Abstract
Background and PurposeBeta adrenergic overstimulation may increase the vascular damage and stroke. However, the underlying mechanisms of beta adrenergic overstimulation in cerebrovascular dysfunctions are not well known. We investigated the possible cerebrovascular dysfunction response to isoproterenol induced beta-adrenergic overstimulation (ISO) in rabbit cerebral arteries (CAs).MethodsISO was induced in six weeks aged male New Zealand white rabbit (0.8–1.0 kg) by 7-days isoproterenol injection (300 μg/kg/day). We investigated the alteration of protein expression in ISO treated CAs using 2DE proteomics and western blot analysis. Systemic properties of 2DE proteomics result were analyzed using bioinformatics software. ROS generation and following DNA damage were assessed to evaluate deteriorative effect of ISO on CAs. Intracellular Ca2+ level change and vascular contractile response to vasoactive drug, angiotensin II (Ang II), were assessed to evaluate functional alteration of ISO treated CAs. Ang II-induced ROS generation was assessed to evaluated involvement of ROS generation in CA contractility.ResultsProteomic analysis revealed remarkably decreased expression of cytoskeleton organizing proteins (e.g. actin related protein 1A and 2, α-actin, capping protein Z beta, and vimentin) and anti-oxidative stress proteins (e.g. heat shock protein 9A and stress-induced-phosphoprotein 1) in ISO-CAs. As a cause of dysregulation of actin-cytoskeleton organization, we found decreased level of RhoA and ROCK1, which are major regulators of actin-cytoskeleton organization. As functional consequences of proteomic alteration, we found the decreased transient Ca2+ efflux and constriction response to angiotensin II and high K+ in ISO-CAs. ISO also increased basal ROS generation and induced oxidative damage in CA; however, it decreased the Ang II-induced ROS generation rate. These results indicate that ISO disrupted actin cytoskeleton proteome network through down-regulation of RhoA/ROCK1 proteins and increased oxidative damage, which consequently led to contractile dysfunction in CA.
Highlights
Background and PurposeBeta adrenergic overstimulation may increase the vascular damage and stroke
Through annotation and categorization of identified proteins, we found that modulated proteins were widely associated with regulation of cytoskeletons (Figure 1C and D)
As for the causes of contractile dysfunction, we found that induced beta-adrenergic overstimulation (ISO) treatment significantly reduced angiotensin II (Ang II)–induced [Ca2+]i transient peak (Figure 6C and D) and prolonged [Ca2+]i elevation (Figure 6E and F and Figure S5B) and reactive oxygen species (ROS) generation rate (Figure 6G and Figure S5C)
Summary
ISO was induced in six weeks aged male New Zealand white rabbit (0.8–1.0 kg) by 7-days isoproterenol injection (300 mg/kg/day). We investigated the alteration of protein expression in ISO treated CAs using 2DE proteomics and western blot analysis. Review Board of Animals, Inje University College of Medicine (approval number: 2011-062). Animals Six weeks aged, male New Zealand white rabbits (0.8–1.0 kg) were purchased from the Orient Bio Inc. After a 7-day administration, isoproterenol-induced bAR stimulation (ISO) on model animal was evaluated by measuring the heart-to-body weight ratio and blood pressure as previously described [8,9]. None of isoproterenol injected rabbit was dead before sacrifice
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