A novel dual-fluorescence strategy for functionally validating microRNA targets in 3-prime untranslated regions: regulation of the inward rectifier potassium channel Kir2.1 by miR-212.

Gene targeting by microRNAs is important in health and disease. We developed a functional assay for identifying microRNA targets and applied it to the K+ channel Kir2.1 (KCNJ2) which is dysregulated in cardiac and vascular disorders. The 3'UTR was inserted downstream of the mCherry red fluorescent protein coding sequence in a mammalian expression plasmid. MicroRNA sequences were inserted into the pSM30 expression vector which provides enhanced green fluorescent protein as an indicator of microRNA expression. HEK293 cells were co-transfected with the mCherry-3'UTR plasmid and a pSM30-based plasmid with a microRNA insert. The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis. The method was validated with miR-1, a known downregulator of Kir2.1 expression, and was used to investigate targeting of the Kir2.1 3'UTR by miR-212. Red/green ratio was lower in miR-212-expressing cells compared to non-targeting controls, an effect that was attenuated by mutating the predicted target site. MiR-212 also reduced inward rectifier current and Kir2.1 protein in HeLa cells. This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.

eNOS overexpression exacerbates vascular closure in the obliterative phase of OIR and increases angiogenic drive in the subsequent proliferative stage

Purpose: In ischemic retinopathies, the misdirection of reparative angiogenesis away from the hypoxic retina leads to pathologic neovascularization. Thus, therapeutic strategies that reverse this trend would be extremely beneficial. Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is an important mediator of vascular endothelial growth factor (VEGF) function facilitating vascular growth and maturation. However, in addition to NO, eNOS can also produce superoxide (O), exacerbating pathology. Here, our aim was to investigate the effect of eNOS overexpression on vascular closure and subsequent recovery of the ischemic retina.

Methods: Mice overexpressing eNOS-GFP were subjected to oxygen-induced retinopathy (OIR) and changes in retinal vascularization quantified. Background angiogenic drive was assessed during vascular development and in aortic rings. NOS activity was measured by Griess assay or conversion of radiolabeled arginine to citrulline, nitrotyrosine (NT), and superoxide by immunolabeling and dihydroethidium fluorescence and VEGF by ELISA.

Results: In response to hyperoxia, enhanced eNOS expression led to increased NOS-derived superoxide and dysfunctional NO production, NT accumulation, and exacerbated vessel closure associated with tetrahydrobiopterin (BH) insufficiency. Despite worse vaso-obliteration, eNOS overexpression resulted in elevated hypoxia-induced angiogenic drive, independent of VEGF production. This correlated with increased vascular branching similar to that observed in isolated aortas and during development. Enhanced recovery was also associated with neovascular tuft formation, which showed defective NO production and increased eNOS-derived superoxide and NT levels.

Conclusions: In hyperoxia, reduced BH bioavailability causes overexpressed eNOS to become dysfunctional, exacerbating vaso-obliteration. In the proliferative phase, however, eNOS has important prorepair functions enhancing angiogenic growth potential and recovery in ischemia. © 2012 The Association for Research in Vision and Ophthalmology, Inc.

Intra-uterine growth restriction and increased risk of hypertension in adult life: A follow-up study of 50-year-olds

OBJECTIVE:

To compare blood pressure between 50-year-old adults who were born at term (37-42 weeks of gestation) with intra-uterine growth restriction (IUGR; birth weight <10th centile) and a control group of similar age born at term without IUGR (birth weight =10th centile).

STUDY DESIGN:

Controlled comparative study.

METHODS:

Participants included 232 men and women who were born at the Royal Maternity Hospital, Belfast, a large regional maternity hospital in Northern Ireland, between 1954 and 1956. One hundred and eight subjects who were born with IUGR were compared with 124 controls with normal birth weight for gestation. The main outcome measures were systolic and diastolic blood pressure at approximately 50 years of age, measured according to European recommendations.

RESULTS:

The IUGR group had higher systolic and diastolic blood pressure than the control group: 131.5 [95% confidence interval (CI) 127.9-135.1] vs 127.1 (95% CI 124.3-129.2) mmHg and 82.3 (95% CI 79.6-85.0) vs 79.0 (95% CI 77.0-81.0) mmHg, respectively. After adjustment for gender, the differences between the groups were statistically significant: systolic blood pressure 4.5 (95% CI 0.3-8.7) mmHg and diastolic blood pressure 3.4 (95% CI 0.2-6.5) mmHg (both P < 0.05). More participants in the IUGR group were receiving treatment for high blood pressure compared with the control group [16 (15%) vs 11 (9%)], although this was not statistically significant. The proportion of subjects with blood pressure >140/90 mmHg or currently receiving antihypertensive treatment was 45% (n = 49) for the IUGR group, and 31% (n = 38) for the control group (odds ratio 1.9, 95% CI 1.1-3.3). Adjustment for potential confounders made little difference.

CONCLUSIONS:

IUGR is associated with higher blood pressure at 50 years of age. Individuals born with IUGR should have regular blood pressure screening and early treatment as required. Hypertension remains underdiagnosed and undertreated in adult life.