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Risk factors for stroke – On pathways involved in vascular receptor regulation by tobacco and smoking

Hardip Sandhu

Summary

Background:Treatment of stroke is a severe challenge for the scientific community, because it is associated withconsiderable morbidity and mortality, and there is no specific treatment. Much investigation into thevascular events needs to be done, in order to advance effective treatment. Stroke incidents arediviede into two groups: thrombo-embolic and haemorrhagic. Thrombo-embolic stroke is the mostcommen type, which is caused by the development of blood clot blockage in a cerebral artery. Thepatient group consists mainly of elderly people. Intracerebral and subarachnoid haemorrhages occurless frequent. Where intracerebral haemorrhage is caused by traumatic or spontaneous braininjuries, subarachnoid haemorrhage occurs often spontaneously by rupture of an aneurysm andbleeding into the subarachnoid space.Risk factors strongly contribute to the development of stroke, and the major risk factors consist ofhypertension, cigarette smoke, and high level of low-density lipoprotein (LDL) in the blood. Theserisk factors change the physiology of the vasculature. The vascular tone of vessels is maintainedthrough a balanced function of the endothelium and smooth muscle cells (SMC). Roughly speaking,the endothelium is responsible for the dilatation and the SMC for the contraction of vessels. Riskfactors can change the function of these vessel parts, resulting in increased vascular contractility.Vessels can mediate contraction through G-protein coupled receptors (GPCR) found in SMC,namely endothelin type A and B receptors (ETA and ETB), serotonin 5-HT1B receptor, andprostanoid TP receptor. Studies have shown that these GPCR’s are expressed as a result of mitogenactivatedprotein kinases (MAPK) pathways activation in both stroke and experimental ischemicmodels.

Hypothesis:Alteration in expression of GPCR in vascular SMC is mediated through MAPK. Blockage of thespecific kinases of MAPK pathways can attenuate the GPCR changes. Cigarette smoke particleshave a harmful effect on the vasculature through GPCR upregulation. Inhibition of the specificMAPKs will reveal what pathway(s) are involved. Swedish moist snus and nicotine also alter theexpression of GPCR.

Method:Organ culture of rat cerebral vessels was used as a model to investigate the expression of GPCR inthe presence of cigarette smoke, Swedish moist snus, or nicotine. Organ culture is an in vitromethod to induce changes in GPCR expression in a pattern seen in vivo. Specific MAPK blockerswere included to examine which MAPK that are involved. Functional studies were performed inmyograph baths, and the results were verified by real time PCR and immunohistochemistry.

Results:Study 1: Organ culture induced upregulation of ETB and 5-HT1B receptors at transcriptional andtranslational levels respectively, and thereby increased their contractions. The TP receptor mediatedcontraction curves was left-wards shifted by organ culture. Administration of MEK1/2 inhibitorU0126 – even 6 h after start of incubation - blocked the organ culture induced elevated receptorcontractions: the ETB receptor mediated contraction was attenuated by U0126 at post-translationallevel or by changing the receptor affinities, and the 5-HT1B and TP receptor protein levels werelowered. Study 2 and 3: Lipid-soluble smoke particles upregulated ETB and TP receptors ontranslational level. The increase in smoke particle mediated ETB and TP receptor contraction wasabolished by U0126. The JNK inhibitor SP600125 also attenuated the smoke particle mediatedupregulation of ETB contraction. Activation of MEK and JNK MAPK-mediated transcription andtranslation resulted in production of new contractile ETB and TP receptors. Study 4: Organ culturewith normal plasma level of nicotine of WSS or DSS lowered the 5-HT1B receptor mediatedcontraction, and in addition lipid-soluble snus shifted the TP receptor mediated contraction curveleft-wards. High dose of nicotine increased the ETB receptor mediated contraction. The 5-HT1Band 5-HT2A receptor mediated contraction was increased and both the 5-HT1B and 5-HT2A andthe TP receptor mediated contraction curves were left-ward shifted by high dose of water- or lipidsolublesnus and nicotine. Only the lipid-soluble snus group showed that the increase of 5-HT1Breceptor mediated contraction was at the transcriptional level.

Conclusions:The MAPK-mediated upregulation of contractile GPCR in cerebral arteries might be apharmacological target for the treatment of cardiovascular diseases and smoke-associated cerebralvascular disease like stroke. Even snus and nicotine alters the GPCR. Given the ability to inhibitGPCR alteration’s at the clinically relevant time-point 6 h post incubation, U0126 makes anattractive therapeutic agent for in vivo studies.