Anna Campain

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.

BRAF Mutation, NRAS Mutation, and the Absence of an Immune-Related Expressed Gene Profile Predict Poor Outcome in Patients with Stage III Melanoma

Prediction of outcome for melanoma patients with surgically resected macroscopic nodal metastases is very imprecise. We performed a comprehensive clinico-pathologic assessment of fresh-frozen macroscopic nodal metastases and the preceding primary melanoma, somatic mutation profiling, and gene expression profiling to identify determinants of outcome in 79 melanoma patients. In addition to disease stage <II at initial presentation, the following clinical and pathologic factors were independent predictors of improved outcome (odds ratios for survival >4 years, 90% confidence interval): the presence of a nodular component in the primary melanoma (6.8, 0.6-76.0), and small cell size (11.1, 0.8-100.0) or low pigmentation (3.0, 0.8-100.0) in the nodal metastases. Absence of BRAF mutation (20.0, 1.0-1000.0) or NRAS mutation (16.7, 0.6-1000.0) were both favorable prognostic factors. A 46-gene expression signature with strong overrepresentation of immune response genes was predictive of better survival (10.9, 0.4-325.6); in the full cohort, median survival was >100 months in those with the signature, but 10 months in those without. This relationship was validated in two previously published independent stage III melanoma data sets. We conclude that the presence of BRAF mutation, NRAS mutation, and the absence of an immune-related expressed gene profile predict poor outcome in melanoma patients with macroscopic stage III disease.

A Randomized Clinical Trial of Intravitreal Bevacizumab versus Intravitreal Dexamethasone for Diabetic Macular Edema: The BEVORDEX Study

OBJECTIVE To report the 12-month results of the first head-to-head comparison of a dexamethasone implant (Ozurdex; Allergan, Inc., Irvine, CA) versus bevacizumab (Avastin; Genentech, South San Francisco, CA) for center-involving diabetic macular edema (DME). DESIGN Phase 2, prospective, multicenter, randomized, single-masked clinical trial (clinicaltrials.gov identifier NCT01298076). PARTICIPANTS We enrolled 88 eyes of 61 patients with center-involving DME. METHODS Forty-two eyes were randomized to receive bevacizumab every 4 weeks and 46 eyes were randomized to receive a dexamethasone implant every 16 weeks, both pro re nata. Results were analyzed using linear regression with generalized estimation equation methods to account for between-eye correlation. MAIN OUTCOME MEASURES The primary outcome was the proportion of eyes that improved vision by 10 logarithm of minimum angle of resolution letters. Secondary outcomes included mean change in best-corrected visual acuity (BCVA), change in central macular thickness (CMT), injection frequency, and adverse events. Patient-reported outcomes were measured using the Impact of Vision Impairment (IVI) questionnaire. RESULTS Improvement in BCVA of 10 or more letters was found in 17 of 42 eyes (40%) treated with bevacizumab compared with 19 of 46 dexamethasone implant-treated eyes (41%; P = 0.83). None of the 42 bevacizumab eyes lost 10 letters or more, whereas 5 of 46 (11%) dexamethasone implant eyes did, mostly because of cataract. Mean CMT decreased by 122 μm for bevacizumab eyes and by 187 μm for dexamethasone implant eyes (P = 0.015). Bevacizumab-treated eyes received a mean of 8.6 injections compared with 2.7 injections for dexamethasone implant eyes. Significant improvement in IVI scores occurred for both treatment groups. CONCLUSIONS Dexamethasone implant achieved similar rates of visual acuity improvement compared with bevacizumab for DME, with superior anatomic outcomes and fewer injections. Both treatments were associated with improvement in visual quality-of-life scores. However, more dexamethasone implant-treated eyes lost vision, mainly because of cataract.

Comparison study of microarray meta-analysis methods

BackgroundMeta-analysis methods exist for combining multiple microarray datasets. However, there are a wide range of issues associated with microarray meta-analysis and a limited ability to compare the performance of different meta-analysis methods.ResultsWe compare eight meta-analysis methods, five existing methods, two naive methods and a novel approach (mDEDS). Comparisons are performed using simulated data and two biological case studies with varying degrees of meta-analysis complexity. The performance of meta-analysis methods is assessed via ROC curves and prediction accuracy where applicable.ConclusionsExisting meta-analysis methods vary in their ability to perform successful meta-analysis. This success is very dependent on the complexity of the data and type of analysis. Our proposed method, mDEDS, performs competitively as a meta-analysis tool even as complexity increases. Because of the varying abilities of compared meta-analysis methods, care should be taken when considering the meta-analysis method used for particular research.

Two-year outcomes of "treat and extend" intravitreal therapy for neovascular age-related macular degeneration.

PURPOSE To report 24-month outcomes of anti-vascular endothelial growth factor (VEGF) therapy for treatment-naïve eyes with neovascular age-related macular degeneration (nAMD) using a treat and extend treatment regimen in routine clinical practice. DESIGN Database observational study. PARTICIPANTS We included treatment-naïve eyes receiving predominantly ranibizumab for nAMD in routine clinical practice treated using a treat and extend regimen that were tracked in the Fight Retinal Blindness observational registry. METHODS A cohort of eyes treated by practitioners using exclusively a treat and extend regimen was extracted from the Fight Retinal Blindness observational registry. MAIN OUTCOME MEASURES Change in visual acuity (VA) over 2 years and number of injections and visits. RESULTS Data from 1198 eyes from 1011 patients receiving anti-VEGF therapy using a treat and extend regimen for treatment-naïve nAMD between January 2007 and December 2012 and with 24-month follow-up were included in the analysis. Mean VA increased by +5.3 logarithm of the minimum angle of resolution letters from 56.5 letters (20/80+1) at initial visit to 61.8 (20/60+2) letters at 24 months. Mean VA gains improved and number of injections increased with successive years from +2.7 letters for eyes commencing in 2007 after a mean of 9.7 injections in 2 years, to +7.8 letters for eyes commencing in 2012 after a mean of 14.2 injections over 2 years. The proportion of eyes with VA >20/40 increased from 27% when starting treatment to 45% after 24 months; the proportion with vision of <20/200 remained unchanged (13% initial, 11% at 24 months). Of the included eyes, 90.5% avoided a vision loss of ≥15 letters. There was an overall mean of 13.0 injections over the 24 months, 7.5 injections in the first year and 5.5 in the second year, with a mean of 14.8 clinic visits. CONCLUSIONS These data indicate that eyes managed in routine clinical practice with a treat and extend regimen can achieve good visual outcomes while decreasing the burden of treatments and clinic visits.

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.

Review and cross-validation of gene expression signatures and melanoma prognosis.

In melanoma, there is an urgent need to identify novel biomarkers with prognostic performance superior to traditional clinical and histological parameters. Gene expression-based prognostic signatures offer promise, but studies have been challenged by sample scarcity, cohort heterogeneity, and doubts about the efficacy of such signatures relative to current clinical practices. Motivated by new studies that have begun to address these challenges, we reviewed prognostic signatures derived from gene expression microarray analysis of human melanoma tissue. We used REMARK-based criteria to select the most relevant studies and directly compared their signature gene lists. Through functional ontology enrichment analysis, we observed that these independent data sets converge in part upon immune response processes and the G-protein signaling NRAS-regulation pathway, both important in melanoma development and progression. The signatures correctly predicted patient outcome in independent gene expression data sets with some notably low misclassification rates, particularly among studies involving more advanced-stage tumors. This successful cross-validation indicates that gene expression analysis-based signatures are becoming translationally relevant to care of melanoma patients, as well as improving understanding of the aspects of melanoma biology that determine patient outcome.

Long-Term Outcomes of Treatment of Neovascular Age-Related Macular Degeneration: Data from an Observational Study

PURPOSE To analyze the long-term outcomes of eyes with neovascular age-related macular degeneration (AMD) starting treatment with vascular endothelial growth factor (VEGF) inhibitors at least 5 years earlier. DESIGN Database observational study. PARTICIPANTS Treatment-naïve eyes with neovascular AMD tracked by the Fight Retinal Blindness outcome registry that received at least 1 anti-VEGF injection. METHODS Locally weighted scatterplot smoothing curves were used to display visual acuity (VA) results. MAIN OUTCOME MEASURES Change in mean VA and number of injections and visits from baseline up to 7 years after initiating treatment. RESULTS The mean follow-up time of all 1212 identified eyes was 53.5 months, and 549 (45%) continued attending after 60 months. Mean VA improved from 55.1 to 61.4 letters after 6 months and remained above the mean presenting VA for approximately 6 years. After 7 years, mean VA was 2.6 letters lower than baseline for the 131 eyes still being followed; 40% had VA ≥70 (20/40) letters, and 18% had VA ≤35 letters (20/200). Of those with 20/40 VA before treatment, 40% had lost it after 7 years. Geographic atrophy affecting the fovea was thought to be the cause of a ≥10-letter loss after 6.5 years in 37% of a subset of such eyes that were retrospectively analyzed. A median of 6 injections and 9 visits were recorded over the first 12 months, and then 5 treatments and 7 to 9 visits per annum thereafter through 7 years. Treatment was discontinued for 663 eyes (53%) within the first 5 years. Despite initial gains in vision, the mean VA of these eyes had deteriorated to baseline or worse around the time treatment was discontinued. The rate of serious adverse events was low. CONCLUSIONS Good long-term outcomes of VEGF inhibition for neovascular AMD were found in this study. These results may be better than other reports because more injections were given to our patients, possibly associated with a greater incentive for the physician to treat. Further studies to determine how to maximize the proportion of eyes that retain the initial VA gains of anti-VEGF are warranted.

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.

Genes Influencing Circadian Differences in Blood Pressure in Hypertensive Mice

Essential hypertension is a common multifactorial heritable condition in which increased sympathetic outflow from the central nervous system is involved in the elevation in blood pressure (BP), as well as the exaggerated morning surge in BP that is a risk factor for myocardial infarction and stroke in hypertensive patients. The Schlager BPH/2J mouse is a genetic model of hypertension in which increased sympathetic outflow from the hypothalamus has an important etiological role in the elevation of BP. Schlager hypertensive mice exhibit a large variation in BP between the active and inactive periods of the day, and also show a morning surge in BP. To investigate the genes responsible for the circadian variation in BP in hypertension, hypothalamic tissue was collected from BPH/2J and normotensive BPN/3J mice at the ‘peak’ (n = 12) and ‘trough’ (n = 6) of diurnal BP. Using Affymetrix GeneChip® Mouse Gene 1.0 ST Arrays, validation by quantitative real-time PCR and a statistical method that adjusted for clock genes, we identified 212 hypothalamic genes whose expression differed between ‘peak’ and ‘trough’ BP in the hypertensive strain. These included genes with known roles in BP regulation, such as vasopressin, oxytocin and thyrotropin releasing hormone, as well as genes not recognized previously as regulators of BP, including chemokine (C-C motif) ligand 19, hypocretin and zinc finger and BTB domain containing 16. Gene ontology analysis showed an enrichment of terms for inflammatory response, mitochondrial proton-transporting ATP synthase complex, structural constituent of ribosome, amongst others. In conclusion, we have identified genes whose expression differs between the peak and trough of 24-hour circadian BP in BPH/2J mice, pointing to mechanisms responsible for diurnal variation in BP. The findings may assist in the elucidation of the mechanism for the morning surge in BP in essential hypertension.