Special Report: Searching for a novel stroke therapy strategy
Acute post-stroke treatment in male rats with the MEK inhibitor U0126 or Raf inhibitor SB386023-b. A) Reduction in infarct damage after transient middle cerebral artery occlusion (MCAO), model of focal ischemic stroke; B) Increased cerebral blood flow after experimental subarachnoid hemorrhage (SAH); Increased survival after transient global forebrain ischemia .
It is important to consider plasticity in the vascular walls following cerebral ischaemia in order to reduce the final brain tissue damage after a stroke because of four decades of failure in neuroprotection strategy. The first paradigm shift to consider is that stroke is a vascular disease and that focus should be directed towards understanding the changes that occur in the vessel wall following a stroke. Below is a sample of key experiments designed to find out the details of the regulation of the cerebrovascular smooth muscle in the situation of a stroke. By understanding these, the brain tissue damage that occurs after both global and focal ischaemia can be reduced; this is a huge paradigm shift in our view on how to help the patients that suffer an acute stroke.
Expressional changes in cerebrovascular receptors after experimental transient forebrain ischaemia
By inducing global frontal lobe ischaemia by a two-vessel occlusion (both common carotid arteries) combined with hypotension – lowering of blood pressure for up to 15 minutes (model of heart stop consequences) – we found that there is marked upregulation of receptors in MCA/ACA (verified by myograph, protein and qPCR) and this is related to neurology damage. These papers have recently been published (Johansson, PLoS One 2012, 2014; Acta Physiologica 2015).
Early MEK1/2 inhibition after global cerebral ischaemia in rats reduces brain damage and improves outcome by preventing delayed vasoconstrictor receptor upregulation. The excessive cerebrovascular expression of contractile receptors correlates with delayed global cerebral ischaemia and likely contributes to the development of neuronal damage. The enhanced cerebrovascular contractility can be prevented by treatment with an MEK1/2 inhibitor which diminishes neuronal damage and improves survival rate. In this method, 50% of vehicle-treated rats had spontaneously died at day seven, while those treated with MEK1/2 inhibitor survived. This suggests a way to treat neurological consequences of global cerebral ischaemia.
MAPK signalling pathways regulate cerebrovascular receptor expression in human cerebral arteries
During the past year we have analysed in detail the effects of different inhibitors of the Raf/MEK/ERK pathway on receptor upregulation in human brain arteries removed in conjunction with tumour or epilepsy operations. The main hypothesis is to understand if the same mechanisms are operating in brain arteries of rat and Man.
The first question has now been published in the manuscript ‘MAPK signalling pathway regulates cerebrovascular receptor expression in human cerebral arteries’ (Ansar, BMC Neurosci 2013). The study demonstrates that there is a clear association between human cerebrovascular receptor upregulation and MAPK transcription. Inhibition of this pathway attenuated the vasoconstriction mediated via endothelin, angiotensin and thromboxane receptors and their molecular expression.
The second question that is being addressed is ‘Male-female differences in upregulation of vasoconstrictor responses in human cerebral arteries’ (Ahnstedt, PLoSOne 2013). The work suggests that the organ culture of human male and female cerebral arteries shows upregulation at mRNA and protein levels, but the enhanced responses to receptor activation are blunted in females when compared to males. This is indeed the first paper to show gender differences in vascular plasticity in human brain vessels. The reason behind the difference in gender receptor coupling is still elusive and under future analysis. However, we have just published that the MEK1/2 inhibition works well in both male and female rats to reduce brain damage after middle cerebral occlusion followed by reperfusion (MCAO) (Ahnstedt, J Cereb Blood Flow Metab 2015).
Role of MAPK signalling in expression of pro-inflammatory molecules in vessels and brain tissue after subarachnoid haemorrhage (SAH)
We have found that there is upregulation of pp38 and pJNK with a somewhat later time course (>24h) than that seen for pERK1/2 (Ansar & Edvinsson, Stroke 2007). This suggests that the inflammation part and the leakiness of the blood-brain barrier will occur at a somewhat later time point than the early activation of MEK1/2. Thus, we are analysing these aspects and follow the changes over time.
It is a well-known fact that after a major SAH, delayed cerebral ischaemia (DCI) is much feared and carries high morbidity and mortality due to the associated much reduced cerebral blood flow, receptor upregulation and the poor neurology outcome. We have reviewed this topic fairly recently (Edvinsson & Povlsen, J Cereb Blood Flow Metab 2011). We have found that the DCI is associated with MAPK upregulation of receptor expression. In an early study we revealed that MEK inhibition can blunt this response and improve the outcome measures (Larsen, J Neurosurg. 2011). In recent studies we aim to analyse further different upstream molecules of MEK to add specificity. In particular we have focused on focal adhesion kinase, which may be the first signal recorded following changes in shear stress that elicit the MAPK activation (Parker, J Cereb Blood Flow Metab 2013).
The role of the inflammation markers in the upregulation process has been addressed in two methods. Using the organ culture method we have examined the effects of exogenous cytokines on the 24h upregulation (Ahnstedt, Acta Physiol. 2012). The receptor upregulation responses were only to a minor extent modified. In vivo (Maddahi, J Neuroinflammation 2012) showed that cytokines are upregulated in the vessel walls after SAH and this is sensitive to MEK1/2 inhibition. We have sampled vessels and brain tissue from the other global ischaemia model: 2-vessel occlusion and hypovolaemia (currently under study).
The third question has been solved in the manuscript ‘MEK1/2 inhibitor but not the endothelin receptor antagonist Clazosentan reduces upregulation of cerebrovascular contractile receptors and delayed cerebral ischaemia after subarachnoid haemorrhage in rats’ (Povlsen and Edvinsson, J Cereb Blood Flow Metab 2015). This provides an explanation of the entire clinical failure of the Clazosentan programme and urges a closer look at the mechanisms involved in the cerebral vascular plasticity principle for novel SAH therapy.
On the miRNA profiling in stroke
In order to find out if there are possibilities to clinically pick up a signal that might reveal aspects of gene regulation that occur after a stroke, analysing miRNA aspects in experimental models of stroke is a future trend. The miRNAs are small, endogenous RNAs that have been shown to change in expression fast after disease onset; they are very stable, have a high conservation between species, and are often secreted from the primary tissue to the blood.
The aim of this project is to identify blood-borne miRNA biomarkers of changes in the cerebral vessels after global and focal stroke. The miRNAs are extracted from the cerebral vessels, cerebrospinal fluid and serum. A qPCR screen of 750 known rat miRNAs revealed that only a select group of miRNAs are regulated in the cerebral vessel walls. The number in the serum is by far numerous. The results have been validated, and the function of some specific miRNAs will be examined (Muller, BMC Genomics 2015). This may truly be a future avenue in stroke research.
Professor Lars Edvinsson
Chairman
Department of Medicine
Lund University
tel: +46 7032 71484
