ABSTRACT
Title
Antioxidant role of prostacyclin in human umbilical vein endothelial cells (HUVEC) from healthy and diabetic women in response to IL-1β
Authors
E. Marcantoni
Dottorato in Scienze Medico-chirurgiche, Cliniche e Sperimentali
Dipartimento di Medicina e Scienze dell’Invecchiamento, Università “G. d’Annunzio”, Chieti
Dottorato in Scienze Medico-chirurgiche, Cliniche e Sperimentali
Dipartimento di Medicina e Scienze dell’Invecchiamento, Università “G. d’Annunzio”, Chieti
Abstract
Cyclooxygenase(COX)-2-dependent prostacyclin(PGI2) is an important pathway to limit the induction of a prothrombotic and proatherogenic phenotype in the endothelium(1). The induction of phase II genes(such as heme oxygenase-1,HO-1),a defense system against oxidative stress, is a key mechanism in the vasoprotective phenotype induced by PGI2(2). The aims of this study were: i)to evaluate the molecular mechanisms of COX-2 expression and PGI2 biosynthesis in HUVEC obtained from healthy and diabetic women in response to IL-1β ;ii)the impact of COX-2-dependent PGI2 inhibition on HO-1 expression in the same experimental conditions.
To realize these aims, we compared COX-2 and HO-1 expression in HUVEC derived from normal(n) and diabetic(d) women(type II diabetes mellitus) cultured under basal conditions or in the presence of IL-1β (5ng/ml) 24 hour. Cell culture medium was assayed for 6-keto-PGF1α (the hydrolysis product of PGI2) by RIA and cell lysates for COX-1, COX-2, microsomal PGES-1(mPGES-1), PGI2 synthase(PGIS) and HO-1 by Western blot. RNAs were extracted and analysed for COX-2 mRNA, microRNA(miR)16(short single-stranded non-coding RNA causing COX-2 mRNA instability) and c-Myc(a regulator of cell proliferation which represses miRNA expression (3)). Moreover, DNA synthesis assessed by [3H]thymidine incorporation was studied as an index of cell proliferation. NS-398(1μ M;a selective COX-2 inhibitor) and the PGI2 receptor(IP) antagonist RO3244794(10μ M) were used in some experiments.
In dHUVEC there was a statistically significant increase of the biosynthesis of PGI2, in response to IL-1β vs nHUVEC(37±10 and 14±2 ng, respectively,P<0.05). After IL-1β treatment, COX-1 and PGIS protein levels were comparable in nHUVEC and dHUVEC, while mPGES-1 levels were undetectable. The protein levels of COX-2 were significantly(P<0.05) higher in dHUVEC vs nHUVEC at 24h(COX-2/β-actin optical density 2.30±0.5 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(COX-2mRNA/GADPH ratio, fold increase:9.16±0.6 vs 3.46±0.7, respectively). Lower levels of COX-2 mRNA, detected in nHUVEC, were associated with the induction of miR16. In contrast, a loss of IL-1β dependent inducibility of miR-16 was detected, in dHUVECs, associated with enhanced COX-2 mRNA. Enhanced c-myc expression and cell proliferation were detected in dHUVEC vs nHUVEC, in response to IL-1β. In nHUVEC stimulated with IL-1β, NS-398 and IP antagonist caused a significant(P<0.05) reduction of HO-1 levels. In contrast, in dHUVEC the same compounds caused the induction of HO-1.
In summary, in nHUVEC, miR-16 is induced in response to IL-β to restrain COX-2 expression. In this scenario, PGI2-dependent induction of HO-1 causes an antioxidant/antiinflammatory effect.
In HUVEC exposed to a diabetic milieu, enhanced c-myc expression represses miR16 induction in response to IL-1β. This translates into overexpression of COX-2 and enhanced PGI2 may counter oxidative stress. In this setting, increased HO-1 levels in the presence of the inhibition of COX-2-dependent PGI2 or IP receptor blockage is presumably a consequence of enhanced oxidative stress.
In conclusion, dysregulated expression of miRNAs may influence COX-2 expression by posttranscriptional mechanisms. In endothelial cells, in the presence of inflammation and oxidative stress, c-myc-dependent repression of miR16 may translate into overexpression of COX-2-dependent PGI2 which may play a role to mitigate oxidative-stress. However, in inflammatory or cancer cells this phenomenon may be associated with pathological consequences, since the major product of COX-2 is PGE2, an inflammatory and tunorigenic mediator.
References:
Grosser et al. (2006) JCI 116: 4-15
Di Francesco et al. (2009) Circ Res104:506-513
Chang et al. (2008) Nature Gen 40 :43-50
To realize these aims, we compared COX-2 and HO-1 expression in HUVEC derived from normal(n) and diabetic(d) women(type II diabetes mellitus) cultured under basal conditions or in the presence of IL-1β (5ng/ml) 24 hour. Cell culture medium was assayed for 6-keto-PGF1α (the hydrolysis product of PGI2) by RIA and cell lysates for COX-1, COX-2, microsomal PGES-1(mPGES-1), PGI2 synthase(PGIS) and HO-1 by Western blot. RNAs were extracted and analysed for COX-2 mRNA, microRNA(miR)16(short single-stranded non-coding RNA causing COX-2 mRNA instability) and c-Myc(a regulator of cell proliferation which represses miRNA expression (3)). Moreover, DNA synthesis assessed by [3H]thymidine incorporation was studied as an index of cell proliferation. NS-398(1μ M;a selective COX-2 inhibitor) and the PGI2 receptor(IP) antagonist RO3244794(10μ M) were used in some experiments.
In dHUVEC there was a statistically significant increase of the biosynthesis of PGI2, in response to IL-1β vs nHUVEC(37±10 and 14±2 ng, respectively,P<0.05). After IL-1β treatment, COX-1 and PGIS protein levels were comparable in nHUVEC and dHUVEC, while mPGES-1 levels were undetectable. The protein levels of COX-2 were significantly(P<0.05) higher in dHUVEC vs nHUVEC at 24h(COX-2/β-actin optical density 2.30±0.5 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(COX-2mRNA/GADPH ratio, fold increase:9.16±0.6 vs 3.46±0.7, respectively). Lower levels of COX-2 mRNA, detected in nHUVEC, were associated with the induction of miR16. In contrast, a loss of IL-1β dependent inducibility of miR-16 was detected, in dHUVECs, associated with enhanced COX-2 mRNA. Enhanced c-myc expression and cell proliferation were detected in dHUVEC vs nHUVEC, in response to IL-1β. In nHUVEC stimulated with IL-1β, NS-398 and IP antagonist caused a significant(P<0.05) reduction of HO-1 levels. In contrast, in dHUVEC the same compounds caused the induction of HO-1.
In summary, in nHUVEC, miR-16 is induced in response to IL-β to restrain COX-2 expression. In this scenario, PGI2-dependent induction of HO-1 causes an antioxidant/antiinflammatory effect.
In HUVEC exposed to a diabetic milieu, enhanced c-myc expression represses miR16 induction in response to IL-1β. This translates into overexpression of COX-2 and enhanced PGI2 may counter oxidative stress. In this setting, increased HO-1 levels in the presence of the inhibition of COX-2-dependent PGI2 or IP receptor blockage is presumably a consequence of enhanced oxidative stress.
In conclusion, dysregulated expression of miRNAs may influence COX-2 expression by posttranscriptional mechanisms. In endothelial cells, in the presence of inflammation and oxidative stress, c-myc-dependent repression of miR16 may translate into overexpression of COX-2-dependent PGI2 which may play a role to mitigate oxidative-stress. However, in inflammatory or cancer cells this phenomenon may be associated with pathological consequences, since the major product of COX-2 is PGE2, an inflammatory and tunorigenic mediator.
References:
Grosser et al. (2006) JCI 116: 4-15
Di Francesco et al. (2009) Circ Res104:506-513
Chang et al. (2008) Nature Gen 40 :43-50