Functional polymorphisms of NOS3 and GUCY1A3 affect both nitric oxide formation and association with hypertensive disorders of pregnancy.

Impaired nitric oxide (NO) formation may be associated with endothelial dysfunction and increased cardiovascular disease risk in preeclampsia (PE). Functional single-nucleotide polymorphisms (SNPs) of nitric oxide synthase 3 (NOS3) (rs3918226) and guanylate cyclase 1, soluble, alpha 3 (GUCY1A3) (rs7692387) increase susceptibility to the adverse consequences due to inadequate generation of NO by the endothelium. However, no previous study has examined whether these SNPs affect NO formation in healthy pregnancy and in gestational hypertension (GH) and PE. Here, we compared the alleles and genotypes of NOS3 (rs3918226) and GUCY1A3 (rs7692387) SNPs in normotensive pregnant women (NP, n = 153), in GH (n = 96) and PE (n = 163), and examined whether these SNPs affect plasma nitrite concentrations (a marker of NO formation) in these groups. We further examined whether the interaction among SNP genotypes is associated with GH and PE. Genotypes were determined using TaqMan allele discrimination assays, and plasma nitrite concentrations were determined by an ozone-based chemiluminescence assay. Multifactor dimensionality reduction was used to examine the interactions among SNP genotypes. Regarding NOS3 rs3918226, the CT genotype (p = 0.046) and T allele (p = 0.020) were more frequent in NP than in GH, and GH patients carrying the CT+TT genotypes showed lower nitrite concentrations than NP carrying the CT+TT genotypes (p < 0.05). Regarding GUCY1A3 rs7692387, the GA genotype (p = 0.013) and A allele (p = 0.016) were more frequent in PE than in NP, and NP women carrying the GG genotype showed higher nitrite concentrations than GH or PE patients carrying the GG genotype (p < 0.05). However, we found no significant interactions among genotypes for these functional SNPs to be associated with GH or PE. Our novel findings suggest that NOS3 rs3918226 and GUCY1A3 rs7692387 may affect NO formation and association with hypertensive disorders of pregnancy.

Plasma eNOS Concentration in Healthy Pregnancy and in Hypertensive Disorders of Pregnancy: Evidence of Reduced Concentrations in Pre-Eclampsia from Two Independent Studies.

Hypertensive disorders of pregnancy (HDP), comprising gestational hypertension (GH) and pre-eclampsia (PE), are leading causes of maternal and perinatal morbidity and mortality. Both GH and PE are characterized by new-onset hypertension, but PE additionally includes proteinuria and/or end-organ damage. Impaired nitric oxide (NO) bioavailability may lead to endothelial dysfunction in GH and PE, and the primary source of vascular NO is endothelial NO synthase (eNOS). However, no previous study has investigated plasma eNOS concentrations in patients with GH and PE. In this study, we compared plasma eNOS concentrations in healthy pregnancies and HDP in two independent cohorts. The primary study included 417 subjects, with 43 non-pregnant (NP) and 156 healthy pregnant (HP) women and 122 patients with GH and 96 with PE. The replication study included 85 pregnant women (41 healthy and 44 pre-eclamptic). Plasma concentrations of eNOS were measured using a commercial ELISA kit provided by R&D Systems, and plasma nitrite concentrations were assessed using two ozone-based chemiluminescence assays. Correlations between plasma eNOS concentrations and plasma nitrite concentrations, as well as clinical and biochemical parameters, were evaluated by either Spearman's or Pearson's tests. In the primary study, NP women and HDP had significantly lower plasma eNOS concentrations compared to HP; concentrations were even lower in PE compared to GH. Plasma eNOS concentrations were reduced but not significant in early-onset PE, PE with severe features, preterm birth, and intrauterine growth restriction. No correlation was observed between plasma eNOS and nitrite levels. In HDP, there was a significant positive correlation between levels of eNOS and hemoglobin (r = 0.1496, p = 0.0336) as well as newborn weight (r = 0.1487, p = 0.0316). Conversely, a negative correlation between eNOS levels and proteinuria was observed (r = -0.2167, p = 0.0179). The replication study confirmed significantly reduced plasma concentrations of eNOS in PE compared to HP. Our findings provide evidence of reduced plasma eNOS concentrations in HDP; they were particularly lower in PE compared to GH and HP in two independent studies.

Effects of ADIPOQ and NOS3 SNPs/haplotypes on blood pressure control in patients with adherence to antihypertensive therapy.

Aim: We examined whether ADIPOQ (rs266729 and rs1501299) and NOS3 (rs3918226 and rs1799983) SNPs or the haplotypes formed by them, affect blood pressure (BP) control in 196 patients with adherence to antihypertensive therapy grouped into controlled (BP <140/90 mmHg) and uncontrolled (BP ≥140/90 mmHg) hypertension. Materials & methods: The average of the three most recent BP measurements was retrieved from the patients' electronic medical records. Adherence to antihypertensive therapy was evaluated using the Morisky-Green test. Haplotype frequencies were estimated using Haplo.stats. Multiple logistic/linear regression analyses were adjusted for the covariates ethnicity, dyslipidemia, obesity, cardiovascular disease and uric acid. Results: ADIPOQ rs266729 genotypes CG (additive model) and CG+GG (dominant model) were associated with uncontrolled hypertension and CG was associated with higher systolic BP and mean arterial pressure (p < 0.05). ADIPOQ haplotypes 'GT' and 'GG' were associated with uncontrolled hypertension and 'GT' was associated with higher diastolic BP and mean arterial pressure (p < 0.05). Conclusion: ADIPOQ SNPs and haplotypes affect BP control in hypertensive patients undergoing treatment.

Longitudinal Study of Plasma Visfatin/Nicotinamide Phosphoribosyltransferase (NAMPT) Levels in Healthy Pregnant Women.

Visfatin/nicotinamide phosphorybosil transferase (NAMPT) is a novel adipocytokine with potential roles in the pathophysiology of metabolic disorders, including gestational disorders. However, there is no clear interpretation regarding the circulating visfatin levels in a healthy pregnancy. Therefore, we conducted the first longitudinal study of plasma visfatin levels that followed up healthy pregnant women until the third trimester, including the postpartum period (PPP). The study recruited healthy women with singleton pregnancy who were not using any drug (including tobacco and alcohol). We have excluded pregnant women who did not attend all scheduled exams and developed gestational diabetes or hypertension, obesity, preeclampsia, or any infections during pregnancy. Nine women were considered eligible and examined during all three trimesters of pregnancy and between 8 and 12 weeks postpartum (PPP). Visfatin/NAMPT concentrations were measured in EDTA-plasma by ELISA. The mean age of pregnant women included was 22±5 years (54% primiparous), and the mean of gestational age at delivery was 40±1.2 weeks. Mean systolic and diastolic blood pressures were 90 and 70 mmHg, respectively. Mean values (± standard error mean) of visfatin concentrations (µg/L) during trimesters were 11.38±1.45 (first, 11-14 weeks), 9.18±1.82 (second, 20-24 weeks), 18.67±2.65 (third, 34-36 weeks), and 10.12±1.49 in the PPP. The value of the third trimester was significantly higher than the second trimester, and significantly reduced in the PPP (p<0.05, ANOVA with Bonferroni's multiple comparison tests). Visfatin/NAMPT levels are significantly lower in the PPP, suggesting that factors stimulating its production would be limited to pregnancy, thereby contributing to its potential application as a biomarker in pregnancy complications.

Circulating levels of tissue inhibitor of metalloproteinase 3, a protein with inhibitory effects on angiogenesis, are increased in pre-eclampsia.

OBJECTIVE: To assess and compare circulating tissue inhibitor of metalloproteinase 3 (TIMP-3) concentrations between women with pre-eclampsia and healthy pregnant women. We also aimed to determine the relationships between circulating TIMP-3 and matrix metalloproteinase 2 (MMP-2), MMP-9, TIMP-1, and TIMP-2 concentrations in pre-eclampsia. METHODS: A primary case-control study included patients with pre-eclampsia (n = 219) and gestational hypertension (n = 118), healthy pregnant women (n = 214), and non-pregnant women (n = 66), and a replication case-control study included patients with pre-eclampsia (n = 177) and healthy pregnant women (n = 124), all from southeastern Brazil. Plasma TIMP-3, MMP-2, MMP-9, TIMP-1, and TIMP-2 concentrations were assessed using commercially available enzyme-linked immunosorbent assay kits, and the relationships between them were analyzed using Spearman's correlation. RESULTS: In our primary study, patients with pre-eclampsia and gestational hypertension exhibited increased TIMP-3 concentrations compared with healthy pregnant women (both P < 0.0001) and non-pregnant women (both P < 0.001). These findings were confirmed in the replication study, showing elevated TIMP-3 concentrations in women with pre-eclampsia versus healthy pregnant women (P < 0.001). We found no difference in TIMP-3 concentrations between early-onset and late-onset pre-eclampsia. Moreover, TIMP-3 concentrations were significantly correlated with plasma concentrations of TIMP-1 (r = 0.2333; P = 0.0086) and MMP-2 (r = 0.2159; P = 0.0156) in pre-eclampsia. CONCLUSIONS: Circulating TIMP-3 concentration is increased in women with pre-eclampsia compared with healthy pregnant women, and it is positively correlated with plasma MMP-2 and TIMP-1 concentrations in pre-eclampsia.

Metformin Treatment Modulates Long Non-Coding RNA Isoforms Expression in Human Cells.

Long noncoding RNAs (lncRNAs) undergo splicing and have multiple transcribed isoforms. Nevertheless, for lncRNAs, as well as for mRNA, measurements of expression are routinely performed only at the gene level. Metformin is the first-line oral therapy for type 2 diabetes mellitus and other metabolic diseases. However, its mechanism of action remains not thoroughly explained. Transcriptomic analyses using metformin in different cell types reveal that only protein-coding genes are considered. We aimed to characterize lncRNA isoforms that were differentially affected by metformin treatment on multiple human cell types (three cancer, two non-cancer) and to provide insights into the lncRNA regulation by this drug. We selected six series to perform a differential expression (DE) isoform analysis. We also inferred the biological roles for lncRNA DE isoforms using in silico tools. We found the same isoform of an lncRNA (AC016831.6-205) highly expressed in all six metformin series, which has a second exon putatively coding for a peptide with relevance to the drug action. Moreover, the other two lncRNA isoforms (ZBED5-AS1-207 and AC125807.2-201) may also behave as cis-regulatory elements to the expression of transcripts in their vicinity. Our results strongly reinforce the importance of considering DE isoforms of lncRNA for understanding metformin mechanisms at the molecular level.

ARG2 single-nucleotide polymorphism rs3742879 affects plasma arginase 2 levels, nitric oxide formation and antihypertensive therapy response in preeclampsia.

Aim: This work examined whether ARG1 (rs2781659, rs2781667, rs2246012 and rs17599586) and ARG2 (rs3742879 and rs10483801) single-nucleotide polymorphisms (SNPs) are associated with antihypertensive therapy responsiveness in preeclampsia (PE) and their effects on arginase isoforms and nitrite concentrations in responsive and nonresponsive patients. Methods: SNP genotypes were determined by TaqMan assays. Plasma arginase levels were measured by ELISA and nitrite concentrations were measured using an ozone-based chemiluminescence assay. Results: The G allele for ARG2 rs3742879 (A>G) was less frequent in nonresponsive compared with responsive patients (15.5% vs 24.7%, respectively) and the G carriers of the nonresponsive subgroup had lower arginase 2 (9.2 ± 7.5 ng/ml vs 19.1 ± 17.3 ng/ml) and higher nitrite concentrations (110.2 ± 52.8 nM vs 78.5 ± 37.9 nM) than carriers of the AA genotype (all p < 0.05). Conclusion: ARG2 SNP rs3742879 is associated with diminished arginase 2 levels and increased nitric oxide formation in nonresponsive PE patients.

Gene-gene interactions in the protein kinase C/endothelial nitric oxide synthase axis impact the hypotensive effects of propofol.

Anaesthesia with propofol is frequently associated with hypotension, which is at least partially attributable to increased nitric oxide (NO) formation derived from the activation of protein kinase C (PKC)/endothelial NO synthase (NOS3) axis. In this cross-sectional study, we tested whether PRKCA (which encodes PKCα) polymorphisms, or haplotypes, and interactions among PRKCA and NOS3 polymorphisms affect the hypotensive responses to propofol. We collected venous blood samples from 164 patients before and 10 min after propofol administration. Genotypes were determined by PCR and haplotype frequencies were estimated. Nitrite and NOx (nitrites+nitrates) levels were measured by using an ozone-based chemiluminescence assay and the Griess reaction, respectively. We used multifactor dimensionality reduction to test interactions among PRKCA and NOS3 polymorphisms. Propofol promoted enhanced blood pressure-lowering effects and increased nitrite levels in subjects carrying GA + AA genotypes for the rs16960228 and TC + CC genotypes for the rs1010544 PRKCA polymorphisms, and the CCG haplotype. Moreover, genotypes for the rs1010544 PRKCA polymorphism were associated with higher or lower blood pressure decreases in response to propofol depending on the genotypes for the rs2070744 NOS3 polymorphism. Our findings suggest that PRKCA genotypes and haplotypes impact the hypotensive responses to propofol, possibly by modifying NO bioavailability, and that PRKCA-NOS3 interactions modify the blood pressure-lowering effects of propofol.

Comprehensive analyses of DNA methylation of the TIMP3 promoter in placentas from early-onset and late-onset preeclampsia.

Preeclampsia (PE) is classified into late-onset (LOPE) or early-onset (EOPE) according to gestational age of onset (≥34 or <34 weeks, respectively), and into preterm and term (delivery at <37 or ≥37 weeks, respectively). An imbalanced expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) impairs proper placentation in PE, and DNA methylation (DNAm) may affect their expression. We performed comprehensive analyses of DNAm and TIMP3 expression in placentas from PE reclassified into EOPE, LOPE, and term PE. We identified significant differentially methylated probes at the TIMP3 promoter in PE (28), EOPE (38), LOPE (20), and term PE (4) compared to controls, and in EOPE vs. LOPE (8). Moreover, we found a hypomethylation >70% in all groups (except EOPE vs. LOPE) and an increased TIMP3 expression in corresponding placental samples from PE, EOPE and LOPE compared to controls (p<0.05). Our findings highlight the role of DNAm of the TIMP3 promoter region regarding an epigenetic mechanism in PE.

Circulating MicroRNAs in the Second Trimester From Pregnant Women Who Subsequently Developed Preeclampsia: Potential Candidates as Predictive Biomarkers and Pathway Analysis for Target Genes of miR-204-5p.

MicroRNAs (miRNAs) play an important role in the pathophysiology of preeclampsia (PE). However, the expression of circulating miRNAs was not analyzed in the second trimester of pregnancy, a period of major relevance to identify predictive biomarkers for PE. Therefore, we examined the expression profiles of 84 circulating miRNAs using a PCR array in plasma collected between 20 and 25 weeks of gestation from pregnant women, who subsequently developed PE and those who remained healthy during pregnancy, randomly selected from a prospective cohort. Overall, 23 miRNAs had a fold change > 2.0 and were considered to be upregulated in plasma from pregnant women who subsequently developed PE, even before the onset of clinical symptoms of PE. However, only miR-204-5p was statistically significant (P = 0.0082). Experimentally validated interactions for the target genes of miR-204-5p extracted from miRTarBase were used in the gene set functional enrichment analysis to identify Reactome pathways. The network connecting the 37 target genes for miR-204-5p revealed pathways of known pathophysiological relevance during the early development of PE and included key genes related to PE, such as BDNF, MMP-9, MALAT1, TGFBR2, and SIRT1. We further depicted downstream targets of SIRT1 that are related to the vascular endothelial function or implicated in the pathophysiology of PE, namely, FOXO1, NFκB, HIF-1α, NOS3, and PPAR-γ. Our novel findings provide for circulating miRNAs upregulated in the second trimester on plasma from pregnant women who subsequently developed PE that is potentially related to the early development of PE, which may guide further studies focused on the validation of potential predictive biomarkers in PE.

NAMPT single-nucleotide polymorphism rs1319501 and visfatin/NAMPT affect nitric oxide formation, sFlt-1 and antihypertensive therapy response in preeclampsia.

Aim: We examined the relationships between visfatin/NAMPT and nitrite concentrations (a marker of nitric oxide [NO] formation) or sFlt-1 levels in 205 patients with preeclampsia (PE) responsive or nonresponsive to antihypertensive therapy, and whether NAMPT SNPs rs1319501 and rs3801266 affect nitrite concentrations in PE and 206 healthy pregnant women. Patients & methods: Circulating visfatin/NAMPT and sFlt-1 levels were measured by ELISA, and nitrite concentrations by using an ozone-based chemiluminescence assay. Results: In nonresponsive PE patients, visfatin/NAMPT levels were inversely related to nitrite concentrations and positively related to sFlt-1 levels. NAMPT SNP rs1319501 affected nitrite concentrations in nonresponsive PE patients and was tightly linked with NAMPT functional SNPs in Europeans. Conclusion:NAMPT SNP rs1319501 and visfatin/NAMPT affect NO formation, sFlt-1 levels and antihypertensive therapy response in PE.

Epigenetic Regulation of the N-Terminal Truncated Isoform of Matrix Metalloproteinase-2 (NTT-MMP-2) and Its Presence in Renal and Cardiac Diseases.

Several clinical and experimental studies have documented a compelling and critical role for the full-length matrix metalloproteinase-2 (FL-MMP-2) in ischemic renal injury, progressive renal fibrosis, and diabetic nephropathy. A novel N-terminal truncated isoform of MMP-2 (NTT-MMP-2) was recently discovered, which is induced by hypoxia and oxidative stress by the activation of a latent promoter located in the first intron of the MMP2 gene. This NTT-MMP-2 isoform is enzymatically active but remains intracellular in or near the mitochondria. In this perspective article, we first present the findings about the discovery of the NTT-MMP-2 isoform, and its functional and structural differences as compared with the FL-MMP-2 isoform. Based on publicly available epigenomics data from the Encyclopedia of DNA Elements (ENCODE) project, we provide insights into the epigenetic regulation of the latent promoter located in the first intron of the MMP2 gene, which support the activation of the NTT-MMP-2 isoform. We then focus on its functional assessment by covering the alterations found in the kidney of transgenic mice expressing the NTT-MMP-2 isoform. Next, we highlight recent findings regarding the presence of the NTT-MMP-2 isoform in renal dysfunction, in kidney and cardiac diseases, including damage observed in aging, acute ischemia-reperfusion injury (IRI), chronic kidney disease, diabetic nephropathy, and human renal transplants with delayed graft function. Finally, we briefly discuss how our insights may guide further experimental and clinical studies that are needed to elucidate the underlying mechanisms and the role of the NTT-MMP-2 isoform in renal dysfunction, which may help to establish it as a potential therapeutic target in kidney diseases.

Effects of arginase genetic polymorphisms on nitric oxide formation in healthy pregnancy and in preeclampsia.

BACKGROUND AND AIMS: Preeclampsia is associated with reduced nitric oxide (NO) bioavailability. Arginase is related to NO synthesis, but relatively unexplored in preeclampsia. However, no previous study has examined whether variations in ARG1 and ARG2 genes affect NO bioavailability and the risk of preeclampsia. Here, we compared the alleles and genotypes of single nucleotide polymorphisms (SNPs) in ARG1 (rs2781659; rs2781667; rs2246012; rs17599586) and ARG2 (rs3742879; rs10483801) in healthy pregnant women and preeclampsia, and examined whether these SNPs affect plasma nitrite concentrations (a marker of NO formation) in these groups. METHODS: Genotypes for the ARG1 and ARG2 SNPs were determined by Taqman probe and plasma nitrite by an ozone-based chemiluminescence assay. RESULTS: Regarding ARG1 SNPs, the GG genotype and G allele frequencies for rs2781659, and the C allele frequencies for rs2246012 were higher in preeclampsia compared to healthy pregnant women. Moreover, the GG genotype for rs2781659 and the TT genotype for rs2781667 were associated with higher plasma nitrite in healthy pregnant. We found no association of ARG2 polymorphisms with preeclampsia or nitrite levels in the study groups. CONCLUSIONS: Our results suggest that SNPs of ARG1 increase the risk of preeclampsia and modulate plasma nitrite levels in healthy pregnant women.

Admixture/fine-mapping in Brazilians reveals a West African associated potential regulatory variant (rs114066381) with a strong female-specific effect on body mass and fat mass indexes.

BACKGROUND/OBJECTIVES: Admixed populations are a resource to study the global genetic architecture of complex phenotypes, which is critical, considering that non-European populations are severely underrepresented in genomic studies. Here, we study the genetic architecture of BMI in children, young adults, and elderly individuals from the admixed population of Brazil. SUBJECTS/METHODS: Leveraging admixture in Brazilians, whose chromosomes are mosaics of fragments of Native American, European, and African origins, we used genome-wide data to perform admixture mapping/fine-mapping of body mass index (BMI) in three Brazilian population-based cohorts from Northeast (Salvador), Southeast (Bambuí), and South (Pelotas). RESULTS: We found significant associations with African-associated alleles in children from Salvador (PALD1 and ZMIZ1 genes), and in young adults from Pelotas (NOD2 and MTUS2 genes). More importantly, in Pelotas, rs114066381, mapped in a potential regulatory region, is significantly associated only in females (p = 2.76e-06). This variant is rare in Europeans but with frequencies of ~3% in West Africa and has a strong female-specific effect (95% CI: 2.32-5.65 kg/m2 per each A allele). We confirmed this sex-specific association and replicated its strong effect for an adjusted fat mass index in the same Pelotas cohort, and for BMI in another Brazilian cohort from São Paulo (Southeast Brazil). A meta-analysis confirmed the significant association. Remarkably, we observed that while the frequency of rs114066381-A allele ranges from 0.8 to 2.1% in the studied populations, it attains ~9% among women with morbid obesity from Pelotas, São Paulo, and Bambuí. The effect size of rs114066381 is at least five times higher than the FTO SNPs rs9939609 and rs1558902, already emblematic for their high effects. CONCLUSIONS: We identified six candidate SNPs associated with BMI. rs114066381 stands out for its high effect that was replicated and its high frequency in women with morbid obesity. We demonstrate how admixed populations are a source of new relevant phenotype-associated genetic variants.

The genetic structure and adaptation of Andean highlanders and Amazonians are influenced by the interplay between geography and culture.

Western South America was one of the worldwide cradles of civilization. The well-known Inca Empire was the tip of the iceberg of an evolutionary process that started 11,000 to 14,000 years ago. Genetic data from 18 Peruvian populations reveal the following: 1) The between-population homogenization of the central southern Andes and its differentiation with respect to Amazonian populations of similar latitudes do not extend northward. Instead, longitudinal gene flow between the northern coast of Peru, Andes, and Amazonia accompanied cultural and socioeconomic interactions revealed by archeology. This pattern recapitulates the environmental and cultural differentiation between the fertile north, where altitudes are lower, and the arid south, where the Andes are higher, acting as a genetic barrier between the sharply different environments of the Andes and Amazonia. 2) The genetic homogenization between the populations of the arid Andes is not only due to migrations during the Inca Empire or the subsequent colonial period. It started at least during the earlier expansion of the Wari Empire (600 to 1,000 years before present). 3) This demographic history allowed for cases of positive natural selection in the high and arid Andes vs. the low Amazon tropical forest: in the Andes, a putative enhancer in HAND2-AS1 (heart and neural crest derivatives expressed 2 antisense RNA1, a noncoding gene related to cardiovascular function) and rs269868-C/Ser1067 in DUOX2 (dual oxidase 2, related to thyroid function and innate immunity) genes and, in the Amazon, the gene encoding for the CD45 protein, essential for antigen recognition by T and B lymphocytes in viral-host interaction.