Abstract
During stroke, cells in the infarct core exhibit rapid failure of their permeability barriers, which releases ions and inflammatory molecules that are deleterious to nearby tissue (the penumbra). Plasma membrane degradation is key to penumbral spread and is mediated by matrix metalloproteinases (MMPs), which are released via vesicular exocytosis into the extracellular fluid in response to stress. DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid) preserves membrane integrity in neurons challenged with an in vitro ischemic penumbral mimic (ischemic solution: IS) and we asked whether this action was mediated via inhibition of MMP activity. In cultured murine hippocampal neurons challenged with IS, intracellular proMMP-2 and -9 expression increased 4–10 fold and extracellular latent and active MMP isoform expression increased 2–22 fold. MMP-mediated extracellular gelatinolytic activity increased ∼20–50 fold, causing detachment of 32.1±4.5% of cells from the matrix and extensive plasma membrane degradation (>60% of cells took up vital dyes and >60% of plasma membranes were fragmented or blebbed). DIDS abolished cellular detachment and membrane degradation in neurons and the pathology-induced extracellular expression of latent and active MMPs. DIDS similarly inhibited extracellular MMP expression and cellular detachment induced by the pro-apoptotic agent staurosporine or the general proteinase agonist 4-aminophenylmercuric acetate (APMA). Conversely, DIDS-treatment did not impair stress-induced intracellular proMMP production, nor the intracellular cleavage of proMMP-2 to the active form, suggesting DIDS interferes with the vesicular extrusion of MMPs rather than directly inhibiting proteinase expression or activation. In support of this hypothesis, an antagonist of the V-type vesicular ATPase also inhibited extracellular MMP expression to a similar degree as DIDS. In addition, in a proteinase-independent model of vesicular exocytosis, DIDS prevented stimulus-evoked release of von Willebrand Factor from human umbilical vein endothelial cells. We conclude that DIDS inhibits MMP exocytosis and through this mechanism preserves neuronal membrane integrity during pathological stress.
Highlights
Cells in the infarct core die within minutes of stroke onset, whereas in the surrounding region death spreads slowly for hours to days post-insult [1,2]
DIDS abolishes pathology-induced vital dye uptake To confirm that the effect of DIDS on pathology-induced blebbing correlated with preservation of membrane integrity as a permeability barrier, we examined the ability of cells to exclude vital dyes and retain adenylate kinase (AK)
We demonstrate that DIDS prevents stress-induced vesicular release of matrix metalloproteinases (MMPs) and subsequent deleterious cleavage of nearby neuronal membranes and cellular detachment from the matrix
Summary
Cells in the infarct core die within minutes of stroke onset, whereas in the surrounding region (the penumbra) death spreads slowly for hours to days post-insult [1,2]. Loss of membrane integrity is a commonly-shared hallmark of cell-death pathways [5] and membrane cleavage facilitates the release of pro-apoptotic and - immunogenic signals, ions, and other debris from dying cells, which accumulate in the local perfusate and initiate inflammatory and/or cell death pathways in adjacent cells [4,6,7] In ischemic pathology, these effects are compounded by reduced cerebral blood flow following stroke, which limits O2 and nutrient delivery [1], and slows the removal of extruded signaling molecules, ions, and metabolically-derived lactate and CO2; thereby enhancing cytotoxicity, ionic imbalance, and acute acidification in the penumbral milieu [7,8,9]. Penumbral cells are exquisitely vulnerable to pro-apoptotic or -inflammatory signals released from ruptured cells in the nearby infarct core; and the mechanisms underlying cell rupture likely play an important role in the spread of cell death and inflammation following stroke
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