Differential post-transcriptional regulation of COX-2 expression in human umbilical vein endothelial cells derived from diabetic and healthy women: role of microR-As

COX-2 is one of the vasoprotective genes up-regulated by steady laminar shear stress (Topper et al. 1996; Di Francesco et al. 2009) and produces the vasoprotective prostacyclin (Grosser et al. 2006; Di Francesco et al. 2009). Early investigations have shown that diabetes markedly alters prostanoid synthesis in the vasculature (Bagi et al. 2006) but studies on the potential involvement of COX-2 in diabetic vascular complications have given controversial results. The aim of this study was to investigate whether the exposure to a diabetic environment in vivo could affect the regulation of endothelial COX-2 expression through post-transcriptional mechanisms. Thus, we compared COX-2 expression in human umbilical vein endothelial cells derived from normal (nHUVEC) and type I diabetic mothers (dHUVEC). Confluent monolayers of cells at passage level 3 were treated with or without IL-1β 5 ng/ml for 6 or 24 h in medium 199/DMEM (50:50) supplemented with ECGF, pen-strep, glutamine, and 5% FCS. The medium was assayed for 6-keto-PGF1α, PGE2, PGF2α, PGD2 by radioimmunoassay or ELISA, cell lysates for COX-1, COX-2, microsomal PGES-1 (mPGES-1), prostacyclin synthase (PGIS), and heme oxygenase (HO)-1 by specific Western blot techniques. RNAs were extracted and analysed for COX-2, microRNA(miR)542-3p, and miR16 by real time-PCR. In dHUVEC there was a statistically significant increase in the biosynthesis of 6-keto-PGF1α, PGE2, and PGD2, but not PGF2α, in response to IL-1β vs nHUVEC. The values of prostanoid generation are reported in the table. Prostanoid levels measured in nHUVEC and dHUVEC after IL-1β (5 ng/ml) for 6 and 24 h (values are reported as ng, mean±SEM, n=5-7; *P<0.05 vs nHUVEC) 6-keto-PGF1α PGE2 PGF2α PGD2 6 h 24 h 6 h 24 h 6 h 24 h 6 h 24 h nHUVEC 2.8 ±0.5 13.7±2.1 3.0±0.7 9.2±3.0 22.7±6.9 122.4±29.8 0.6±0.1 1.1±0.3 dHUVEC 9.9±3.6* 37.7±10.5* 5.4±1.0 23.2±5.2* 35.2±14.7 132.1±54.6 0.8±0.3 2.4±0.7* In both types of HUVEC, prostanoids were generated by COX-2; in fact, pretreatment with 1 μM NS-398 (a selective inhibitor of COX-2) caused an almost complete inhibition of prostanoid biosynthesis. After IL-1β treatment, Western blot analysis showed that COX-1 and PGIS protein levels were comparable in nHUVEC and dHUVEC while mPGES-1 levels were undetectable both in nHUVEC and dHUVEC. The protein levels of COX-2 were significantly (P<0.05) higher in dHUVEC vs nHUVEC at 24 h (COX-2/β-actin optical density 2.30±0.50 vs 0.88±0.12, respectively). mRNA levels of COX-2 in response to IL-1β were significantly (P<0.01) higher in dHUVEC than in nHUVEC (17.98±1.50 vs 5.91±0.60 at 6 h; 9.16±0.60 vs 3.46±0.70 at 24 h, respectively). Experiments of mRNA stability performed in the presence of actynomycin D (0.65 μg/ml), to inhibit transcription, demonstrated that COX-2 mRNA was more stable in dHUVEC than in nHUVEC after IL-1β stimulation; in fact, at 3h there was 30% vs 13% of COX-2 mRNA remaining in dHUVEC and nHUVEC, respectively (P<0.01). We analysed the levels of two different miRNAs involved in the destabilization of COX-2 mRNA through their binding in the 3’UTR: miR542-3p and miR16. In nHUVEC at 24 h with IL-1β, miR542-3p and miR16 levels were significantly (P<0.05) increased vs unstimulated cells (relative miRNA levels: miR542-3p, 2.2±0.8 vs 1.00±0.02; miRNA 16, 1.71±0.26 vs 1.04±0.02, respectively). On the contrary, in dHUVEC stimulated with IL-1β for 24 h, miR542-3p and miR16 levels were lower than in unstimulated cells. Interestingly, miR542-3p and miR16 levels were lower in dHUVEC vs nHUVEC, at 24 h with IL1β (0.53±0.20 and 0.74±0.20, respectively; P<0.05). HO-1 expression was higher (P<0.01) in HUVEC from diabetic than healthy women, in response to IL-1β and NS- 398 reduced HO-1 levels both in nHUVEC and dHUVEC. In summary, IL-1β induces COX-2-dependent prostanoids in HUVEC. Prostacyclin is a dominant autocrine prostanoid. HUVEC exposed to a diabetic environment express higher levels of COX-2 through post-transcriptional mechanisms. Loss of IL-1β- dependent inducibility of miRNA-542-3p and miR-16 in dHUVECs is associated with enhanced COX-2 whose expression was associated with higher levels of HO-1. In conclusion, targeting of miRNAs in endothelial cells may represent a new therapeutic strategy to modulate COX-2-dependent prostacyclin. References Topper JN et al. (1996) Proc Natl Acad Sci U.S.A. 93:10417 Di Francesco L et al. (2009) Circ Res 104:506 Grosser T et al. (2006) J Clin Invest 116:4 Bagi Z et al. (2006) Pharmacol Rep 58 Suppl:52