Inhibition of Matrix Metalloproteinases-2/-9 Transiently Reduces Pre-Oligodendrocyte Loss during Lipopolysaccharide- but Not Tumour Necrosis Factor-alpha-Induced Inflammation in Fetal Ovine Glial Culture

Abstract

To determine whether increased matrix metalloproteinase (MMP) proteolytic activity plays a pathological role in infection/inflammation-induced preterm brain injury, primary cultures of preterm (day 90 of gestation; term 145 days) fetal ovine mixed glia were exposed to 24-96 h of lipopolysaccharide (LPS, 1 μg/ml) or tumour necrosis factor-α (TNF-α, 100 ng/ml). MMP-2 mRNA levels were significantly increased after TNF-α (96 h) and LPS exposure (48 and 96 h), and MMP-9 mRNA levels were significantly increased at 48 and 96 h after TNF-α. On zymography, the active form of secreted MMP-2 was significantly increased 24 h after LPS, but not TNF-α. Both active and latent forms of MMP-9 gelatinolytic activity were significantly increased by TNF-α (96 h) and LPS (72 and 96 h). On reverse zymography, inhibitory activity of TIMP-1 but not TIMP-2 was significantly increased by TNF-α and LPS. SB-3CT-mediated MMP-2 and MMP-9 inhibition transiently reduced LPS-induced oligodendrocyte cell death but had no effect during TNF-α exposure. Collectively, these observations suggest a limited, transient effect of MMPs on immature white matter damage associated with infection but not TNF-α-mediated inflammation.

Lipopolysaccharide-Induced Preconditioning Attenuates Apoptosis and Differentially Regulates TLR4 and TLR7 Gene Expression after Ischemia in the Preterm Ovine Fetal Brain

“…Stability of expression among the housekeeping genes was assessed using the algorithm BestKeeper software application [50]; of the 4 putative housekeeping genes tested, GAPDH had the most stable level of expression. For TaqMan-based detection, 18S ribosomal RNA was chosen as the housekeeping gene since it was previously validated for use [51,52]. …”

Ischemic-Reperfusion Injury Increases Matrix Metalloproteinases and Tissue Metalloproteinase Inhibitors in Fetal Sheep Brain

“…The purpose of the present study was to examine changes in the protein expression of gelatinases (MMP-2 and -9), collagenases (MMP-8 and -13), and their endogenous tissue inhibitors (TIMP-1, -2, -3, and -4) in the cerebral cortices of fetal sheep at 85% of gestation that were exposed to ischemia with reperfusion for 4, 24, or 48 h. Although the presence of basal levels of proteolytic activity for MMP-2 and -9, and TIMP-1 and -2 have been previously detected in primary mixed glial cultures from preterm ovine fetuses at 90 days of gestation [42], MMPs and TIMPs have not been systematically examined after brain ischemia in a large mammalian animal species. Six main findings can be derived from our study: (1) MMP-2, -9, -8, and -13, and TIMP-1, -2, -3, and -4 are expressed endogenously in the cerebral cortex in fetal life both under basal conditions and after exposure to ischemic insults; (2) protein expression of MMP-2 and -9 does not change in the fetal cerebral cortex after ischemic injury; in contrast, the activity of pro-MMP-2 increases after ischemia; (3) MMP-8 expression gradually increases after ischemia reaching a significant difference 48 h after ischemia; (4) protein expression of TIMP-1, -2, and -3 increases after ischemic brain injury; (5) changes in MMP-8 and -13 protein expression show direct correlations with changes in TIMP-1, -2, and -3, suggesting that there is a balance between MMP-8 and -13 and some of the TIMP inhibitors of MMPs; and (6) correlations were not observed between increases in BBB permeability measured with the blood-brain transfer constant K i and increases in MMP-8 and TIMP-1, -2, and -3 protein expression after ischemia in the fetal sheep brain.…”

“…We visualized the signals using enhanced chemiluminescence (ECL Prime; GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) before exposure to autoradiographic film (Phenix, Candler, NC, USA). We detected pro-MMP-2, cleaved-MMP-2, MMP-8, MMP-13, TIMP-1, TIMP-2, TIMP-3, TIMP-4, β-actin, and vinculin bands at 72,64,53,54,26,24,26,25,42, and 117 kDa, respectively. Omission of the primary antibodies eliminated the chemiluminescent signals, thereby establishing the specificity of the primary antibodies to detect the specific protein bands.…”

A simple, xeno-free method for oligodendrocyte generation from human neural stem cells derived from umbilical cord: engagement of gelatinases in cell commitment and differentiation

“…The interesting data obtained from either in vitro or in vivo studies indicate diversified changes in the MMP expression, localization and activity during oligodendrocyte maturation. It has been shown, for instance, that matrix metalloproteinase‐9 is engaged in controlling OPC proliferation, which occurs after spinal cord injury (Liu and Shubayev, ), infection/inflammation‐induced preterm brain injury (Weaver‐Mikaere et al ., ) and in the acute phase after adult brain injury (Pham et al ., ; Seo et al ., ). Gelatinase B is also supposed to contribute to the pathogenesis of multiple sclerosis, due to its ability to degrade myelin protein (Latronico et al ., ; Zhao et al ., ).…”