Enhanced Vasoconstriction in Sickle Cell Disease is Mediated by ET A Receptor‐Dependent Induction of alpha 1A ‐Adrenergic Receptor Expression

Abstract

Sickle cell disease (SCD) patients are at a significant risk for developing the potentially fatal vaso-occlusive crisis, which has been associated with stressful stimuli. We previously reported that SCD mice display enhanced alpha-1 adrenergic receptor mediated vasoconstriction along with increased alpha-1A receptor expression in the aorta. Also, we have demonstrated SCD mice have higher levels endothelin-1 (ET-1), a potent vasoconstrictor mediating actions via the ETA receptor as well as increased reactive oxygen species (ROS). The mechanisms underlying the enhanced vasoconstriction in SCD mice remains largely unknown. We hypothesized that the enhanced vasoconstrictor response to alpha-1 adrenergic receptor stimulation is meditated through an ET-1/ETA receptor and ROS axis. Male 16-week-old humanized SCD (HbSS) and genetic control (HbAA) mice were utilized for all experiments. Vascular reactivity of isolated thoracic aortic (100–150 μm diameter) rings to phenylephrine (PE) and potassium chloride (KCl) were examined using wire myography. Mice were pre-treated for two weeks with the specific ETA receptor antagonist, ambrisentan (AMB). Thoracic aortic rings were also pre-incubated with superoxide dismutase-polyethylene glycol (PEG-SOD) for 30 minutes ex vivo at 100 units/ml. HbSS mice treated with AMB demonstrated a significantly blunted maximum vasoconstrictive response to PE when compared to control HbSS mice (132.3 ± 3.69 vs. 113.5 ± 4.59 %KCl, p=0.003). HbSS mice treated with ex vivo PEG-SOD alone did not demonstrate a blunted maximum vasoconstrictive response to PE stimulation (132.3 ± 3.69 vs. 121.6 ± 5.36 %KCl, p>0.05). However, HbSS mice treated with a combination of AMB and ex vivo PEG-SOD demonstrated an overall significant decrease response to PE stimulation compared to HbSS control and HbSS treated with AMB alone (132.3 ± 3.69 vs. 113.5 ± 4.59 vs. 95.6 ± 11.3 %KCl, p=0.006, p=0.015, respectively). HbAA mice showed similar vasoconstrictive response across treatments (84.9 ± 5.38 vs. 78.2 ± 6.89 vs. 75.1 ± 9.92 %KCl, p>0.05). Graded concentration responses to KCl were similar between genotypes and pharmacologic treatment. To demonstrate the translational nature of this enhanced vasoconstriction response, flow mediated dilation was used in human subjects with SCD. A subset of the human subjects were treated with AMB or placebo for 3 months. SCD patients treated with AMB for 3 months did show a significant increase in baseline diameter compared to placebo (1.54 % vs -2.05 % change in diameter, p = 0.012). However, there was no increase in flow-mediated dilation supporting a role for the ET-1/ETA pathway and an enhanced vasoconstrictor response in SCD in humans. These findings suggest the enhancement of alpha-1 adrenergic mediated vasoconstriction is, in part, mediated through an ETA receptor dependent mechanism. These data suggest that reactive oxygen species contribute to this enhancement and may work synergistically with the ETA receptor pathway to produce the enhanced vasoconstrictive phenotype seen in SCD.