Effect of Age and Allele Variants of CYP3A5, CYP3A4, and POR Genes on the Pharmacokinetics of Cyclosporin A in Pediatric Renal Transplant Recipients From Serbia.

BACKGROUND: The interindividual variability of cyclosporin A (CsA) pharmacokinetics might be explained by heterogeneity in the cytochrome P450 3A (CYP3A) subfamily. Altered CYP3A enzyme activity was associated with variant allele of P450 oxidoreductase gene (POR*28). The aim of this study was to assess the impact of age, CYP3A5*3, CYP3A4*22, and POR*28 alleles on CsA pharmacokinetics in pediatric renal transplant recipients. METHODS: Renal transplant patients receiving CsA (n = 47) were genotyped for CYP3A5*3, CYP3A4*22, and POR*28. RESULTS: CYP3A5 nonexpressers had higher overall dose-adjusted predose concentration (C0/dose; ng/mL per mg/kg) compared with expressers (31.48 ± 12.75 versus 22.44 ± 7.12, P = 0.01). CYP3A5 nonexpressers carrying POR*28 allele had a lower overall dose-adjusted concentration (C2/dose) than those with POR*1/*1 genotype (165.54 ± 70.40 versus 210.55 ± 79.98, P = 0.02), with age as covariate. Children aged 6 years and younger had a lower overall C0/dose (18.82 ± 4.72 versus 34.19 ± 11.89, P = 0.001) and C2/dose (106.75 ± 26.99 versus 209.20 ± 71.57, P < 0.001) compared with older children. Carriers of CYP3A5*3 allele aged ≤6 years required higher dose of CsA and achieved lower C0/dose and C2/dose, at most time points, than older carriers of this allele. Carriers of POR*28 allele aged ≤6 years required higher doses of CsA, whereas they achieved lower C0/dose and C2/dose, at most time points, in comparison to older carriers of this allele. The significant effect of age (P < 0.002) and CYP3A5 variation (P < 0.02) was shown for overall C0/dose, whereas age (P < 0.00001) and POR variation (P = 0.05) showed significant effect on C2/dose. Regression summary for overall C2/dose in patients aged 6 years younger showed a significant effect of both CYP3A5 and POR variations (P < 0.016). CONCLUSIONS: Younger age, POR*28 allele, and CYP3A5*3 allele were associated with higher CsA dosing requirements and lower concentration/dose ratio. Pretransplant screening of relevant polymorphisms in accordance with age should be considered to adjust therapy.

A vascularized breast cancer spheroid platform for the ranked evaluation of tumor microenvironment-targeted drugs by light sheet fluorescence microscopy.

Targeting the supportive tumor microenvironment (TME) is an approach of high interest in cancer drug development. However, assessing TME-targeted drug candidates presents a unique set of challenges. We develop a comprehensive screening platform that allows monitoring, quantifying, and ranking drug-induced effects in self-organizing, vascularized tumor spheroids (VTSs). The confrontation of four human-derived cell populations makes it possible to recreate and study complex changes in TME composition and cell-cell interaction. The platform is modular and adaptable for tumor entity or genetic manipulation. Treatment effects are recorded by light sheet fluorescence microscopy and translated by an advanced image analysis routine in processable multi-parametric datasets. The system proved to be robust, with strong interassay reliability. We demonstrate the platform's utility for evaluating TME-targeted antifibrotic and antiangiogenic drugs side-by-side. The platform's output enabled the differential evaluation of even closely related drug candidates according to projected therapeutic needs.

Polyphenol-Rich Aronia melanocarpa Juice Consumption Affects LINE-1 DNA Methylation in Peripheral Blood Leukocytes in Dyslipidemic Women.

Cardiovascular disease (CVD) is associated with alterations in DNA methylation and polyunsaturated fatty acid (PUFA) profile, both modulated by dietary polyphenols. The present parallel, placebo-controlled study (part of the original clinical study registered as NCT02800967 at www.clinicaltrials.gov) aimed to determine the impact of 4-week daily consumption of polyphenol-rich Aronia melanocarpa juice (AMJ) treatment on Long Interspersed Nucleotide Element-1 (LINE-1) methylation in peripheral blood leukocytes and on plasma PUFAs, in subjects (n = 54, age range of 40.2 ± 6.7 years) at moderate CVD risk, including an increased body mass index, central obesity, high normal blood pressure, and/or dyslipidemia. The goal was also to examine whether factors known to affect DNA methylation (folate intake levels, MTHFR C677T gene variant, anthropometric and metabolic parameters) modulated the LINE-1 methylation levels upon the consumption of polyphenol-rich aronia juice. Experimental analysis of LINE-1 methylation was done by MethyLight method. MTHFR C677T genotypes were determined by the polymerase chain reaction-restriction fragment length polymorphism method, and folate intake was assessed by processing the data from the food frequency questionnaire. PUFAs were measured by gas-liquid chromatography, and serum lipid profile was determined by using Roche Diagnostics kits. The statistical analyses were performed using Statistica software package. In the comparison after vs. before the treatment period, in dyslipidemic women (n = 22), we observed significant decreases in LINE-1 methylation levels (97.54 ± 1.50 vs. 98.39 ± 0.86%, respectively; P = 0.01) and arachidonic acid/eicosapentaenoic acid ratio [29.17 ± 15.21 vs. 38.42 (25.96-89.58), respectively; P = 0.02]. The change (after vs. before treatment) in LINE-1 methylation directly correlated with the presence of MTHFR 677T allele, average daily folate intake, and the change in serum low-density lipoprotein cholesterol but inversely correlated with the change in serum triacylglycerols (R = 0.72, R 2 = 0.52, adjusted R 2 = 0.36, P = 0.03). The current results imply potential cardioprotective effects of habitual polyphenol-rich aronia juice consumption achieved through the modifications of DNA methylation pattern and PUFAs in subjects at CVD risk, which should be further confirmed. Hence, the precision nutrition-driven modulations of both DNA methylation and PUFA profile may become targets for new approaches in the prevention of CVD.

Fructose-rich diet differently affects angiotensin II receptor content in the nucleus and a plasma membrane fraction of visceral adipose tissue.

The adipose tissue renin-angiotensin system (RAS) is proposed to be a pathophysiological link between adipose tissue dysregulation and metabolic disorders induced by a fructose-rich diet (FRD). RAS can act intracellularly. We hypothesized that adipocyte nuclear membranes possess angiotensin receptor types 1 and 2 (AT1R and AT2R), which couple to nuclear signaling pathways and regulate oxidative gene expression under FRD conditions. We analyzed the effect of consumption of 10% fructose solution for 9 weeks on biochemical parameters, adipocyte morphology, and expression of AT1R, AT2R, AT1R-associated protein (ATRAP), NADPH oxidase 4 (NOX4), matrix metalloproteinase-9 (MMP-9), and manganese superoxide dismutase (MnSOD) in adipose tissue of Wistar rats. We detected AT1R and AT2R in the nuclear fraction. FRD reduced the level of angiotensin receptors in the nucleus, while increased AT1R and decreased AT2R levels were observed in the plasma membrane. FRD increased the ATRAP mRNA level and decreased MnSOD mRNA and protein levels. No significant differences were observed for MMP-9 and NOX4 mRNA levels. These findings coincided with hyperleptinemia, elevated blood pressure and triglycerides, and unchanged visceral adipose tissue mass and morphology in FRD rats. Besides providing evidence for nuclear localization of angiotensin receptors in visceral adipose tissue, this study demonstrates the different effects of FRD on AT1R expression in different cellular compartments. Elevated blood pressure and decreased antioxidant capacity in visceral fat of fructose-fed rats were accompanied by an increased AT1R level in the plasma membrane, while upregulation of ATRAP and a decrease of nuclear membrane AT1R suggest an increased capacity for attenuation of excessive AT1R signaling and visceral adiposity.

Fructose-rich diet and insulin action in female rat heart: Estradiol friend or foe?

Increased intake of fructose in humans and laboratory animals is demonstrated to be a risk factor for development of metabolic disorders (insulin resistance, metabolic syndrome, type 2 diabetes) and cardiovascular diseases. On the other hand, estradiol is emphasized as a cardioprotective agent. The main goal of this review is to summarize recent findings on damaging cardiac effects of fructose-rich diet in females, mostly experimental animals, and to evaluate protective capacity of estradiol. Published results of our and other research groups indicate mostly detrimental effects of fructose-rich diet on cardiac insulin signaling molecules, glucose and fatty acid metabolism, nitric oxide production and ion transport, as well as renin-angiotensin system and inflammation. Some of these processes are involved in cardiac insulin signal transmission, others are regulated by insulin or have an influence on insulin action. Administration of estradiol to ovariectomized female rats, exposed to increased intake of fructose, was mostly beneficial to the heart, but sometimes it was ineffective or even detrimental, depending on the particular processes. We believe that these data, carefully translated to human population, could be useful for clinicians dealing with postmenopausal women susceptible to metabolic diseases and hormone replacement therapy.

Angiotensin receptor type 1 polymorphism A1166C is associated with altered AT1R and miR-155 expression in carotid plaque tissue and development of hypoechoic carotid plaques.

BACKGROUND AND AIMS: The principal biologic effects of the renin-angiotensin system are mediated by activation of the AT1R receptor. The microRNA miR-155 regulates AT1R expression, with both its, and AT1R's activity, linked to atherosclerosis. Target sites for miR-155 lie within the 3' UTR of the human AT1R gene, and include the AT1R A1166C polymorphism. Thus far, only levels of circulating miR-155 have been investigated with respect to A1166C genotypes. We hypothesized that the A1166C polymorphism could correlate with different, ultra-sonographically defined plaque phenotypes, as well as with an altered expression of AT1R mRNA and protein in human carotid plaques (CP), and altered expression of miR-155 in patients with advanced atherosclerosis. METHODS: Our study cohort comprised 411 patients with advanced carotid atherosclerosis (298 hyperechoic; 113 hypoechoic plaques). PCR analyses identified A1166C genotypes; quantitative real-time PCR determined AT1R and miR-155 expression levels, with AT1R protein expression evaluated by western blot. RESULTS: Genotypes containing the C allele bore a significant association with the hypoechoic plaque phenotype (adjusted OR 1.87, 95% CI 1.16-3.00, p = 0.01). The expression of AT1R mRNA and miR-155 were significantly up-regulated in the CPs of CC genotype carriers compared to the AA/AC genotypes (p = 0.032, p = 0.015, respectively). AT1R protein expression was also significantly higher for CC genotypes (p < 0.01). CONCLUSION: Our results indicate that the AT1R A1166C polymorphism impacts an ultrasonographically-defined human plaque phenotype, with intra-plaque AT1R and miR-155 expression altered in advanced carotid atherosclerosis. Validation and replication of these data should contribute to an improved personalized therapy with which to prevent carotid atherosclerosis.

Fructose-rich diet induces gender-specific changes in expression of the renin-angiotensin system in rat heart and upregulates the ACE/AT1R axis in the male rat aorta.

INTRODUCTION: The cardiovascular renin-angiotensin system (RAS) could be affected by gender and dietary regime. We hypothesized that male rats will be more susceptible to activation of RAS in the heart and aorta, as a response to a fructose-rich diet (FRD). MATERIALS AND METHODS: Both male and female Wistar rats were given a 10% (w/v) fructose solution for 9 weeks. We measured the biochemical parameters, blood pressure (BP) and heart rate. We used Western blot and real-time polymerase chain reaction (PCR) to quantify protein and gene expression. RESULTS: In the male rats, the FRD elevated BP and expression of cardiac angiotensin-converting enzyme (ACE), while the expression of angiotensin-converting enzyme 2 (ACE2) and angiotensin II Type 2 receptor (AT2R) were significantly decreased. In female rats, there were no changes in cardiac RAS expression due to FRD. Furthermore, the ACE/AT1R axis was overexpressed in the FRD male rats' aortae, while only AT1R was upregulated in the FRD female rats' aortae. ACE2 expression remained unchanged in the aortae of both genders receiving the FRD. CONCLUSIONS: The FRD induced gender-specific changes in the expression of the RAS in the heart and aortae of male rats. Further investigations are required in order to get a comprehensive understanding of the underlying mechanisms of gender-specific fructose-induced cardiovascular pathologies.