Cytochrome P450 (CYP) epoxygenases catalyze endothelial EET biosynthesis. These potent vasodilators are rapidly hydrolyzed by sEH, and pharmacological inhibition of sEH potentiates the effect of EETs.1,2,4
This study identified a significant association between functional genetic variants in EPHX2
, the gene which encodes sEH, and vascular function in humans. Specifically, the Arg55 variant allele was associated with significantly lower FBF and higher FVR in response to vasodilators in white Americans, while the Gln287 variant allele was associated with higher FBF and lower FVR at baseline and in response to vasodilators in black Americans. Similar genotype-phenotype relationships were observed in response to bradykinin, methacholine and sodium nitroprusside, suggesting that the mechanism underlying the functional link between sEH and forearm vascular responsiveness in humans is both bradykinin receptor-and endothelium-independent. Collectively, these data demonstrate a potentially important role for sEH in the regulation of vascular function in humans, and offer important mechanistic insight into previously reported associations between the EPHX2
Lys55Arg and Arg287Gln polymorphisms and cardiovascular and cerebrovascular disease risk.
The nonsynonymous Lys55Arg polymorphism exhibits higher sEH metabolic activity in vitro14,15
and in vivo
and has been associated with higher risk of coronary artery disease and ischemic stroke events in individuals of European ancestry.11,12
Consistent with higher sEH metabolic activity and EET hydrolysis, white American carriers of the Arg55 variant allele exhibited lower vasodilation responses compared to wild-type individuals. Lower vasodilation responses were also observed in carriers of the Arg55-tagged haplotype, suggesting that Lys55Arg is the functional allele driving these associations. We cannot rule out, however, that Lys55Arg is simply a marker in linkage disequilibrium with the true causative locus. In contrast, there was no association between Lys55Arg genotype and either resting or agonist-stimulated changes in FBF or FVR in black American subjects. Although the mechanism underlying the racial differences in this genotype-phenotype relationship is not known, these findings are consistent with the prior observation that Lys55Arg genotype was associated with sEH metabolic activity in vivo
and cardiovascular disease risk in white, but not black, Americans enrolled in the Atherosclerosis Risk in Communities (ARIC) study.11
Multiple studies have demonstrated that the Arg287Gln polymorphism exhibits significantly lower sEH metabolic activity and EET hydrolysis in vitro
although its functional impact on sEH metabolic activity in vivo
remains unknown. Epidemiological studies provide inconsistent data regarding the presence and direction of a significant relationship between the Arg287Gln polymorphism and risk of cardiovascular and cerebrovascular events.8–13,17
Consistent with the vascular protective effects associated with sEH inhibition in preclinical models,4
the Gln287 variant allele was recently associated with a significantly lower risk of ischemic stroke in a Chinese population.8
In contrast, the Gln287 variant allele was associated with a higher prevalence and extent of coronary artery calcification in black Americans9
and insulin resistance in Japanese type 2 diabetics.23
Similarly, in contrast to the neuronal protective effects conferred by the Gln287 variant allele in vitro
an association with significantly higher risk of ischemic stroke was reported in a white European population.10
No association with coronary artery disease and ischemic stroke risk has been observed in other white or black American11,13
and European populations.12,17
Collectively, these conflicting data demonstrate that the functional relevance of the Arg287Gln polymorphism in humans remains unclear and requires further study.
Consistent with the hypothesis that decreased endogenous sEH metabolic activity enhances the vasodilator effects of EETs, Gln287 variant allele carriers demonstrated higher FBF and lower FVR under basal and agonist-stimulated conditions compared to wild-type individuals in the current investigation. These data are also consistent with the observation that administration of pharmacological inhibitors of sEH cause vasodilation in preclinical models and isolated human vessels.4–6
The observed relationship between the Arg287Gln polymorphism and vasodilator responses was more pronounced in black American subjects compared to the white American subjects studied. Although the current study was underpowered to investigate genotype-by-genotype interactions in each racial group, an interaction between the Lys55Arg and Arg287Gln polymorphisms appears unlikely to account for the observed racial differences.
The Lys55Arg and Arg287Gln polymorphisms were associated with altered vasodilation responses to both bradykinin and methacholine. These endothelium-dependent vasodilators cause vascular relaxation in part by stimulating the endothelial formation of EETs, which subsequently hyperpolarize vascular smooth muscle.2
Somewhat unexpectedly, these functional variants in EPHX2
also affected the endothelium-independent vasodilator response to sodium nitroprusside. Although it is well-established that endothelial-derived EETs act as paracrine mediators of vasodilation,2 EPHX2
deletion and pharmacological sEH inhibition also attenuate vascular remodeling in preclinical models by potentiating the anti-proliferative and anti-inflammatory effects of EETs.24,25
Our findings suggest that chronic inter-individual differences in EET exposure based on Lys55Arg and Arg287Gln genotype could contribute to physiological differences in forearm vascular responsiveness via an effect on vascular remodeling. An important limitation of our work, however, is the lack of functional data demonstrating that Arg55 and Gln287 variant allele carriers, respectively, exhibit higher and lower EET hydrolysis in vivo
. Further studies will be necessary to characterize the direct effects of exogenous EET administration and sEH inhibition on vasodilation and vascular remodeling in humans, and define the underlying mechanisms. Moreover, although in vitro
studies have demonstrated that the Arg55 and Gln287 variant alleles elicit lower and higher sEH phosphatase activity, respectively,26,27
the potential contribution of sEH phosphatase activity to the association between EPHX2
polymorphisms, vascular function, and cardiovascular and cerebrovascular risk remains unknown and requires further investigation.
This study examined the vascular effects of EPHX2
polymorphisms in one of the largest cohorts that directly measured vasodilation responses to infused agonists. Nevertheless, the relatively small sample size represents a limitation since we may have been underpowered to detect an association, particularly in the black Americans studied. Moreover, due to the very low number of homozygous Arg55 and Gln287 carriers, we were only powered to characterize these relationships using a dominant genetic model of inheritance. In the bradykinin studies, there was excellent power to detect a 30% difference in FVR across the Lys55Arg (β=0.97) and Arg287Gln (β=0.94) genotype groups in the white American subset. The power to detect these differences in the black American subset was considerably less (Lys55Arg: β=0.67; Arg287Gln: β=0.52). We also made multiple comparisons and there is a possibility of false positive associations. In order to account for this possibility, we calculated a FDR q
-value for each comparison. Although no gold-standard q
-value threshold has been established to identify “true” associations, incorporation of this statistical approach into candidate gene association studies has become an increasingly recognized method to account for multiple comparisons and enhance confidence in observed associations.28
Since all q
-values were estimated to be <0.08, we have a higher level of confidence in our reported findings. Importantly, validation in a well-powered, independent population will be ultimately necessary to confirm presence of a functional relationship between genetic variation in EPHX2
and vasodilator responses in humans.