Genetic and seasonal determinants of vitamin D status in Confederated Salish and Kootenai Tribes (CSKT) participants

Background: Vitamin D is a hormone produced in the skin upon ultraviolet B (UVB) radiation. Vitamin D is a crucial regulator of calcium and phosphate levels for bone mineralization and other physiological roles. Vitamin D levels vary globally in human populations due to genetics, geography, and other demographic factors. It is estimated that 20-85 % of the variability in vitamin D levels is driven by genetic variation. To improve our understanding of contributors to vitamin D levels, we conducted a candidate-gene study in partnership with the Confederated Salish and Kootenai Tribes (CSKT). Methods: We recruited 472 CSKT study participants on the Flathead Reservation in Montana. Demographic factors included age, BMI, and gender (185 male and 287 female; ≥ 18 years old). Genomic DNA and plasma were isolated from whole blood. We sequenced 14 vitamin D regulatory candidate genes: CASR, CUBN, CYP2R1, CYP3A4,CYP24A1, CYP27B1, DHCR7, GC, RXRA, RXRB, RXRG, SULT2A1, UGT1A4, and VDR. We also measured plasma levels of vitamin D and vitamin D metabolites by liquid chromatography/mass-spectrometry (LC/MS), including the clinical marker of vitamin D status, 25-hydroxyvitamin D3 [25(OH)D3]. We tested demographic factors as well as common and rare genetic variants for statistical associations with vitamin D levels using bioinformatics software and R statistical programming language code. Results: We identified 7,370 total genetic variants with 8% (n = 585) of them being novel. We identified 60 genetic variants that may be of clinical significance (disease associated or predicted to influence medication response). Vitamin D levels were below sufficiency [25(OH)D3 + 25(OH)D2 levels \u3c 20 ng/mL] in 56 % of CSKT participants across the year. We observed seasonal vitamin D and metabolite level fluctuations in a seasonal, sinusoidal statistical model with peak concentrations in June – August and trough concentrations in December – February. In linear regression analysis, we found that age, BMI, season, and 5 variants in CUBN and CYP3A4 were significantly associated with 25(OH)D3 concentration (p-value\u3c 0.05). In logistic regression, we found that 4 variants in CUBN, CYP3A4, and UGT1A4 were associated with 25(OH)D sufficiency status [25(OH)D3 + 25(OH)D2 levels of 20 ng/mL] (p-value\u3c 0.05). Multivariate linear regression analysis revealed that genetic variation alone explained ~13% of the variability in 25(OH)D3 concentration in CSKT participants. Genetic variation and environmental factors together explained ~23 % of the variability in 25(OH)D3 concentration in CSKT participants. It is likely that genetic variation in additional genes and other environmental factors (e.g., dietary vitamin D intake) that were not included in this study explain the remaining variability in 25(OH)D3 concentration. Conclusion: This research addresses the need for increased inclusion of American Indian and Alaska Natives in precision medicine health research. We are the first to describe the contribution of season and genetics to vitamin D levels in an American Indian population. Our next steps will be to use these findings to perform mechanistic studies and develop interventional strategies for the CSKT people

Role of Epoxyeicosatrienoic Acids in Protecting Against Ischemic Cardiomyopathy

Thesis (Ph.D.)--University of Washington, 2018Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid (AA) epoxidation with important cardioprotective and signaling properties. AA is a twenty carbon ω-6 polyunsaturated fatty acid containing four cis-double bonds. Like other PUFAs, both free and membrane-bound AA undergo autoxidation in the presence of initiators such as air, light, heat, and transition metal ions. While autoxidation yields both cis- and trans-EETs with preference to the later, cytochrome P450 (CYP) epoxygenases, especially CYP2J2, exclusively catalyze the formation of all regioisomer of cis-EETs. The overall goals of this dissertation were to elucidate the formation of enzymatic and non-enzymatic EETs during P450 incubations and in biological systems and determine the protective role EETs play during ischemic cardiomyopathy. To achieve these goals, we used in vitro systems, a mouse model that overexpresses human CYP2J2 in the heart cardiomyocytes, and tissue from diseased and control human subjects. In in vitro work, formation of EETs in free radical initiated reactions of AA in benzene and in liposomes exhibited time- and AA concentration-dependence and favored the formation of trans-EETs over cis-EETs. Experimental conditions were optimized to minimize non-enzymatic EET formation during P450 reactions by adding pyruvate, a hydrogen peroxide scavenger, and an iron chelating agent. In the in vivo mouse model, stressors such as age and disease altered EET levels in transgenic (Tr) mice. EET levels in erythrocyte membranes increased with age while alterations appeared to be regioisomer-specific for cardiac EETs. Effects of acute ischemic cardiac events were evaluated in both a mouse model and human sudden cardiac arrest (SCA) subjects. Acute myocardial infarction in the mouse model increased both erythrocyte membrane and cardiac tissue cis- and trans-EETs in Tr mice, while human SCA cases had significantly lower EET levels in their erythrocyte membrane compared to the control group. In ventricular cardiac tissue obtained from patients with cardiovascular disease and controls, EET levels were significantly higher in control tissue compared to diseased. Lower EET levels in diseased cardiac tissue were associated with lower protein expression of CYP2J2, NADPH-Cytochrome P450 oxidoreductase and soluble epoxide hydrolase. Finally, specific activity of CYP2J2 in hydroxylating the probe substrate, terfenadine was shown to be significantly decreased with lower levels of CYP2J2, the main cardiac epoxygenase, in diseased cardiac tissue. These results suggest that higher levels of EETs, specifically cis-EETs are cardioprotective, and that erythrocyte membrane of cis-EETs could potentially serve as surrogate markers to report on cis-EET levels in cardiac tissue

Higher Epoxyeicosatrienoic Acids in Cardiomyocytes-Specific CYP2J2 Transgenic Mice Are Associated with Improved Myocardial Remodeling

Elevated cis-epoxyeicosatrienoic acids (EETs) are known to be cardioprotective during ischemia-reperfusion injury in cardiomyocyte-specific overexpressing cytochrome P450 2J2 (CYP2J2) transgenic (Tr) mice. Using the same Tr mice, we measured changes in cardiac and erythrocyte membranes EETs following myocardial infarction (MI) to determine if they can serve as reporters for cardiac events. Cardiac function was also assessed in Tr vs. wild-type (WT) mice in correlation with EET changes two weeks following MI. Tr mice (N = 25, 16 female, nine male) had significantly higher cardiac cis- and trans-EETs compared to their WT counterparts (N = 25, 18 female, seven male). Total cardiac cis-EETs in Tr mice were positively correlated with total cis-EETs in erythrocyte membrane, but there was no correlation with trans-EETs or in WT mice. Following MI, cis- and trans-EETs were elevated in the erythrocyte membrane and cardiac tissue in Tr mice, accounting for the improved cardiac outcomes observed. Tr mice showed significantly better myocardial remodeling following MI, evidenced by higher % fractional shortening, smaller infarct size, lower reactive oxygen species (ROS) formation, reduced fibrosis and apoptosis, and lower pulmonary edema. A positive correlation between total cardiac cis-EETs and total erythrocyte membrane cis-EETs in a Tr mouse model suggests that erythrocyte cis-EETs may be used as predictive markers for cardiac events. All cis-EET regioisomers displayed similar trends following acute MI; however, the magnitude of change for each regioisomer was markedly different, warranting measurement of each individually

Regulation of CYP2J2 and EET Levels in Cardiac Disease and Diabetes

Cytochrome P450 2J2 (CYP2J2) is a known arachidonic acid (AA) epoxygenase that mediates the formation of four bioactive regioisomers of cis-epoxyeicosatrienoic acids (EETs). Although its expression in the liver is low, CYP2J2 is mainly observed in extrahepatic tissues, including the small intestine, pancreas, lung, and heart. Changes in CYP2J2 levels or activity by xenobiotics, disease states, or polymorphisms are proposed to lead to various organ dysfunctions. Several studies have investigated the regulation of CYP2J2 and EET formation in various cell lines and have demonstrated that such regulation is tissue-dependent. In addition, studies linking CYP2J2 polymorphisms to the risk of developing cardiovascular disease (CVD) yielded contradictory results. This review will focus on the mechanisms of regulation of CYP2J2 by inducers, inhibitors, and oxidative stress modeling certain disease states in various cell lines and tissues. The implication of CYP2J2 expression, polymorphisms, activity and, as a result, EET levels in the pathophysiology of diabetes and CVD will also be discussed