Mechanism of direct regulation of cerebral artery contractility by statins

Abstract

Statins are amongst the most widely prescribed drugs in the world with a range of vascular effects that have been primarily attributed to the inhibition of cholesterol and mevalonate biosynthesis, and the inhibition of mevalonate-dependent Rho/ROCK signaling upon long-term treatment. However, no studies have investigated the direct effects of acute statin application on fresh isolated resistance cerebral arteries using therapeutic concentrations of statins. We examined acute vascular effects of therapeutically relevant concentrations (0.01-10nM) of rosuvastatin and simvastatin on Sprague Dawley rat cerebral arteries and underlying molecular mechanisms. Cannulated arterial segments were maintained at 37°C in a perfusion chamber and intraluminal pressure slowly increased to 60 mmHg. For calcium imaging, the isolated arteries were preincubated in Fura-2AM (10μM) and 0.02% Kolliphor solution at room temperature for two hours before mounting in the perfusion chamber. At 60mmHg, cerebral arteries developed ~36% myogenic tone, after which increasing concentrations of statins were applied. Our data showed that the application of 1nM rosuvastatin and simvastatin constricted cerebral arteries by ~26 μm and ~24μm, respectively, within 2-3 minutes of drug application. Such statin-induced vasoconstriction remained unaltered upon endothelium denudation (intact ~23μm vs denuded ~25μm), suggesting an endothelium-independent mechanism. Co-application of mevalonate did not alter the vasoconstriction either (control ~28μm vs mevalonate ~29 μm), indicating that the effect is HMG-CoA reductase-independent. However, removal of extracellular Ca2+ with EGTA or the application of nimodipine, a selective blocker of smooth muscle cell voltage-gated Ca2+ channel, CaV1.2, each abolished cerebral artery vasoconstriction by statins, indicating that the Ca2+ entry through CaV1.2 plays a critical role here. Since Ca2+ entry into smooth muscle cells induces Ca2+ release from intracellular Ca2+ stores such as sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER), we next examined the role of these Ca2+ release pathways. We found that co-application of ryanodine, a blocker of ryanodine receptor-mediated Ca2+ release, had no effects on statin-induced constriction. In contrast, statin-evoked vasoconstriction of cerebral arteries was significantly attenuated upon co-application of thapsigargin, a blocker of SR/ER membrane Ca2+-ATPase pump (SERCA), highlighting the involvement of thapsigargin-sensitive Ca2+ stores in regulating [Ca2+]i and vasoconstriction. Simultaneous measurement of arterial Ca2+ fluorescence and diameter further confirmed the involvement of CaV1.2 channel in mediating Ca2+ entry and subsequent Ca2+ release, leading to cerebral artery vasoconstriction. Altogether, our data suggests that smooth muscle cell CaV1.2 opening and Ca2+ influx is the primary mechanism underlying statin-induced constriction of cerebral arteries.