swelling, we studied primary astrocyte cultures from newborn mouse and analyzed them for expression of functional water channels. Immunocytochemical analysis of mouse brain confirms the presence of AQP4 location in astrocytic endfeet with a polarized pattern, as found in rat. Using Southern blot PCR and Western blot analysis, we demonstrate that primary astrocyte cultures from mouse express the AQP4 water channel at both the RNA and protein levels. Two polypeptides, of 30 kDa and 32 kDa, were identified in the astrocytes. Densitometric analysis demonstrates that the 32‐kDa form represents 25% of the total AQP4 protein. Moreover, immunofluorescence experiments show strong surface membrane expression of AQP4 protein in cultured cells, even though the polarity of the expression is not maintained. Furthermore, functional studies indicate that cultured astrocytes manifest rapid and temperature‐independent volume changes in response to osmotic gradients, in agreement with a channel‐mediated water transport. Water movement was found to be HgCl2 insensitive, suggesting AQP4 and AQP7 as putative water channels. Using Western blot and PCR experiments, we exclude the presence of AQP7 in astrocytes, indicating that only AQP4 is responsible for the rapid water movement. Altogether, the results indicate that primary astrocyte cultures are a valid cell model for further investigation of the molecular mechanism of water movement in the brain and its physiological regulation. GLIA 31:29–38, 2000.

Extracting and purifying R-phycoerythrin from Mediterranean red algae Corallina elongata Ellis & Solander

R-Phycoerythrin (R-PE) is a protein acting as a photosynthetic accessory pigment in red algae (Rodophyta). This protein has gained importance in many biotechnological applications in food science, immunodiagnostic, therapy, cosmetics, protein and cell labelling, and analytical processes. In this paper we report on a new, one step procedure for the extraction and purification of R-PE from a new source: the Mediterranean red algae Corallina elongata Ellis & Solander. This red algae contains mainly R-PE and is suitable for the production in culture. No other contaminating phycobiliproteins could be detected in the extracts. The method we propose for the purification is based on the use of hydroxyapatite, a chromatographic resin that can be produced in the laboratory at very low cost and can be used batch-wise with large amounts of extracts, alternative to chromatography, and therefore can be scaled up. Both the yield and the purity of R-PE are very good.

Increased activity of matrix metalloproteinases in the cerebrospinal fluid of patients with HIV-associated neurological diseases

Matrix metalloproteinases (MMPs) have been identified as mediators of brain injury in HIV-associated neurological diseases. The activity of the 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) was detected by zymography in the cerebrospinal fluid (CSF) of 138 HIV-infected patients (40 with AIDS dementia, 83 with brain opportunistic infections and 15 neurologically asymptomatic), 26 HIV-seronegative individuals with inflammatory neurological diseases (IND) and 12 HIV-seronegative subjects with noninflammatory neurological diseases (NIND). MMP-2 was present in all CSF samples from HIV-seropositive and HIV-seronegative individuals, including those of subjects with NIND. On the contrary, MMP-9 was absent in the CSF of NIND controls, whereas the activity of this MMP was found in the 77 - 100% of CSF samples from HIV-infected patients, including those with HIV dementia, central nervous system (CNS) opportunistic infections or neurologically asymptomatic subjects. The highest levels of MMP-9 were found in the CSF of patients with cryptococcosis, cytomegalovirus encephalitis and tuberculous meningitis and were comparable with those found in the CSF of HIV-negative patients with multiple sclerosis or meningitis. A significant correlation between CSF MMP-9 activity and CSF cell count was found only in patients with HIV dementia. The increased CSF activity of MMPs capable to degrade components of the extracellular matrix of blood-brain barrier may contribute to the transendothelial migration of virus-infected cells into the CNS and development of HIV-associated neurologic damage.

Interferon-beta inhibits the expression of metalloproteinases in rat glial cell cultures: implications for multiple sclerosis pathogenesis and treatment

Matrix metalloproteinases (MMPs) have been identified as mediators of brain injury in multiple sclerosis (MS) and it has recently been reported that treatment of MS patients with interferon-beta (IFN-b) reduces MMP-9 serum levels and in vitro release from monocytes. We investigated whether IFN-b is able to modulate the expression of MMPs in glial cell cultures. Rat microglial and astrocyte cultures were treated with different doses of IFN-b, then activated by exposure to LPS. In another set of experiments cells were simultaneously activated with LPS and treated with IFN-b. C ulture supernatants collected from astrocytes and microglia were subjected to zymography for the assessment of MMP-2 and MMP-9. Increased amounts of MMP-9 and MMP-2 were observed in supernatants from LPS-treated astrocytes in comparison with supernatants from nontreated control cells. MMP-9 also increased in LPS-treated microglia. The treatment of astrocytes and microglia with IFN-b inhibited dose-dependently the expression of both MMP-2 and MMP-9 in LPS-treated astrocytes and of MMP-9 in LPS-treated microglia. These results demonstrate a modulating effect of IFN-b on the release of MMPs from C NS cells. This effect represents an additional mechanism by which IFN-b may decrease the development of new C NS lesions in the course of MS.

Inhibitory Effect of Polyunsaturated Fatty Acids on MMP-9 Release from Microglial Cells—Implications for Complementary Multiple Sclerosis Treatment

We investigated whether polyunsaturated fatty acids (PUFA), which might be a useful complementary therapy among patients with multiple sclerosis (MS), are able to modulate matrix metalloproteinase (MMP) production in microglial cultures. MMPs are myelinotoxic factors. Primary cultures of rat microglia were treated with different doses of omega-3 (ω-3) PUFA or purified fish oil, containing a mixture of ω-3 and ω-6 PUFA, and simultaneously activated by exposure to lipopolysaccharide (LPS). Culture supernatants were subjected to zymography and Western blot analysis for the assessment of MMP-2 and MMP-9 levels. Increased amounts of MMP-9, but not of the constitutively expressed MMP-2, were observed in supernatants from LPS-treated microglia in comparison with non-treated control cells. The treatment with both ω-3 PUFA and fish oil dose-dependently inhibited the LPS-induced production of MMP-9. Our results suggest that a low fat diet supplemented with ω-3 PUFA may become recommended for the well being of MS patients under therapy.

Adhesion molecules and matrix metalloproteinases in Multiple Sclerosis: effects induced by Interferon-beta

In Multiple Sclerosis (MS) pathology, early inflammation involves leukocyte migration across the blood-brain barrier (BBB) within the central nervous system. In this process, adhesion molecules (AMs), both membrane-bound and soluble-circulating forms, and matrix metalloproteinases (MMPs) certainly play a regulatory role. In MS, recombinant Interferon-beta (rIFNbeta) is effective in reducing gadolinium contrast-enhancing lesions on magnetic resonance imaging and this suggests that it may reduce BBB damage or even restore its integrity by different mechanisms that include interference with both AM and MMP pathways. This review will highlight the effects induced by rIFNbeta, both in vitro and in vivo, on cell-bound and soluble forms of AMs and on MMPs.

May Diet and Dietary Supplements Improve the Wellness of Multiple Sclerosis Patients? A Molecular Approach

Multiple sclerosis is a complex and multifactorial neurological disease, and nutrition is one of the environmental factors possibly involved in its pathogenesis. At present, the role of nutrition is unclear, and MS therapy is not associated to a particular diet. MS clinical trials based on specific diets or dietary supplements are very few and in some cases controversial. To understand how diet can influence the course of MS and improve the wellness of MS patients, it is necessary to identify the dietary molecules, their targets and the molecular mechanisms involved in the control of the disease. The aim of this paper is to provide a molecular basis for the nutritional intervention in MS by evaluating at molecular level the effect of dietary molecules on the inflammatory and autoimmune processes involved in the disease.