Plasmin Generation Potential and Recanalization in Acute Ischaemic Stroke; an Observational Cohort Study of Stroke Biobank Samples

Rationale More than half of patients who receive thrombolysis for acute ischaemic stroke fail to recanalize. Elucidating biological factors which predict recanalization could identify therapeutic targets for increasing thrombolysis success. Hypothesis We hypothesize that individual patient plasmin potential, as measured by in vitro response to recombinant tissue-type plasminogen activator (rt-PA), is a biomarker of rt-PA response, and that patients with greater plasmin response are more likely to recanalize early. Methods This study will use historical samples from the Barcelona Stroke Thrombolysis Biobank, comprised of 350 pre-thrombolysis plasma samples from ischaemic stroke patients who received serial transcranial-Doppler (TCD) measurements before and after thrombolysis. The plasmin potential of each patient will be measured using the level of plasmin-antiplasmin complex (PAP) generated after in-vitro addition of rt-PA. Levels of antiplasmin, plasminogen, t-PA activity, and PAI-1 activity will also be determined. Association between plasmin potential variables and time to recanalization [assessed on serial TCD using the thrombolysis in brain ischemia (TIBI) score] will be assessed using Cox proportional hazards models, adjusted for potential confounders. Outcomes The primary outcome will be time to recanalization detected by TCD(defined as TIBI ≥4). Secondary outcomes will be recanalization within 6-h and recanalization and/or haemorrhagic transformation at 24-h. This analysis will utilize an expanded cohort including ∼120 patients from the Targeting Optimal Thrombolysis Outcomes (TOTO) study. Discussion If association between proteolytic response to rt-PA and recanalization is confirmed, future clinical treatment may customize thrombolytic therapy to maximize outcomes and minimize adverse effects for individual patients

LDL receptor blockade reduces mortality in a mouse model of ischaemic stroke without improving tissue-type plasminogen activator-induced brain haemorrhage: towards pre-clinical simulation of symptomatic ICH

Abstract Background Symptomatic intracerebral haemorrhage (sICH) following tissue-type plasminogen activator (rt-PA) administration is the most feared and lethal complication of thrombolytic therapy for ischaemic stroke, creating a significant obstacle for a broader uptake of this beneficial treatment. rt-PA also undermines cerebral vasculature stability in a multimodal process which involves engagement with LDL receptor-related protein 1 (LRP-1), potentially underlying the development of sICH. Aims and methods We aimed to simulate rt-PA-induced haemorrhagic transformation (HT) in a mouse model of stroke and to assess if it drives symptomatic neurological deterioration and whether it is attenuated by LDL receptor blockade. rt-PA (10 mg/kg) or its vehicle, with or without the LDL receptor antagonist, receptor-associated protein (RAP; 2 mg/kg), were intravenously injected at reperfusion after 0.5 or 4 h of middle cerebral artery occlusion (MCAo). Albumin and haemoglobin content were measured in the perfused mouse brains 24 h post MCAo as indications of blood–brain barrier (BBB) compromise and HT, respectively. Results rt-PA did not elevate brain albumin and haemoglobin levels in sham mice or in mice subjected to 0.5 h MCAo. In contrast, administration of rt-PA after prolonged MCAo (4 h) caused a marked increase in HT (but similar changes in brain albumin) compared to vehicle, mimicking the clinical shift from a safe to detrimental intervention. Interestingly, this HT did not correlate with functional deficit severity at 24 h, suggesting that it does not play a symptomatic role in our mouse stroke model. Co-administration of RAP with or without rt-PA reduced mortality and neurological scores but did not effectively decrease brain albumin and haemoglobin levels. Conclusion Despite the proven causative relationship between severe HT and neurological deterioration in human stroke, rt-PA-triggered HT in mouse MCAo does not contribute to neurological deficit or simulate sICH. Model limitations, such as the long duration of occlusion required, the type of HT achieved and the timing of deficit assessment may account for this mismatch. Our results further suggest that blockade of LDL receptors improves stroke outcome irrespective of rt-PA, blood–brain barrier breakdown and HT

Selective inhibition of brain endothelial Rho-kinase-2 provides optimal protection of an <i>in vitro</i> blood-brain barrier from tissue-type plasminogen activator and plasmin

<div><p>Rho-kinase (ROCK) inhibition, broadly utilised in cardiovascular disease, may protect the blood-brain barrier (BBB) during thrombolysis from rt-PA-induced damage. While the use of nonselective ROCK inhibitors like fasudil together with rt-PA may be hindered by possible hypotensive side-effects and inadequate capacity to block detrimental rt-PA activity in brain endothelial cells (BECs), selective ROCK-2 inhibition may overcome these limitations. Here, we examined ROCK-2 expression in major brain cells and compared the ability of fasudil and KD025, a selective ROCK-2 inhibitor, to attenuate rt-PA-induced BBB impairment in an <i>in vitro</i> human model. ROCK-2 was highly expressed relative to ROCK-1 in all human and mouse brain cell types and particularly enriched in rodent brain endothelial cells and astrocytes compared to neurons. KD025 was more potent than fasudil in attenuation of rt-PA- and plasminogen-induced BBB permeation under normoxia, but especially under stroke-like conditions. Importantly, only KD025, but not fasudil, was able to block rt-PA-dependent permeability increases, morphology changes and tight junction degradation in isolated BECs. Selective ROCK-2 inhibition further diminished rt-PA-triggered myosin phosphorylation, shape alterations and matrix metalloprotease activation in astrocytes. These findings highlight ROCK-2 as the key isoform driving BBB impairment and brain endothelial damage by rt-PA and the potential of KD025 to optimally protect the BBB during thrombolysis.</p></div

Selective ROCK-2 inhibition blocks rt-PA-induced MMP-2 activation in SVG human astrocytes, but not in BECs.

<p>Representative gelatine zymogram (left panels) and quantitation by densitometry (right panels) of active MMP-2, pro-MMP-2 (<b>A</b>) or their ratio (<b>B</b>) in media harvested from the luminal (endothelial; <b>A</b>) or the abluminal (astrocytic; <b>B</b>) chambers of the <i>in vitro</i> BBB 6 h post stimulation with DMSO (control) or with rt-PA+plasminogen (t-PA+Plgn; 25nM+100nM, respectively), in the presence or absence of KD025 (20μM). In (B) ratios obtained in the t-PA+Plgn group were assigned a value of 100%. KD025 reduces MMP activation in SVG, but not in BECs. n = 4. ****<i>P</i><0.0001 against all other groups by one-way ANOVA, **<i>P</i><0.01 against control or KD025 by two-way ANOVA with Tukey’s post hoc analysis.</p

Only KD025, but not fasudil, protects brain endothelial cells from rt-PA and plasmin.

<p><b>(A)</b> KD025 concentration-dependently blocks permeability changes in primary human brain microvascular endothelial cells (BECs; cultured without astrocytes) treated for 6 h with DMSO as control or with rt-PA (25nM) and human plasminogen (plgn; 100nM), with or without KD025 (2 and 20μM). n = 4. <b>(B)</b> Comparison of selective ROCK-2 inhibition by KD025 (20μM) <i>versus</i> non-selective ROCK inhibition by fasudil (HA1077; 2, 20 and 100μM) against rt-PA+plasminogen (25nM+100nM, respectively) in BECs, as assessed 6 h post stimulation. HA1077 displays no protective capacity in BECs. n = 3. Bars represent mean±SEM. **<i>P</i><0.01 compared to all other groups, ***<i>P</i><0.001 compared to t-PA+Plgn by one-way ANOVA with Tukey’s post hoc analysis. ## <i>P</i><0.01 compared to DMSO control and <i>P</i> = 0.06 by two-tailed paired t-test. <b>(C)</b> Representative phase-contrast micrographs (top panels) and double immunofluorescence images of zonula occludens 1 (ZO-1, representing tight junctions (TJs); white) and nuclei (Hoechst; red) (bottom panels) of BECs stimulated for 6 h with DMSO (control) or with rt-PA+plasminogen (25nM+100nM, respectively), in the presence or absence of KD025 (20μM). KD025 attenuates morphological changes (arrows) and preserves TJs in endothelial cells. Asterisks depict areas with degraded TJs. Scale bars = 200μm.</p

ROCK-2 is the primary isoform expressed in cells of the neurovascular unit.

<p>Real-time RT-PCR (<b>A</b>; n = 4 for each cell type) and western blot analysis <b>(B)</b> showing higher steady-state levels of ROCK-1 and similar levels of ROCK-2 (per equal quantities of total RNA and proteins) in unstimulated human SVG astrocytes compared to human brain microvascular endothelial cells (hBECs). <b>(C)</b> Comparative qPCR analysis (by ΔΔCt, normalising to HPRT) in hBECs and SVGs (n = 4 each) demonstrating that ROCK-2 is the principal ROCK transcript expressed in each human cell type. <b>(D)</b> Real-time RT-PCR in equal quantities of RNA from unstimulated primary mouse neurons (mNeuro; n = 4) and primary mouse astrocytes (mAstro; n = 4) relative to primary mouse brain endothelial cells (mBECs; n = 3). mBECs are enriched in ROCK-1 and ROCK-2 transcripts while mouse neurons contains the least. <b>(E)</b> Western blot analysis of ROCK-1 and ROCK-2 protein levels in unstimulated mouse BECs, astrocytes and neurons. <b>(F)</b> Comparative qPCR analysis of ROCK-1 <i>versus</i> ROCK-2 mRNA relative to HPRT within primary mouse BECs (n = 3), astrocytes (n = 4) and neurons (n = 4). The ROCK-2 isoform is dominant also within each of the mouse brain cell types. In all panels bars represent mean±SEM. *<i>P</i><0.05, **<i>P</i><0.01, ****<i>P</i><0.0001 compared to the respective reference column (on the left) by two-tailed unpaired (A) or paired (B, D) t-tests. # <i>P</i><0.05, ## <i>P</i><0.01, ### <i>P</i><0.001, #### <i>P</i><0.0001 (compared to all other groups if unspecified) by one-way ANOVA with Tukey’s post hoc analysis. In panels (B) and (E) the letters A and B represent independent cultures.</p

Selective ROCK-2 inhibition by KD025 blocks rt-PA-induced effects on BBB permeability under stroke-like conditions.

<p><b>(A)</b> Permeability changes in the <i>in vitro</i> human BBB 7.5 h post stimulation under oxygen-glucose deprivation (OGD) with DMSO as control or with rt-PA (25nM) and human plasminogen (plgn; 100nM), with or without KD025 (0.2, 2 and 20μM, added to both luminal and abluminal chambers). Data is presented relative to DMSO control under normoxia. n = 3, Bars represent mean±SEM. *<i>P</i><0.05, **<i>P</i><0.01 compared to rt-PA+Plgn by one-way ANOVA with Newman–Keuls post hoc analysis. #<i>P</i><0.05 compared to DMSO OGD control by two-tailed t-test. <b>(B)</b> Comparison of selective ROCK-2 inhibition by KD025 (20μM) <i>versus</i> non-selective ROCK inhibition by fasudil (HA1077; 20μM) against rt-PA+plasminogen (25nM+100nM, respectively) 6 h and 7.5 h after treatment under OGD (relative to DMSO control under OGD). n = 3 for 6 h, n = 4 for 7.5 h. Data points represent mean±SEM. *<i>P</i><0.05 by one-way ANOVA with Tukey’s post hoc analysis. #<i>P</i><0.05 compared to rt-PA+Plgn by one-tailed paired t-test.</p

Selective ROCK-2 inhibition effectively blocks early, but not late rt-PA-induced increases in BBB permeability under normal conditions.

<p><b>(A, B)</b> Permeability changes in the <i>in vitro</i> human BBB 6 h (<b>A</b>; n = 4) or 24 h (<b>B</b>; n = 4–10) post stimulation under normal conditions with DMSO as control or with rt-PA (25nM) and human plasminogen (plgn; 100nM), with or without KD025 (2 and 20μM, added to both luminal and abluminal chambers). <b>(C)</b> Comparison of selective ROCK-2 inhibition by KD025 (20μM) <i>versus</i> non-selective ROCK inhibition by fasudil (HA1077; 20μM) against rt-PA+plasminogen (25nM+100nM, respectively) 6 h and 24 h after treatment under normal conditions. KD025, but not HA1077, displays strong protective capacity at 6 h, but not at 24 h. n = 3–4. Bars represent mean±SEM. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 by one-way ANOVA with Tukey’s post hoc analysis. # <i>P</i><0.05 compared to rt-PA+Plgn, ## <i>P</i><0.01 compared to DMSO control and specified <i>P</i> values are by two-tailed paired t-test.</p

Selective ROCK-2 inhibition blocks rt-PA-induced morphological changes and ROCK signalling in human and mouse astrocytes.

<p><b>(A)</b> Representative phase-contrast images (top panels) and double immunofluorescence images of the actin cytoskeleton (phalloidin; grey) and nuclei (Hoechst; red) (bottom panels) of SVG human astrocytes demonstrating blockade of morphology changes (arrows) by KD025 after 6 h treatment with DMSO (control) or with rt-PA+plasminogen (t-PA+Plgn; 25nM+100nM), in the presence or absence of KD025 (20μM). Scale bars = 50μm. <b>(B)</b> Representative phase-contrast images of primary mouse astrocytes treated overnight (13–16 h) as stipulated in A. n = 3. Scale bars = 200μm. Arrows depict changes in cell morphology. <b>(C)</b> Representative western blot analysis (left panel) and quantification (right panel) of phosphorylated myosin light chain levels (pMLC per total MLC) in primary mouse astrocytes treated for two hours with rt-PA+Plgn (50nM+100nM, respectively), with or without KD025 (20μM). Ratios obtained in the rt-PA+Plgn group were assigned a value of 100%. KD025 fully abolishes rt-PA- and plasmin-triggered MLC phosphorylation in mouse astrocytes. n = 3. Bars represent mean±SEM. ****<i>P</i><0.0001 compared to all other groups, *<i>P</i><0.05 compared to DMSO control by one-way ANOVA with Tukey’s post hoc analysis.</p

The Influence of Differentially Expressed Tissue-Type Plasminogen Activator in Experimental Autoimmune Encephalomyelitis: Implications for Multiple Sclerosis

<div><p>Tissue type plasminogen activator (t-PA) has been implicated in the development of multiple sclerosis (MS) and in rodent models of experimental autoimmune encephalomyelitis (EAE). We show that levels of t-PA mRNA and activity are increased ~4 fold in the spinal cords of wild-type mice that are mice subjected to EAE. This was also accompanied with a significant increase in the levels of pro-matrix metalloproteinase 9 (pro-MMP-9) and an influx of fibrinogen. We next compared EAE severity in wild-type mice, t-PA^-/- mice and T4+ transgenic mice that selectively over-express (~14-fold) mouse t-PA in neurons of the central nervous system. Our results confirm that t-PA deficient mice have an earlier onset and more severe form of EAE. T4+ mice, despite expressing higher levels of endogenous t-PA, manifested a similar rate of onset and neurological severity of EAE. Levels of proMMP-9, and extravasated fibrinogen in spinal cord extracts were increased in mice following EAE onset regardless of the absence or over-expression of t-PA wild-type. Interestingly, MMP-2 levels also increased in spinal cord extracts of T4+ mice following EAE, but not in the other genotypes. Hence, while the absence of t-PA confers a more deleterious form of EAE, neuronal over-expression of t-PA does not overtly protect against this condition with regards to symptom onset or severity of EAE.</p></div