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

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

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

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

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-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

Activity of versican promoter luciferase reporter constructs in normoxia and hypoxia in HMDM.

<p><b>(A)</b> Putative transcription binding sites for hypoxia inducible factor (HIF), cAMP responsive element binding (CREB), activator Protein 1 (AP1), SP1, nuclear factor 1 (NF-1) and E2F within the 240 bp (-56+184) versican promoter sequence. The -56 to -26 and +54 to +104 sequences which are important for high level expression are in bold. <b>(B)</b> Schematic diagram of versican promoter or random 26mer luciferase reporter constructs used. <b>(C)</b> activity of versican promoter or random 26mer luciferase reporter constructs in HMDM after 5d incubation in normoxia (20.9% O₂), or hypoxia (0.2% O₂). Data from an average of 6 independent experiments with each construct, minimum n = 3 for each construct, are expressed as means ± SEM. Luciferase data were normalized to protein levels. Data assessed for significant increase in hypoxia compared to random construct control using two-tailed t tests, *** = p <0.001 ** p < 0.01,* = p <0.05, ns = not significant.</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

Quantitation of versican protein expression in monocytes / macrophages by flow cytometry.

<p><b>(A and D)</b> Dot plot analysis of PBMC after 5 days in normoxia (20.9% O₂; A) and hypoxia (0.2% O₂; D). Monocyte/macrophages are subdivided into 3 regions R3-R1 in respect of increasing cell size (forward scatter). Lymphocytes are included in region 4. Region 5 encompasses all monocyte macrophages in Regions 1, 2, and 3. A representative example of 5 independent experiments is shown. <b>(B and E)</b> Percentage of the total monocyte/macrophage population (R5) present in regions R1, R2, and R3 in normoxia (B) and hypoxia (E). Data from 5 independent experiments are expressed as means ± SEM. <b>(C and F)</b> Versican mean fluorescent intensity in regions 1, 2, 3 and 4 in Normoxia (C) and Hypoxia (F). Data from 5 independent experiments are expressed as means ± SEM. <b>(G)</b> Histogram of the fluorescent intensity with a versican specific antibody (black fill) compared to the isotype control antibody (white line) in region R1 cells in Normoxia and Hypoxia. A representative example of 5 independent experiments is shown. <b>(H)</b> Histogram analysis of the versican fluorescent intensity in region R1 cells in Normoxia (shaded) and Hypoxia (clear). A representative example of 5 independent experiments is shown. <b>(I)</b> Versican protein fold induction in cells region R1 in normoxia and hypoxia. Data from 5 independent experiments are expressed as means ± SEM. For panels B, E, C, F, and I, the normoxic value in each experiment was assigned an arbitrary value of 1. Data were further analyzed using two-tailed, paired t-tests. ** p < 0.01,* = p <0.05.</p