Mannan binding lectin-associated serine protease-2 (MASP-2) critically contributes to post-ischemic brain injury independent of MASP-1.

BACKGROUND: Complement activation via the lectin activation pathway (LP) has been identified as the key mechanism behind post-ischemic tissue inflammation causing ischemia-reperfusion injury (IRI) which can significantly impact the clinical outcome of ischemic disease. This work defines the contributions of each of the three LP-associated enzymes-mannan-binding lectin-associated serine protease (MASP)-1, MASP-2, and MASP-3-to ischemic brain injury in experimental mouse models of stroke. METHODS: Focal cerebral ischemia was induced in wild-type (WT) mice or mice deficient for defined complement components by transient middle cerebral artery occlusion (tMCAO) or three-vessel occlusion (3VO). The inhibitory MASP-2 antibody was administered systemically 7 and 3.5 days before and at reperfusion in WT mice in order to assure an effective MASP-2 inhibition throughout the study. Forty-eight hours after ischemia, neurological deficits and infarct volumes were assessed. C3 deposition and microglia/macrophage morphology were detected by immunohistochemical, immunofluorescence, and confocal analyses. RESULTS: MASP-2-deficient mice (MASP-2(-/-)) and WT mice treated with an antibody that blocks MASP-2 activity had significantly reduced neurological deficits and histopathological damage after transient ischemia and reperfusion compared to WT or control-treated mice. Surprisingly, MASP-1/3(-/-) mice were not protected, while mice deficient in factor B (fB(-/-)) showed reduced neurological deficits compared to WT mice. Consistent with behavioral and histological data, MASP-2(-/-) had attenuated C3 deposition and presented with a significantly higher proportion of ramified, surveying microglia in contrast to the hypertrophic pro-inflammatory microglia/macrophage phenotype seen in the ischemic brain tissue of WT mice. CONCLUSIONS: This work demonstrates the essential role of the low-abundant MASP-2 in the mediation of cerebral ischemia-reperfusion injury and demonstrates that targeting MASP-2 by an inhibitory therapeutic antibody markedly improved the neurological and histopathological outcome after focal cerebral ischemia. These results contribute to identifying the key lectin pathway component driving brain tissue injury following cerebral ischemia and call for a revision of the presently widely accepted view that MASP-1 is an essential activator of the lectin pathway effector component MASP-2

Mechanisms of hypoxic up-regulation of versican gene expression in macrophages

Hypoxia is a hallmark of many pathological tissues. Macrophages accumulate in hypoxic sites and up-regulate a range of hypoxia-inducible genes. The matrix proteoglycan versican has been identified as one such gene, but the mechanisms responsible for hypoxic induction are not fully characterised. Here we investigate the up-regulation of versican by hypoxia in primary human monocyte-derived macrophages (HMDM), and, intriguingly, show that versican mRNA is up-regulated much more highly (>600 fold) by long term hypoxia (5 days) than by 1 day of hypoxia (48 fold). We report that versican mRNA decay rates are not affected by hypoxia, demonstrating that hypoxic induction of versican mRNA is mediated by increased transcription. Deletion analysis of the promoter identified two regions required for high level promoter activity of luciferase reporter constructs in human macrophages. The hypoxia-inducible transcription factor HIF-1 has previously been implicated as a key potential regulator of versican expression in hypoxia, however our data suggest that HIF-1 up-regulation is unlikely to be principally responsible for the high levels of induction observed in HMDM. Treatment of HMDM with two distinct specific inhibitors of Phosphoinositide 3-kinase (PI3K), LY290042 and wortmannin, significantly reduced induction of versican mRNA by hypoxia and provides evidence of a role for PI3K in hypoxic up-regulation of versican expression

The Lectin Pathway of Complement Activation in Cerebral Ischaemia and Reperfusion Injury

The complement system constitutes a critical component of the innate immune response. The lectin pathway is one of the three activation pathways of the complement activation cascade that can recognise and respond to structures on oxygen deprived cells and contribute to ischaemia and reperfusion injury (IRI). Cerebral IRI mediated inflammation is known to be responsible for secondary damage in the penumbra region surrounding the initial area of infarct and the prevention of IRI-mediated secondary damage provides an attractive target for therapeutic intervention. Mannose binding lectin associated serine protease 2 (MASP-2) is the key effector enzyme of the lectin pathway, since depletion of this enzyme completely ablates lectin pathway function or activity. This study assessed the impact of MASP-2 deficiency on cerebral IRI and to what extent MASP-2 targeting can reduce the secondary inflammatory damage following an ischaemic insult. The 3 vessel occlusion (3-VO) model of stroke was found to be the most appropriate model to use in this study, as it was shown to have a lower degree of variability than the middle cerebral artery occlusion (MCAO) stroke model. TTC staining revealed that MASP-2 -/- mice were significantly protected from cerebral damage, showing statistically significant smaller infarct sizes when compared to age and sex matched wild type controls. MASP-2 deficient mice showed reduced C3 deposition and a lower degree of astrocytic activation in brain sections from mice undergoing 3-VO and showed higher mRNA abundance of anti-inflammatory mediators (such as IL-10) and lower abundance of pro-inflammatory mediators (such as MIP-2) when compared to wild type control mice. Subsequently, a recombinant inhibitory anti-MASP-2 antibody, AbD04211, a murine specific MASP-2 inhibitor, was assessed for the therapeutic utility of MASP-2 inhibition in the 3-VO cerebral IRI model of stroke. The results revealed that the use of MASP-2 inhibitors at a dose of 5mg/kg of body weight achieved a statistically significant protective effect, with infarct sizes reduced by up to 30% in the anti-MASP-2 treated animals

miRNAs-19b, -29b-2* and -339-5p Show an Early and Sustained Up-Regulation in Ischemic Models of Stroke

<div><p>Stroke, the loss of neurons after ischemic insult to the brain, is one of the leading causes of death and disability worldwide. Despite its prevalence and severity, current therapy is extremely limited, highlighting the importance of further understanding the molecular events underlying ischemia-induced neuronal cell death. An ischemic area can be subdivided into two separate pathophysiological regions: the rapidly dying necrotic core, and the potentially salvageable apoptotic penumbra. Understanding molecular events occurring in the apoptotic ischemic penumbra may give greater insight into mechanisms controlling this salvageable tissue. miRNAs are known to have key roles in the regulation of gene expression in numerous pathological conditions, including the modulation of distinct pathways in stroke. However, previous studies have profiled miRNAs in the whole ischemic infarct, and do not differentiate between miRNA regulation in the necrotic core versus the apoptotic penumbra. We asked if there were unique miRNAs that are differentially regulated following ischemic insults in the salvageable apoptotic penumbra. miRNA expression profiles were compared in the whole infarct from <i>in vivo</i> stroke models, using the three vessel occlusion approach, to an <i>in vitro</i> model of the ischemic penumbra, prior to apoptotic induction. Multiple miRNAs were found to be differentially regulated following ischemic insults in each system. However, miR-19b, miR-29b-2* and miR-339-5p were significantly up-regulated in both model systems. Further, we confirmed these results in a neuroblastoma cell line subjected to a penumbra-like ischemic insult that induced the apoptotic cell death pathway. The data show that miR-19b, miR-29b-2* and miR-339-5p are up-regulated following ischemic insults and may be regulating gene expression to control important cellular pathways in the salvageable ischemic penumbra. Further investigation of their role and mRNA target identification may lead to new insights into the molecular mechanisms taking place in the salvageable apoptotic penumbra.</p></div

miR-29b-2*, miR-339-5p and miR-19b are up-regulated in response to <i>in vivo</i> and <i>in vitro</i> ischemia.

<p>A. Comparing both <i>in vivo</i> and <i>in vitro</i> data identified 4 miRNAs common to both models, which are differentially regulated in response to ischemia. B. Taqman Assay q-PCR confirms the up-regulation of miR-29b-2*, -339-5p and -19b following ischemic insults both <i>in vivo</i> (n = 3) and <i>in vitro</i> (n = 4).</p

Ischemia induced the differential regulation of miRNAs both <i>in</i> vitro and <i>in</i> vivo.

<p>MiRNAs were selected on the basis that their expression changed by at least 1.5-fold (linear fold change) and are present in all biological repeats (n = 3) and each internal microarray technical repeat (4), <i>in vitro</i> (A) and <i>in vivo</i> (B). Data represents mean ± SD. Ns  =  non-significant. Significance accepted at p<0.05.</p

OGD in N2As induces the up-regulation of miR-29b-2*, miR-339-5p and miR-19b.

<p>Taqman q-PCR analysis of RNA collected 2 h post-OGD termination was analysed for the expression of selected miRNA candidates. The data shows that the miRNAs are up-regulated: by 1.42±0.28, 1.84±0.10 (P = 0.0013) and 2.22±0.39-fold (P = 0.035), for miR-29b-2*, miR-19b and miR-339-5p respectively (n = 3). Data represent mean ± SEM.</p

OGD-induced cell death in rat cortical neurons.

<p>A. OGD caused an increase in neuronal cell death levels, analysed by sytox/hoechst imaging, which correlated with increased durations of OGD. Basal levels of cell death within our cultures, under normal culture conditions, were determined as 23.7±2% (n = 3). Neurons were placed in EBSS with or without oxygen and glucose. Cells with EBSS plus oxygen and glucose (EBSS+Oxygen+Glucose) only demonstrated a significant increase in levels of death, in comparison to controls, following 6 h of exposure. 2 h of OGD did not increase cell death levels above those in EBSS+Oxygen+Glucose controls. However, 4 and 6 h of OGD increased cell death levels significantly to 62.4±8.2% (p = 0.026, n = 5) and 84.9±3.2% (p<0.0001, n = 5) respectively, compared to EBSS+Oxygen+Glucose controls. B. Cell death induced by 4 h of OGD becomes apparent between 8 and 24 h post-OGD termination. Analysis of the time course of OGD-induced cell death using Calcein AM time-lapse assay. Time-lapse imaging of Calcein AM stained cells indicated that OGD-induced cell death became apparent at 8 h and reached maximum levels at 24 h post-OGD termination. Levels of cell death became significantly higher than controls at 10 h post- OGD termination, indicated by *, where cell death levels were 18.7±2.4% and 39.7±6.4% for control and OGD, respectively (p = 0.045, n = 3). Neuronal cell death levels, following OGD reach 75±2% at 24 h post-OGD termination. Data represent mean ± SEM.</p

OGD induced cell death demonstrates characteristics of apoptotic cell death.

<p>A. The pan-caspase inhibitor, zVAD.fmk at a concentration of 100 µM, was able to significantly reduce levels of OGD-induced cell death from 70.6±2%, in naïve cells, to 54.3±5% in zVAD.fmk treated cells (p = 0.0459, n = 3) at 24 h post-OGD termination, thus indicating cell death is caspase dependent. B. Western blot analysis shows that OGD induces cytochrome C release into the cytosolic fraction of cell lysates. OGD also induces activation of Caspase 3, as indicated by the presence of the cleaved 17 kDa fragment. OGD-treated rat cortical neurons display PARP that has been cleaved to form the classic 89 kDa apoptosis fragment. The 50 kDa fragment of PARP that is found during necrosis is not detected. C. Quantification of the Western blots demonstrates an increase of cytochrome C intensity of 0.9±0.3, in controls, to 21±3.7 following OGD (p = 0.0056, n = 3). Quantification of Caspase 3 signal intensity shows a significant increase in OGD samples reaching 36.6±5.4 (p = 0.0046, n = 3). Quantification revealed that levels of the 89 kDa PARP fragment changed from 13.3±11 in control cells, to 135.6±16 in OGD treated rat cortical neurons (p = 0.0032, n = 3).Data represent Mean ± SEM.</p

Real Time RT-PCR analysis of the effect of PI3K inhibitors on induction of versican and GLUT-1 mRNAs by 18h of exposure to hypoxia (0.2% O₂).

<p>LY290042 was used at 2μM and wortmannin at 300μM. ^^; p<0.05 compared to DMSO control, *; p<0.05 compared to untreated control, ratio paired t test, one tailed. Data from 5 independent experiments using HMDM from different donors, expressed as means ± SEM.</p