Effects of Ile164 Polymorphism of Beta2-Adrenergic Receptor Gene on Coronary Artery Disease
Federico Piscione, MD*,
Guido Iaccarino, MD,
Gennaro Galasso, MD,
Ersilia Cipolletta, MD,
Maria Assunta Rao, MD,
Gregorio Brevetti, MD,
Raffaele Piccolo, MD,
Bruno Trimarco, MD and
Massimo Chiariello, MD
Department of Clinical Medicine, Cardiovascular and Immunology Sciences, Federico II University School of Medicine, Naples, Italy
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Figure 1 Observed Event-Free Survival Curve at Follow-Up
Patients harboring the ThrIle164 allele of the beta2-adrenergic receptor gene (group 2) had a higher incidence of major adverse cardiac events with reduced event-free follow-up time as indicated by the Kaplan-Meier analysis.
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Figure 2 Carotid Ultrasound Analysis
Patients harboring the ThrIle164 allele showed a diffuse carotid atherosclerotic disease with a significantly higher incidence of atherosclerotic plaque. (A) In accordance with a more severe atherosclerotic carotid disease, intima-media thickness (IMT) was significantly higher in ThrIle164 patients. (B) Plaque composition was analyzed with integrated backscatter (IBS) ultrasound technique. Patients with Thr164Thr polymorphism presented lesions with a significantly lower IBS value in the carotid artery as an index of different plaque composition between groups with a higher presence of "soft tissue" in ThrIle164 patients. Values are reported as mean ± SD.
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Figure 3 In Vitro Effects of B2AR Stimulation on VSMC Biology
Panel A shows the proliferative response in subconfluent vascular smooth muscle cells (VSMCs). Adenoviral beta2-adrenergic receptor–wild type (Ad B2AR-WT) (open circles) but not the Ad B2AR-Ile164 mutation (in which isoleucine is located at position 164) (open triangles) produced a proliferative response (*p < 0.05 vs. control [CTR]; analysis of variance [ANOVA], with Bonferroni post-hoc test). Isoproterenol (ISO) (10–7 mol/l) caused an increase of cell numbers over time (solid symbols). Panel B shows deoxyribonucleic acid (DNA) synthesis by [3H]-thymidine incorporation. The activation of B2AR by ISO (10–7 mol/l) caused increased DNA synthesis. The same effect was obtained in Ad B2AR-WT but not in Ad B2AR-Ile164. The maximal response was observed in ISO-stimulated Ad B2AR-WT cells (*p < 0.05 vs. no ISO; #p < 0.05 vs. control; ANOVA, with Bonferroni post-hoc test; n = 3 in duplicate). Panels C and D show the progression in cell cycle assessed by retinoblastoma protein (Rb) phosphorylation. This protein regulates cell-cycle progression through the restriction point within the G1 phase. By Western blot at 24 h of stimulation, ISO (10–7 mol/l), caused Rb phosphorylation (P-Rb). Equal amount of proteins were confirmed via blotting for actin. Densitometric analysis (bar graph) shows that B2AR stimulation caused Rb activation, and Ad B2AR-WT treatment induced Rb activation. The ISO (10–7 mol/l) response was enhanced. These amounts were measured in arbitrary densitometry units (ADU); *p < 0.05 versus control; #p < 0.05 versus the ThrIle164 polymorphism (threonine is replaced by isoleucine at position 164) ANOVA, with Bonferroni post-hoc test (n = 3 in duplicate). Panels E and F show extracellular signal-related kinase/mitogen-activated protein kinase (ERK/MAPK) activation. Western blot of activated phosphorylated ERK (pERK) after ISO treatment. Equal amount of proteins were confirmed via blotting for total ERK. Representative blots are presented in the inset. Densitometric analysis (bar graph) shows that B2AR stimulation caused ERK/MAPK activation, and that Ad B2AR-WT treatment induced ERK activation. The ISO (10–7mol/l) response was enhanced (*p < 0.05 vs. no ISO; #p < 0.05 vs. basal ANOVA, with Bonferroni post-hoc test; n = 3 in duplicate).
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