Francine Z. Marques

Gene Expression Profiling Reveals Renin mRNA Overexpression in Human Hypertensive Kidneys and a Role for MicroRNAs

The kidney has long been invoked in the etiology of essential hypertension. This could involve alterations in expression of specific genes and microRNAs (miRNAs). The aim of the present study was to identify, at the transcriptome-wide level, mRNAs and miRNAs that were differentially expressed between kidneys of 15 untreated hypertensive and 7 normotensive white male subjects of white European ancestry. By microarray technology we found 14 genes and 11 miRNAs that were differentially expressed in the medulla. We then selected and confirmed by real-time quantitative PCR expression differences for NR4A1, NR4A2, NR4A3, PER1, and SIK1 mRNAs and for the miRNAs hsa-miR-638 and hsa-let-7c. Luciferase reporter gene experiments in human kidney (HEK293) cells confirmed the predicted binding of hsa-let-7c to the 3′ untranslated region of NR4A2 mRNA. In the renal cortex we found differential expression of 46 genes and 13 miRNAs. We then confirmed expression differences for AIFM1, AMBP, APOE, CD36, EFNB1, NDUFAF1, PRDX5, REN, RENBP, SLC13A1, STX4, and TNNT2 mRNAs and for miRNAs hsa-miR-21, hsa-miR-126, hsa-miR-181a, hsa-miR-196a, hsa-miR-451, hsa-miR-638, and hsa-miR-663. Functional experiments in HEK293 cells demonstrated that hsa-miR-663 can bind to the REN and APOE 3′ untranslated regions and can regulate REN and APOE mRNA levels, whereas hsa-miR-181a regulated REN and AIFM1 mRNA. Our data demonstrated for the first time that miRNAs can regulate renin expression. The observed downregulation of 2 miRNAs in hypertension could explain the elevation in intrarenal renin mRNA. Renin, CD36, and other mRNAs, as well as miRNAs and associated pathways identified in the present study, provide novel insights into hypertension etiology.

Resveratrol: cellular actions of a potent natural chemical that confers a diversity of health benefits.

Resveratrol is a polyphenolic flavonoid with potent antioxidant activity. It is found in a diversity of plants, notably berry fruit, and is attracting increased attention due to its health benefits, especially in common age-related diseases such as cancer, type 2 diabetes, cardiovascular disease, and neurological conditions. Resveratrol has positive effects on metabolism and can increase the lifespan of various organisms. Its effects arise from its capacity to interact with multiple molecular targets involved in diverse intracellular pathways. Most well known is the ability of resveratrol to activate sirtuins, a class of NAD(+)-dependent deacetylases that affect multiple transcription factors and other protein targets. More potent sirtuin activators have now been discovered by large-scale screening programs. Resveratrol and the new compounds are the subject of clinical trials to determine their consumer safety and suitability for the prevention and treatment of most common diseases of aging.

Exercise: Putting Action into Our Epigenome

Most human phenotypes are influenced by a combination of genomic and environmental factors. Engaging in regular physical exercise prevents many chronic diseases, decreases mortality risk and increases longevity. However, the mechanisms involved are poorly understood. The modulating effect of physical (aerobic and resistance) exercise on gene expression has been known for some time now and has provided us with an understanding of the biological responses to physical exercise. Emerging research data suggest that epigenetic modifications are extremely important for both development and disease in humans. In the current review, we summarise findings on the effect of exercise on epigenetic modifications and their effects on gene expression. Current research data suggest epigenetic modifications (DNA methylation and histone acetylation) and microRNAs (miRNAs) are responsive to acute aerobic and resistance exercise in brain, blood, skeletal and cardiac muscle, adipose tissue and even buccal cells. Six months of aerobic exercise alters whole-genome DNA methylation in skeletal muscle and adipose tissue and directly influences lipogenesis. Some miRNAs are related to maximal oxygen consumption (VO2max) and VO2max trainability, and are differentially expressed amongst individuals with high and low VO2max. Remarkably, miRNA expression profiles discriminate between low and high responders to resistance exercise (miR-378, -26a, -29a and -451) and correlate to gains in lean body mass (miR-378). The emerging field of exercise epigenomics is expected to prosper and additional studies may elucidate the clinical relevance of miRNAs and epigenetic modifications, and delineate mechanisms by which exercise confers a healthier phenotype and improves performance.

Meta-Analysis of Genome-Wide Gene Expression Differences in Onset and Maintenance Phases of Genetic Hypertension

Gene expression differences accompany both the onset and established phases of hypertension. By an integrated genome-transcriptome approach we performed a meta-analysis of data from 74 microarray experiments available on public databases to identify genes with altered expression in the kidney, adrenal, heart, and artery of spontaneously hypertensive and Lyon hypertensive rats. To identify genes responsible for the onset of hypertension we used a statistical approach that sought to eliminate expression differences that occur during maturation unrelated to hypertension. Based on this adjusted fold-difference statistic, we found 36 genes for which the expression differed between the prehypertensive phase and established hypertension. Genes having possible relevance to hypertension onset included Actn2, Ankrd1, ApoE, Cd36, Csrp3, Me1, Myl3, Nppa, Nppb, Pln, Postn, Spp1, Slc21a4, Slc22a2, Thbs4, and Tnni3. In established hypertension 102 genes exhibited altered expression after Bonferroni correction (P<0.05). These included Atp5o, Ech1, Fabp3, Gnb3, Ldhb, Myh6, Lpl, Pkkaca, Vegfb, Vcam1, and reduced nicotinamide-adenine dinucleotide dehydrogenases. Among the genes identified, there was an overrepresentation of gene ontology terms involved in energy production, fatty acid and lipid metabolism, oxidation, and transport. These could contribute to increases in reactive oxygen species. Our meta-analysis has revealed many new genes for which the expression is altered in hypertension, so pointing to novel potential causative, maintenance, and responsive mechanisms and pathways.

A Novel Interaction Between Sympathetic Overactivity and Aberrant Regulation of Renin by miR-181a in BPH/2J Genetically Hypertensive Mice

Genetically hypertensive mice (BPH/2J) are hypertensive because of an exaggerated contribution of the sympathetic nervous system to blood pressure. We hypothesize that an additional contribution to elevated blood pressure is via sympathetically mediated activation of the intrarenal renin–angiotensin system. Our aim was to determine the contribution of the renin–angiotensin system and sympathetic nervous system to hypertension in BPH/2J mice. BPH/2J and normotensive BPN/3J mice were preimplanted with radiotelemetry devices to measure blood pressure. Depressor responses to ganglion blocker pentolinium (5 mg/kg IP) in mice pretreated with the angiotensin-converting enzyme inhibitor enalaprilat (1.5 mg/kg IP) revealed a 2-fold greater sympathetic contribution to blood pressure in BPH/2J mice during the active and inactive period. However, the depressor response to enalaprilat was 4-fold greater in BPH/2J compared with BPN/3J mice, but only during the active period (P=0.01). This was associated with 1.6-fold higher renal renin messenger RNA (mRNA; P=0.02) and 0.8-fold lower abundance of micro-RNA-181a (P=0.03), identified previously as regulating human renin mRNA. Renin mRNA levels correlated positively with depressor responses to pentolinium (r=0.99; P=0.001), and BPH/2J mice had greater renal sympathetic innervation density as identified by tyrosine hydroxylase staining of cortical tubules. Although there is a major sympathetic contribution to hypertension in BPH/2J mice, the renin–angiotensin system also contributes, doing so to a greater extent during the active period and less during the inactive period. This is the opposite of the normal renin–angiotensin system circadian pattern. We suggest that renal hyperinnervation and enhanced sympathetically induced renin synthesis mediated by lower micro-RNA-181a contributes to hypertension in BPH/2J mice.

The transcardiac gradient of cardio-microRNAs in the failing heart

Differential microRNA expression in peripheral blood has been observed in patients with heart failure, suggesting their value as potential biomarkers and likely contributors to disease mechanisms. In the present study, we aimed to evaluate the transcardiac gradient of 84 cardio‐microRNAs in healthy and failing hearts to determine which microRNAs are released or absorbed by the myocardium in heart failure.

Fetal sex affects expression of renin-angiotensin system components in term human decidua

The maternal decidua expresses the genes of the renin-angiotensin system (RAS). Human decidua was collected at term either before labor (i.e. cesarean delivery) or after spontaneous labor. The mRNA for prorenin (REN), prorenin receptor (ATP6AP2), angiotensinogen (AGT), angiotensin-converting enzymes 1 and 2 (ACE1 and ACE2), angiotensin II type 1 receptor (AGTR1), and angiotensin 1-7 receptor (MAS1) were measured by quantitative real-time RT-PCR. Decidual explants were cultured in duplicate for 24 and 48 h, and all RAS mRNA, and the secretion of prorenin, angiotensin II, and angiotensin 1-7 was measured using quantitative real-time RT-PCR, ELISA, and radioimmunoassay, respectively. In the decidua collected before labor, REN mRNA levels were higher if the fetus was female. In addition, REN, ATP6AP2, AGT, and MAS1 mRNA abundance was greater in decidual explants collected from women carrying a female fetus, as was prorenin protein. After 24 h, ACE1 mRNA was higher in the decidual explants from women with a male fetus, whereas after 48 h, both ACE1 and ACE2 mRNA was higher in decidual explants from women with a female fetus. Angiotensin II was present in all explants, but angiotensin 1-7 levels often registered below the lower limits of sensitivity for the assay. After labor, decidua, when compared with nonlaboring decidua, demonstrated lower REN expression when the fetus was female. Therefore, the maternal decidual RAS is regulated in a sex-specific manner, suggesting that it may function differently when the fetus is male than when it is female.

Resveratrol, by Modulating RNA Processing Factor Levels, Can Influence the Alternative Splicing of Pre- mRNAs

Alternative pre-mRNA splicing defects can contribute to, or result from, various diseases, including cancer. Aberrant mRNAs, splicing factors and other RNA processing factors have therefore become targets for new therapeutic interventions. Here we report that the natural polyphenol resveratrol can modulate alternative splicing in a target-specific manner. We transfected minigenes of several alternatively spliceable primary mRNAs into HEK293 cells in the presence or absence of 1, 5, 20 and 50 µM resveratrol and measured exon levels by semi-quantitative PCR after separation by agarose gel electrophoresis. We found that 20 µg/ml and 50 µg/ml of resveratrol affected exon inclusion of SRp20 and SMN2 pre-mRNAs, but not CD44v5 or tau pre-mRNAs. By Western blotting and immunofluorescence we showed that this effect may be due to the ability of resveratrol to change the protein level but not the localization of several RNA processing factors. The processing factors that increased significantly were ASF/SF2, hnRNPA1 and HuR, but resveratrol did not change the levels of RBM4, PTBP1 and U2AF35. By means of siRNA-mediated knockdown we depleted cells of SIRT1, regarded as a major target of resveratrol, and showed that the effect on splicing was not dependent on SIRT1. Our results suggest that resveratrol might be an attractive small molecule to treat diseases in which aberrant splicing has been implicated, and justify more extensive research on the effects of resveratrol on the splicing machinery.

The molecular basis of longevity, and clinical implications.

The determinants of length of life are multifactorial and involve complex processes, most of which are not as yet understood completely. Tremendous advances have, however, been made in recent times in understanding some of the key molecular mechanisms that influence ageing and lifespan. Herein we highlight many of the more important findings and their potential clinical implications. Most of the intracellular factors involved in the ageing process, such as members of the sirtuin family, as well as insulin and insulin-like growth factor-I and their genes, are part of interconnected pathways. The manipulation of these and other genes in animal models can increase or decrease lifespan. Transcriptional and post-transcriptional regulatory mechanisms, some of which involve microRNAs, as well as modifications to chromatin and histones, can influence longevity. A decline in the function of stem cells might also be responsible for some aspects of mammalian ageing. Calorie restriction, polyphenols such as resveratrol, rapamycin, spermidine and angiotensin I converting enzyme inhibitor, are able to increase lifespan by modulation of branches of the longevity pathways. Molecular genetic studies of long-lived subjects have identified several potential candidate genes, but genetic research on ageing is in its infancy. Large genome-wide association studies should provide insights. Although new biomarkers for ageing and health, such as ones that might reveal telomere dysfunction, have been described, advances in the genetics and molecular biology of longevity will require interdisciplinary approaches if the much-hoped for success in alleviating the diseases of ageing, and an extension of both lifespan and healthspan is to be achieved.

Global identification of the genes and pathways differentially expressed in hypothalamus in early and established neurogenic hypertension.

The hypothalamus has an important etiological role in the onset and maintenance of hypertension and stress responses in the Schlager high blood pressure (BP) (BPH/2J) mouse, a genetic model of neurogenic hypertension. Using Affymetrix GeneChip Mouse Gene 1.0 ST Arrays we identified 1,019 hypothalamic genes whose expression differed between 6 wk old BPH/2J and normal BP (BPN/3J) strains, and 466 for 26 wk old mice. Of these, 459 were in 21 mouse BP quantitative trait loci. We validated 46 genes by qPCR. Gene changes that would increase sympathetic outflow at both ages were: Dynll1 encoding dynein light chain LC8-type 1, which physically destabilizes neuronal nitric oxide synthase, decreasing neuronal nitric oxide, and Hcrt encoding hypocretin and Npsr1 encoding neuropeptide S receptor 1, each involved in sympathetic response to stress. At both ages we identified genes for inflammation, such as CC-chemokine ligand 19 (Ccl19), and oxidative stress. Via reactive oxygen species generation, these could contribute to oxidative damage. Other genes identified could be responding to such perturbations. Atp2b1, the major gene from genome-wide association studies of BP variation, was underexpressed in the early phase. Comparison of profiles of young and adult BPH/2J mice, after adjusting for maturation genes, pointed to the proopiomelanocortin-α gene (Pomc) and neuropeptide Y gene (Npy), among others, as potentially causative. The present study has identified a diversity of genes and possible mechanisms involved in hypertension etiology and maintenance in the hypothalamus of BPH/2J mice, highlighting both common and divergent processes in each phase of the condition.